Inflammation-responsive hydrogel microspheres, and preparation method and application thereof

Abstract

The application discloses an inflammation-responsive hydrogel microsphere and a preparation method and application thereof, and particularly relates to the technical field of medical materials.The method comprises the following steps: firstly, mixing a natural macromolecule and an inflammation-responsive group crosslinking agent according to a proportion to obtain an aqueous phase W; then, mixing mineral oil and an amphoteric molecule, i.e., Span 80, according to a volume ratio to obtain an oil phase O; stirring and mixing the aqueous phase W and the oil phase O according to a proportion to obtain a mixed solution C; washing the mixed solution C with isopropyl alcohol, centrifuging, discarding a supernatant, and obtaining precipitated microspheres; finally, soaking the microspheres obtained in the above step in a drug solution, and then transferring to a freeze dryer to completely freeze dry to obtain the inflammation-responsive hydrogel microspheres.The hydrogel microspheres are crosslinked by the inflammation-responsive group inside, the crosslinking degree of the related groups is limited, appropriate responsive rupture occurs in the physiological environment of inflammation, the microspheres are damaged, and the drug is further released intelligently.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to an inflammation-responsive hydrogel microsphere and a preparation method and application thereof. BACKGROUND

[0002] Inflammation is a pathological process mainly for defense in response to external stimuli and damage, and its occurrence and development involve multiple regulation mechanisms. When physical / chemical / biological damage / abnormal immune response causes tissue necrosis, the immune system is activated and up-regulates the expression of NF-κB through the Toll-like receptor (TLR4 / MyD88 signaling pathway), leading to the cascade release of pro-inflammatory factors such as TNF-α and IL-6, and at the same time, NADPH oxidase is activated to produce excessive ROS, causing oxidative stress damage and leading to related pathological manifestations such as redness, swelling, pain, and tissue exudate. Inflammation can be divided into acute inflammation and chronic inflammation according to the residence time. Acute inflammation can gradually heal and subside with the body's own immunity, while chronic inflammation persists and causes significant economic pressure and mental burden to patients.

[0003] At present, drug treatment is still one of the main ways to treat inflammation, and direct drug delivery to the inflammation site by injection, dropwise addition, etc. has the risks of low drug use efficiency, burst release, high concentration side effects and toxicity, etc. Based on this, various drug delivery systems have been developed in clinical practice, such as sponges, gauze, hydrogels, powders, etc. These dressings have their own advantages, but also have unavoidable defects, one of which is the inability to intelligently release drugs according to the changes in the inflammatory environment, and the inability to meet the needs of in vivo and in vitro use. Based on this, various inflammation-responsive hydrogels have been developed and designed, which can intelligently release drugs according to the in vivo and in vitro inflammatory environment by adding inflammation-responsive groups or physical structures, prolonging the drug action time and improving the drug efficacy. However, these hydrogel dressings cannot well absorb the exudate in some high-osmotic liquid inflammatory environments, and also have the risk of hindering subsequent healing.

[0004] In addition, biodegradability and biotoxicity are also important factors that need to be considered for dressings. Traditional dressings prepared from synthetic macromolecules have the risk of foreign body reaction caused by long-term degradation or difficulty in degradation, further exacerbating inflammation, and the small molecular fragments after degradation also have potential physiological toxicity. Natural macromolecules have good biocompatibility with living organisms because they are derived from living organisms, which can effectively avoid physiological toxicity, and some natural macromolecules even have special physiological functions, such as hyaluronic acid has anti-inflammatory and proliferation-promoting functions, and dextran has collagen deposition-promoting functions. These special physiological functions provide assistance for further regulation of inflammation.

[0005] Chinese patent CN116808286A, a kind of inflammation responsive on-demand anti-inflammatory hydrogel medical dressing for skin wound healing and its preparation method, the patent is the patent of inventor in advance, the patent belongs to traditional hydrogel, and the hydrophobic drug needs to be modified to hydrophobic drug, and the process is complex;Traditional hydrogel has limited liquid absorption rate, and the inflammation responsive group in this patent is only redox group, which limits the range of inflammation responsive group. SUMMARY

[0006] To this end, the present application provides an inflammation responsive hydrogel microsphere and its preparation method and application to solve the problems in the prior art.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0008] According to the preparation method of the inflammation responsive hydrogel microsphere provided by the first aspect of the present application, the method comprises:

[0009] Step one, preparation of aqueous phase

[0010] After mixing natural macromolecule, inflammation responsive group crosslinking agent in proportion, mixed aqueous solution is obtained, which is denoted as aqueous phase W;

[0011] Step two, preparation of oil phase

[0012] Mix mineral oil and amphoteric molecule Span 80 in proportion to obtain oil phase O;

[0013] Step three, preparation of microspheres

[0014] Mix the aqueous phase W and the oil phase O in proportion, and obtain the mixed solution C;

[0015] After washing with isopropyl alcohol, centrifugation, discarding the supernatant, the precipitated microspheres are obtained;

[0016] Step four, drug loading

[0017] The microspheres obtained in step three are soaked in the drug solution, and then transferred to the freeze dryer for complete freeze-drying to obtain the inflammation responsive hydrogel microspheres.

[0018] Further, in the step one, the concentration of natural macromolecule is 1-5 wt%, and the molecular weight ratio of natural macromolecule to inflammation responsive group crosslinking agent is 1:0.5-1:2.

[0019] Further, the natural macromolecule includes one or more of starch, cellulose, hyaluronic acid, dextran, chitosan, alginic acid, agarose, carrageenan, heparin, chondroitin sulfate, chitin, pectin, gelatin, collagen, albumin, silk fibroin.

