CN114276563A

CN114276563A - Preparation method of medical hydrogel, hydrogel and dressing

Info

Publication number: CN114276563A
Application number: CN202110348693.3A
Authority: CN
Inventors: 何一燕; 宋吉亮; 苑明; 秦加骏; 姜于航; 彭万佳; 顾忠伟
Original assignee: Jiangsu Ruiying New Material Technology Development Co ltd; Nanjing Tech University
Current assignee: Jiangsu Ruiying New Material Technology Development Co ltd; Nanjing Tech University
Priority date: 2020-12-14
Filing date: 2021-03-31
Publication date: 2022-04-05
Anticipated expiration: 2041-03-31
Also published as: CN114276563B

Abstract

The invention provides a method for preparing medical hydrogel, which comprises the steps of respectively modifying LA on HEP and MA-HHA in an esterification mode to prepare precursors of the hydrogel, namely LA-HEP and LA-MA-HHA. And adding a catalytic amount of reducing agent into the precursor mixed solution to trigger the disulfide bond part in the lipoic acid to be disconnected to form sulfhydryl, and forming disulfide bond again to trigger the two precursors to be crosslinked to form hydrogel through a sulfhydryl-disulfide bond dynamic exchange reaction. The invention also provides a medical hydrogel and a dressing comprising the hydrogel. The hydrogel is prepared by taking heparin sodium, lipoic acid, hyaluronic acid and mannose as raw materials of the composite material and adopting an activity controllable crosslinking strategy of a sulfhydryl-disulfide bond dynamic exchange reaction, and has the functions of continuously resisting inflammation and promoting repair in batches.

Description

Preparation method of medical hydrogel, hydrogel and dressing

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of medical hydrogel and a medical dressing containing the hydrogel.

Background

Diabetic food syndrome is one of the major health problems of diabetic patients worldwide, diabetes being a chronic hyperglycemia and generally divided into two prevalent types, type 1 diabetes and type 2 diabetes (T2D), wherein T2D is non-insulin dependent diabetes caused by autoimmune insulin deficiency and insulin resistance, whereas diabetic patients have T2D accounting for 90-95%. Wound healing is a complex, highly regulated process that is critical to maintaining skin barrier function, and diabetic wounds have slow or even no healing conditions, resulting in diabetic feet and amputations, which are major complications leading to high disability rates in diabetes and can be life threatening in severe cases. The healing of surface wounds requires the synergistic action of multiple factors to restore the barrier function of the injured skin, and the mechanisms causing the difficulty in healing diabetic wounds are complex, including oxidative stress, chronic long-term inflammation, reduction of new blood vessels, peripheral neuropathy, extracellular matrix accumulation, and imbalance in remodeling.

The persistent inflammatory response and excess Reactive Oxygen Species (ROS) in the wounds of diabetic patients are one of the major causes of delayed wound healing in diabetic patients. In T2D, excessive inflammatory factors and ROS produced by leukocytes are not normally eliminated in the acute inflammatory phase of diabetic wounds due to impaired glucose metabolism, resulting in cytotoxicity and slow wound healing, and thus, it is very important to eliminate excessive ROS and inflammatory factors in the wound healing process of diabetic patients.

The hydrogel for promoting the healing of the diabetic wounds on the market at present is mainly realized by a medicament-carrying mode, and the hydrogel does not have obvious anti-inflammatory performance. In the drug-loaded hydrogel, heparin is more doped in the drug-loaded hydrogel in the form of small medical molecules, so that the overall anti-inflammatory capability of the material is limited by the encapsulation rate, drug loading rate and release rate of the hydrogel, and the release of the heparin is carried out by a certain carrier without realizing the direct contact of the anti-inflammatory drug and inflammatory factors, so that the anti-inflammatory capability of the drug-loaded hydrogel is reduced, and for the repair of slow-healing wounds such as diabetes and the like, if a large amount of heparin sodium is used on the surface of the wound, the blood coagulation mechanism of the wound is reduced, so that the wound is subjected to long-term bleeding to cause other diseases. High molecular weight hyaluronic acid and mannose are common substances, are widely applied in the field of biomedical polymers due to good biocompatibility, and have been proved to have good effects on inducing macrophage polarization in recent years through research, but cannot be widely applied due to long time required for inducing the macrophage polarization.

