Preparation method of dendrobium officinale polysaccharide-based dual-responsive drug-release hydrogel

Abstract

The invention discloses a preparation method of dendrobium officinale polysaccharide-based dual-responsive drug-release hydrogel, and relates to a preparation method of drug-release hydrogel. The invention aims to solve the problem of difficult healing of diabetic wounds. The preparation method comprises the following steps: firstly synthesizing hyaluronic acid-3-aminophenylboronic acid, connecting the hyaluronic acid-3-aminophenylboronic acid with resveratrol, and finally doping the hyaluronic acid-3-aminophenylboronic acid with dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate, so as to prepare the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel. The borate bond in the hydrogel can be broken in a high glucose or high active oxygen environment, so that the responsive release of the active substance resveratrol is realized. The prepared hydrogel has the characteristics of being injectable, self-healing and very sensitive to the pathological environment responding to diabetic wounds, can effectively inhibit the ROS level in cells and regulate and control M2 polarization of macrophages, and has an obvious proliferation effect on fibroblasts, so that wound gap closure is promoted, and the healing time of the wounds is shortened.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a method for preparing a drug-releasing hydrogel. Background Art

[0002] Diabetic wounds have complex pathophysiological mechanisms, making wound healing difficult. Without proper or timely treatment, they can lead to infection, gangrene, and even amputation, which brings huge economic and psychological burdens to patients. Traditional wound dressings have a single function and are not adaptable to the wound environment, making it difficult to meet the complex needs of the healing process.

[0003] Hydrogel wound dressing is one of the new dressings currently used in clinical practice. The hydrophilic three-dimensional network structure of hydrogel is similar to the extracellular matrix (ECM), which can provide cells with a suitable living environment. At present, the hydrogel wound dressings used in clinical practice have relatively single functions, which not only limits the use effect of hydrogel dressings, but also prolongs the healing time of wounds. The drug-loaded hydrogel wound dressings that are currently studied more often use animal-derived polysaccharides (such as chitosan, gelatin, etc.) or algae-derived polysaccharides (such as sodium alginate) as skeleton materials and load small molecule drugs, which often lead to sudden release or irregular release of small molecule drugs during use, and cannot dynamically release drugs according to changes in the wound microenvironment, thereby causing adverse reactions or reducing the therapeutic effect. On the other hand, hydrogel skeleton materials that do not have specific activity do not provide biochemical signals related to tissue repair. Therefore, although the dressing is in contact with the wound tissue, it cannot effectively support cell proliferation or differentiation, resulting in poor wound repair effect. In addition, clinical wound dressings are mostly based on patches, which lack flexibility, cannot adapt to the shape of the wound and fill it, resulting in insufficient tightness between the hydrogel and the wound tissue.

[0004] Dendrobium officinale polysaccharide (DOP) is one of the main components of the traditional Chinese medicine Dendrobium officinale, and has high medicinal value. Dendrobium officinale polysaccharide has a good pharmacological effect of promoting fibroblast proliferation and migration. Resveratrol (Resveratrol, Res) is a natural polyphenol compound with multiple biological activities. Its role in scavenging reactive oxygen species, regulating macrophage polarization, and regulating the expression of inflammatory factors has been confirmed. At present, there is no report on the application of hydrogel dressings with DOP as the hydrogel skeleton and loaded with Res in the treatment of diabetic wounds. Summary of the invention

[0005] In order to solve the problem of difficult wound healing in diabetes, the present invention provides a method for preparing a dual-responsive drug-releasing hydrogel based on Dendrobium officinale polysaccharide. Hyaluronic acid-3-aminophenylboronic acid is first synthesized, and resveratrol is connected, and finally Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate is added, thereby preparing Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel. The boronate bond in the hydrogel can be broken in an environment of high glucose or high active oxygen, thereby realizing the responsive release of the active substance resveratrol.

[0006] The preparation method of the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel of the present invention is carried out according to the following steps:

[0007] Step 1: Preparation of hyaluronic acid-3-aminophenylboronic acid-resveratrol:

[0008] ①, hyaluronic acid and 3-aminophenylboronic acid are mixed, added to an aqueous solution of dimethyl sulfoxide, and then a dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added, the pH is adjusted to 4.5 with an aqueous HCl solution, and the mixture is reacted at 30° C. for 24 hours to obtain a hyaluronic acid-3-aminophenylboronic acid solution; after dialysis with pure water, the mixture is freeze-dried to obtain a hyaluronic acid-3-aminophenylboronic acid flocculent;

[0009] The mass ratio of the hyaluronic acid to 3-aminophenylboronic acid is (3-1): (0.5-2);

[0010] The mass ratio of the hyaluronic acid to the volume of the aqueous solution of dimethyl sulfoxide is 1 g:70 mL; the mass ratio of dimethyl sulfoxide to water in the aqueous solution of dimethyl sulfoxide is 2:5;

[0011] The mass ratio of the hyaluronic acid to the mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1.52;

[0012] The molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1;

[0013] The mass fraction of N-hydroxysuccinimide in the dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1.9%;

[0014] ②, dispersing the prepared hyaluronic acid-3-aminophenylboronic acid flocs in pure water to obtain hyaluronic acid-3-aminophenylboronic acid gel;

[0015] The ratio of the mass of the hyaluronic acid-3-aminophenylboronic acid floccules to the volume of pure water is 0.09 g: 2 mL;

[0016] ③, dispersing resveratrol in anhydrous ethanol to obtain a resveratrol solution;

[0017] ④, mixing the prepared resveratrol solution with the prepared hyaluronic acid-3-aminophenylboronic acid gel to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol;

[0018] The ratio of the volume of the resveratrol solution to the mass of the hyaluronic acid-3-aminophenylboronic acid floccules is 300 μL: 0.09 g;