[0020] Further, the inflammation-responsive group crosslinking agent comprises one or more of redox groups, matrix metalloproteinase-responsive groups, and pH-responsive groups; wherein the redox groups comprise one or more of diselenide bonds, disulfide bonds, phenylboronic acid ester bonds, thioether bonds, oxalic acid polyesters, acylpyrrolidine-2-carboxamide groups, ferrocene-cyclodextrin self-assembly structure groups, ketone thiol, and concanavalin A structure; the matrix metalloproteinase-responsive groups comprise one or more of matrix metalloproteinase-responsive peptide chains and gelatin structures; and the pH-responsive groups comprise one or more of phenylboronic acid ester bonds, Schiff base bonds, pH-responsive peptide chains, and glycyrrhizic acid self-assembly structures.

[0021] Further, in the second step, the volume ratio of the mineral oil to the amphoteric molecule Span 80 is 1:50-1:100.

[0022] Further, in the third step, the volume ratio of the aqueous phase W to the oil phase O is 1:3-1:10; the stirring speed is 150-500 rpm; and the reaction time is 6-24 h.

[0023] Further, in the third step, the volume ratio of the mixed solution C to isopropyl alcohol is 1:50-1:100; the centrifugation condition is a rotation speed of 1000-2500 rpm and a centrifugation time of 3-10 min.

[0024] Further, in the fourth step, the mass ratio of the microspheres to the drug solution is 1:50-1:200; the drug concentration is 5-20 wt%; the drug solution is a chemical drug or a cytokine; and the freeze-drying machine temperature is -20--60℃.

[0025] Further, in order to improve the reaction between the crosslinking agent and the backbone molecule, one or more of the activators 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride hydrochloride can be added to promote the reaction.

[0026] As an example, the chemical drug comprises one or more of gallic acid, epigallocatechin gallate, astragaloside IV, asiaticoside, hydroxyasiaticoside, triamcinolone acetonide, berberine, curcumin, tea polyphenol, rhein, glycyrrhizic acid, quercetin, myricetin, dihydroquercetin, tannic acid, polydopamine, shikonin, lidocaine, and amoxicillin; the cytokine comprises one or more of matrix metalloproteinase inhibitors, interleukins, vascular endothelial growth factors, insulin, vascular endothelial growth factors, lipoxins, amino acids, polypeptides, and cell exosomes; and the cumulative release amount of the drug within 24 h is 50%-91%.

[0027] According to a second aspect of the present invention, the inflammatory-responsive hydrogel microspheres prepared by the method described above are characterized in that the hydrogel microspheres use natural macromolecules as the microsphere skeleton, are cross-linked through inflammatory-responsive groups, and are internally loaded with anti-inflammatory drugs; the average diameter of the hydrogel microspheres is 10–500 micrometers, the drug loading rate is 30%–80%, the degree of cross-linking is 50%–100%, and the degradation time is 3–14 days. The inflammatory-responsive hydrogel microspheres can absorb liquids and undergo responsive destruction in response to inflammation-related physiological factors, thereby releasing the internal drugs and further cross-linking to form macroscopic hydrogels. The liquids that the inflammatory-responsive hydrogel microspheres of the present invention can absorb include one or more of aqueous solutions, wound exudates, subcutaneous exudates, and blood; the mass of the absorbed liquid is 1–5 times the bulk mass.

[0028] The mass of the hydrogel formed after the inflammatory responsive hydrogel microspheres of the present invention is 50-500 times that of the initial microspheres after liquid absorption and destruction, the maximum deformation is 300%-1000%, the modulus is 100-3000 Pascals, and the in vivo degradation time is 3-14 days.

[0029] The inflammatory responsive hydrogel microspheres of the present invention undergo responsive destruction through one or more of the following mechanisms: chemical bond breaking, molecular chain rearrangement, and microsphere structure deformation.

[0030] According to a third aspect of the present invention, the application of the aforementioned inflammatory-responsive hydrogel microspheres in the preparation of a medicament for treating and healing inflammatory wounds is provided. The inflammatory wounds include skin surface wounds or wounds at sites of inflammation within the body. Physiological factors associated with inflammation include one or more of the following: reactive oxygen species, matrix metalloproteinases, and pH within the organism.

[0031] The inflammatory-responsive hydrogel microspheres of the present invention can be directly applied to exposed wound environments in a freeze-dried state, or dispersed in a suspension and injected subcutaneously or into inflamed sites in the body, absorbing inflammatory fluid and continuously releasing drugs in response.

[0032] The present invention has the following advantages:

[0033] 1) The hydrogel microspheres of the present invention are cross-linked with inflammatory responsive groups. By limiting the degree of cross-linking of the relevant groups, appropriate responsive cleavage can occur in the physiological environment of inflammation, so that the microspheres are destroyed and the drug is released intelligently.

[0034] 2) The hydrogel microspheres of the present invention are in the form of freeze-dried microspheres, which can absorb exudate from the inflamed site and reduce the retention of inflammatory exudate in the inflamed site.

[0035] 3) The hydrogel microspheres of the present invention can expand after inflammatory response disruption to further absorb inflammatory exudate. At the same time, after expansion, they can cross-link to form macro-hydrogels to protect the inflammatory site.

[0036] 4) The framework of the hydrogel microspheres of the present invention is a natural macromolecule, which has good biocompatibility and biodegradability, avoiding foreign body reactions and degradation toxicity caused by traditional macromolecular microspheres.

[0037] 5) The hydrogel microspheres of the present invention can adjust the drug release rate according to the strength of the reaction at the inflammatory site, so as to realize intelligent drug release. They can not inhibit the normal inflammatory response, but can also release anti-inflammatory drugs when the inflammatory response is strong, so as to exert the anti-inflammatory function.

