CN119818716A - Preparation method of dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel - Google Patents
Preparation method of dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel Download PDFInfo
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Abstract
A preparation method of dendrobium candidum polysaccharide-based dual-response liquid-releasing gel relates to a preparation method of liquid-releasing gel. The invention aims to solve the problem of difficult healing of diabetic wounds. According to the preparation method, hyaluronic acid-3-aminophenylboronic acid is synthesized, resveratrol is connected, and dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate is finally doped, so that dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel is prepared. The boric acid ester bond in the hydrogel can be broken in the environment of high glucose or high active oxygen, so that the active substance resveratrol can be released in a responsive way. The prepared hydrogel has the characteristics of injectability, self-healing and sensitivity to pathological environments responding to diabetes wounds, can effectively inhibit the level of ROS in cells, regulate and control the polarization of macrophage M2, and has obvious proliferation effect on fibroblasts, so that wound gap closure is promoted, and the healing time of wounds is shortened.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of a liquid medicine releasing gel.
Background
The diabetes wound is difficult to heal due to complex pathophysiological mechanism, and if not properly or timely treated, infection, gangrene and even amputation can be caused, so that great economic and psychological burden is brought to patients. Traditional wound dressing function is single, and wound environment suitability is not enough, is difficult to satisfy the complicated demand of healing process.
Hydrogel wound dressings are one of the new types of dressing currently in clinical use. The hydrophilic three-dimensional network structure of hydrogels is similar to the extracellular matrix (ECM) and can provide a suitable living environment for cells. At present, the hydrogel wound dressing used clinically has relatively single effect, which not only limits the use effect of the hydrogel dressing, but also prolongs the healing time of the wound. In the current research, many hydrogel wound dressings take animal-derived polysaccharide (such as chitosan, gelatin and the like) or algae-derived polysaccharide (such as sodium alginate) as a framework material and load small molecule drugs, which often causes sudden release or irregular release of the small molecule drugs in the using process, and dynamic drug release according to the change of the microenvironment of the wound cannot be realized, so that adverse reaction is caused or the treatment effect is reduced. On the other hand, hydrogel matrix materials having no specific activity do not provide biochemical signals related to tissue repair, and thus the dressing, although in contact with wound tissue, cannot effectively support cell proliferation or differentiation, resulting in poor wound repair effects. In addition, most of the clinical wound dressings are mainly patches, lack of flexibility, cannot adapt to the shape of a wound and are filled, and the hydrogel is not tightly combined with wound tissues.
The dendrobium candidum polysaccharide (Dendrobium officinale polysaccharide, DOP) is one of the main components of traditional Chinese medicine dendrobium candidum, and has high medicinal value. Dendrobium candidum polysaccharide has better pharmacological action for promoting proliferation and migration of fibroblast. Resveratrol (Resveratrol, res) is a natural polyphenol compound with various biological activities, and the effects of scavenging active oxygen, regulating macrophage polarization and regulating inflammatory factor expression are currently confirmed. At present, DOP is used as a hydrogel skeleton, and Res-loaded hydrogel dressing is applied to treatment of diabetes wounds.
Disclosure of Invention
The invention provides a preparation method of dendrobium candidum polysaccharide-based dual-response liquid-releasing gel for solving the problem of difficult healing of diabetic wounds. Firstly synthesizing hyaluronic acid-3-aminophenylboronic acid, connecting resveratrol, and finally doping dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate, thereby preparing dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel. The boric acid ester bond in the hydrogel can be broken in the environment of high glucose or high active oxygen, so that the active substance resveratrol can be released in a responsive way.
The preparation method of the dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel comprises the following steps:
Step one, preparing hyaluronic acid-3-aminophenylboronic acid-resveratrol:
① . Mixing hyaluronic acid and 3-aminophenylboric acid, adding the mixture into an aqueous solution of dimethyl sulfoxide, adding a dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and reacting for 24 hours by adjusting the pH value to be 4.5,30 ℃ with an aqueous solution of HCl to obtain a hyaluronic acid-3-aminophenylboric acid solution;
The mass ratio of the hyaluronic acid to the 3-aminophenylboronic acid is (3-1) (0.5-2);
the ratio of the mass of the hyaluronic acid to the volume of the aqueous solution of the dimethyl sulfoxide is 1g to 70mL, and the mass ratio of the dimethyl sulfoxide to the water in the aqueous solution of the 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 the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 1:1;
the mass fraction of N-hydroxysuccinimide in the dimethyl sulfoxide solution of the mixture of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide was 1.9%;
② . Dispersing the prepared hyaluronic acid-3-aminophenylboronic acid floccule in pure water to obtain hyaluronic acid-3-aminophenylboronic acid gel;
the ratio of the mass of the hyaluronic acid-3-aminophenylboronic acid floc to the volume of the pure water is 0.09 g/2 mL;
③ . Dispersing resveratrol in absolute ethyl alcohol to obtain 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 floc is 300 mu L and 0.09g;
step two, preparing dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel:
① . Adding dendrobium candidum polysaccharide into NaOH solution to obtain dendrobium candidum polysaccharide solution, adding 1, 4-butanediol diglycidyl ether, and obtaining dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel after reaction is completed at 30 ℃;
the mass ratio of the dendrobium candidum polysaccharide to the 1, 4-butanediol diglycidyl ether is 0.48:0.1-1;
② . Mixing a sodium polyacrylate aqueous solution with dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel, sequentially dialyzing by using an ethanol aqueous solution and pure water as dialysis media, and adjusting the pH to 7.45-8.2 to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel;
The volume ratio of the sodium polyacrylate solution to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel is (2-1) (1-3);
Step three, mixing hyaluronic acid-3-aminophenylboronic acid-resveratrol and dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel;
the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is (1-3) (3-6).