[0019] Step 2: Preparation of Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel:

[0020] ①, adding Dendrobium officinale polysaccharide to NaOH solution to obtain Dendrobium officinale polysaccharide solution, adding 1,4-butanediol diglycidyl ether, and after the reaction is completed at 30°C, obtaining Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel;

[0021] The mass ratio of the Dendrobium officinale polysaccharide to 1,4-butanediol diglycidyl ether is 0.48:0.1-1;

[0022] ②, mixing the sodium polyacrylate aqueous solution with the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel, dialyzing with ethanol aqueous solution and pure water as dialysis media in turn, adjusting the pH to 7.45-8.2, and obtaining the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel;

[0023] The volume ratio of the sodium polyacrylate solution to the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel is (2-1): (1-3);

[0024] Step 3, mixing hyaluronic acid-3-aminophenylboronic acid-resveratrol and Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel;

[0025] The volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol and the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is (1-3): (3-6).

[0026] The beneficial effects of the present invention are:

[0027] 1. The hydrogel prepared by the present invention has the properties of diabetic wound microenvironment responsive drug release, injectability, self-healing, swelling and the like. 3-aminophenylboronic acid is modified on the hyaluronic acid molecular chain to synthesize hyaluronic acid-3-aminophenylboronic acid, and resveratrol is then connected to hyaluronic acid-3-aminophenylboronic acid to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol. The self-crosslinked hydrogel of Dendrobium officinale polysaccharide is mixed with sodium polyacrylate to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel; the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is co-incubated with hyaluronic acid-3-aminophenylboronic acid-resveratrol to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel. On the one hand, the boronate bonds in the hydrogel break in a high glucose or reactive oxygen environment, thereby achieving a responsive release of resveratrol and avoiding disordered release of resveratrol. On the other hand, the hydrogel has injectable and self-healing properties, which can increase the binding area between the hydrogel and the wound tissue and isolate the wound from the external environment.

[0028] 2. The Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel prepared by the present invention has the functions of anti-oxidation, promoting macrophage M2 polarization, and anti-inflammatory, and has a significant proliferation effect on fibroblasts, thereby promoting wound gap closure and shortening wound healing time.

[0029] 3. The preparation of the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel of the present invention is easy to implement; and the sources of Dendrobium officinale polysaccharide and resveratrol are extensive and low in cost.

[0030] 4. The present invention connects small molecule active substances to the polysaccharide skeleton from traditional Chinese medicine through an environmentally sensitive linker arm. The small molecule and polysaccharide are used together to directly act on the wound site, dually respond to the diabetic wound microenvironment (glucose, reactive oxygen) to release drugs, and realize the combined application of small molecules and traditional Chinese medicine polysaccharides to inhibit inflammation and oxidative stress in diabetic wounds, and promote the proliferation and migration of fibroblasts in the proliferative stage, playing a role in continuous treatment. At present, there is no literature reporting the use of Dendrobium officinale polysaccharide-based hydrogels for the treatment of diabetic wounds.

[0031] 5. Traditional Chinese medicine polysaccharides usually have good biocompatibility, but hydrogels formed by simply relying on self-crosslinking often have poor mechanical strength, while artificially synthesized polymers have good mechanical properties. The present invention combines artificially synthesized sodium polyacrylate with traditional Chinese medicine polysaccharides to improve the problem of poor mechanical properties of the simple traditional Chinese medicine polysaccharide hydrogel skeleton. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a scanning electron micrograph of the DOP-BDDE-PAAs / HA-BA-Res hydrogel prepared in Example 1;

[0033] Figure 2 This is a scanning electron micrograph of the DOP-BDDE-PAAs / HA-BA hydrogel prepared in Example 2;

[0034] Figure 3 This is a scanning electron micrograph of the DOP-BDDE-PAAs / Res hydrogel prepared in Example 3;

[0035] Figure 4 This is a scanning electron micrograph of the BDDE-PAAs / HA-BA-Res hydrogel prepared in Example 4;

[0036] Figure 5 This is a scanning electron micrograph of the BDDE-PAAs / HA-BA hydrogel prepared in Example 5;

[0037] Figure 6 is the swelling ratio diagram of different groups of hydrogels;

[0038] Figure 7 is a graph of the self-healing, injectability and adhesion properties of the DOP-BDDE-PAAs / HA-BA-Res hydrogel prepared in Example 1;

[0039] Figure 8 is a graph showing the glucose and active oxygen response characteristics of the DOP-BDDE-PAAs / HA-BA-Res hydrogel prepared in Example 1;

[0040] Fig. 9 Figure 2 shows the antioxidant activity of different groups of hydrogels;

[0041] Fig.10 It is a bar graph of the relative proliferation number of fibroblasts in different groups after 24 h of incubation;

[0042] Fig.11 It is a bar graph of the relative proliferation number of fibroblasts in different groups after 48 h of incubation;

[0043] Fig.12 Figure 1 shows the fibroblast migration experiment of different groups of hydrogels;

[0044] Fig.13 This is a graph showing the ability of different groups of hydrogels to remove ROS in fibroblasts;

[0045] Fig.14 Fluorescence images of different groups of hydrogels regulating macrophage M2 polarization. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any reasonable combination of the specific implementation modes.

