CN114316087B - Hyaluronic acid cross-linking active material, preparation method and application thereof - Google Patents
Hyaluronic acid cross-linking active material, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a hyaluronic acid crosslinking active material, the hyaluronic acid crosslinking active material prepared by the method and application of the hyaluronic acid crosslinking active material in the field of medicine. The preparation method comprises the following steps: (1) Reacting side chain carboxyl of a hyaluronic acid raw material with an active reagent and a cross-linking agent simultaneously under the action of carbodiimide to generate an amido bond, thereby obtaining a hyaluronic acid cross-linking material; (2) And reacting the hyaluronic acid cross-linking material with a reducing agent, wherein the disulfide bonds in the diamine or diacyl trap containing the disulfide bonds are reduced to sulfydryl, and purifying to obtain the hyaluronic acid cross-linking active material. The preparation method is a continuous process, can introduce the active sulfydryl while realizing the stable cross-linking structure, and has the advantages of simple preparation process, flexible and controllable preparation parameters, easy industrial production and the like.
Description
Technical Field
The invention relates to a preparation method of a hyaluronic acid crosslinking active material, in particular to a preparation method of a sulfhydryl-containing crosslinking hyaluronic acid crosslinking active material, and further relates to a hyaluronic acid crosslinking active material and application thereof in the field of medicines.
Background
Biocompatible polymers have many important physiological functions, and hyaluronic acid has significant effects in clinical medicine such as adhesion promotion treatment of arthritis, promotion of wound healing, and the like. However, generally, biocompatible polymers are metabolized rapidly or easily in body fluids, which greatly limits their application in many clinical medicine, for example, hyaluronic acid is used for viscosity-increasing treatment of arthritis, and the course of treatment is once-weekly intra-articular injection administration for three to five weeks continuously, which not only causes inconvenience to patients and medical workers, but also increases the risk of infection for patients. The chemical modification/crosslinking of the biocompatible polymer is an effective means for delaying the metabolism of the biocompatible polymer in vivo and reducing the solubility, and the application of the biocompatible polymer in clinical medicine is remarkably expanded. For example, for the tackification treatment of arthritis, the curative effect of three to five times of intra-articular injection administration of the non-crosslinked sodium hyaluronate can be achieved by one intra-articular injection administration of the crosslinked sodium hyaluronate; meanwhile, the cross-linked hyaluronic acid is widely applied to skin beautifying purposes of plastic surgery such as dermal fillers.
Although crosslinking/modification expands the application of hyaluronic acid in clinical medicine, the current hyaluronic acid crosslinked materials are prepared in a single manner and are difficult to simultaneously introduce biocompatible functional groups with specific activity during the preparation of stable crosslinked structures (vercruyse et al, bioconjugate Chemistry 1997, 8. For example, cross-linked sodium hyaluronate gel for orthopedic surgery, conventionally used is divinyl sulfoxide (DVS) or 1,4-butanediol diglycidyl ether (BDDE), not only does not introduce a biocompatible functional group of specific activity, but also may have a large amount of unreacted single-terminal functional groups, which are chemically bonded to the cross-linked material, cannot be removed by a purification process, and may induce various toxic side reactions in clinical use (Edsman et al, dermotol Surg 2012, 38.
In the normal metabolism process of human body, there is dynamic balance in the oxidation process and reduction process. However, under the action of various endogenous factors (such as chronic or acute infection) and exogenous factors (such as environmental pollution, ultraviolet radiation and the like), oxidation reaction may dominate, and oxidative stress appears, so that various harmful active oxygen radicals are generated; active oxygen radicals have strong oxidizing properties, attack lipids, proteins, DNA, etc., and may damage tissues and cells of the body, thereby causing various chronic diseases and aging effects (Gutteridge et al, british Medical Bulletin 1999, 55. For example, studies have shown that up to 80% of the skin aging phenomenon is associated with reactive oxygen radicals. The cells and extracellular matrix maintaining the skin balance are destroyed and reduced due to the destruction of active oxygen radicals, and the immune repair ability is rapidly decreased, resulting in various skin aging and pathological symptoms such as dryness, roughness, darkness, laxity, wrinkles, etc.