The invention has the beneficial effects that:
1. The hydrogel prepared by the invention has the performances of responding to drug release, injectability, self-healing property, swelling property and the like of the microenvironment of the diabetes wound. And modifying 3-aminophenylboronic acid on a hyaluronic acid molecular chain to synthesize hyaluronic acid-3-aminophenylboronic acid, and connecting resveratrol with the hyaluronic acid-3-aminophenylboronic acid to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol. Mixing the dendrobium candidum polysaccharide self-crosslinked hydrogel with sodium polyacrylate to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel, and incubating the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel with hyaluronic acid-3-aminophenylboronic acid-resveratrol to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel. On one hand, the borate ester bond in the hydrogel is broken in a high glucose or active oxygen environment, so that the responsive release of resveratrol is realized, and the disordered release of resveratrol is avoided. On the other hand, the hydrogel has injectable and self-healing properties, can increase the bonding area of the hydrogel and wound tissues and isolate the wound from the external environment.
2. The dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel prepared by the invention has the functions of resisting oxidation, promoting M2 polarization of macrophages and resisting inflammation, and has obvious proliferation effect on fibroblasts, thereby promoting wound gap closure and shortening wound healing time.
3. The dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel is easy to manufacture, and the dendrobium candidum polysaccharide and the resveratrol are wide in sources and low in cost.
4. According to the invention, the small molecule active substance is connected with the traditional Chinese medicine source polysaccharide skeleton through the environment-sensitive connecting arm, the small molecule and the polysaccharide are used as drugs which directly act on the wound site, and the dual-response diabetes wound microenvironment (glucose and active oxygen) releases drugs, so that the small molecule and the traditional Chinese medicine polysaccharide are jointly applied to inflammation and oxidative stress inhibition of the diabetes wound, and the proliferation and migration of fibroblast cells in the proliferation stage are promoted, and the continuous treatment effect is realized. At present, a document for applying dendrobium candidum polysaccharide-based hydrogel to diabetes wound treatment is not reported.
5. The traditional Chinese medicine polysaccharide generally has good biocompatibility, but hydrogel formed by self-crosslinking is poor in mechanical strength, and the artificially synthesized polymer has good mechanical properties. The invention combines the artificially synthesized sodium polyacrylate and the traditional Chinese medicine polysaccharide, and solves the problem of poor mechanical property of the pure traditional Chinese medicine polysaccharide hydrogel skeleton.
Drawings
FIG. 1 is a scanning electron microscope image of DOP-BDDE-PAAs/HA-BA-Res hydrogels prepared in example 1;
FIG. 2 is a scanning electron microscope image of DOP-BDDE-PAAs/HA-BA hydrogels prepared in example 2;
FIG. 3 is a scanning electron microscope image of DOP-BDDE-PAAs/Res hydrogels prepared in example 3;
FIG. 4 is a scanning electron microscope image of BDDE-PAAs/HA-BA-Res hydrogels prepared in example 4;
FIG. 5 is a scanning electron microscope image of BDDE-PAAs/HA-BA hydrogels prepared in example 5;
FIG. 6 is a graph of swelling ratios for different sets of hydrogels;
FIG. 7 is a graph showing the self-healing, injectability and adhesive properties of DOP-BDDE-PAAs/HA-BA-Res hydrogels prepared in example 1;
FIG. 8 is a graph showing the glucose vs. active oxygen response characteristics of DOP-BDDE-PAAs/HA-BA-Res hydrogels prepared in example 1;
FIG. 9 is a graph of antioxidant activity of different sets of hydrogels;
FIG. 10 is a bar graph of the relative proliferation count of fibroblasts in different groups after 24h incubation;
FIG. 11 is a bar graph of the relative proliferation count of fibroblasts in different groups after 48h incubation;
FIG. 12 is a graph of fibroblast migration experiments for different sets of hydrogels;
FIG. 13 is a graph of ROS scavenging ability of various sets of hydrogels against fibroblasts;
FIG. 14 is a fluorescence image of M2 polarization of different sets of hydrogel regulatory macrophages.
Detailed Description
The technical scheme of the invention is not limited to the specific embodiments listed below, and also comprises any reasonable combination of the specific embodiments.