[0047] Specific implementation method 1: The preparation method of the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel in this implementation method is carried out according to the following steps:

[0048] Step 1: Preparation of hyaluronic acid-3-aminophenylboronic acid-resveratrol:

[0049] ①, hyaluronic acid and 3-aminophenylboronic acid are mixed, added to an aqueous solution of dimethyl sulfoxide, and then a dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added, the pH is adjusted to 4.5 with an aqueous HCl solution, and the mixture is reacted at 30° C. for 24 hours to obtain a hyaluronic acid-3-aminophenylboronic acid solution; after dialysis with pure water, the mixture is freeze-dried to obtain a hyaluronic acid-3-aminophenylboronic acid flocculent;

[0050] The mass ratio of the hyaluronic acid to 3-aminophenylboronic acid is (3-1): (0.5-2);

[0051] The mass ratio of the hyaluronic acid to the volume of the aqueous solution of dimethyl sulfoxide is 1 g:70 mL; the mass ratio of dimethyl sulfoxide to water in the aqueous solution of dimethyl sulfoxide is 2:5;

[0052] The mass ratio of the hyaluronic acid to the mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1.52;

[0053] The molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1;

[0054] The mass fraction of N-hydroxysuccinimide in the dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1.9%;

[0055] ②, dispersing the prepared hyaluronic acid-3-aminophenylboronic acid flocs in pure water to obtain hyaluronic acid-3-aminophenylboronic acid gel;

[0056] The ratio of the mass of the hyaluronic acid-3-aminophenylboronic acid floccules to the volume of pure water is 0.09 g: 2 mL;

[0057] ③, dispersing resveratrol in anhydrous ethanol to obtain a resveratrol solution;

[0058] ④, mixing the prepared resveratrol solution with the prepared hyaluronic acid-3-aminophenylboronic acid gel to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol;

[0059] The ratio of the volume of the resveratrol solution to the mass of the hyaluronic acid-3-aminophenylboronic acid floccules is 300 μL: 0.09 g;

[0060] Step 2: Preparation of Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel:

[0061] ①, adding Dendrobium officinale polysaccharide to NaOH solution to obtain Dendrobium officinale polysaccharide solution, adding 1,4-butanediol diglycidyl ether, and after the reaction is completed at 30°C, obtaining Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel;

[0062] The mass ratio of the Dendrobium officinale polysaccharide to 1,4-butanediol diglycidyl ether is 0.48:0.1-1;

[0063] ②, mixing the sodium polyacrylate aqueous solution with the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel, dialyzing with ethanol aqueous solution and pure water as dialysis media in turn, adjusting the pH to 7.45-8.2, and obtaining the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel;

[0064] The volume ratio of the sodium polyacrylate solution to the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel is (2-1): (1-3);

[0065] Step 3, mixing hyaluronic acid-3-aminophenylboronic acid-resveratrol and Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel (DOP-BDDE-PAAs / HA-BA-Res);

[0066] The volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol and the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is (1-3): (3-6).

[0067] This embodiment has the following beneficial effects:

[0068] 1. The hydrogel prepared in this embodiment has the properties of diabetic wound microenvironment responsive drug release, injectability, self-healing, swelling, etc. 3-aminophenylboronic acid is modified on the hyaluronic acid molecular chain to synthesize hyaluronic acid-3-aminophenylboronic acid, and resveratrol is connected with hyaluronic acid-3-aminophenylboronic acid to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol. The self-crosslinked hydrogel of Dendrobium officinale polysaccharide is mixed with sodium polyacrylate to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel; Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is co-incubated with hyaluronic acid-3-aminophenylboronic acid-resveratrol to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel. On the one hand, the boronate bonds in the hydrogel break in a high glucose or reactive oxygen environment, thereby achieving a responsive release of resveratrol and avoiding disordered release of resveratrol. On the other hand, the hydrogel has injectable and self-healing properties, which can increase the binding area between the hydrogel and the wound tissue and isolate the wound from the external environment.

[0069] 2. The Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel prepared in this embodiment has the functions of anti-oxidation, promoting macrophage M2 polarization, and anti-inflammatory, and has a significant proliferation effect on fibroblasts, thereby promoting wound gap closure and shortening wound healing time.

[0070] 3. The preparation of the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel in this embodiment is easy to implement; and the sources of Dendrobium officinale polysaccharide and resveratrol are extensive and low in cost.

[0071] 4. This embodiment connects the small molecule active substance to the polysaccharide skeleton from traditional Chinese medicine through an environmentally sensitive linker. The small molecule and polysaccharide are used together to directly act on the wound site, dually respond to the diabetic wound microenvironment (glucose, reactive oxygen) to release drugs, and realize the combined application of small molecules and traditional Chinese medicine polysaccharides to inhibit inflammation and oxidative stress in diabetic wounds, and promote the proliferation and migration of fibroblasts in the proliferative stage, playing a role in continuous treatment. At present, there is no literature reporting the use of Dendrobium officinale polysaccharide-based hydrogels for the treatment of diabetic wounds.

[0072] 5. Traditional Chinese medicine polysaccharides usually have good biocompatibility, but hydrogels formed by simply relying on self-crosslinking often have poor mechanical strength, while artificially synthesized polymers have good mechanical properties. This embodiment uses artificially synthesized sodium polyacrylate and traditional Chinese medicine polysaccharides together to improve the problem of poor mechanical properties of the simple traditional Chinese medicine polysaccharide hydrogel skeleton.

[0073] Specific implementation method 2: This implementation method is different from specific implementation method 1 in that: in step 1①, the concentration of the HCl aqueous solution is 1 mol / L.

[0074] Specific implementation method three: This implementation method is different from specific implementation methods one or two in that: in step 1③, the mass fraction of resveratrol in the resveratrol solution is 1-5%.

[0075] Specific implementation method 4: This implementation method is different from any one of specific implementation methods 1 to 3 in that: in step 2①, the mass fraction of the Dendrobium officinale polysaccharide solution is 15-25%.

[0076] Specific implementation method five: This implementation method is different from any one of specific implementation methods one to four in that: the mass fraction of the NaOH solution in step two ① is 1 to 3%.