The introduction of the biocompatible functional group with special activity into the hyaluronic acid cross-linked material is beneficial to enhancing the clinical treatment effect and has important significance. For example, sulfhydryl is a biocompatible functional group widely present in the body, and can effectively inhibit oxidative stress directly caused by active oxygen free radicals, and can prevent damage to lipids, proteins and DNA; in addition, formation of hydroxyl radicals and the like can also be indirectly inhibited by complexing with divalent iron ions. Therefore, the inventor considers that if sulfydryl can be introduced into the stably crosslinked hyaluronic acid crosslinking material, the application of hyaluronic acid in clinical medicine is expanded, meanwhile, the hyaluronic acid crosslinking material can effectively inhibit oxidative stress, quench free radicals, control inflammatory reaction, and has positive prospects in various medical applications such as arthritis tackifying treatment, skin reshaping and beautifying, wound repair and the like. However, such a preparation method does not exist in the prior art.
Accordingly, the present inventors have aimed to provide a method for preparing a hyaluronic acid crosslinking active material, which solves the above-mentioned technical problems.
Disclosure of Invention
The invention aims to provide a preparation method of a hyaluronic acid crosslinking active material, which can introduce active sulfydryl on one hand, can simultaneously form a stable crosslinking structure on the other hand, has simple preparation process and flexible and controllable preparation parameters, and is easy for industrial continuous production.
The present invention also provides a hyaluronic acid cross-linking active material prepared according to the above preparation method of the present invention, which has a thiol group with a specific activity, can effectively inhibit oxidative stress, quench free radicals, control inflammatory reaction, and simultaneously has a stable cross-linked structure, can delay the metabolism of a biocompatible polymer in vivo and reduce solubility.
The invention also aims to provide an application of the hyaluronic acid cross-linked material in the field of medicines. The hyaluronic acid cross-linked material can meet the application requirements of wider medical fields.
The preparation method of the hyaluronic acid active material provided by the invention comprises the following steps:
reacting side chain carboxyl of a hyaluronic acid raw material with an active reagent and a cross-linking agent simultaneously under the action of carbodiimide to generate an amide bond to obtain a hyaluronic acid cross-linking material, or reacting the side chain carboxyl of the hyaluronic acid raw material with an intermediate reagent to form a relatively stable intermediate activated product under the action of the carbodiimide, and then reacting the intermediate product with the active reagent and the cross-linking agent simultaneously to generate the amide bond to obtain the hyaluronic acid cross-linking material;
wherein the active agent is a diamine or dihydrazide containing a disulfide bond; or a primary amine containing a free thiol group, or a thiol-protected primary amine;
wherein the crosslinker does not contain a disulfide linkage but contains a polyhydrazide with two or more hydrazide functional groups;
wherein the hyaluronic acid raw material is selected from hyaluronic acid, a form of a salt thereof and/or a modified derivative thereof;
step (2) when the active reagent is diamine or dihydrazide containing disulfide bonds, reacting the hyaluronic acid crosslinking material in the step (1) with a reducing agent, wherein the disulfide bonds in the diamine or dihydrazide containing disulfide bonds are reduced to sulfydryl, and purifying to obtain the hyaluronic acid crosslinking active material; or, when the active reagent is primary amine containing free sulfydryl, purifying the hyaluronic acid cross-linking material obtained in the step (1) to obtain the hyaluronic acid cross-linking active material, or, when the active reagent is primary amine with sulfydryl protecting groups, removing the sulfydryl protecting groups from the hyaluronic acid cross-linking material obtained in the step (1) and then obtaining the hyaluronic acid cross-linking active material through a purification process.
Preferably, the polyhydrazides employed in the present invention are one or more components selected from the group consisting of: succinic dihydrazide, adipic dihydrazide, suberic dihydrazide, terephthalic dihydrazide, thiodipropionic dihydrazide, tartaric dihydrazide, polyethylene glycol dihydrazide.
Preferably, the polyhydrazides employed in the present invention are those based on ammonia.
Preferably, the diamine or dihydrazide containing a disulfide bond as an active agent in the present invention is one or more components selected from the group consisting of: cystamine, cystine dimethyl ester, cystine diethyl ester, dithiodipropylhydrazide and dithiodibutylhydrazine. More preferably, the diamine containing a disulfide bond is preferably cystamine, cystine dimethyl ester, cystine diethyl ester, or the like. The dihydrazide having a disulfide bond is preferably dithiodipropylhydrazide, dithiodibutylhydrazide or the like.
Preferably, the carbodiimide used in the present invention is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
Preferably, the reducing agent employed in the present invention is dithiothreitol andor tris (2-carboxyethyl) phosphine hydrochloride.