Disclosure of Invention

Aiming at the defect of slow healing of the existing diabetes wound treatment, the invention takes heparin sodium, lipoic acid, hyaluronic acid and mannose as raw materials, adopts an activity controllable crosslinking strategy of a sulfhydryl-disulfide bond dynamic exchange reaction, and reversibly activates or terminates the sulfhydryl-disulfide bond dynamic exchange reaction by controlling external response stimulation, thereby realizing controllable construction of hydrogel structure and performance in macroscopic and micro/nano multi-scale and preparing the hydrogel material with environmental responsiveness.

The invention also aims to provide a medical dressing containing the hydrogel material, based on the hydrogel with inherent anti-inflammatory performance and environmental response, nano-scale heparin sodium can better adsorb inflammatory factors and promote wound repair, meanwhile, mannose-modified hyaluronic acid induces macrophage polarization to be an anti-inflammatory M2 phenotype, the inflammatory factors are reduced from the source, and the chitosan-modified hyaluronic acid and the heparin sodium act synergistically to realize continuous batch anti-inflammation.

According to a first aspect of the object of the present invention, a process for preparing a hydrogel is proposed, comprising the following steps:

s1: preparation of LA-HEP precursor

Grafting LA to HEP in an esterification mode to prepare an LA-HEP precursor, wherein carboxyl of lipoic acid is activated by CDI, and then carrying out esterification condensation reaction on the activated lipoic acid and hydroxyl of activated heparin sodium to obtain a lipoic acid modified heparin sodium precursor LA-HEP;

s2: preparation of LA-MA-HHA precursor

Grafting lipoic acid onto mannose-modified hyaluronic acid in an esterification mode to prepare a lipoic acid and mannose-bifunctional hyaluronic acid precursor, wherein carboxyl of the lipoic acid is activated by CDI, and then carrying out esterification condensation reaction on the activated lipoic acid and hydroxyl of the mannose-modified hyaluronic acid to prepare the lipoic acid and mannose-bifunctional hyaluronic acid precursor LA-MA-HHA;

s3: preparation of hydrogels

Preparing a certain amount of LA-HEP precursor prepared in the step S1 into a first solution, preparing a certain amount of LA-MA-HHA precursor prepared in the step S2 into a second solution, mixing the first solution and the second solution, adding a catalytic amount of reducing agent, triggering the part of disulfide bonds in the lipoic acid to be broken to form sulfhydryl groups, and regenerating the disulfide bonds in LA-HEP and LA-MA-HHA through a dynamic exchange reaction of the sulfhydryl-disulfide bonds to form hydrogel.

Further, the specific preparation of step S1 includes the following steps:

adding lipoic acid into the first reaction bottle, vacuumizing, and introducing nitrogen for protection;

measuring a proper amount of CDI and DMAC, injecting into a first reaction bottle by using a needle tube, stirring, and reacting at normal temperature for 30-60 min;

dissolving heparin sodium into FA in a second reaction bottle, and cooling the second reaction bottle to room temperature;

adding the product in the first reaction bottle into the second reaction bottle, stirring and reacting for 4-6h under the condition of DMAP, and allowing the obtained crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Further, the specific preparation of step S2 includes the following steps:

adding lipoic acid into the third reaction bottle, vacuumizing, and introducing nitrogen for protection;

measuring a proper amount of CDI and DMAC, injecting into a third reaction bottle by using a needle tube, stirring, and reacting at normal temperature for 30-60 min;

dissolving mannose-modified hyaluronic acid into FA in a fourth reaction flask, and cooling the fourth reaction flask to room temperature;

adding the product in the third reaction bottle into the fourth reaction bottle, stirring and reacting for 4-6h under the condition of DMAP, and allowing the obtained crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Furthermore, the molar mass ratio of the lipoic acid to the CDI is (1:0.1) - (1: 10).

Further, the molar ratio of the carboxyl group to the hydroxyl group is (1:0.1) - (1: 10).

Further, in step S3, the reducing agent added is dithiothreitol or glutathione.

Further, in step S3, the molar mass ratio of the reducing agent to lipoic acid is (0.1:10) - (1: 10).

Further, in step S3, the mass ratio of LA-HEP to LA-MA-HHA is (5:1) - (1: 5).

Further, the mannose-modified hyaluronic acid of step S2 is prepared as follows: dissolving hyaluronic acid in deionized water, and then adding a carboxyl activating reagent and a condensing agent to activate the carboxyl of the hyaluronic acid; adding mannose into activated hyaluronic acid, controlling the pH value of a reaction system to be between 4 and 8, performing esterification reaction for a certain time at normal temperature, then dropwise adding a sodium hydroxide solution to increase the pH value of the reaction system to be between 5.5 and 8, and reacting for a period of time to obtain a reaction solution; transferring the reaction solution into a dialysis bag, dialyzing in sodium chloride solution, dialyzing in deionized water, and freeze-drying to obtain mannose-modified hyaluronic acid MA-HHA.