[0038] 6) The hydrogel microspheres of the present invention can completely fill the gaps of the wound through injection and other methods, achieve contact with the wound surface, absorb the exudate at the wound, and keep the wound moisturized for a long time. They also have good mechanical properties, meeting the needs of hydrogel dressings in the wound healing process.

[0039] 7) The product formed by the hydrogel microspheres of the present invention after absorbing inflammatory exudate and being destroyed is a hydrogel with good self-healing properties and suitable mechanical modulus. It can effectively cover the wound inflammatory environment and meet the requirements for use in dynamic physiological environments.

[0040] 8) The hydrogel microspheres of the present invention can intelligently regulate the release of drugs, avoiding the drug toxicity and low efficiency caused by direct dripping or injection of drugs into the in vitro and in vivo inflammatory environment, while effectively prolonging the drug release time and reducing the pain caused to patients when changing dressings.

[0041] 9) The hydrogel microspheres of the present invention do not require modification of the drug or limitation of the drug properties, and the hydrogel microspheres of the present invention can further broaden the scope of use of inflammatory responsive groups. Attached Figure Description

[0042] 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.

[0043] 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.

[0044] Figure 1 Scanning electron microscope photos of inflammation-responsive hydrogel microspheres provided for Example 1 of the present application;

[0045] Figure 2 Photos of the process of in vitro simulation of swelling and cross-linking to form hydrogels by absorbing inflammatory exudates provided for Experimental Example 1 of the present application;

[0046] Figure 3 Photos of in vitro simulation of swelling and cross-linking by absorbing inflammatory exudates taken by a polarizing microscope provided for Experimental Example 2 of the present application;

[0047] Figure 4 Photos taken after staining by a cell activity and cytotoxicity detection kit after co-culturing Example 1 and human keratinocytes for 24 hours provided for Experimental Example 5 of the present application;

[0048] Figure 5 Photos of Example 1 on the 10th day of treatment of full-thickness wounds on the back of diabetic mice provided for Experimental Example 6 of the present application. DETAILED DESCRIPTION

[0049] The embodiments of the present application will be described in detail by the following specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the contents disclosed in the specification. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0050] Example 1

[0051] In this embodiment, hyaluronic acid is prepared as a skeleton, a redox group diselenide bond is prepared as an inflammation-responsive group, and a hydrogel microsphere delivering gallic acid is prepared, with an average particle size of 80 microns and a drug loading rate of 43%.

[0052] The specific steps are as follows:

[0053] S1. Prepare a 1.5 wt% hyaluronic acid solution, and then prepare the aqueous phase w by dissolving and mixing according to a molar ratio of hyaluronic acid (CAS No. 9004-61-9): selenocystamine (CAS No. 2697-61-2): activator (1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimide, CAS No. 1892-57-5) = 1:0.5:1;

[0054] S2. Mix mineral oil (CAS No. 8042-47-5) and amphoteric molecule Span 80 (CAS No. 1338-43-8) uniformly according to a volume ratio of 1:80 to obtain the oil phase o;

[0055] S3. Mix the water phase w and the oil phase o with a volume ratio of 20:80 to obtain a mixed solution c, the stirring speed is 300 rpm, and the reaction time is 12 h;

[0056] S4. After the reaction is completed, the mixed solution c is mixed with isopropyl alcohol (CAS No.: 67-63-0) at a volume ratio of 1:100, washed, and then transferred to a container for centrifugation at a speed of 1500 rpm for 3 min, the liquid is discarded, and the precipitated microspheres are retained;

[0057] S5. The centrifuged microspheres are soaked in a gallic acid (CAS No.: 149-91-7) solution with a mass ratio of 1:50, and then transferred to a freeze dryer for complete freeze-drying to obtain inflammation-responsive hydrogel microspheres, wherein the concentration of the gallic acid solution is 10wt%, and the temperature of the freeze dryer is -55°C;

[0058] S6. The freeze-dried microspheres are screened with a screen mesh, wherein the upper limit of the hole size of the screen mesh is 150 microns, and the lower limit is 50 microns.

[0059] The scanning electron microscope photograph of the inflammation-responsive hydrogel microspheres is shown in Figure 1 As can be seen from the photograph, Figure 1 The surface of the microspheres is rough and porous, which helps the microspheres to absorb exudate, and the size distribution of the microspheres is uniform, indicating that the preparation method is relatively stable and has potential for large-scale application.

[0060] Example 2

[0061] In this example, hydrogel microspheres are prepared with hyaluronic acid as the skeleton, diselenide bond as the inflammation-responsive group, and gallic acid as the delivery agent, with an average particle size of 280 microns and a drug loading rate of 55%.

[0062] The specific preparation steps are as follows:

[0063] Steps S1-S5 are the same as in Example 1;

[0064] S6. The freeze-dried microspheres are screened with a screen mesh, wherein the lower limit of the hole size of the screen mesh is 220 microns, and the upper limit is 330 microns.

[0065] Example 3

[0066] In this example, hydrogel microspheres are prepared with gelatin as the skeleton, which responds to matrix metalloproteinase by itself structure, and deliver tea polyphenols (CAS No.: 84650-60-2), with an average particle size of 250 microns and a drug loading rate of 35%.