Preferably, the intermediate reagent employed in the present invention is N-hydroxysuccinimide and/or N-hydroxythiosuccinimide.
Preferably, in the present invention, the molecular weight of the hyaluronic acid starting material employed is generally between 1 and 1000 kilodaltons, more preferably between 10 and 300 kilodaltons, and particularly preferably between 20 and 100 kilodaltons.
In the present invention, the hyaluronic acid raw material includes the form of its salt (such as sodium salt, potassium salt, etc.), and also includes the derivative obtained by chemical modification thereof, such as carboxymethyl hyaluronic acid, acetylated hyaluronic acid, etc.
In the invention, the adopted cross-linking agent does not contain a disulfide bond structure but contains two or more hydrazide functional groups, and has good biocompatibility.
Preferably, the crosslinking agent containing two hydrazide functional groups is selected from one or more of the following groups: succinic dihydrazide, adipic dihydrazide, suberic dihydrazide, terephthalic dihydrazide, thiodipropionic dihydrazide, tartaric dihydrazide, polyethylene glycol dihydrazide. The chemical structure is as follows:
more preferably, the crosslinking agent containing two hydrazide functional groups in the present invention is succinic dihydrazide, adipic dihydrazide, suberic dihydrazide.
Preferably, the crosslinking agent containing two or more hydrazide functional groups used in the present invention is selected from the group consisting of ammonia-based polyhydrazides having the following structure, the chemical structure of which is shown below:
in step (1) of the production method according to the present invention, the reaction of the side chain carboxyl group of the hyaluronic acid raw material with the crosslinking agent, and the reaction with the active agent, which occur simultaneously. The above two reactions are detailed separately as follows.
The side chain carboxyl of the hyaluronic acid raw material and the hydrazide functional group of the cross-linking agent which does not contain a disulfide bond structure but contains two or more hydrazide functional groups are subjected to coupling reaction to form an amide bond cross-linking structure. The common mode of the coupling reaction is that the side chain carboxyl of the raw material of the hyaluronic acid generates an amido bond crosslinking structure with the hydrazide functional group of the crosslinking agent under the activation of carbodiimide.
Wherein, the side chain carboxyl of the hyaluronic acid raw material can also form a relatively stable intermediate activated product with an intermediate reagent (such as N-hydroxysuccinimide, N-hydroxythiosuccinimide and the like) under the activation of carbodiimide, and then react with the cross-linking agent to generate an amido bond, so that an amido bond cross-linking structure can also be obtained.
In the preparation method according to the present invention, taking the crosslinking agent containing two hydrazide functional groups as an example, the amide bond crosslinking structure formed according to step 1) of the present invention has a crosslinking structure of the following general formula (I):
wherein HA is a hyaluronic acid residue; r' is the residue of the aforementioned polyhydrazide functional group crosslinking agent and includes alkylene groups, substituted alkylene groups, aromatic groups, polyether groups, and the like.
Meanwhile, in the step (1) of the preparation method, the side chain carboxyl of the hyaluronic acid is subjected to coupling reaction with the amino or hydrazide functional group of the active reagent adopted by the invention to form an amide bond connection. The coupling reaction is usually carried out in such a way that the side chain carboxyl of hyaluronic acid forms an amide bond with the amino or hydrazide functional group of the active reagent under the activation of carbodiimide, and in the step (2), the disulfide bond in the active reagent is reduced to a sulfhydryl group under the action of a reducing agent.
In another embodiment according to the present invention, in step (1) of the preparation method of the present invention, the side chain carboxyl group of hyaluronic acid may also be activated by carbodiimide to form a relatively stable intermediate activated product with an intermediate reagent (e.g., N-hydroxysuccinimide, N-hydroxythiosuccinimide, etc.), and then reacted with the above-mentioned active reagent to form an amide bond.
In another embodiment according to the present invention, in step (1) of the above-mentioned preparation method according to the present invention, the reaction of step (1) may be further carried out using a primary amine containing a free thiol group or a thiol-protected primary amine instead of the diamine or dihydrazide containing a disulfide bond in the above scheme as an active reagent, in which case, in step (2), the technical scheme of the primary amine containing a free thiol group is employed, thus directly obtaining the hyaluronic acid crosslinking active material prepared according to the present invention through step (1).