Furthermore, the molecular weight of the hyaluronic acid is more than or equal to 1500 KDa.

According to a second aspect of the present invention, there is also provided a medical hydrogel prepared by the method for preparing a medical hydrogel, wherein the medical hydrogel is prepared from lipoic acid modified heparin sodium and lipoic acid and mannose dual-functionalized hyaluronic acid composite

According to a third aspect of the object of the present invention, there is also proposed a medical dressing having the aforementioned hydrogel, in particular a medical dressing for the treatment of slow healing of diabetic wounds.

According to the technical scheme, the invention has the beneficial effects that:

1. the invention takes heparin sodium, lipoic acid, hyaluronic acid and mannose as raw materials to prepare an environment-responsive material, and can control the starting and the stopping of self-assembly. In the preparation process, the preparation of the hydrogel material is realized in a dynamic covalent bond crosslinking way by adopting an activity controllable crosslinking strategy of a sulfhydryl-disulfide bond dynamic exchange reaction and by the characteristic that the hydrogel material can be reversibly activated or terminated by external responsive stimulation; the addition of the reducing agent enables the thioctic acid modified heparin sodium and the disulfide bond on the thioctic acid and mannose bifunctional hyaluronic acid to be broken, the disulfide bond on the thioctic acid and mannose bifunctional hyaluronic acid is changed into unstable sulfydryl on the terminal group, the sulfydryl on the thioctic acid and the mannose bifunctional hyaluronic acid is changed into the disulfide bond to be connected together under the stimulation of the environment, and the disulfide bond is formed again through the dynamic exchange reaction of the sulfydryl and the disulfide bond to trigger the two precursors to be crosslinked to form the hydrogel.

2. The hydrogel constructed by the invention directly acts on a wound, has good anti-inflammatory effect, and avoids the problem of reduced blood coagulation mechanism of the wound caused by the large-scale use of heparin sodium by multiple anti-inflammatory effects on the premise of ensuring the anti-inflammatory effect, and the time for inducing macrophage polarization by the mannose-modified hyaluronic acid is also reduced due to the synergistic effect of the mannose-modified hyaluronic acid and the heparin sodium; the hydrogel directly absorbs inflammatory factors from the material layer at the initial stage of inflammation, and realizes continuous anti-inflammatory and repair promotion by inducing macrophage polarization to be an anti-inflammatory M2 phenotype in the process and the later repair process. In the repair of chronic wounds, the inflammation is severe at the initial stage of wound repair, and heparin sodium can better absorb inflammatory chemokines such as MCP-1, IL-8 and the like from the chronic wounds and reduce the concentration of the inflammatory chemokines in the chronic wounds, so that the migration of neutrophils and monocytes can be inhibited, and the generation of inflammation is reduced and inhibited; meanwhile, the reduction of inflammatory factors such as IL-8 and the like can promote macrophages to secrete anti-inflammatory factors such as IL-4 and the like, and the anti-inflammatory factors such as IL-4 and the like can mutually promote the process of inducing macrophage polarization with mannose-modified hyaluronic acid, so that the polarization of M0 type macrophages can be better and faster caused, the macrophages are polarized into M2 type macrophages capable of inhibiting inflammation and promoting wound repair, and the repair of wounds is fundamentally promoted.

It should be understood that all combinations of the foregoing concepts and additional concepts described in greater detail below can be considered as part of the inventive subject matter of this disclosure unless such concepts are mutually inconsistent. In addition, all combinations of claimed subject matter are considered a part of the presently disclosed subject matter.

The foregoing and other aspects, embodiments and features of the present teachings can be more fully understood from the following description taken in conjunction with the accompanying drawings. Additional aspects of the present invention, such as features and/or advantages of exemplary embodiments, will be apparent from the description which follows, or may be learned by practice of specific embodiments in accordance with the teachings of the present invention.

Drawings

The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. Embodiments of various aspects of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:

fig. 1 is a carboxy activation roadmap for lipoic acid of the present invention.

Fig. 2 is a synthesis route diagram of sodium heparin-modified lipoic acid of the present invention.

FIG. 3 is a scheme showing the synthesis of mannose-modified hyaluronic acid according to the invention.

Fig. 4 is a synthesis scheme of mannose and lipoic acid bi-functionalized hyaluronic acid of the present invention.

FIG. 5 is a schematic diagram of the synthesis of a hydrogel of the invention.