[0067] The specific steps are as follows:

[0068] S1. Prepare a 2 wt% gelatin (CAS No: 9000-70-8) solution, then heat the solution to 40°C to completely dissolve the gelatin to prepare the aqueous phase w;

[0069] S2. Mix the mineral oil and the amphoteric molecule Span 80 uniformly at a volume ratio of 1.5:75 to obtain the oil phase o in a 40°C environment;

[0070] S3. Mix the aqueous phase w and the oil phase o at a volume ratio of 25:75 in a 40°C environment to obtain the mixed solution c, the stirring speed is 350 rpm, and the reaction time is 6 h;

[0071] S4. Cool the reaction to room temperature, after the reaction is completed, mix the mixed solution c with isopropyl alcohol at a volume ratio of 1:150, then transfer to a container for centrifugation, the speed is 1500 rpm, the centrifugation time is 5 min, discard the liquid, and retain the precipitated microspheres;

[0072] S5. Soak the centrifuged microspheres in a tea polyphenol solution with a mass ratio of 1:80, then transfer to a freeze dryer for complete freeze-drying to obtain the inflammation-responsive hydrogel microspheres, wherein the tea polyphenol solution concentration is 5 wt%, and the freeze dryer temperature is -50°C;

[0073] S6. Screen the freeze-dried microspheres with a screen, wherein the lower limit of the screen hole is 220 microns and the upper limit is 330 microns.

[0074] Example 4

[0075] This example prepares a hydrogel microsphere with hyaluronic acid and chitosan as the skeleton, relying on the Schiff base bond formed by the crosslinking of the aldehyde group of oxidized hyaluronic acid and the amino group on chitosan to construct a pH-responsive group, and delivering myricetin (CAS No: 529-44-2), with an average particle size of 120 microns and a drug loading rate of 30%.

[0076] The specific steps are as follows:

[0077] S1. Prepare a 2 wt% hyaluronic acid solution and a 2 wt% chitosan (CAS No: 9012-76-4) solution, then mix them at a volume ratio of 1:1 to prepare the aqueous phase w;

[0078] S2. Mix the mineral oil and the amphoteric molecule Span 80 uniformly at a volume ratio of 1:80 to obtain the oil phase o;

[0079] S3. Mix the mixed aqueous phase w and the oil phase o at a volume ratio of 20:80 to obtain the mixed solution c, the stirring speed is 250 rpm, and the reaction time is 6 h;

[0080] S4. After the reaction is completed, the mixed solution c is mixed with isopropyl alcohol at a volume ratio of 1:150 for washing, and then transferred to a container for centrifugation at a speed of 1500 rpm for 6 min, the liquid is discarded, and the precipitated microspheres are retained;

[0081] S5. The centrifuged microspheres are soaked in a myricetin solution at a mass ratio of 1:60, and then transferred to a freeze dryer for complete freeze-drying to obtain the inflammation-responsive hydrogel microspheres, wherein the concentration of the myricetin solution is 5 wt%, and the temperature of the freeze dryer is -55°C;

[0082] S6. The freeze-dried microspheres are screened by a screen mesh, wherein the upper limit of the screen mesh hole is 150 microns, and the lower limit is 50 microns.

[0083] Example 5

[0084] In this example, hyaluronic acid is prepared as a skeleton, and the pH response of the self-assembled structure of glycyrrhizic acid is used to deliver glycyrrhizic acid. The average particle size of the hydrogel microspheres is 210 microns, and the drug loading rate is 47%.

[0085] The specific steps are as follows:

[0086] S1. A mixed solution of 2 wt% hyaluronic acid and 10 wt% glycyrrhizic acid (CAS No.: 1405-86-3) is prepared, and then the solution is heated to 60°C to completely dissolve the glycyrrhizic acid to prepare an aqueous phase w;

[0087] S2. In a 60°C environment, mineral oil and amphoteric molecule Span 80 are mixed at a volume ratio of 1:80 to obtain an oil phase o;

[0088] S3. In a 60°C environment, the aqueous phase w and the oil phase o are mixed at a volume ratio of 20:80 to obtain a mixed solution c, and the stirring speed is 300 rpm, and the reaction time is 12 h;

[0089] S4. After the reaction is completed, the mixed solution c is mixed with isopropyl alcohol at a volume ratio of 1:100 for washing, and then transferred to a container for centrifugation at a speed of 1000 rpm for 3 min, the liquid is discarded, and the precipitated microspheres are retained;

[0090] S5. The centrifuged microspheres are soaked in a tea polyphenol solution at a mass ratio of 1:80, and then transferred to a freeze dryer for complete freeze-drying to obtain the inflammation-responsive hydrogel microspheres, wherein the concentration of the tea polyphenol solution is 5 wt%, and the temperature of the freeze dryer is -50°C;

[0091] S6. The freeze-dried microspheres are screened by a screen mesh, wherein the lower limit of the screen mesh hole is 220 microns, and the upper limit is 330 microns.

[0092] Example 6

[0093] This embodiment uses hyaluronic acid as the backbone, and grafts ferrocene structure (CAS No.: 12176-38-4) and cyclodextrin structure (CAS No.: 12619-70-4), relies on ferrocene-cyclodextrin self-assembly to construct an oxidation-responsive group, and delivers interleukin-10 (CAS No.: 453656-85-4) hydrogel microspheres with an average particle size of 350 microns and a drug loading rate of 35%.

[0094] The specific steps are as follows:

[0095] S1. Prepare a 4 wt% ferrocene structure and cyclodextrin structure hyaluronic acid solution, and then mix to prepare an aqueous phase w in a volume ratio of 1:1, wherein the detailed preparation steps are described in the previous Chinese patent CN116808286A;

[0096] S2. Mix mineral oil and amphoteric molecule Span 80 uniformly to obtain an oil phase o in a volume ratio of 4:80;

[0097] S3. Mix the mixed aqueous phase w and the oil phase o quickly to obtain a mixed liquid c in a volume ratio of 15:85, the stirring speed is 350 rpm, and the reaction time is 12 h;

[0098] S4. After the reaction is completed, the mixed liquid c is mixed with isopropanol in a volume ratio of 1:100 for washing, and then transferred to a container for centrifugation at a speed of 1000 rpm for 3 min, the liquid is discarded, and the precipitated microspheres are retained;

[0099] S5. The centrifuged microspheres are soaked in an interleukin-10 solution with a mass ratio of 1:40, and then transferred to a freeze dryer for complete freeze-drying to obtain inflammation-responsive hydrogel microspheres, wherein the interleukin solution has a concentration of 0.1 wt%, and the freeze dryer has a temperature of -55°C;

[0100] S6. The freeze-dried microspheres are screened with a screen mesh, wherein the upper limit of the screen mesh hole is 440 microns, and the lower limit is 330 microns.