According to another embodiment of the present invention, in the step (1) of the preparation method according to the present invention, a primary amine having a thiol group protection may be used instead of the diamine or dihydrazide containing a disulfide bond in the above scheme as an active reagent to perform the reaction of the step (1), and after the thiol group protection group is removed from the obtained hyaluronic acid crosslinked material through the reaction and purification of the step (1), the thiol group linked to the side chain carboxyl group of hyaluronic acid may also be obtained, that is, the hyaluronic acid crosslinked material prepared according to the present invention may be obtained.
In the present invention, the thiol group is linked to the side chain carboxyl group of hyaluronic acid via an amide bond, and has a structure represented by the following general formula (II) or (III):
wherein HA is a hyaluronic acid residue; r is the residue of the aforementioned active agent and includes alkylene, substituted alkylene, aromatic, polyether, and the like.
In addition, the hyaluronic acid crosslinking active material with a specific structure can be prepared by adjusting parameters such as the structure, concentration, proportion, reaction temperature, reaction time and the like of each reactant.
The preparation method is a continuous process, can introduce active sulfydryl while realizing stable cross-linking structure, and has the advantages of simple preparation process, flexible and controllable preparation parameters, easy industrial production and the like.
The hyaluronic acid crosslinking active material prepared by the invention not only has the amido bond crosslinking structure (shown as a general formula I), but also contains sulfydryl (shown as general formulas II and III) connected with a hyaluronic acid side chain. The cross-linked structure of the hyaluronic acid cross-linked active material has good stability: on one hand, the amido bond is a stable chemical bond and is not easy to hydrolyze; on the other hand, the cross-linked structure does not contain disulfide bonds, can not be reduced and dissociated by a reducing agent under the conventional condition, can not be oxidized and damaged by an oxidizing agent, and has good stability.
The sulfhydryl contained in the hyaluronic acid crosslinking active material prepared by the invention is a biocompatible functional group widely existing in a living body, can effectively inhibit oxidative stress directly caused by active oxygen free radicals, and can prevent damages to lipid, protein and DNA; further, formation of hydroxyl radicals and the like can also be indirectly inhibited by complexing with ferrous ions.
The hyaluronic acid cross-linking active material prepared by the invention has stable cross-linking structure and active sulfydryl, not only expands the clinical application of hyaluronic acid, but also can effectively inhibit oxidative stress, quench free radicals, control inflammatory reaction, reduce toxic and side reaction and improve biocompatibility, and has positive prospect in various medical applications such as arthritis tackifying treatment, skin reshaping and beautifying, wound repair and the like.
In addition, if necessary, the thiol group contained in the hyaluronic acid cross-linking active material prepared by the invention can be further appropriately oxidized to form a disulfide bond, thereby further enhancing the cross-linking strength. The oxidant used may be dissolved oxygen, hydrogen peroxide, etc.
The hyaluronic acid cross-linking active material prepared according to the invention is generally in the form of a hydrogel, having a water content generally greater than 95% (weight/volume percentage, g/ml), preferably greater than 98%, particularly preferably greater than 99%. The hydrogel refers to a substance having a three-dimensional cross-linked network structure containing a large amount of water, which is between a liquid state and a solid state, and has no fluidity.
The hyaluronic acid cross-linking active material prepared by the invention can also be prepared into various solid forms such as films, sponges and the like after being dried or freeze-dried.
The sulfhydryl content of the hyaluronic acid crosslinking active material prepared by the invention can be flexibly adjusted, and is usually less than 350 mu mol/g, preferably 80-150 mu mol/g.
In addition, the preparation process of the preparation method according to the invention is greatly simplified because the introduction of the active thiol group and the stabilization of the cross-linking structure are simultaneously accomplished.
The invention has the beneficial effects that:
the preparation method is a continuous process, can introduce active sulfydryl while realizing stable cross-linking structure, and has the advantages of simple preparation process, flexible and controllable preparation parameters, easy industrial production and the like; the prepared hyaluronic acid crosslinking active material has stable crosslinking structure and active sulfydryl, can effectively inhibit oxidative stress, quench free radicals and control inflammatory reaction, has positive prospect in a plurality of medical applications such as arthritis tackifying treatment, skin reshaping beauty, wound repair and the like, and can simultaneously meet the requirements of crosslinking stability, biocompatibility and hyaluronic acid material with special activity in the fields of medicine and beauty.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
Example 1: (Prior Art) preparation of hyaluronic acid Cross-Linked gel (adipic acid dihydrazide Cross-linking)
Example 1 is a prior art example.