FIG. 6 is a nuclear magnetic spectrum of LA-HEP in example 2.

FIG. 7 is an IR spectrum of HEP, LA-HEP, LA from example 2.

FIG. 8 is an IR spectrum of MA-HHA in example 2.

FIG. 9 shows the results of in vitro anti-inflammatory experiments on the hydrogel prepared in example 2.

FIG. 10 shows the results of in vitro anti-inflammatory experiments on the hydrogel prepared in example 2.

Detailed Description

In order to better understand the technical content of the present invention, specific embodiments are described below with reference to the accompanying drawings.

In this disclosure, aspects of the present invention are described with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. Embodiments of the present disclosure are not necessarily intended to include all aspects of the invention. It should be appreciated that the various concepts and embodiments described above, as well as those described in greater detail below, may be implemented in any of numerous ways, as the disclosed concepts and embodiments are not limited to any one implementation. In addition, some aspects of the present disclosure may be used alone, or in any suitable combination with other aspects of the present disclosure.

With reference to fig. 1-3, aiming at the defect of slow healing of the existing diabetic wound, heparin sodium, lipoic acid, hyaluronic acid and mannose are used as raw materials, a controllable cross-linking strategy of the activity of a sulfhydryl-disulfide bond dynamic exchange reaction is adopted, and the sulfhydryl-disulfide bond exchange reaction is reversibly activated or terminated by controlling external response stimulation, so that the controllable construction of the gel structure and the performance under macroscopic scale and micro/nano multi-scale is realized, and the hydrogel material with environmental responsiveness is prepared. The self-assembled hydrogel realizes continuous anti-inflammatory and repair promotion through the inflammation synergistic effect of heparin sodium adsorption wounds.

In an exemplary embodiment, Lipoic Acid (LA) is prepared by esterification grafted onto sodium Heparin (HEP), and mannose-modified hyaluronic acid (MA-HHA) to prepare a precursor.

For example, carboxyl of lipoic acid is activated by N, N' -Carbonyl Diimidazole (CDI), then the activated product is condensed with hydroxyl of HEP or MA-HHA, and finally the two are connected together through ester bond to obtain lipoic acid-heparin sodium (LA-HEP) and lipoic acid and mannose dual-functional hyaluronic acid (LA-MA-HHA) precursor.

Then, a certain amount of LA-HEP is prepared into a first solution, a certain amount of LA-MA-HHA is prepared into a second solution, the first solution and the second solution are mixed, a catalytic amount of reducing agent Dithiothreitol (DTT) or Glutathione (GSH) is added, the disulfide bond in the lipoic acid is triggered to be partially broken to form sulfhydryl, the disulfide bond is formed again through the dynamic exchange reaction of the sulfhydryl-disulfide bond, and two precursors are triggered to be crosslinked to form hydrogel, wherein the mass ratio of the LA-HEP to the LA-MA-HHA is (5:1) - (1: 5).

The specific preparation of the LA-HEP precursor comprises the following steps:

measuring a proper amount of CDI and N, N-Dimethylacetamide (DMAC) (DMAC is used as a solvent), injecting the mixture into a first reaction bottle by using a needle tube, stirring, and reacting at normal temperature for 30-60 min;

dissolving HEP into Formamide (FA) in a second reaction bottle, and cooling the second reaction bottle to room temperature;

adding the product in the first reaction bottle into the second reaction bottle, stirring and reacting for 4-6h under the condition of 4-Dimethylaminopyridine (DMAP), and allowing the obtained crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

The specific preparation of the LA-MA-HHA precursor comprises the following processes:

dissolving mannose-modified hyaluronic acid into formamide in a fourth reaction flask, and cooling the fourth reaction flask to room temperature;

Wherein, in the preparation of the two precursors, the molar mass ratio of the CDI to the lipoic acid is (1:0.1) - (1: 10).

The added reducing agent is DTT or GSH, and the molar mass ratio of the reducing agent to the lipoic acid is (0.1:10) - (1: 10).

Wherein, the molar ratio of the carboxyl of the lipoic acid to the hydroxyl of the heparin sodium is (1:0.1) - (1:10), and the molar ratio of the carboxyl of the lipoic acid to the hydroxyl of the mannose-high molecular weight hyaluronic acid is (1:0.1) - (1: 10).

FIG. 1 is a schematic representation of the carboxyl activation scheme of lipoic acid, from which we can see that the lipoic acid activation process is a spontaneous process and that CO can be generated by the reaction process₂The amount of activated lipoic acid is deduced.