[0077] Specific implementation method 6: This implementation method is different from any one of specific implementation methods 1 to 5 in that: the reaction time at 30° C. in step 2① is 8 to 24 hours.

[0078] Specific implementation method 7: This implementation method is different from any one of specific implementation methods 1 to 6 in that the mass fraction of the sodium polyacrylate aqueous solution described in step 2② is 1 to 5%.

[0079] Specific implementation eight: This implementation differs from any one of specific implementations one to seven in that: the mass fraction of the ethanol aqueous solution in step two ② is 15%.

[0080] Specific embodiment nine: This embodiment differs from any one of specific embodiments one to eight in that: in step three, the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol and the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is 1:6.

[0081] Specific embodiment ten: This embodiment differs from any one of specific embodiments one to nine in that: in step three, the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol and the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is 2.3:5.

[0082] Example 1

[0083] The preparation method of the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel (DOP-BDDE-PAAs / HA-BA-Res) of this embodiment is carried out according to the following steps:

[0084] Step 1: Preparation of hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res):

[0085] ①, 1g hyaluronic acid (HA) and 0.68g 3-aminophenylboronic acid (PBA) were mixed and added to 70mL dimethyl sulfoxide (DMSO) aqueous solution, and then 30mL 0.57g N-hydroxysuccinimide (NHS) and 0.95g 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) dimethyl sulfoxide solution was added, and pH was adjusted to 4.5 with 1mol / L HCl aqueous solution, and reacted at 30°C for 24h to obtain hyaluronic acid-3-aminophenylboronic acid solution; after dialysis with pure water, freeze-dried to obtain hyaluronic acid-3-aminophenylboronic acid flocs (HA-BA);

[0086] The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;

[0087] ②, Disperse the prepared 0.09 g HA-BA in 2 mL pure water to obtain HA-BA gel;

[0088] ③, dispersing resveratrol (Res) in anhydrous ethanol to obtain a Res solution; the Res fraction in the Res solution is 2%;

[0089] ④, mixing the prepared 300 μL Res solution with the prepared HA-BA gel to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res);

[0090] Step 2: Preparation of Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs):

[0091] ①, 0.48g of Dendrobium officinale polysaccharide (DOP) was added to 2mL of NaOH solution to obtain a DOP solution, 0.5g of 1,4-butanediol diglycidyl ether (BDDE) was added, and after reacting at 30°C for 16h, a Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel (DOP-BDDE) was obtained; the mass fraction of the NaOH solution was 2%;

[0092] ②, 2 mL of sodium polyacrylate (PAAs) aqueous solution was mixed with 2.5 mL of DOP-BDDE, and dialyzed using ethanol aqueous solution and pure water as dialysis media in turn, and the pH was adjusted to 7.45-8.2 to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs);

[0093] The mass fraction of the PAAs aqueous solution is 3%;

[0094] The mass fraction of the ethanol aqueous solution is 15%;

[0095] Step 3: Mix HA-BA-Res and DOP-BDDE-PAAs to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel (DOP-BDDE-PAAs / HA-BA-Res).

[0096] The volume ratio of the HA-BA-Res and DOP-BDDE-PAAs is 2.3:5.

[0097] Example 2

[0098] The preparation method of DOP-BDDE-PAAs / HA-BA hydrogel in this embodiment is carried out according to the following steps:

[0099] Step 1: Preparation of hyaluronic acid-3-aminophenylboronic acid (HA-BA):

[0100] ①, 1g hyaluronic acid (HA) and 0.68g 3-aminophenylboronic acid (PBA) were mixed and added to 70mL dimethyl sulfoxide (DMSO) aqueous solution, and then 30mL 0.57g N-hydroxysuccinimide (NHS) and 0.95g 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) dimethyl sulfoxide solution was added, and pH was adjusted to 4.5 with 1mol / L HCl aqueous solution, and reacted at 30°C for 24h to obtain hyaluronic acid-3-aminophenylboronic acid solution; after dialysis with pure water, freeze-dried to obtain hyaluronic acid-3-aminophenylboronic acid flocs (HA-BA);

[0101] The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;

[0102] ②, Disperse the prepared 0.09 g HA-BA in 2 mL pure water to obtain HA-BA gel;

[0103] Step 2: Preparation of Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs):

[0104] ①, 0.48g of Dendrobium officinale polysaccharide (DOP) was added to 2mL of NaOH solution to obtain a DOP solution, 0.5g of 1,4-butanediol diglycidyl ether (BDDE) was added, and after reacting at 30°C for 16h, a Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel (DOP-BDDE) was obtained; the mass fraction of the NaOH solution was 2%;

[0105] ②, 2 mL of sodium polyacrylate (PAAs) aqueous solution was mixed with 2.5 mL of DOP-BDDE, and dialyzed using ethanol aqueous solution and pure water as dialysis media in turn, and the pH was adjusted to 7.45-8.2 to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs);

[0106] The mass fraction of the PAAs aqueous solution is 3%;

[0107] The mass fraction of the ethanol aqueous solution is 15%;

[0108] Step 3, HA-BA and DOP-BDDE-PAAs are mixed to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate / hyaluronic acid-3-aminophenylboronic acid hydrogel (DOP-BDDE-PAAs / HA-BA);

[0109] The volume ratio of the HA-BA and DOP-BDDE-PAAs hydrogel is 2:5.