Sodium hyaluronate (molecular weight 50 kilodaltons) 1g was dissolved in 10ml of distilled water at room temperature to give a clear and transparent solution. 0.087g of adipic acid dihydrazide was added to the above solution, and dissolved by stirring. Then the pH value of the solution is adjusted to 4.75 by using 0.1mol/L hydrochloric acid, and 0.192g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added to keep the pH value of the solution at 4.75; the solution viscosity increased and gel gradually formed. The resulting gel was allowed to stand overnight, and then soaked with a physiological saline solution of not less than 20 times in volume for 12 hours, with fresh physiological saline being replaced every 2 hours. And purifying to obtain the gel, namely the hyaluronic acid crosslinked gel (adipic acid dihydrazide crosslinked).
Example 2: preparation of thiol-group-containing hyaluronic acid Cross-Linked reactive gel (adipic acid dihydrazide Cross-Linked)
Sodium hyaluronate (molecular weight 50 kilodaltons) 1g was dissolved in 10ml of distilled water at room temperature to give a clear and transparent solution. 0.087g of adipic acid dihydrazide and 0.0595g of dithiodipropylhydrazide were added to the above solution, respectively, and dissolved by stirring. Then the pH value of the solution is adjusted to 4.75 by using 0.1mol/L hydrochloric acid, and 0.288g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added to keep the pH value of the solution at 4.75; the solution viscosity increased and gel gradually formed. The resulting gel was allowed to stand overnight and then soaked with 20 volumes of 1% dithiothreitol solution (pH 8.5) for 4 hours; and finally, soaking the soaked materials in a normal saline solution with the volume not less than 20 times for removing the substances for 12 hours, replacing fresh normal saline every 2 hours, and adjusting the pH value of the soaking solution to 3.5 by using 0.1mol/L hydrochloric acid. And purifying to obtain gel which is the thiol-containing hyaluronic acid crosslinking active gel (adipic acid dihydrazide crosslinking).
The thiol-group-containing hyaluronic acid crosslinking active gel (adipic acid dihydrazide crosslinked) prepared above was immersed in distilled water to remove salts, lyophilized and hydrolyzed with 0.1mol/L hydrochloric acid, and then the thiol content was measured to be 213. Mu. Mol/g by using a modified Ellman method (Biomacromolecules 2002, 3.
Example 3: antioxidant effect of sulfhydryl-containing hyaluronic acid crosslinking active gel (adipic acid dihydrazide crosslinking) for scavenging free radicals
The crosslinked activated gels prepared in examples 1 (prior art) and 2 (invention) were desalted by immersion in distilled water, lyophilized and hydrolyzed with 0.1mol/L hydrochloric acid, and then free radical scavenging was determined using the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) test method reported by Wang et al (CN 110279610A).
The DPPH radical scavenging efficiency of the cross-linked reactive gel prepared in example 2 was increased by about 35 times under the same conditions as the cross-linked gel prepared in example 1. The test result shows that the mercapto group contained in the cross-linking active gel prepared in the example 2 has good functions of eliminating free radicals and resisting oxidation.
Example 4: preparation of thiol-group-containing hyaluronic acid Cross-Linked reactive gel (adipic acid dihydrazide Cross-Linked, different thiol contents)
Sodium hyaluronate (molecular weight 50 ten thousand daltons) 1g was dissolved in 10ml of distilled water at room temperature to give a clear and transparent solution. 0.087g of adipic acid dihydrazide and an appropriate amount of dithiodipropylhydrazide (0.0149 g, 0.0298g, 0.0595g or 0.119 g) were added to the above solution, and dissolved by stirring. Then the pH value of the solution is adjusted to 4.75 by using 0.1mol/L hydrochloric acid, and 0.384g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added to keep the pH value of the solution at 4.75; the solution viscosity increased and a gel gradually formed. The gel was allowed to stand overnight and then soaked in a 20-fold volume of 1% dithiothreitol solution (pH 8.5) for 4 hours; and finally, soaking the soaked materials in a normal saline solution with the volume not less than 20 times for removing the substances for 12 hours, replacing fresh normal saline every 2 hours, and adjusting the pH value of the soaking solution to 3.5 by using 0.1mol/L hydrochloric acid. And purifying to obtain gel which is the thiol-containing hyaluronic acid crosslinking active gel (adipic acid dihydrazide crosslinking).