FIG. 2 is a scheme showing the synthesis of LA-HEP, from which we can see that HEP is esterified with lipoic acid activated in the first step with DMAP activation, and finally successfully synthesize the LA-HEP product.

FIG. 4 is a scheme showing the synthesis scheme of LA-MA-HHA, from which we can see that MA-HHA is esterified with lipoic acid activated in the first step under the activation of DMAP, and finally, we successfully synthesize LA-MA-HHA as a product.

FIG. 5 is a schematic diagram of a LA-HEP and LA-MA-HHA hydrogel synthesis process in which LA-HEP and LA-MA-HHA self-assemble under the action of a reducing agent to form the hydrogel of the present invention.

In a preferred embodiment, the mannose-modified hyaluronic acid is prepared as follows:

dissolving hyaluronic acid in deionized water, and then adding a carboxyl activating reagent and a condensing agent to activate the carboxyl of the hyaluronic acid; adding mannose into activated hyaluronic acid, controlling the pH value of a reaction system to be between 4 and 8, performing esterification reaction for a certain time at normal temperature, then dropwise adding a sodium hydroxide solution to increase the pH value of the reaction system to be between 5.5 and 8, and reacting for a period of time to obtain a reaction solution; transferring the reaction solution into a dialysis bag, dialyzing in sodium chloride solution, dialyzing in deionized water, and freeze-drying to obtain mannose-modified hyaluronic acid MA-HHA.

Wherein the molecular weight of the hyaluronic acid is more than or equal to 1500 KDa.

The synthesis scheme of MA-HHA is shown in FIG. 3, and mannose and hyaluronic acid are subjected to esterification reaction of mannose hydroxyl and hyaluronic acid carboxyl under the action of a condensing agent (EDC/HOBT), and finally MA-HHA is successfully synthesized.

In one embodiment, the medical hydrogel prepared according to the preparation method of the medical hydrogel is formed by compounding and crosslinking lipoic acid modified heparin sodium and lipoic acid and mannose bifunctional hyaluronic acid.

In another embodiment, a medical dressing having the aforementioned hydrogel is provided, particularly a medical dressing for the treatment of slow healing diabetic wounds.

In other embodiments, the aforementioned hydrogels can be used for the repair of arthritis, can be injected directly into a joint, lubricate the joint and promote the repair of joint inflammation.

Particularly, the hydrogel can be used as a drug delivery material, the cavity of the hydrogel can carry drug loading, the drug can be delivered to a target part, the purpose of treatment is achieved, and the hydrogel has good cell compatibility.

In further embodiments, it may also be used as a stent, drug delivery, and diagnostic application.

The above preparation process and the prepared hydrogel were subjected to experimental tests in the following with reference to specific examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents, and the like used in the following embodiments are commercially available unless otherwise specified.

[ example 1 ]

Preparation of LA-HEP precursor

A25 mL reaction flask was prepared, washed and packaged with tinfoil. Adding 0.5g of lipoic acid into the reaction bottle, vacuumizing, and introducing nitrogen for protection. 3.9g of CDI and 2.5mL of DMAC (dimethylacetamide) are weighed, injected into a reaction bottle by a needle tube, stirred and reacted for 30min at normal temperature.

0.65g of HEP was dissolved in 30mL of formamide, and the solution was dissolved by heating in an oil bath at 90 ℃ and, after completion of the dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of MA-HHA

0.5000g of sodium hyaluronate was weighed out and dissolved in 100mL of deionized water (0.5%), 0.0312g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC.HCl was added, 0.0220g of 1-hydroxybenzotriazole monohydrate HOBt was weighed out and dissolved in 2mL of DMSO, and added to the above hyaluronic acid solution and activated for half an hour. Weighing 0.0218g of mannose, adding 1N hydrochloric acid solution, controlling the pH value of a reaction system to be 4.75, reacting for 24 hours at normal temperature, dropwise adding 1N sodium hydroxide solution to increase the pH value of a reaction solution to 7.0, finishing the reaction, transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing for 1.5 days in 100mM sodium chloride solution, dialyzing for 2 days in deionized water, and freeze-drying to obtain MA-HHA.

Synthesis of LA-MA-HHA precursor

Dissolving 1.4g of mannose-modified hyaluronic acid in 150mL of formamide, transferring the solution to a 250mL reaction flask, heating the solution to dissolve the solution at 90 ℃ in an oil bath, and cooling the reaction flask to room temperature after complete dissolution. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of hydrogels

0.82g of the prepared LA-HEP and 0.788g of LA-MA-HHA were mixed, and 1mmoL/L of DTT was added thereto to induce hydrogel formation.