[0101] Example 7

[0102] This embodiment uses oxidized hyaluronic acid as the backbone, and uses quercetin (CAS No.: 80460-73-7) modified by p-aminobenzoic acid as a pH-responsive and oxidation-responsive crosslinking agent with a phenylboronic acid ester bond as the responsive group, and delivers quercetin (CAS No.: 117-39-5) hydrogel microspheres with an average particle size of 250 microns and a drug loading rate of 65%.

[0103] The specific steps are as follows:

[0104] S1. Prepare a 2 wt% oxidized hyaluronic acid solution to obtain an aqueous phase w;

[0105] S2. Mix the mineral oil and the amphoteric molecule Span 80 uniformly according to a volume ratio of 1:80 to obtain an oil phase o;

[0106] S3. Mix the mixed water phase w and the oil phase o quickly according to a volume ratio of 20:80 to obtain a mixed liquid c, the stirring speed is 500 rpm, and the reaction time is 6 h;

[0107] S4. After the reaction is completed, the mixed liquid c is mixed with isopropanol according to a volume ratio of 1:100, and then transferred to a container for centrifugation at a speed of 1500 rpm for 3 min, the liquid is discarded, and the precipitated microspheres are retained;

[0108] S5. Mix quercetin and p-aminobenzoic acid to obtain a p-aminobenzoic acid modified quercetin solution, and then immerse the centrifuged microspheres in the modified quercetin solution at a mass ratio of 1:100, and then transfer to a freeze dryer for complete freeze-drying to obtain the inflammation-responsive hydrogel microspheres, wherein the concentration of the quercetin solution is 5 wt%, and the temperature of the freeze dryer is -55°C;

[0109] S6. Screen the freeze-dried microspheres with a screen, wherein the upper limit of the screen hole is 330 microns, and the lower limit is 220 microns.

[0110] Comparative Example 1

[0111] The comparative example prepared hyaluronic acid as the skeleton, redox group diselenide bond as the inflammation-responsive group, and gallic acid delivery hydrogel microspheres, the average particle size is 550 microns, and the drug loading rate is 3%.

[0112] The specific preparation steps are as follows:

[0113] S1-S5 steps are the same as Example 1;

[0114] S6. Screen the freeze-dried microspheres with a screen, wherein the lower limit of the screen hole is 500 microns, and the upper limit is 650 microns.

[0115] Comparative Example 2

[0116] The comparative example prepared hyaluronic acid as the skeleton, redox group diselenide bond as the inflammation-responsive group, and gallic acid delivery hydrogel microspheres, the average particle size is 5 microns, and the drug loading rate is 62%. The specific preparation steps are as follows:

[0117] S1-S5 steps are the same as Example 1;

[0118] S6. Screen the freeze-dried microspheres with a screen, wherein the upper limit of the screen hole is 10 microns.

[0119] Comparative Example 3

[0120] The comparative example prepared a hydrogel microsphere with polyacrylic acid as the skeleton, a redox group diselenium bond as the inflammation-responsive group, and gallic acid as the delivery. The average particle size of the microsphere was 95 microns, and the drug loading rate was 15%. The specific preparation steps are as follows:

[0121] Steps S1-S6 are the same as Example 1, except that the hyaluronic acid is replaced with a polyacrylic acid solution.

[0122] Comparative Example 4

[0123] This example prepared a hydrogel microsphere with hyaluronic acid as the skeleton, a redox group diselenium bond as the inflammation-responsive group, and gallic acid as the delivery. The average particle size of the microsphere was 94 microns, and the drug loading rate was 41%. The specific preparation steps are as follows:

[0124] S1. A 1.5 wt% hyaluronic acid solution was prepared, and then a water phase w was prepared by mixing and dissolving in a molar ratio of hyaluronic acid: selenocystamine: activator (1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimide) = 1:0.2:1.

[0125] Steps S2-S6 are the same as Example 1.

[0126] The crosslinking degree of the microsphere obtained in this comparative example is 39%, which is less than the protection scope of the present application.

[0127] Comparative Example 5

[0128] This example is based on the previous patent CN116808286A, and a hydrogel with hyaluronic acid as the skeleton, a redox group diselenium bond as the inflammation-responsive group, and gallic acid as the delivery was prepared. The drug loading rate was 12%. The specific preparation steps are as follows:

[0129] S1. A 1.5 wt% hyaluronic acid solution was prepared, and then a water phase w was prepared by mixing and dissolving in a molar ratio of hyaluronic acid: selenocystamine: activator (1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimide, CAS number: 1892-57-5) = 1:0.5:1.

[0130] S2. Pour the water phase w into the mold and react for 24 hours, then obtain the gel a.

[0131] S3. Soak the gel a in the same drug solution as Example 1 for 12 hours, then take it out.

[0132] Comparative Example 6

[0133] This comparative example prepared a hydrogel microsphere with hyaluronic acid as the skeleton, crosslinked by hexanediamine, and delivered gallic acid. This microsphere does not have responsiveness, the average particle size of the microsphere is 85 microns, and the drug loading rate is 35%. The specific preparation steps are as follows:

[0134] Steps S1 to S6 are the same as in Example 1, except that selenocysteine ​​is replaced with hexamethylenediamine.