[0110] Example 3

[0111] The preparation method of DOP-BDDE-PAAs / Res hydrogel in this embodiment is carried out according to the following steps:

[0112] Step 1: Preparation of Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs):

[0113] ①, 0.48g of Dendrobium officinale polysaccharide (DOP) was added to 2mL of NaOH solution to obtain a DOP solution, 0.5g of 1,4-butanediol diglycidyl ether (BDDE) was added, and after reacting at 30°C for 16h, a Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel (DOP-BDDE) was obtained; the mass fraction of the NaOH solution was 2%;

[0114] ②, 2mL of sodium polyacrylate (PAAs) aqueous solution was mixed with 2.5mL of DOP-BDDE hydrogel, and dialyzed using ethanol aqueous solution and pure water as dialysis media in turn, and the pH was adjusted to 7.45-8.2 to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs);

[0115] Step 2, dispersing resveratrol (Res) in anhydrous ethanol to obtain a resveratrol solution; the mass fraction of Res in the Res solution is 2%;

[0116] Step 3: Mix the Res solution with DOP-BDDE-PAAs to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel / resveratrol (DOP-BDDE-PAAs / Res).

[0117] The volume ratio of the Res solution to DOP-BDDE-PAAs is 0.3:5.

[0118] Example 4

[0119] The preparation method of BDDE-PAAs / HA-BA-Res hydrogel in this embodiment is carried out according to the following steps:

[0120] ①, 1g hyaluronic acid (HA) and 0.68g 3-aminophenylboronic acid (PBA) were mixed and added to 70mL dimethyl sulfoxide (DMSO) aqueous solution, and then 30mL 0.57g N-hydroxysuccinimide (NHS) and 0.95g 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) dimethyl sulfoxide solution was added, and pH was adjusted to 4.5 with 1mol / L HCl aqueous solution, and reacted at 30°C for 24h to obtain hyaluronic acid-3-aminophenylboronic acid solution; after dialysis with pure water, freeze-dried to obtain hyaluronic acid-3-aminophenylboronic acid flocs (HA-BA);

[0121] The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;

[0122] ②, Disperse the prepared 0.09 g HA-BA in 2 mL pure water to obtain HA-BA gel;

[0123] ③, dispersing resveratrol (Res) in anhydrous ethanol to obtain a Res solution; the mass fraction of Res in the Res solution is 2%;

[0124] ④, mixing the prepared 300 μL Res solution with the prepared HA-BA gel to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res);

[0125] Step 2: Preparation of 1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs):

[0126] ①, add 0.5g 1,4-butanediol diglycidyl ether (BDDE) into 2mL NaOH solution, react at 30°C for 16h to obtain BDDE alkaline solution; the mass fraction of the NaOH solution is 2%;

[0127] ②, 2 mL of sodium polyacrylate (PAAs) aqueous solution was mixed with 2.5 mL of BDDE alkaline solution, and dialyzed using ethanol aqueous solution and pure water as dialysis media in turn, and the pH was adjusted to 7.45-8.2 to obtain 1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs);

[0128] The mass fraction of the PAAs aqueous solution is 3%;

[0129] The mass fraction of the ethanol aqueous solution is 15%;

[0130] Step 3: Mix HA-BA-Res and BDDE-PAAs to obtain 1,4-butanediol diglycidyl ether-sodium polyacrylate / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel (BDDE-PAAs / HA-BA-Res).

[0131] The volume ratio of the HA-BA-Res and BDDE-PAAs is 2.3:5.

[0132] Example 5

[0133] The preparation method of BDDE-PAAs / HA-BA hydrogel in this embodiment is carried out according to the following steps:

[0134] Step 1: Preparation of hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res):

[0135] ①, 1g hyaluronic acid (HA) and 0.68g 3-aminophenylboronic acid (PBA) were mixed and added to 70mL dimethyl sulfoxide (DMSO) aqueous solution, and then 30mL 0.57g N-hydroxysuccinimide (NHS) and 0.95g 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) dimethyl sulfoxide solution was added, and pH was adjusted to 4.5 with 1mol / L HCl aqueous solution, and reacted at 30°C for 24h to obtain hyaluronic acid-3-aminophenylboronic acid solution; after dialysis with pure water, freeze-dried to obtain hyaluronic acid-3-aminophenylboronic acid flocs (HA-BA);

[0136] The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;

[0137] ②, Disperse the prepared 0.09 g HA-BA in 2 mL pure water to obtain HA-BA gel;

[0138] Step 2: Preparation of 1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs):

[0139] ①, add 0.5g 1,4-butanediol diglycidyl ether (BDDE) to 2mL NaOH solution, react at 30°C for 16h to obtain BDDE alkaline solution; the mass fraction of the NaOH solution is 2%;

[0140] ②, 2 mL of sodium polyacrylate (PAAs) aqueous solution was mixed with 2.5 mL of BDDE alkaline solution, and dialyzed using ethanol aqueous solution and pure water as dialysis media in turn, and the pH was adjusted to 7.45-8.2 to obtain 1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs);

[0141] The mass fraction of the PAAs aqueous solution is 3%;

[0142] The mass fraction of the ethanol aqueous solution is 15%;

[0143] Step 3: Mix HA-BA gel and BDDE-PAAs to obtain 1,4-butanediol diglycidyl ether-sodium polyacrylate / hyaluronic acid-3-aminophenylboronic acid hydrogel (BDDE-PAAs / HA-BA).

[0144] The volume ratio of the HA-BA gel to BDDE-PAAs is 2:5.

[0145] The products of Examples 1 to 5 were subjected to the following analytical tests:

[0146] 1. The morphology of the hydrogels obtained in Experimental Examples 1 to 5 was observed using a scanning electron microscope. After the fresh hydrogels were freeze-dried, the hydrogels were placed on a sample stage and sprayed with gold. The hydrogels were then observed using a scanning electron microscope to obtain scanning electron microscope images of Examples 1 to 5. Figures 1 to 5 As shown, it can be observed that the five groups of hydrogels all have interconnected porous network structures, which can be used for the transport of oxygen and nutrients.