The preparation method of the dendrobium candidum polysaccharide-based dual-response release hydrogel comprises the following steps:
Step one, preparing hyaluronic acid-3-aminophenylboronic acid-resveratrol:
① . Mixing hyaluronic acid and 3-aminophenylboric acid, adding the mixture into an aqueous solution of dimethyl sulfoxide, adding a dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and reacting for 24 hours by adjusting the pH value to be 4.5,30 ℃ with an aqueous solution of HCl to obtain a hyaluronic acid-3-aminophenylboric acid solution;
The mass ratio of the hyaluronic acid to the 3-aminophenylboronic acid is (3-1) (0.5-2);
the ratio of the mass of the hyaluronic acid to the volume of the aqueous solution of the dimethyl sulfoxide is 1g to 70mL, and the mass ratio of the dimethyl sulfoxide to the water in the aqueous solution of the 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 the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 1:1;
the mass fraction of N-hydroxysuccinimide in the dimethyl sulfoxide solution of the mixture of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide was 1.9%;
② . Dispersing the prepared hyaluronic acid-3-aminophenylboronic acid floccule in pure water to obtain hyaluronic acid-3-aminophenylboronic acid gel;
the ratio of the mass of the hyaluronic acid-3-aminophenylboronic acid floc to the volume of the pure water is 0.09 g/2 mL;
③ . Dispersing resveratrol in absolute ethyl alcohol to obtain 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 floc is 300 mu L and 0.09g;
step two, preparing dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel:
① . Adding dendrobium candidum polysaccharide into NaOH solution to obtain dendrobium candidum polysaccharide solution, adding 1, 4-butanediol diglycidyl ether, and obtaining dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel after reaction is completed at 30 ℃;
the mass ratio of the dendrobium candidum polysaccharide to the 1, 4-butanediol diglycidyl ether is 0.48:0.1-1;
② . Mixing a sodium polyacrylate aqueous solution with dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel, sequentially dialyzing by using an ethanol aqueous solution and pure water as dialysis media, and adjusting the pH to 7.45-8.2 to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel;
The volume ratio of the sodium polyacrylate solution to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel is (2-1) (1-3);
Step three, mixing hyaluronic acid-3-aminophenylboronic acid-resveratrol and dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel (DOP-BDDE-PAAs/HA-BA-Res);
the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is (1-3) (3-6).
The present embodiment has the following advantageous effects:
1. The hydrogel prepared by the embodiment has the properties of drug release, injectability, self-healing property, swelling property and the like in response to the microenvironment of the diabetes wound. And modifying 3-aminophenylboronic acid on a hyaluronic acid molecular chain to synthesize hyaluronic acid-3-aminophenylboronic acid, and connecting resveratrol with the hyaluronic acid-3-aminophenylboronic acid to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol. Mixing the dendrobium candidum polysaccharide self-crosslinked hydrogel with sodium polyacrylate to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel, and incubating the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel with hyaluronic acid-3-aminophenylboronic acid-resveratrol to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel. On one hand, the borate ester bond in the hydrogel is broken in a high glucose or active oxygen environment, so that the responsive release of resveratrol is realized, and the disordered release of resveratrol is avoided. On the other hand, the hydrogel has injectable and self-healing properties, can increase the bonding area of the hydrogel and wound tissues and isolate the wound from the external environment.
2. The dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel prepared by the embodiment has the functions of resisting oxidation, promoting M2 polarization of macrophages and resisting inflammation, and has obvious proliferation effect on fibroblasts, so that wound gap closure is promoted, and wound healing time is shortened.
3. The dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel is easy to manufacture, and the dendrobium candidum polysaccharide and the resveratrol are wide in sources and low in cost.
4. According to the embodiment, the micromolecular active substances are connected with the traditional Chinese medicine source polysaccharide skeleton through the environment-sensitive connecting arm, and the micromolecular active substances and the polysaccharide are used as drugs directly acting on the wound part, double-response diabetes wound microenvironment (glucose and active oxygen) release drugs, so that the combined application of the micromolecular active substances and the traditional Chinese medicine polysaccharide in inflammation and oxidative stress inhibition of the diabetes wound is realized, the proliferation and migration of fibroblast cells in the proliferation stage are promoted, and the continuous treatment effect is realized. At present, a document for applying dendrobium candidum polysaccharide-based hydrogel to diabetes wound treatment is not reported.
5. The traditional Chinese medicine polysaccharide generally has good biocompatibility, but hydrogel formed by self-crosslinking is poor in mechanical strength, and the artificially synthesized polymer has good mechanical properties. According to the embodiment, the artificially synthesized sodium polyacrylate and the traditional Chinese medicine polysaccharide are combined, so that the problem of poor mechanical property of the pure traditional Chinese medicine polysaccharide hydrogel skeleton is solved.
In the second embodiment, the difference between the first embodiment and the second embodiment is that the concentration of the HCl aqueous solution in the first step ① is 1mol/L.
The third embodiment is different from the first embodiment or the second embodiment in that the mass fraction of resveratrol in the resveratrol solution in the first ③ is 1-5%.
In a fourth embodiment, the difference between the fourth embodiment and one or three embodiments is that the mass fraction of the dendrobium candidum polysaccharide solution in the second ① is 15-25%.
In a fifth embodiment, the difference between the fifth embodiment and the first to fourth embodiments is that the mass fraction of the NaOH solution in the second ① is 1-3%.
In a sixth embodiment, the difference between the present embodiment and one of the first to fifth embodiments is that the reaction time at 30 ℃ in the second ① is 8 to 24 hours.
In a seventh embodiment, the difference between the seventh embodiment and one of the first to sixth embodiments is that the mass fraction of the sodium polyacrylate aqueous solution in the second ② is 1-5%.