The thiol-group-containing hyaluronic acid crosslinking reactive gel (adipic acid dihydrazide crosslinked) prepared above was immersed in distilled water to remove salts, lyophilized and hydrolyzed with 0.1mol/L hydrochloric acid, and then the thiol content was measured by using a modified Ellman method (Biomacromolecules 2002, 3. The thiol content of dithiodipropylhydrazide is 89 mu mol/g, 148 mu mol/g, 213 mu mol/g and 336 mu mol/g when the feeding amount of dithiodipropylhydrazide is 0.0149g, 0.0298g, 0.0595g and 0.119g respectively.
Example 5: antioxidant effect of sulfhydryl-containing hyaluronic acid crosslinking active gel (adipic acid dihydrazide crosslinking, different sulfhydryl contents) for scavenging free radicals
The cross-linked reactive gels prepared in examples 1 and 4 and having different mercapto contents were desalted by soaking in distilled water, dried and hydrolyzed with 0.1mol/L hydrochloric acid, and then the radical scavenging rate (CN 110279610A) was measured by the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) test method reported by Wang et al.
Under the same conditions, the DPPH free radical clearance of the cross-linked reactive gel prepared in example 4 is improved by about 15-46 times compared with that of the cross-linked gel prepared in example 1, and the clearance of the free radical is in positive correlation with the content of the sulfhydryl in the cross-linked reactive gel.
Example 6: sulfhydryl-containing hyaluronic acid crosslinking active gel (adipic acid dihydrazide crosslinking, different hyaluronic acid concentrations)
Sodium hyaluronate (molecular weight 20 kilodaltons) 3g is dissolved in 10ml, 15ml or 30ml of distilled water at room temperature to give a clear and transparent solution of 1%, 2% or 3% concentration. To the above solution, 0.261g of adipic acid dihydrazide and 0.1785g of dithiodipropylhydrazide were added, and the mixture was dissolved with stirring. Then the pH value of the solution is adjusted to 4.75 by using 0.1mol/L hydrochloric acid, and 0.864g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added to keep the pH value of the solution at 4.75; the solution viscosity increased and gel gradually formed. The resulting gel was allowed to stand overnight and then soaked with 20 volumes of 1% dithiothreitol solution (pH 8.5) for 4 hours; and finally, soaking the soaked materials in a normal saline solution with the volume not less than 20 times for removing the substances for 12 hours, replacing fresh normal saline every 2 hours, and adjusting the pH value of the soaking solution to 3.5 by using 0.1mol/L hydrochloric acid. The gel obtained after purification is the hyaluronic acid crosslinking active gel (adipic acid dihydrazide crosslinking, different hyaluronic acid concentrations) containing sulfhydryl.
At initial concentrations of 1%, 2% and 3% sodium hyaluronate, the water content of the prepared hyaluronic acid cross-linked active gel was 99.4%, 98.7% and 97.9% (weight/volume percentage, g/ml), respectively.
Example 7: preparation of thiol-group-containing hyaluronic acid Cross-linking active film and sponge (adipic acid dihydrazide Cross-linking)
Drying and dehydrating the cross-linked active gel prepared in the embodiments 2, 4 and 6 at room temperature to obtain the thiol-group-containing hyaluronic acid cross-linked active film.
The cross-linking active gel prepared in the examples 2, 4 and 6 is freeze-dried and dehydrated to obtain the thiol-containing hyaluronic acid cross-linking active sponge.
Example 8: preparation of thiol-group-containing hyaluronic acid Cross-Linked active gel (Dithiohydrazide Cross-Linked)
Sodium hyaluronate (molecular weight 100 ten thousand daltons) 1g was dissolved in 10ml of distilled water at room temperature to give a clear and transparent solution. To the above solution were added 0.073g of succinic dihydrazide and 0.0665g of dithiodibutyrylhydrazine, respectively, and the resulting mixture was dissolved with stirring. Then the pH value of the solution is adjusted to 4.75 by using 0.1mol/L hydrochloric acid, and 0.288g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added to keep the pH value of the solution at 4.75; the solution viscosity increased and gel gradually formed. The resulting gel was allowed to stand overnight and then soaked with 20 volumes of 1% dithiothreitol solution (pH 8.5) for 4 hours; and finally, soaking the soaked materials in a normal saline solution with the volume not less than 20 times for removing the substances for 12 hours, replacing fresh normal saline every 2 hours, and adjusting the pH value of the soaking solution to 3.5 by using 0.1mol/L hydrochloric acid. And purifying to obtain gel (succinic dihydrazide crosslinking) containing sulfhydryl and hyaluronic acid crosslinking active gel.