[ example 2 ]

Preparation of LA-HEP precursor

A25 mL reaction flask was prepared, washed and packaged with tinfoil. Adding 0.5g of lipoic acid into the reaction bottle, vacuumizing, and introducing nitrogen for protection. 0.39g of CDI and 2.5mL of DMAC were weighed out, injected into a reaction flask through a needle tube, stirred, and reacted at room temperature for 60 min.

0.65g of HEP was dissolved in 30mL of formamide, and the solution was dissolved by heating in an oil bath at 90 ℃ and, after completion of the dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP, continuously stirring and reacting for 6h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

Dissolving 1.4g of mannose-modified hyaluronic acid in 150mL of formamide, transferring the solution to a 250mL reaction flask, heating the solution to dissolve the solution at 90 ℃ in an oil bath, and cooling the reaction flask to room temperature after complete dissolution. Adding the product of the first step into HEP, adding 0.015g DMAP, continuously stirring and reacting for 6h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of hydrogels

0.82g of LA-HEP prepared as described above and 1.576g of LA-MA-HHA were mixed, and DTT was added thereto at a concentration of 1mmoL/L to induce hydrogel formation.

[ example 3 ]

Preparation of LA-HEP precursor

A25 mL reaction flask was prepared, washed and packaged with tinfoil. Adding 0.5g of lipoic acid into the reaction bottle, vacuumizing, and introducing nitrogen for protection. 1.56g of CDI and 2.5mL of DMAC were weighed, injected into a reaction flask through a needle, stirred, and reacted at room temperature for 30 min.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

A25 mL reaction flask was prepared, washed and packaged with tinfoil. Adding 0.5g of lipoic acid into the reaction bottle, vacuumizing, and introducing nitrogen for protection. 1.56g of CDI and 2.5mL of DMAC were weighed, injected into a reaction flask through a needle, stirred, and reacted at room temperature for 60 min.

Synthesis of hydrogels

0.82g of the prepared LA-HEP and 2.364g of LA-MA-HHA are mixed, and GSH of 1mmoL/L is added to the mixture to induce the formation of hydrogel.

[ example 4 ]

Preparation of LA-HEP precursor

A25 mL reaction flask was prepared, washed and packaged with tinfoil. Adding 0.5g of lipoic acid into the reaction bottle, vacuumizing, and introducing nitrogen for protection. 0.039g of CDI and 2.5mL of DMAC (dimethylacetamide) were weighed, injected into a reaction flask through a needle tube, stirred, and reacted at normal temperature for 30 min.

Dissolving 0.65g HEP in 30mL formamide, heating at 90 deg.C in oil bath for dissolving completelyAfter that, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

Synthesis of hydrogels

1.64g of the prepared LA-HEP and 0.788g of LA-MA-HHA were mixed, and 5mmoL/L of DTT was added thereto to induce hydrogel formation.

[ example 5 ]

Preparation of LA-HEP precursor

A25 mL reaction flask was prepared, washed and packaged with tinfoil. Adding 0.5g of lipoic acid into the reaction bottle, vacuumizing, and introducing nitrogen for protection. 0.156g of CDI and 2.5mL of DMAC (dimethylacetamide) were weighed, injected into a reaction flask through a needle tube, stirred, and reacted at normal temperature for 30 min.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

Synthesis of hydrogels

1.64g of the prepared LA-HEP and 2.364g of LA-MA-HHA were mixed, and 5mmoL/L of DTT was added thereto to induce hydrogel formation.

[ example 6 ]

Preparation of LA-HEP precursor

A25 mL reaction flask was prepared, washed and packaged with tinfoil. Adding 0.5g of lipoic acid into the reaction bottle, vacuumizing, and introducing nitrogen for protection. 0.39g of CDI and 2.5mL of DMAC were weighed, injected into a reaction flask through a needle, stirred, and reacted at room temperature for 30 min.

0.065g of HEP was dissolved in 30mL of formamide, and the solution was dissolved by heating in an oil bath at 90 ℃ and, after completion of the dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

0.14g of mannose-modified hyaluronic acid was dissolved in 150mL of formamide, transferred to a 250mL reaction flask, heated and dissolved in an oil bath at 90 ℃ and, after complete dissolution, the reaction flask was cooled to room temperature. Will be provided withAdding the product of the first step reaction into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4 hours, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of hydrogels

0.82g of the prepared LA-HEP and 0.788g of LA-MA-HHA were mixed, and GSH at 5mmoL/L was added thereto to induce hydrogel formation.