In a eighth embodiment, the difference between the first embodiment and the seventh embodiment is that the mass fraction of the aqueous ethanol solution in the second ② is 15%.
The difference between the embodiment and one of the embodiments from one to eight is that the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel in the step three is 1:6.
The tenth embodiment is different from one of the first to ninth embodiments in that the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel in the third step is 2.3:5.
Example 1
The preparation method of the dendrobium candidum polysaccharide-based dual-response liquid release gel (DOP-BDDE-PAAs/HA-BA-Res) comprises the following steps:
Step one, preparing hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res):
① .1g of Hyaluronic Acid (HA) is mixed with 0.68g of 3-aminophenylboric acid (PBA), added into 70mL of an aqueous solution of dimethyl sulfoxide (DMSO), added with 30mL of a dimethyl sulfoxide solution of a mixture of 0.57g of N-hydroxysuccinimide (NHS) and 0.95g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), and reacted for 24 hours by adjusting the pH to be = 4.5,30 ℃ with 1mol/L of aqueous solution of HCl to obtain a hyaluronic acid-3-aminophenylboric acid solution, dialyzed with pure water and freeze-dried to obtain hyaluronic acid-3-aminophenylboric acid floccule (HA-BA);
The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;
② . Dispersing the prepared 0.09g HA-BA in 2mL pure water to obtain HA-BA gel;
③ . Dispersing resveratrol (Res) in absolute ethyl alcohol to obtain a Res solution, wherein the Res fraction in the Res solution is 2%;
④ . Mixing the prepared solution 300 mu LRes with the prepared HA-BA gel to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res);
Step two, preparing dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs):
① . Adding 0.48g of dendrobium candidum polysaccharide (DOP) into 2mLNaOH g of solution to obtain DOP solution, adding 0.5g of 1, 4-butanediol diglycidyl ether (BDDE), and reacting at 30 ℃ for 16 hours to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel (DOP-BDDE), wherein the mass fraction of the NaOH solution is 2%;
② . Mixing 2mL of sodium Polyacrylate (PAAs) aqueous solution with 2.5mLDOP-BDDE, dialyzing sequentially with ethanol aqueous solution and pure water as dialysis medium, and adjusting pH to 7.45-8.2 to obtain Dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs);
The mass fraction of the PAAs aqueous solution is 3%;
the mass fraction of the ethanol water solution is 15%;
And step three, mixing the HA-BA-Res and the DOP-BDDE-PAAs to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel (DOP-BDDE-PAAs/HA-BA-Res).
The volume ratio of HA-BA-Res to DOP-BDDE-PAAs is 2.3:5.
Example 2
The preparation method of the DOP-BDDE-PAAs/HA-BA hydrogel comprises the following steps:
step one, preparing hyaluronic acid-3-aminophenylboronic acid (HA-BA):
① . 1g of Hyaluronic Acid (HA) is mixed with 0.68g of 3-aminophenylboric acid (PBA), added into 70mL of an aqueous solution of dimethyl sulfoxide (DMSO), added with 30mL of a dimethyl sulfoxide solution of a mixture of 0.57g of N-hydroxysuccinimide (NHS) and 0.95g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), and reacted for 24 hours by adjusting the pH to be = 4.5,30 ℃ with 1mol/L of aqueous solution of HCl to obtain a hyaluronic acid-3-aminophenylboric acid solution, dialyzed with pure water and freeze-dried to obtain hyaluronic acid-3-aminophenylboric acid floccule (HA-BA);
The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;
② . Dispersing the prepared 0.09g HA-BA in 2mL pure water to obtain HA-BA gel;
Step two, preparing dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs):
① . Adding 0.48g of dendrobium candidum polysaccharide (DOP) into 2mL of NaOH solution to obtain DOP solution, adding 0.5g of 1, 4-butanediol diglycidyl ether (BDDE), and reacting at 30 ℃ for 16 hours to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel (DOP-BDDE), wherein the mass fraction of the NaOH solution is 2%;
② . Mixing 2mL of sodium Polyacrylate (PAAs) aqueous solution with 2.5mLDOP-BDDE, dialyzing sequentially with ethanol aqueous solution and pure water as dialysis medium, and adjusting pH to 7.45-8.2 to obtain Dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs);
The mass fraction of the PAAs aqueous solution is 3%;
the mass fraction of the ethanol water solution is 15%;
Step three, mixing HA-BA and DOP-BDDE-PAAs to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate/hyaluronic acid-3-aminophenylboronic acid hydrogel (DOP-BDDE-PAAs/HA-BA);
The volume ratio of the HA-BA to the DOP-BDDE-PAAs hydrogel is 2:5.