[0147] 2. After freeze-drying the DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, DOP-BDDE-PAAs / Res, BDDE-PAAs / HA-BA-Res and BDDE-PAAs / HA-BA hydrogels obtained in Examples 1 to 5, the freeze-dried hydrogels were immersed in PBS with a pH of 7.4, and then placed in a 37° C. incubator for 24 hours before being taken out. The surface moisture of the hydrogels was absorbed with filter paper and the hydrogels were weighed. The swelling rate formula is as follows:

[0148] Swelling rate (%) = (W 0 -W t ) / W 0 ×100%

[0149] Note: t and W 0 is the weight of the composite hydrogel at 24h and 0h.

[0150] In tissue engineering, the swelling ratio of hydrogels is an important indicator of biomaterials, so we observed the swelling behavior of hydrogels in PBS at 37°C. Figure 6 As shown in the figure (* represents significant difference, data represents mean ± standard deviation *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.), the hydrogel samples were taken out after being soaked in PBS for 24 hours. After 24 hours, the hydrogels in each group reached equilibrium, and the swelling rate formed a denser cross-linked network with the addition of Dendrobium officinale polysaccharide, which improved its water absorption performance. The results show that DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, and DOP-BDDE-PAAs / Res hydrogels all have good swelling capacity, effectively absorb tissue exudate, reduce the possibility of wound infection, and accelerate wound healing.

[0151] 3. Cut the DOP-BDDE-PAAs / HA-BA-Res hydrogel in half, dye them in different colors, place them together for 30 minutes, pick up the hydrogel with tweezers, and check whether the hydrogel is broken. Put the hydrogel into a 1mL syringe, observe the injectability of the hydrogel, and observe how easy it is to inject. Stick the prepared hydrogel on the finger joints, bend the finger angle, observe whether the hydrogel moves or falls, and test the viscosity of the hydrogel.

[0152] The ideal diabetic wound dressing should have good self-healing, adhesion and injectability properties. We selected DOP-BDDE-PAAs / HA-BA-Res hydrogel for characterization. Figure 7As shown, we prepared a rectangular hydrogel sample, cut it into two halves, and dyed one half red. After the sample was in contact for 30 minutes, the whole hydrogel after healing was picked up with tweezers. We observed that there was no break at the seam, and it did not crack when stretched to both sides with tweezers, which was attributed to the interaction of boronate bonds in the hydrogel matrix. We put the DOP-BDDE-PAAs / HA-BA-Res hydrogel into a 1mL syringe, and the English letters "QMU" can be easily written, and it is insoluble when injected into water. Then the hydrogel was injected into a star mold and demolded to form a star pattern. By bending the finger angle, we found that the DOP-BDDE-PAAs / HA-BA-Res hydrogel can adhere tightly to the skin without falling off, and the DOP-BDDE-PAAs / HA-BA-Res hydrogel can still adhere tightly to the skin when changing the adhesion material, such as fingers, wood, plastic, rubber and glass. These results show that the DOP-BDDE-PAAs / HA-BA-Res hydrogel has good injectability, injectability and adhesion.

[0153] 4. The DOP-BDDE-PAAs / HA-BA-Res hydrogel obtained in Example 1 was placed in PBS buffer with 20 mM glucose, 1 mM H 2 O 2 PBS buffer and PBS buffer; take out 1 mL of release medium at each time interval and add 1 mL of new release medium. Measure the UV absorbance of the release medium at 306 nm and use the corresponding UV standard curve to determine the release amount.

[0154] In diabetic chronic wounds with high blood sugar and reactive oxygen levels, hydrogels that respond to glucose and ROS can achieve sustained release of active substances and promote wound repair. Figure 8As shown, compared with PBS medium, the cumulative release of Res in high glucose medium increased by about 40%. This indicates that glucose can compete with the 3-aminophenylboronic acid groups cross-linked by Res in DOP-BDDE-PAAs / HA-BA-Res hydrogel to achieve the release of Res under glucose stimulation. ROS can cleave dynamic boronate bonds to achieve ROS-sensitive drug release performance. The cumulative release of Res in ROS medium increased by about 20% compared with that in PBS medium. This indicates that the boronate bonds of the hydrogel are more likely to release Res in a high sugar environment. We can also observe that in the PBS medium with glucose and reactive oxygen, the release of Res increased by about 50% compared with the PBS medium. It can be seen that in a high sugar environment, the cumulative release of Res from DOP-BDDE-PAAs / HA-BA-Res hydrogel is significantly higher than that in a high reactive oxygen environment. This indicates that compared with PBS containing glucose and PBS containing active oxygen, the hydrogel has the highest cumulative drug release in PBS containing glucose and active oxygen. Diabetic wounds just meet the required high-sugar and high-active oxygen environment, allowing DOP-BDDE-PAAs / HA-BA-Res hydrogel to release drugs better on demand.

[0155] 5. Place the DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, DOP-BDDE-PAAs / Res, BDDE-PAAs / HA-BA-Res and BDDE-PAAs / HA-BA hydrogels (50 mg) obtained in Examples 1 to 5 into a centrifuge tube, add DPPH-anhydrous ethanol (200 mM, 2 mL), place in a shaker at 37°C for reaction for 30 min, and measure the absorbance at 517 nm using a microplate reader.

[0156] In this study, a chemical colorimetric method was used to determine the scavenging ability of the hydrogel for DPPH. Fig. 9 As shown in the figure, compared with the control group, the scavenging rates of DPPH free radicals by DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, DOP-BDDE-PAAs / Res, BDDE-PAAs / HA-BA-Res and BDDE-PAAs / HA-BA hydrogels were 89.80±0.52%, 62.82±3.20%, 90.06±1.06%, 71.37±7.87% and 38.35±7.49%, respectively. These results indicate that both DOP and Res have good scavenging effects on DPPH free radicals, but the scavenging effect of Res is higher than that of DOP, and the combination of the two has an enhanced effect on scavenging DPPH.