[ example 7 ]

Preparation of LA-HEP precursor

6.5g of HEP was dissolved in 30mL of formamide, transferred to a 100mL reaction flask, heated and dissolved in an oil bath at 90 ℃ and, after complete dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

14g of mannose-modified hyaluronic acid was dissolved in 150mL of formamide, transferred to a 250mL reaction flask, heated and dissolved in an oil bath at 90 ℃ and, after complete dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of hydrogels

0.82g of the prepared LA-HEP and 0.788g of LA-MA-HHA were mixed, and 10mmoL/L of DTT was added thereto to induce hydrogel formation.

[ example 8 ]

Preparation of LA-HEP precursor

0.26g of HEP was dissolved in 30mL of formamide, and the solution was dissolved by heating in an oil bath at 90 ℃ and, after completion of the dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

0.56g of mannose-modified hyaluronic acid was dissolved in 150mL of formamide, transferred to a 250mL reaction flask, heated and dissolved in an oil bath at 90 ℃ and, after complete dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of hydrogels

[ example 9 ]

Preparation of LA-HEP precursor

2.6g of HEP was dissolved in 30mL of formamide, and the solution was dissolved by heating in an oil bath at 90 ℃ and, after completion of the dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

5.6g of mannose-modified hyaluronic acid was dissolved in 150mL of formamide, transferred to a 250mL reaction flask, heated and dissolved in an oil bath at 90 ℃ and, after complete dissolution, the reaction flask was cooled to room temperature. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of hydrogels

0.82g of the prepared LA-HEP and 0.788g of LA-MA-HHA are mixed, and GSH of 10mmoL/L is added to the mixture to induce the formation of hydrogel.

[ example 10 ]

Preparation of LA-HEP precursor

Synthesis of MA-HHA

Synthesis of LA-MA-HHA precursor

Dissolving 1.4g of mannose-modified high molecular weight hyaluronic acid into 150mL of formamide, transferring the dissolved hyaluronic acid into a 250mL reaction flask, heating the dissolved hyaluronic acid to dissolve the hyaluronic acid in oil bath at 90 ℃, and cooling the reaction flask to room temperature after complete dissolution. Adding the product of the first step into HEP, adding 0.015g DMAP and continuously stirring for reacting for 4h, and allowing the crude product to pass through KH₂PO₄Neutralizing, dialyzing, purifying, and freeze-drying for recovery.

Synthesis of hydrogels

[ characterizations and tests ]

The hydrogels and intermediates prepared in example 2 were used for the following characterization and testing

[ Nuclear magnetic Hydrogen Spectrum ]

Weighing 10mg of LA-HEP hydrogel, dissolving the LA-HEP hydrogel in heavy water, and scanning 16 times in 400 megaBruk to obtain a nuclear magnetic hydrogen spectrum.

FIG. 6 is the nuclear magnetic hydrogen spectrum of the hydrogel, and from the spectrum we can see that the labeled 1, 2, 3, 4 respectively correspond to the hydrogen on the methylene group of lipoic acid, and the labeled 5, 6, 7 respectively correspond to the hydrogen on the five-membered ring of lipoic acid, which indicates that LA-HEP is successfully prepared.

[ Infrared Spectroscopy of LA-HEP and MA-HHA ]

To further verify the successful preparation of LA-HEP, 1mg of HEP, LA and LA-HEP, respectively, were subjected to infrared testing, flaked under KBr, and then scanned for infrared spectra.

FIG. 7 shows the IR spectra of HEP, LA, LA-HEP. From the spectrum, it can be seen that the LA-HEP synthesized by the present invention contains both the spectrum of HEP and LA, and the spectrum of LA-HEP is 1710cm^-1The peak shows that ester bonds newly generated by HEP and LA connection reaction exist, so that LA-HEP can be successfully prepared; by comparing the three patterns, only LA-HEP was found to be at 1710cm^-1Peaks are shown, demonstrating the appearance of new groups, and further demonstrating the successful synthesis of LA-HEP.

FIG. 8 is an IR spectrum of MA-HHA, which is found at 1710cm^-1The presence of the ester group in the ester group indicates that MA-HHA is indeed linked by esterification, and it can be seen from FIG. 7 and FIG. 8 that LA is grafted by esterification with MA-HHA.