Example 3
The preparation method of the DOP-BDDE-PAAs/Res hydrogel comprises the following steps:
Step one, preparing dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs):
① . Adding 0.48g of dendrobium candidum polysaccharide (DOP) into 2mL of NaOH solution to obtain DOP solution, adding 0.5g of 1, 4-butanediol diglycidyl ether (BDDE), and reacting at 30 ℃ for 16 hours to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel (DOP-BDDE), wherein the mass fraction of the NaOH solution is 2%;
② . Mixing 2mL of sodium Polyacrylate (PAAs) aqueous solution with 2.5mLDOP-BDDE hydrogel, sequentially dialyzing with ethanol aqueous solution and pure water as dialysis medium, and adjusting pH to 7.45-8.2 to obtain Dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (DOP-BDDE-PAAs);
dispersing resveratrol (Res) in absolute ethyl alcohol to obtain a resveratrol solution, wherein the Res mass fraction in the Res solution is 2%;
And thirdly, mixing the Res solution with DOP-BDDE-PAAs to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel/resveratrol (DOP-BDDE-PAAs/Res).
The volume ratio of Res solution to DOP-BDDE-PAAs was 0.3:5.
Example 4
The preparation method of the BDDE-PAAs/HA-BA-Res hydrogel comprises the following steps:
① .1g of Hyaluronic Acid (HA) is mixed with 0.68g of 3-aminophenylboric acid (PBA), added into 70mL of an aqueous solution of dimethyl sulfoxide (DMSO), added with 30mL of a dimethyl sulfoxide solution of a mixture of 0.57g of N-hydroxysuccinimide (NHS) and 0.95g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), and reacted for 24 hours by adjusting the pH to be = 4.5,30 ℃ with 1mol/L of aqueous solution of HCl to obtain a hyaluronic acid-3-aminophenylboric acid solution, dialyzed with pure water and freeze-dried to obtain hyaluronic acid-3-aminophenylboric acid floccule (HA-BA);
The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;
② . Dispersing the prepared 0.09g HA-BA in 2mL pure water to obtain HA-BA gel;
③ . Dispersing resveratrol (Res) in absolute ethyl alcohol to obtain a Res solution, wherein the Res mass fraction in the Res solution is 2%;
④ . Mixing the prepared solution 300 mu LRes with the prepared HA-BA gel to obtain hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res);
step two, preparing 1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs):
① . Adding 0.5g of 1, 4-butanediol diglycidyl ether (BDDE) into the 2mLNaOH solution, and reacting for 16 hours at 30 ℃ to obtain a BDDE alkaline solution, wherein the mass fraction of the NaOH solution is 2%;
② . Mixing 2mL of sodium Polyacrylate (PAAs) aqueous solution with 2.5mLBDDE alkaline solution, dialyzing sequentially by using ethanol aqueous solution and pure water as dialysis media, and regulating pH to 7.45-8.2 to obtain 1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs);
The mass fraction of the PAAs aqueous solution is 3%;
the mass fraction of the ethanol water solution is 15%;
And step three, mixing the HA-BA-Res and BDDE-PAAs to obtain the 1, 4-butanediol diglycidyl ether-sodium polyacrylate/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel (BDDE-PAAs/HA-BA-Res).
The volume ratio of HA-BA-Res to BDDE-PAAs was 2.3:5.
Example 5
The preparation method of the BDDE-PAAs/HA-BA hydrogel comprises the following steps:
Step one, preparing hyaluronic acid-3-aminophenylboronic acid-resveratrol (HA-BA-Res):
① . 1g of Hyaluronic Acid (HA) is mixed with 0.68g of 3-aminophenylboric acid (PBA), added into 70mL of an aqueous solution of dimethyl sulfoxide (DMSO), added with 30mL of a dimethyl sulfoxide solution of a mixture of 0.57g of N-hydroxysuccinimide (NHS) and 0.95g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), and reacted for 24 hours by adjusting the pH to be = 4.5,30 ℃ with 1mol/L of aqueous solution of HCl to obtain a hyaluronic acid-3-aminophenylboric acid solution, dialyzed with pure water and freeze-dried to obtain hyaluronic acid-3-aminophenylboric acid floccule (HA-BA);
The mass ratio of DMSO to water in the DMSO aqueous solution is 2:5;
② . Dispersing the prepared 0.09g HA-BA in 2mL pure water to obtain HA-BA gel;
step two, preparing 1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs):
① . Adding 0.5g of 1, 4-butanediol diglycidyl ether (BDDE) into 2mL of NaOH solution, and reacting for 16 hours at 30 ℃ to obtain BDDE alkaline solution, wherein the mass fraction of the NaOH solution is 2%;
② . Mixing 2mL of sodium Polyacrylate (PAAs) aqueous solution with 2.5mLBDDE alkaline solution, dialyzing sequentially by using ethanol aqueous solution and pure water as dialysis media, and regulating pH to 7.45-8.2 to obtain 1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel (BDDE-PAAs);
The mass fraction of the PAAs aqueous solution is 3%;
the mass fraction of the ethanol water solution is 15%;
and step three, mixing the HA-BA gel and BDDE-PAAs to obtain the 1, 4-butanediol diglycidyl ether-sodium polyacrylate/hyaluronic acid-3-aminophenylboronic acid hydrogel (BDDE-PAAs/HA-BA).
The volume ratio of the HA-BA gel to the BDDE-PAAs is 2:5.
The following analytical tests were performed on the products of examples 1 to 5:
1. The morphology of the hydrogels obtained in experimental examples 1 to 5 was observed by a scanning electron microscope, and after freeze-drying of fresh hydrogels, the hydrogels were placed on a sample stage and subjected to metal spraying, and the scanning electron microscope was used for observation, so that the scanning electron microscope images of examples 1 to 5 were obtained. As shown in fig. 1-5, five groups of hydrogels were observed to have interconnected porous network structures that could be used for oxygen and nutrient transport.