[0157] 6. The DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, DOP-BDDE-PAAs / Res, BDDE-PAAs / HA-BA-Res and BDDE-PAAs / HA-BA hydrogel samples obtained in Examples 1 to 5 were incubated in 1 mL of DMEM medium for 24 h to obtain extracts for evaluating cell proliferation and cell migration tests. 100 μL (2.5×10 3 L929 cells (100 cells / well) were inoculated in a 96-well plate for 24 hours. The supernatant was then replaced with the extract and incubated for 24 hours and 48 hours. At 24 hours and 48 hours, 20 μL of MTT solution was added and incubated for 4 hours. The supernatant was removed and 100 μL of DMSO was added. The plate was shaken for 1 minute and the absorbance was measured at 490 nm.

[0158] The cell compatibility and blood compatibility of hydrogels are basic properties of biomaterials in clinical applications.

[0159] The MTT method was used to quantitatively evaluate the effect of hydrogel on the proliferation of fibroblasts (L929). Fig.10 and Fig.11 As shown, the proliferation rates of the experimental groups were higher than those of the control group, indicating that the hydrogels had no cytotoxicity. The proliferation rates of the DOP-BDDE-PAAs / HA-BA-Res and DOP-BDDE-PAAs / HA-BA hydrogel groups within 48 hours were 143.93±3.22% and 138.07±2.23%, respectively, which were significantly higher than those of the control group (P<0.0001). There was no difference between the BDDE-PAAs / HA-BA-Res hydrogel group and the control group within 24 hours and 48 hours, indicating that the DOP in the hydrogel can promote the growth of fibroblasts and further promote wound tissue repair.

[0160] 7. The DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, DOP-BDDE-PAAs / Res, BDDE-PAAs / HA-BA-Res and BDDE-PAAs / HA-BA hydrogel samples obtained in Examples 1 to 5 were incubated in 2 mL of 2 mL 1% fetal bovine serum (FBS) DMEM medium for 24 h to obtain an extract, and then 2 mL of L929 cell suspension was planted in a 6-well plate for 24 h, and a 200 μL pipette tip was used to draw a mark in each well. Then the supernatant was removed and washed 3 times with PBS solution and replaced with 2 mL 1% fetal bovine serum (FBS) DMEM medium extract and 2 mL 1% fetal bovine serum (FBS) DMEM medium were incubated for 24 h and 48 h. Fluorescence images were obtained using an inverted fluorescence microscope and analyzed using imageJ software.

[0161] In vitro scratch test results Fig.12 As shown, as time goes by, L929 cells gradually migrate to the scratch area, and the migration induced by DOP-BDDE-PAAs / HA-BA-Res is significantly higher than that in the control group, indicating that L929 cells exhibit better migration ability, which is beneficial to wound healing.

[0162] 8. Add 500 μL (2.5×10 5 cells / well) L929 cells were cultured in 24-well plates for 24 h. The experiment was divided into 7 groups, 5 experimental groups (DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, DOP-BDDE-PAAs / Res, BDDE-PAAs / HA-BA-Res and BDDE-PAAs / HA-BA hydrogel groups), ROSup group and Control group. ROSup drugs can induce cells to produce highly reactive oxygen species, so we added ROSup solution to the experimental group and ROSup group for 4 h. After that, the supernatant was removed from all wells, washed 3 times with PBS, and the hydrogel extraction solution was added to the experimental group, and complete culture medium was added to the ROSup group and Control group. Washed 3 times with PBS, stained with 2',7'-dichlorodihydrofluorescein (DCFH-DA) solution (10μM) in the dark for 40 min. Fluorescence images were obtained by laser confocal microscopy and analyzed with imageJ software.

[0163] Hyperglycemia patients have disordered blood sugar metabolism, which leads to limited expression of the antioxidant system in the body, excessive production of reactive oxygen species, causing normal cell death and tissue damage, and reducing the healing rate of diabetic wounds. Therefore, regulating the homeostasis of reactive oxygen species at the wound is also the key to promoting diabetic wound healing. The expression level of DCFH-DA in L929 cells was detected by immunofluorescence staining. Fig.13 As shown, the ROSup group showed obvious green fluorescence, indicating that ROSup successfully stimulated the reactive oxygen species in the cells. Compared with the ROSup group, there was no significant difference in the green fluorescence of the hydrogel in the BDDE-PAAs / HA-BA group, while the green fluorescence of DOP-BDDE-PAAs / HA-BA and BDDE-PAAs / HA-BA-Res was significantly weakened. The green fluorescence of the DOP-BDDE-PAAs / HA-BA-Res and DOP-BDDE-PAAs / Res hydrogel treatment groups has approached that of the Control and cannot be observed by the naked eye. It proves that the hydrogel containing DOP and Res is a good ROS scavenger.

[0164] 9. Add 1.5 mL (1.5 × 10 5After cultured with a suspension of macrophages (RAW 264.7) cells / well in a laser confocal dish for 24 h, the control group was treated with 3 mL of complete medium; the LPS group was treated with 3 mL of lipopolysaccharide (LPS, 1 ng / mL) + complete medium; the experimental group was treated with 3 mL of lipopolysaccharide (LPS, 1 ng / mL) + hydrogel (DOP-BDDE-PAAs / HA-BA-Res, DOP-BDDE-PAAs / HA-BA, DOP-BDDE-PAAs / Res, BDDE-PAAs / HA-BA-Res and BDDE-PAAs / HA-BA hydrogel groups). The cells were fixed with paraformaldehyde (1mL 4%) for 15min; washed with PBS, and 500μL Triton X-100 was added to break the membrane for 30min; washed with PBS, blocked with bovine serum albumin (5%, 500μL) solution for 1h; washed with PBS, M2 macrophages were labeled with CD206 (1:400, 500μL), and placed at 4℃ overnight. Washed with PBS, IgG (1:200, 500μL) was added and incubated at 37℃ in the dark for 2h, and then 300μL DAPI was used to stain the cell nucleus for positioning, and fluorescence images were obtained using a laser confocal microscope (CLSM).