[ in vitro cell compatibility of hydrogel ]

In order to prove that the hydrogel has better cell compatibility, the prepared LA-HEP is co-cultured for 24h, 48h and 72h according to the concentration of 25, 12.5, 6.25 and 3.125mg/mL and the L929 cells respectively, and the cell survival rate is detected by a CCK-8 kit (cell proliferation and toxicity detection kit). Experimental results prove that the cell survival rates of the prepared LA-HEP at the concentrations of 25mg/mL, 12.5 mg/mL, 6.25 mg/mL and 3.125mg/mL are respectively 90%, 93%, 95% and 96%, namely the cell survival rate is integrally higher than 90%, and the prepared hydrogel is proved to have good cell compatibility.

[ cell experiment ]

In a cell experiment using primary macrophages (BMDM), macrophages were isolated and extracted from C57BL/6 mouse bone marrow and induced to differentiate into BMDM with macrophage colony stimulating factor (M-CSF).

(1) Anti-inflammatory testing of diabetic wounds

Lipopolysaccharide (LPS) is adopted to stimulate BMDM cells to construct a slow-healing inflammation model of a diabetic wound, then the prepared hydrogel is added into the model, cell supernatant is collected after 4-6 hours, and an ELISA kit is used for detecting the secretion level of the cell factors.

As shown in FIG. 9, the result shows that the addition of the hydrogel obviously reduces the levels of proinflammatory factors such as TNF-alpha, IL-1 beta, IL-6, IL-8, MCP-1 and the like, and is accompanied by the increase of anti-inflammatory factors of IL-4 and IL-10, and the hydrogel prepared by the method has better anti-inflammatory capability on diabetic wounds, and heparin sodium absorbs inflammatory chemokines from chronic wounds and promotes macrophages to secrete the anti-inflammatory factors.

(2) Macrophage polarization assay

After co-culturing the hydrogel prepared by us and BMDM for 24h, cell supernatants were collected separately and examined for cytokine changes using ELISA kits.

As shown in figure 10, the result shows that after the hydrogel is added, the secretion of anti-inflammatory cytokines such as IL-4 and IL-10 is obviously increased compared with that of untreated BMDM, which proves that BMDM is polarizing to M2 subtype, the process that anti-inflammatory factors such as IL-4 and mannose modified hyaluronic acid induce macrophage polarization mutually promotes, the M0 macrophage is better and faster induced to polarize to M2 macrophage, and the hydrogel material can better promote the repair of T2D wound.

From the above, the hydrogel prepared by the invention can directly absorb inflammatory factors from the material layer, and can realize continuous anti-inflammatory and repair promotion by inducing macrophage polarization to have an anti-inflammatory M2 phenotype in the process and the later repair process.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method of making a medical hydrogel comprising the steps of:

s1: preparation of LA-HEP precursor

s2: preparation of LA-MA-HHA precursor

s3: preparation of hydrogels

2. The method according to claim 1, wherein the specific preparation of step S1 includes the following steps:

3. The method according to claim 1, wherein the specific preparation of step S2 includes the following steps:

4. A method according to claim 2 or 3, characterized in that: the molar mass ratio of the lipoic acid to the CDI is (1:0.1) - (1: 10).

5. A method according to claim 2 or 3, characterized in that: the molar ratio of carboxyl groups to hydroxyl groups is (1:0.1) - (1: 10).

6. The method of claim 2, wherein: in step S3, the reducing agent added is dithiothreitol or glutathione.

7. The method of claim 6, wherein: in step S3, the molar mass ratio of the reducing agent to lipoic acid is (0.1:10) - (1: 10).

8. The method of claim 1, wherein: in step S3, the mass ratio of LA-HEP to LA-MA-HHA is (5:1) - (1: 5).

9. The method of claim 1, wherein the mannose-modified hyaluronic acid of step S2 is prepared by the following method: dissolving hyaluronic acid in deionized water, and then adding a carboxyl activating reagent and a condensing agent to activate the carboxyl of the hyaluronic acid; adding mannose into activated hyaluronic acid, controlling the pH value of a reaction system to be between 4 and 8, performing esterification reaction for a certain time at normal temperature, then dropwise adding a sodium hydroxide solution to increase the pH value of the reaction system to be between 5.5 and 8, and reacting for a period of time to obtain a reaction solution; transferring the reaction solution into a dialysis bag, dialyzing in sodium chloride solution, dialyzing in deionized water, and freeze-drying to obtain mannose-modified hyaluronic acid MA-HHA.

10. The method of claim 9, wherein the molecular weight of the hyaluronic acid is at least 1500 kDa.

11. A medical hydrogel prepared by the method of any one of claims 1 to 10, wherein: the medical hydrogel is formed by crosslinking lipoic acid modified heparin sodium and lipoic acid and mannose dual-functionalized hyaluronic acid.

12. A medical dressing characterized by: comprising the medical hydrogel of claim 9.