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 having pH=7.4, and then taken out after being placed in a 37 ℃ incubator for 24 hours, the surface moisture of the hydrogels was sucked up with filter paper and the hydrogel was weighed to swell ratio formula was as follows:
Swelling ratio (%) = (W 0-Wt)/W0 ×100%
Note that W t and W 0 are the weight of the composite hydrogels at 24h and 0 h.
In tissue engineering, the swelling ratio of hydrogels is an important indicator of biomaterials, and therefore we observe the swelling behavior of hydrogels in PBS at 37 ℃. As shown in fig. 6 (graph represents significant differences, data represents mean ± standard deviation of p <0.05, p <0.01, p <0.001, p < 0.0001.) hydrogel samples were taken out after 24h of immersion in PBS, each group of hydrogels reached equilibrium after 24h, swelling rate formed a denser crosslinked network with the addition of dendrobium candidum polysaccharide, improving water absorption properties. The results show that the DOP-BDDE-PAAs/HA-BA-Res, DOP-BDDE-PAAs/HA-BA and DOP-BDDE-PAAs/Res hydrogels have good swelling capacity, effectively absorb tissue exudates, reduce the possibility of wound infection and accelerate wound healing.
3. DOP-BDDE-PAAs/HA-BA-Res hydrogel was cut in half, dyed in different colors, combined and left for 30min, the hydrogel was held up with forceps, and checked for breakage. The hydrogel was placed in a 1mL syringe, and the injectability of the hydrogel was observed, and the ease of injection was observed. The prepared hydrogel is stuck on the joint of the finger, and the viscosity of the hydrogel is tested by bending the finger to see whether the hydrogel moves or falls.
The ideal diabetic wound dressing should have good self-healing, adhesion and injectability properties, and we selected DOP-BDDE-PAAs/HA-BA-Res hydrogels for characterization. As shown in fig. 7, we prepared a rectangular hydrogel sample, which was cut in half, one half of which was colored red. After a prolonged 30min contact of the sample, the healed whole hydrogel was lifted with forceps, and we observed that no break occurred at the seam and that stretching to both sides with forceps did not crack due to the interaction of the borate ester bonds in the hydrogel matrix. We gel DOP-BDDE-PAAs/HA-BA-Res into a 1mL syringe, the English letter "QMU" can be written easily, and is insoluble in injected water. And then injecting the hydrogel into a star mold, and demolding to form a star pattern. By bending the finger angle, it was found that DOP-BDDE-PAAs/HA-BA-Res hydrogels could be tightly adhered to the skin without falling off and that the DOP-BDDE-PAAs/HA-BA-Res hydrogels could still be tightly adhered by changing the adhesive materials such as fingers, wood, plastic, rubber and glass. These results indicate that DOP-BDDE-PAAs/HA-BA-Res hydrogels have good injectability, and adhesion.
4. The DOP-BDDE-PAAs/HA-BA-Res hydrogels obtained in example 1 were placed in 20mM glucose in PBS buffer, 1mM H 2O2 in PBS buffer, respectively, 1mL of release medium was removed at each time interval, and 1mL of fresh release medium was added. The UV absorbance of the release medium at 306nm was measured and the release was determined using a corresponding UV standard curve.
In diabetic chronic wounds with high blood glucose and reactive oxygen species levels, hydrogels responsive to glucose and ROS can achieve sustained release of active substances, promoting wound repair. As shown in fig. 8, the cumulative release of Res in the high sugar medium was increased by about 40% compared to the PBS medium. It was demonstrated that glucose could compete for Res-crosslinked 3-aminophenylboronic acid groups in DOP-BDDE-PAAs/HA-BA-Res hydrogels to effect Res release upon glucose stimulation. ROS can cleave dynamic borate ester bonds to achieve drug release properties that are sensitive to ROS. The cumulative release of Res in ROS medium was about 20% greater than in PBS medium. Indicating that the borate ester bond of the hydrogel releases Res more easily in a high sugar environment. We can also observe an increase in Res release in PBS medium with glucose and active oxygen of about 50% compared to PBS medium, and can see that in high sugar environments the cumulative release of Res from DOP-BDDE-PAAs/HA-BA-Res hydrogels is significantly higher than in high active oxygen environments. Compared with PBS containing glucose and PBS containing active oxygen, the hydrogel HAs the highest accumulated and released amount of the drug in the PBS containing glucose and active oxygen, and the diabetes wound just meets the required high-sugar and high-active oxygen environment, so that the DOP-BDDE-PAAs/HA-BA-Res hydrogel can release the drug better according to the requirement.
5. 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 were placed in centrifuge tubes, DPPH-absolute ethanol (200 mM, 2 mL) was added, and reacted in a shaking table at 37℃for 30min, and absorbance was measured with a microplate reader at 517 nm.