[0165] DOP and Res have been shown to have anti-inflammatory effects; however, in the field of diabetic wound repair, it has not yet been studied whether the combination of DOP and Res has a synergistic anti-inflammatory effect. Natural anti-inflammatory substances have always been a hot topic in the field of biomedical materials. Therefore, we studied the in vitro anti-inflammatory effects of DOP and Res. Immune system disorders can interfere with the natural healing of wounds, and persistent inflammatory responses can lead to wound ulcers and necrosis. Therefore, effective regulation of macrophage phenotype is crucial to promoting diabetic wound healing. Lipopolysaccharide (LPS) can induce M0 macrophages to differentiate into M1 (pro-inflammatory) macrophages and reduce the expression of CD206, a marker of M2 (anti-inflammatory) macrophages. Fig.14 As shown, the green fluorescence of the LPS group was weaker than that of the control group, indicating that the induction of M0→M1 was successful (indicating that lipopolysaccharide can induce the differentiation of M0 macrophages into M1 macrophages). The CD206 expression of the DOP-BDDE-PAAs / HA-BA-Res hydrogel and DOP-BDDE-PAAs / Res hydrogel treatment groups was significantly higher than that of the control group (increased green fluorescence). Especially the DOP-BDDE-PAAs / HA-BA-Res hydrogel, indicating that DOP-BDDE-PAAs / HA-BA-Res has the effect of promoting the polarization of macrophages from M1 to M2 morphology.

Claims

1. A method for preparing a Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel, characterized in that: The preparation method of the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel is carried out according to the following steps: Step 1: Preparation of hyaluronic acid-3-aminophenylboronic acid-resveratrol: ①, hyaluronic acid and 3-aminophenylboronic acid are mixed, added to an aqueous solution of dimethyl sulfoxide, and then a dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added, the pH is adjusted to 4.5 with an aqueous HCl solution, and the mixture is reacted at 30° C. for 24 hours to obtain a hyaluronic acid-3-aminophenylboronic acid solution; after dialysis with pure water, the mixture is freeze-dried to obtain a hyaluronic acid-3-aminophenylboronic acid flocculent; The mass ratio of the hyaluronic acid to 3-aminophenylboronic acid is (3-1): (0.5-2); The mass ratio of the hyaluronic acid to the volume of the aqueous solution of dimethyl sulfoxide is 1 g:70 mL; the mass ratio of dimethyl sulfoxide to water in the aqueous solution of dimethyl sulfoxide is 2:5; The mass ratio of the hyaluronic acid to the mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1.52; The molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1; The mass fraction of N-hydroxysuccinimide in the dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1.9%; ②, dispersing the prepared hyaluronic acid-3-aminophenylboronic acid flocs in pure water to obtain hyaluronic acid-3-aminophenylboronic acid gel; The ratio of the mass of the hyaluronic acid-3-aminophenylboronic acid floccules to the volume of pure water is 0.09 g: 2 mL; ③, dispersing resveratrol in anhydrous ethanol to obtain a resveratrol solution; ④, mixing the prepared resveratrol solution with the prepared hyaluronic acid-3-aminophenylboronic acid gel to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol; The ratio of the volume of the resveratrol solution to the mass of the hyaluronic acid-3-aminophenylboronic acid floccules is 300 μL: 0.09 g; Step 2: Preparation of Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel: ①, adding Dendrobium officinale polysaccharide to NaOH solution to obtain Dendrobium officinale polysaccharide solution, adding 1,4-butanediol diglycidyl ether, and after the reaction is completed at 30°C, obtaining Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel; The mass ratio of the Dendrobium officinale polysaccharide to 1,4-butanediol diglycidyl ether is 0.48:0.1-1; ②, mixing the sodium polyacrylate aqueous solution with the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel, dialyzing with ethanol aqueous solution and pure water as dialysis media in turn, adjusting the pH to 7.45-8.2, and obtaining the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel; The volume ratio of the sodium polyacrylate solution to the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether hydrogel is (2-1): (1-3); Step 3, mixing hyaluronic acid-3-aminophenylboronic acid-resveratrol and Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel to obtain Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether / hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel; The volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol and the Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is (1-3): (3-6).

2. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: Step 1① The concentration of the HCl aqueous solution is 1 mol / L.

3. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: Step 1③ The mass fraction of resveratrol in the resveratrol solution is 1-5%.

4. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: Step 2① The mass fraction of the Dendrobium officinale polysaccharide solution is 15-25%.

5. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: Step 2① The mass fraction of the NaOH solution is 1-3%.

6. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: In step 2①, the reaction time at 30° C. is 8 to 24 hours.

7. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: The mass fraction of the sodium polyacrylate aqueous solution described in step 2② is 1-5%.

8. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: The mass fraction of the ethanol aqueous solution in step 2② is 15%.

9. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: In step 3, the volume ratio of hyaluronic acid-3-aminophenylboronic acid-resveratrol and Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is 1:

6.

10. The method for preparing the Dendrobium officinale polysaccharide-based dual-responsive drug-releasing hydrogel according to claim 1, characterized in that: In step 3, the volume ratio of hyaluronic acid-3-aminophenylboronic acid-resveratrol and Dendrobium officinale polysaccharide-1,4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is 2.3:5.

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