[0135] Experimental Example 1

[0136] The microspheres from Example 1 were used to simulate reactive oxygen species at the site of inflammation using a 500 μM hydrogen peroxide solution. Figure 2 Macroscopic images of the in vitro simulated absorption of oxidized inflammatory exudate and the gradual cross-linking to form a hydrogel show that the microspheres can rapidly absorb the oxidized liquid and gradually cross-link to form a hydrogel within 24 hours. When 10 mM hydrogen peroxide solution is added after 24 hours, the hydrogel microspheres absorb the excess oxidized liquid and can degrade spontaneously, avoiding foreign body reactions caused by prolonged retention. At the same time, it can prolong the drug treatment time compared to multiple administrations.

[0137] Experimental Example 2

[0138] The microspheres from Example 1 were soaked in a 500 μM hydrogen peroxide solution, and the damage of reactive oxygen species to the microspheres at the simulated inflammation site was observed. The process of the microspheres obtained in Example 1 absorbing and oxidizing inflammatory exudate in vitro and gradually cross-linking to form a hydrogel was simulated. The images taken under a polarizing microscope are shown below. Figure 3 As shown, the microspheres can rapidly absorb oxidizing liquids and gradually expand, break down, cross-link, and eventually form a hydrogel.

[0139] Experimental Example 3

[0140] The microspheres from Examples 1-7 and Comparative Examples 1-5 were degraded using relevant enzymes, and the drug loading was then tested using a microplate reader or ELISA kit. The microspheres were implanted into organisms, and their mass was weighed at different time points. The degradation time of the microspheres was also measured. The drug loading rate and degradation time of different microspheres are shown in Table 1.

[0141] Table 1

[0142]

[0143] For drug-loaded dressings, excellent drug loading capacity is essential. Insufficient drug loading can lead to unsatisfactory therapeutic effects. Therefore, it is necessary to adjust the drug loading capacity of microspheres by modifying their size, specific surface area, and other physical properties. At the same time, the size of the microspheres should also be adapted to the requirements of the application environment.

[0144] When the microspheres are too small, they are difficult to prepare and will degrade too quickly; when the microspheres are too large, they cannot be injected into the body or effectively cover exposed wounds. Therefore, it is necessary to screen the size of the microspheres.

[0145] Meanwhile, the degradation time of the microspheres is also crucial for biological safety. When the microspheres degrade too fast, the effective drug loading cannot be achieved; when the degradation is too slow, it will cause foreign body reactions or other biological safety problems. Therefore, the degradation time of the microspheres needs to be limited.

[0146] As can be seen from Table 1, the sizes of Comparative Example 1 and Comparative Example 2 are greater than the specified diameter (500 microns) and less than the specified diameter (10 microns), respectively. By comparing the drug loading rate and the degradation time, it can be concluded that as the diameter of the microspheres increases, the drug loading rate of the microspheres decreases, which cannot meet the needs of in vivo injection, and the degradation time is prolonged. In the case of using natural macromolecules as the framework, when the diameter of the microspheres is too large (Comparative Example 1, 550 microns), the drug loading amount is significantly reduced to 3% due to the reduction of the specific surface area, and the degradation time is prolonged to 14 days, but it is still within the range acceptable by the organism. When the diameter of the microspheres is too small (Comparative Example 2, 5 microns), the specific surface area of the microspheres increases, and the drug loading amount increases to 62%, but the degradation time is shortened to 2 days, which is obviously not conducive to the long-term use of the drug.

[0147] For the hydrogel microspheres with non-degradable macromolecules as the framework, although the diameter of the microspheres and the drug loading amount meet the requirements (Comparative Example 3, 95 microns, drug loading rate 13%), the degradation time is greater than 30 days, which will cause the organism to produce fibrosis, inflammation and other foreign body reactions, hindering the healing of inflammation.

[0148] As shown in Comparative Example 4, for the hydrogel microspheres with too low crosslinking degree, although the diameter of the microspheres and the drug loading amount meet the requirements, the degradation time is only 1 day, which obviously does not meet the requirements for use, which will cause the loaded drug to be unable to be effectively released and act for a long time.

[0149] Comparative Example 5 shows the performance of the hydrogel prepared according to the previous patent. It can be seen that when the drug is loaded according to the previous method, the hydrogel contains a large amount of water, which causes the drug solution to be unable to effectively enter the interior of the gel, resulting in a drug loading rate of only 12%, which is obviously lower than Examples 1-7. Meanwhile, since the hydrogel has become a macroscopic gel state, it cannot enter the wound tissue area by injection, affecting the use.

[0150] Therefore, according to the test results of Comparative Examples 1-5, it can be known that: 1) the larger the volume of the hydrogel microspheres, the smaller the specific surface area, the lower the drug loading rate, and the longer the degradation time, which is also more unable to meet the requirements for injection.

[0151] 2) The synthesis of macromolecules as the framework of the hydrogel will cause non-degradable foreign body reactions, while the natural macromolecule hydrogel microspheres have excellent biocompatibility.

[0152] 3) The cross-linking degree of the hydrogel microspheres is too low, which can cause the degradation speed of the microspheres to be obviously accelerated, and the long-term drug effect cannot be met.

[0153] 4) The macro hydrogel contains a large amount of water, which causes the drug solution to be unable to effectively enter the inside of the hydrogel, the drug loading rate is low, and the injectable use mode cannot be met.

[0154] The hydrogel microspheres constructed in examples 1-7 have a suitable diameter (80-350 microns), can meet the requirement of 30%-65% drug loading rate, and can meet the dual treatment mode of direct coverage in vitro and injection treatment in vivo, and have good applicability. Meanwhile, the hydrogel microspheres can be completely degraded within 3-14 days, and do not cause foreign body reactions.