The present study uses chemochromic methods to determine the DPPH scavenging capacity of hydrogels. As shown in FIG. 9, 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 cleared DPPH free radical at 89.80.+ -. 0.52%, 62.82.+ -. 3.20%, 90.06.+ -. 1.06%, 71.37.+ -. 7.87%, 38.35.+ -. 7.49%, respectively. These results indicate that both DOP and Res have good DPPH radical scavenging effect, but Res has higher DPPH scavenging effect than DOP, and the combination of both has enhanced DPPH scavenging effect.
6. 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 1mL of DMEM medium for 24 hours to obtain extracts for evaluation of cell proliferation and cell migration assays. 100. Mu.L (2.5X10 3 cells/well) of L929 cells were seeded in 96-well plates for 24h. The supernatant was then replaced with the extract and incubated for 24h, 48h. 20. Mu.L of MTT solution was added at 24h and 48h for incubation for 4h, and 100. Mu.L of DMSO was added to the supernatant removed. The absorbance was measured at 490nm by shaking for 1 min.
The cytocompatibility and hemocompatibility of hydrogels are fundamental properties of biological materials in clinical applications.
The effect of hydrogels on the proliferative capacity of fibroblasts (L929) was quantitatively evaluated using MTT. As shown in FIGS. 10 and 11, the proliferation rates of the experimental groups were higher than that of the control group, indicating that the hydrogels were not cytotoxic, and 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). The BDDE-PAAs/HA-BA-Res hydrogel groups within 24h and 48h are not different from the control group, which shows that DOP in the hydrogel can promote the growth of fibroblasts and further promote the repair of wound tissues.
7. 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 1to 5 were incubated in 2mL of 1% Fetal Bovine Serum (FBS) DMEM medium for 24 hours to obtain an extract, 2mL of L929 cell suspension was seeded in 6-well plates for 24 hours, and traces were streaked out on each well with a 200. Mu.L pipette tip. The supernatant was then removed and washed 3 times with PBS solution and replaced with DMEM medium extract containing 2ml of 1% Fetal Bovine Serum (FBS) and incubated with 2ml of 1% Fetal Bovine Serum (FBS) DMEM medium for 24h, 48h. Fluorescence images were obtained using an inverted fluorescence microscope and analyzed using imageJ software.
The results of in vitro scratch experiments are shown in FIG. 12, L929 cells gradually migrate to the scratch area along with the extension of time, and DOP-BDDE-PAAs/HA-BA-Res induced migration is obviously higher than that of the control group, which shows that the L929 cells exhibit better migration capacity and are beneficial to wound healing.
8. Mu.L (2.5X10 5 cells/well) of L929 cells were cultured in 24-well plates for 24 hours. The experiments were divided into 7 groups, 5 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 groups, control group. ROSup the drug induced high reactive oxygen species in cells, so we added ROSup solution to the experimental group and ROSup group for 4h. After all the plates were removed the supernatant, washed 3 times with PBS, and the hydrogel extraction solution was added to the experimental group, ROSup and Control groups, and complete medium was added. Washed 3 times with PBS and stained with 2',7' -dichlorofluorescein (DCFH-DA) solution (10. Mu.M) for 40min in the dark. Fluorescence images were obtained using laser confocal and analyzed using imageJ software.
The hyperglycemic patient has limited expression of an in vivo antioxidant system due to the disorder of blood glucose metabolism, so that active oxygen is overproduced, normal cell death and tissue injury are caused, and the healing speed of a diabetic wound surface is reduced, so that the regulation of the active oxygen steady state at the wound is also a key for promoting the healing of the diabetic wound. The expression level of DCFH-DA in L929 cells was detected by immunofluorescent staining, as shown in fig. 13, and group ROSup showed a clear green fluorescence, indicating that ROSup successfully stimulated intracellular reactive oxygen species. Compared with ROSup, the BDDE-PAAs/HA-BA group hydrogel HAs no obvious difference in green fluorescence, while DOP-BDDE-PAAs/HA-BA and BDDE-PAAs/HA-BA-Res have obviously reduced green fluorescence. The green fluorescence of the DOP-BDDE-PAAs/HA-BA-Res and DOP-BDDE-PAAs/Res hydrogel treatment groups HAs approached Control and is not visible to the naked eye. Hydrogels containing DOP and Res proved to be a good ROS scavenger.
9. After 1.5mL (1.5X10 5 cells/well) of macrophage (RAW 264.7) suspension was cultured in a laser confocal dish for 24 hours, the control group was 3mL of complete medium, the LPS group was 3mL of lipopolysaccharide (LPS, 1 ng/mL) +complete medium, the experimental group was 3mL 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 group). Cells were fixed by paraformaldehyde (1 mL 4%) for 15min, washed with PBS, added with 500. Mu.L Triton X-100 for 30min, washed with PBS, blocked with bovine serum albumin (5%, 500. Mu.L) solution for 1h, washed with PBS, CD206 (1:400, 500. Mu.L) labeled M2 macrophages, and left at 4℃overnight. After washing with PBS and adding IgG (1:200,500. Mu.L) at 37℃for 2h in the dark, nuclei were stained with 300. Mu.L DAPI for localization, and fluorescence images were obtained using a confocal laser microscope (CLSM).