The thiol-group-containing hyaluronic acid crosslinking active gel (succinic dihydrazide crosslinked) prepared above was soaked in distilled water to remove salts, lyophilized and hydrolyzed with 0.1mol/L hydrochloric acid, and then the thiol content was measured to be 195. Mu. Mol/g by using a modified Ellman method (Biomacromolecules 2002, 3.
Example 9: preparation of thiol-group-containing hyaluronic acid crosslinking reactive gel (N-3 propionyl hydrazine crosslinking)
Sodium hyaluronate (molecular weight 100 kilodaltons) 1g was dissolved in 10ml of distilled water at room temperature to give a clear and transparent solution. 0.0917g N-3 propionohydrazide and 0.0665g dithiodibutyrhydrazide are added to the above solution, and dissolved by stirring. Then the pH value of the solution is adjusted to 4.75 by using 0.1mol/L hydrochloric acid, and 0.288g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added to keep the pH value of the solution at 4.75; the solution viscosity increased and a gel gradually formed. The resulting gel was allowed to stand overnight and then soaked with 20 volumes of 1% dithiothreitol solution (pH 8.5) for 4 hours; and finally, soaking the soaked materials in a normal saline solution with the volume not less than 20 times for removing the substances for 12 hours, replacing fresh normal saline every 2 hours, and adjusting the pH value of the soaking solution to 3.5 by using 0.1mol/L hydrochloric acid. And purifying to obtain gel (N-3 propionyl hydrazine cross-linking) containing thiol and hyaluronic acid cross-linking activity.
The thiol-group-containing hyaluronic acid cross-linked active gel (N-3 propionylhydrazine cross-linked) prepared above was immersed in distilled water to remove salts, lyophilized and hydrolyzed with 0.1mol/L hydrochloric acid, and then the thiol content was measured to be 174. Mu. Mol/g by using a modified Ellman method (Biomacromolecules 2002, 3.
Example 10: preparation of hyaluronic acid Cross-Linked gel (Dithiodipropylhydrazide Cross-Linked)
Example 10 is a prior art example.
Sodium hyaluronate (molecular weight 50 kilodaltons) 1g was dissolved in 10ml of distilled water at room temperature to give a clear and transparent solution. To the above solution, 0.119g of dithiodipropionazide was added and dissolved by stirring. Then the pH value of the solution is adjusted to 4.75 by using 0.1mol/L hydrochloric acid, and 0.192g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added to keep the pH value of the solution at 4.75; the solution viscosity increased and gel gradually formed. The resulting gel was allowed to stand overnight, and then soaked with a physiological saline solution of not less than 20 times in volume for 12 hours, with fresh physiological saline being replaced every 2 hours. And purifying to obtain the gel, namely the hyaluronic acid cross-linked gel (dithiodipropylhydrazide cross-linked), wherein the cross-linked structure of the gel contains disulfide bonds.
Example 11: preparation of disulfide-bond crosslinked hyaluronic acid gel
Example 11 is a prior art example.
The thiol-modified hyaluronic acid derivative is prepared by using sodium hyaluronate with molecular weights of 300KDa and 1,500KDa as a raw material by a method reported by Wang et al (Wang et al, J Mater chem. B,2015,3, 7546-7553), and the thiol contents of the thiol-modified hyaluronic acid derivative are 103 mu mol/g and 75 mu mol/g respectively.
Dissolving the thiol-modified hyaluronic acid derivative to obtain water solutions with contents of 8mg/mL and 10mg/mL, adjusting pH to 7.4, transferring into glass container, and performing wet heat sterilization; keeping for 4 weeks at room temperature, sealing and standing, and allowing the solution to lose fluidity and form disulfide bond crosslinked hyaluronic acid gel.