[0155] Therefore, the hydrogel microspheres constructed in the application can obtain excellent drug loading rate by screening suitable size and cross-linking degree, and can meet the requirements of in-vivo and in-vitro treatment, and can be degraded at a suitable time during in-vivo treatment, thereby avoiding foreign body reactions.

[0156] Comparative example 6 and the experimental examples to be explained have nothing to do with the problems, and will not be described here.

[0157] Experimental example 4

[0158] The microspheres are soaked in a simulated inflammatory exudate environment, and then the drug release amount is tested by an enzyme marker or an Elisa kit at different time periods. The microspheres are soaked in PBS buffer and a simulated inflammatory exudate environment, and the microsphere mass is weighed at different time periods, and the liquid absorption rate of the microspheres is determined. The drug release amount and the liquid absorption rate of the microspheres at different time periods are shown in table 2.

[0159] Table 2

[0160]

[0161] The traditional direct drug administration mode has the risk of drug burst release and physiological toxicity, and the drug added directly on the treatment dressing cannot meet the dynamic treatment demand of the dynamic inflammatory environment of the wound, and can cause poor treatment effect of the drug during the treatment stage, so the dressing needs to be designed to intelligently respond to the release of the drug, and meet the treatment demand of the inflammation response.

[0162] Meanwhile, many inflammatory exudates are produced during the inflammation process, which further hinders the healing of the inflammation. Therefore, the treatment dressing also needs to have the ability to absorb the exudate. The traditional absorption of inflammatory exudate only focuses on the inflammatory exudate of the body surface wound, and ignores the exudate of the inflammatory site in the body, so the simple absorption of the exudate is not enough, and the specific absorption of the inflammatory exudate also needs to be concerned.

[0163] From Table 2, it can be seen that the hydrogel microspheres constructed by Comparative Example 6 do not have inflammation responsiveness, and therefore the amount of drug released in the inflammatory environment only increases by 15% from the 3rd hour to the 24th hour, which cannot meet the high demand for drugs in the actual treatment of severe inflammatory environment. At the same time, due to the absence of responsive cleavage leading to swelling and liquid absorption, the liquid absorption amount in the PBS environment and the inflammatory exudate absorption environment can only reach 52% and 53%, with no significant difference. At the same time, the absorption amount at the 6th hour and the 12th hour has no significant difference (within 8%), and the liquid absorption reaches equilibrium at the 6th hour.

[0164] The hydrogel microspheres constructed in Examples 1-7 can release drugs on demand by responsive regulation in a severe inflammatory environment, with a release rate of 50% or more in 24 hours, which breaks the disadvantage that the drug in Comparative Example 4 cannot be intelligently released from the hydrogel microspheres, and the drug release amount is increased by 1-2 times compared with Comparative Example 4.

[0165] At the same time, under inflammatory conditions, the liquid absorption rate of Examples 1-7 is significantly improved, and the exudate absorption amount is increased by 2-4 times compared with PBS buffer at the 6th hour, and by 4-5 times at the 12th hour, which can effectively absorb inflammatory liquid and has the ability to specifically absorb inflammatory exudate.

[0166] Comparative Examples 1-5 do not relate to the problems of the experimental examples, and will not be described here.

[0167] Experimental Example 5

[0168] The preferred embodiment 1 of the present application was tested for cell compatibility. The hydrogel microspheres of Example 1 were co-cultured with human keratinocytes for 24 hours, and then the cells were stained with a cell viability and cytotoxicity detection kit (Bi Yun Tian, item number C2015M) to stain live cells and dead cells, respectively, and counted. The results are shown in Figure 4 As can be seen, the number of live cells is significantly more than the number of dead cells, and after counting, the survival rate of the cells is 99.6%, indicating that the material has excellent biocompatibility.

[0169] Experimental Example 6

[0170] In view of the ability of preferred embodiment 1 to release drugs in response to wound inflammation to intervene in wound healing, a c57bl / 6j mouse model of diabetic full-thickness defect wound was constructed. The diabetic wound is difficult to heal due to severe inflammation caused by the sustained high glucose environment. Therefore, it is necessary to release anti-inflammatory drugs in the inflammatory period to relieve inflammation and effectively promote wound healing.

[0171] The blank diabetic mouse group, commercially available sodium alginate dressing group (Sofu Kang Rui, item number: 202310318840) and preferred embodiment 1 three groups were constructed, and the materials were placed at the wound site and wrapped with 3M film on the outside to prevent the materials from detaching from the tissue. The tissues on the 4th day were collected, and the content of the tissue inflammatory factor TNF-α was determined after homogenization. The wound photos were recorded by taking photos, and the results are shown in Figure 5 Fig. 2, and further the healing rate of the wound was statistically analyzed, and the results are shown in Table 3.

[0172] Table 3

[0173]

[0174] The diabetic wound is caused by persistent severe inflammation, resulting in a significant increase in the level of inflammatory factors such as TNF-α, which in turn hinders wound healing and reduces the wound healing rate. Therefore, it is necessary to design a material that can effectively release anti-inflammatory drugs according to the verification level, thereby reducing the content of inflammatory factors and effectively promoting wound healing.

[0175] As can be seen from Table 3, the diabetic mouse has a high level of inflammation, and the level of TNF-α factor is very high, about 455 pg / mL, and the wound healing rate on the 10th day is only 54%. The sodium alginate dressing is a commercially available product, which has good ability to absorb exudate. However, the sodium alginate dressing does not contain components to reduce inflammation, so the level of inflammation in the wound tissue is not effectively reduced, and the level of inflammatory factors is 413 pg / mL, and the wound healing rate is only increased by 8%.