DOP and Res have been shown to have anti-inflammatory effects, however, in the field of diabetic wound repair, it has not been investigated whether DOP and Res in combination have synergistic anti-inflammatory effects. Natural anti-inflammatory substances have been a hotspot in the biomedical material field. Thus, we studied the anti-inflammatory effects of DOP and Res in vitro. Disorders of the immune system interfere with the inability of the wound to heal naturally, while persistent inflammatory responses lead to ulceration and necrosis of the wound, so effective regulation of macrophage phenotype is critical to promote diabetic wound healing. Lipopolysaccharide (LPS) is capable of inducing M0 macrophages to differentiate into M1 (pro-inflammatory) macrophages, reducing the expression of CD206, a marker of M2 (anti-inflammatory) macrophages. As shown in fig. 14, the LPS group showed weaker green fluorescence than the control group, indicating successful m0→m1 induction (indicating that lipopolysaccharide was able to induce M0 macrophages to differentiate into M1 macrophages). CD206 expression was significantly higher in DOP-BDDE-PAAs/HA-BA-Res hydrogels than in DOP-BDDE-PAAs/Res hydrogel treated groups compared to control groups (increased green fluorescence). In particular, DOP-BDDE-PAAs/HA-BA-Res hydrogels, indicate that DOP-BDDE-PAAs/HA-BA-Res HAs the effect of promoting the polarization of macrophages from the M1 to the M2 morphology.
Claims (10)
1. The preparation method of the dendrobium candidum polysaccharide-based dual-response liquid-released gel is characterized by comprising the following steps of:
Step one, preparing hyaluronic acid-3-aminophenylboronic acid-resveratrol:
① . Mixing hyaluronic acid and 3-aminophenylboric acid, adding the mixture into an aqueous solution of dimethyl sulfoxide, adding a dimethyl sulfoxide solution of a mixture of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and reacting for 24 hours by adjusting the pH value to be 4.5,30 ℃ with an aqueous solution of HCl to obtain a hyaluronic acid-3-aminophenylboric acid solution;
The mass ratio of the hyaluronic acid to the 3-aminophenylboronic acid is (3-1) (0.5-2);
the ratio of the mass of the hyaluronic acid to the volume of the aqueous solution of the dimethyl sulfoxide is 1g to 70mL, and the mass ratio of the dimethyl sulfoxide to the water in the aqueous solution of the 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 the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 1:1;
the mass fraction of N-hydroxysuccinimide in the dimethyl sulfoxide solution of the mixture of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide was 1.9%;
② . Dispersing the prepared hyaluronic acid-3-aminophenylboronic acid floccule in pure water to obtain hyaluronic acid-3-aminophenylboronic acid gel;
the ratio of the mass of the hyaluronic acid-3-aminophenylboronic acid floc to the volume of the pure water is 0.09 g/2 mL;
③ . Dispersing resveratrol in absolute ethyl alcohol to obtain 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 floc is 300 mu L and 0.09g;
step two, preparing dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel:
① . Adding dendrobium candidum polysaccharide into NaOH solution to obtain dendrobium candidum polysaccharide solution, adding 1, 4-butanediol diglycidyl ether, and obtaining dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel after reaction is completed at 30 ℃;
the mass ratio of the dendrobium candidum polysaccharide to the 1, 4-butanediol diglycidyl ether is 0.48:0.1-1;
② . Mixing a sodium polyacrylate aqueous solution with dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel, sequentially dialyzing by using an ethanol aqueous solution and pure water as dialysis media, and adjusting the pH to 7.45-8.2 to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel;
The volume ratio of the sodium polyacrylate solution to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether hydrogel is (2-1) (1-3);
Step three, mixing hyaluronic acid-3-aminophenylboronic acid-resveratrol and dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel to obtain dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether/hyaluronic acid-3-aminophenylboronic acid-resveratrol hydrogel;
the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is (1-3) (3-6).
2. The method for preparing dendrobium candidum polysaccharide-based dual-response release hydrogel of claim 1, wherein the concentration of the HCl aqueous solution in the first ① step is 1mol/L.
3. The method for preparing dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel of claim 1, wherein the mass fraction of resveratrol in the resveratrol solution in the step one ③ is 1-5%.
4. The preparation method of the dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel of claim 1, which is characterized in that the mass fraction of the dendrobium candidum polysaccharide solution in the second ① is 15-25%.
5. The preparation method of the dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel of claim 1, wherein the mass fraction of the NaOH solution in the second ① is 1-3%.
6. The preparation method of the dendrobium candidum polysaccharide-based dual-response liquid release gel of claim 1, wherein the reaction time of the second ① at 30 ℃ is 8-24 hours.
7. The preparation method of the dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel, which is characterized in that the mass fraction of the sodium polyacrylate aqueous solution in the step two ② is 1-5%.
8. The preparation method of the dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel of claim 1, wherein the mass fraction of the ethanol aqueous solution in the second ② is 15%.
9. The preparation method of the dendrobium candidum polysaccharide-based dual-responsiveness release hydrogel, which is characterized in that the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel is 1:6.
10. The preparation method of the dendrobium candidum polysaccharide-based dual-response liquid-release gel of claim 1, wherein the volume ratio of the hyaluronic acid-3-aminophenylboronic acid-resveratrol to the dendrobium candidum polysaccharide-1, 4-butanediol diglycidyl ether-sodium polyacrylate hydrogel in the step three is 2.3:5.
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