Example 12: stability of the Cross-Linked hyaluronic acid gel of the present invention
1g of the crosslinked gel prepared in examples 2, 4, 6, 8, 9, 10, 11 was added to 10ml of a tris (2-carboxyethyl) phosphine hydrochloride (reducing agent) solution (concentration: 2.0% w/v), and the reaction was stirred for 24 hours. The crosslinked gels prepared in examples 2, 4, 6, 8 and 9 maintained the stable structure of the gel. The crosslinked gels prepared in examples 10 and 11 gradually dissolved away to give clear and transparent solutions in which the disulfide bonds in the crosslinked structure were reduced by the reducing agent and the crosslinked structure was destroyed.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (15)
1. A method for preparing a hyaluronic acid cross-linking active material, comprising the steps of:
reacting side chain carboxyl of a hyaluronic acid raw material with an active reagent and a cross-linking agent simultaneously under the action of carbodiimide to generate an amido bond, thereby obtaining a hyaluronic acid cross-linking material; or side chain carboxyl of the hyaluronic acid raw material reacts with an intermediate reagent under the action of carbodiimide to form a relatively stable intermediate activated product, and then the intermediate product reacts with the active reagent and the cross-linking agent simultaneously to generate an amido bond, so that the hyaluronic acid cross-linked material is obtained;
wherein the active agent is a diamine or dihydrazide containing a disulfide bond; or a primary amine containing a free thiol group, or a thiol-protected primary amine;
wherein the crosslinker does not contain a disulfide linkage but contains a polyhydrazide with two or more hydrazide functional groups;
wherein the hyaluronic acid raw material is selected from hyaluronic acid, a form of a salt thereof and/or a modified derivative thereof;
step (2) when the active reagent is diamine or dihydrazide containing disulfide bonds, reacting the hyaluronic acid crosslinking material in the step (1) with a reducing agent, wherein the disulfide bonds in the diamine or dihydrazide containing disulfide bonds are reduced to sulfydryl, and purifying to obtain the hyaluronic acid crosslinking active material; or, when the active reagent is primary amine containing free sulfydryl, purifying the hyaluronic acid cross-linking material in the step (1) to obtain the hyaluronic acid cross-linking active material, or, when the active reagent is primary amine with sulfydryl protecting groups, removing the sulfydryl protecting groups from the hyaluronic acid cross-linking material in the step (1) and then obtaining the hyaluronic acid cross-linking active material through a purification process.
2. The method for preparing a hyaluronic acid cross-linking active material of claim 1, wherein the polyhydrazide is one or more selected from the group consisting of: succinic dihydrazide, adipic dihydrazide, suberic dihydrazide, terephthalic dihydrazide, thiodipropionic dihydrazide, tartaric dihydrazide, polyethylene glycol dihydrazide.
3. The method for preparing a hyaluronic acid cross-linking active material of claim 1, wherein said polyhydrazide is an ammonia-based polyhydrazide.
4. The method for preparing a hyaluronic acid cross-linking active material of claim 1, wherein the diamine or dihydrazide containing disulfide bonds is one or more components selected from the group consisting of: cystamine, cystine dimethyl ester, cystine diethyl ester, dithiodipropylhydrazide and dithiodibutylhydrazine.
5. The method of claim 1, wherein the carbodiimide is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
6. The method for preparing a hyaluronic acid cross-linking active material of claim 1, wherein said reducing agent is dithiothreitol and/or tris (2-carboxyethyl) phosphine hydrochloride.
7. The method for preparing a hyaluronic acid cross-linking active material of claim 1, wherein said intermediate agent is N-hydroxysuccinimide and/or N-hydroxythiosuccinimide.
8. A hyaluronic acid cross-linking active material prepared by the method of any of claims 1-7.
9. The hyaluronic acid cross-linking active material of claim 8, wherein the hyaluronic acid cross-linking active material is in a solid form comprising a film or a sponge.
10. The hyaluronic acid cross-linking active material of claim 8, wherein the hyaluronic acid cross-linking active material is a hydrogel.
11. The hyaluronic acid crosslinking-active material of claim 10, wherein the hydrogel has a water content of 98% or more, and the water content is weight/volume%.
12. The hyaluronic acid crosslinking-active material of claim 11, wherein the hydrogel has a water content of 99% or more, the water content being weight/volume%.
13. The hyaluronic acid crosslinking-active material of claim 8, wherein the content of the thiol group in the hyaluronic acid crosslinking-active material is less than 350 μmol/g.
14. The hyaluronic acid cross-linking active material of claim 13, wherein the thiol group content is 80-150 μmol/g.
15. Use of the hyaluronic acid cross-linking active material according to any of claims 8-14 in the medical field.
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