[0176] Preferred embodiment 1 has good ability to absorb exudate, and can effectively release anti-inflammatory drug gallic acid, effectively reduce the level of inflammatory factors, and reduce the level of inflammatory factors in diabetic mice by more than 50%, and the wound healing rate is increased to 88%, combined with the photos in Figure 5 , it can be seen that the wound of the mouse of embodiment 1 is almost completely closed, which is obviously better than the other groups.

[0177] The above results show that preferred embodiment 1 can effectively reduce the inflammation level of the tissue and promote the healing of the diabetic wound, and its functionality and therapeutic effect are even better than the existing commercially available products.

[0178] Based on the above results, it can be seen that the hydrogel microspheres prepared by the present application can regulate the release rate of the drug according to the inflammatory environment, when the inflammation is aggravated, the release rate of the drug is also accelerated, and the inflammation is inhibited by a higher dose of the drug; at the same time, it can effectively absorb inflammatory exudate, avoid the contact between the inflammatory exudate and the tissue, and synergistically promote the treatment of inflammation.

[0179] The hydrogel microspheres of the present application have excellent biocompatibility, degradability, responsive drug release and the ability to specifically absorb inflammatory exudates, and have better potential for treating inflammation in in-vivo and in-vitro environments compared to the comparative examples. These excellent anti-inflammatory properties are mainly attributed to:

[0180] 1) Examples 1-7 have suitable particle sizes, and by adjusting the size, they can meet the simple and quick operation mode of spraying in-vitro, and benefit from the suitable specific surface area to have excellent drug loading rate. At the same time, the hydrogel microspheres have a natural macromolecular skeleton, which can be degraded in a physiological environment in time, avoiding foreign body reactions and other physiological toxicities.

[0181] 2) Examples 1-7 can be responsive to destruction in an inflammatory environment, thereby releasing anti-inflammatory drugs to meet the on-demand treatment in an inflammatory environment; at the same time, the hydrogel microspheres can effectively absorb inflammatory exudates while being destroyed in the inflammatory environment, avoiding further irritation of the inflammatory exudates to the damaged site.

[0182] Although the present application has been described in detail in the foregoing description with general principles and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection required by the present application.

Claims

1. A method for preparing inflammation-responsive hydrogel microspheres, characterized by, The method comprises: Step one, preparation of water phase After mixing the natural macromolecule and the inflammation-responsive group crosslinking agent in proportion, a mixed aqueous solution is obtained, denoted as water phase W; wherein the concentration of the natural macromolecule is 1-5 wt%; Step two, preparation of oil phase Mineral oil and amphoteric molecule Span 80 are mixed uniformly in a volume ratio of 1:50-1:100 to obtain oil phase O; Step three, preparation of microspheres Water phase W and oil phase O are stirred and mixed in a volume ratio of 1:3-1:10 to obtain a mixed solution C; After the mixed solution C is mixed and washed with isopropyl alcohol, centrifugation is performed, the supernatant is discarded, and precipitated microspheres are obtained; Step four, drug loading The microspheres obtained in step three are soaked in a drug solution with a drug concentration of 5-20 wt%, the mass ratio of the microspheres to the drug solution being 1:50-1:200, and then transferred to a freeze dryer for complete freeze-drying, and screened with a screen, to obtain inflammation-responsive hydrogel microspheres; the average diameter of the hydrogel microspheres is 10-500 microns, and the crosslinking degree is 50%-100%.

2. The method of claim 1, wherein the inflammation-responsive hydrogel microspheres are prepared by the method of claim 1. The natural macromolecule includes one or more of starch, cellulose, hyaluronic acid, dextran, chitosan, alginic acid, agarose, carrageenan, heparin, chondroitin sulfate, chitin, pectin, gelatin, collagen, albumin, and silk fibroin.

3. The method for preparing an inflammatory-responsive hydrogel microsphere according to claim 1, characterized in that, The inflammation-responsive group crosslinking agent includes one or more of redox groups, matrix metalloproteinase-responsive groups, and pH-responsive groups; wherein the redox groups include one or more of diselenide bonds, disulfide bonds, phenylboronic acid ester bonds, thioether bonds, oxalic acid polyesters, acyl pyrrolidine-2-carboxamide groups, ferrocene-cyclodextrin self-assembly structure groups, ketone thiol, and concanavalin A structure; the matrix metalloproteinase-responsive groups include one or more of matrix metalloproteinase-responsive peptide chains and gelatin structures; and the pH-responsive groups include one or more of phenylboronic acid ester bonds, Schiff base bonds, pH-responsive peptide chains, and glycyrrhizic acid self-assembly structures.

4. The method of claim 1, wherein the inflammation-responsive hydrogel microspheres are prepared by the method comprising: In step three, the stirring speed is 150-500 rpm, and the reaction time is 6-24 h.

5. The method for preparing an inflammatory-responsive hydrogel microsphere according to claim 1, characterized in that, In step three, the volume ratio of the mixed solution C to isopropyl alcohol is 1:50-1:100, and the centrifugation conditions are a rotation speed of 1000-2500 rpm and a centrifugation time of 3-10 min.

6. The method for preparing an inflammatory-responsive hydrogel microsphere according to claim 1, characterized in that, In step four, the drug solution is a chemical drug or a cytokine.

7. The inflammation-responsive hydrogel microspheres prepared by the method of any one of claims 1-6, characterized in that, The hydrogel microspheres have a natural macromolecule as a microsphere skeleton, load anti-inflammatory drugs inside through inflammation-responsive group crosslinking, and have a drug loading rate of 30%-80%.

8. Use of the inflammation-responsive hydrogel microspheres according to claim 7 in the preparation of a drug for treating inflammatory wound healing.

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