CN114773608A - Long-acting hyaluronic acid for treating osteoarthritis and preparation method thereof - Google Patents
Long-acting hyaluronic acid for treating osteoarthritis and preparation method thereof Download PDFInfo
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- CN114773608A CN114773608A CN202210439051.9A CN202210439051A CN114773608A CN 114773608 A CN114773608 A CN 114773608A CN 202210439051 A CN202210439051 A CN 202210439051A CN 114773608 A CN114773608 A CN 114773608A
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- hyaluronic acid
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- osteoarthritis
- polyethylene glycol
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 89
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
Abstract
The invention relates to a long-acting hyaluronic acid for treating osteoarthritis and a preparation method thereof. Specifically, the preparation method comprises the following steps: the modified hyaluronic acid is prepared by blending and modifying the branched macromolecules, the thiolated hyaluronic acid and a solvent, wherein the double bonds in the branched macromolecules and the thiol functional groups on the hyaluronic acid are subjected to Michael addition reaction to obtain a required hyaluronic acid modified product; and removing unreacted branched macromolecules in the mixed solution by a dialysis method, and freeze-drying to obtain a modified hyaluronic acid product. The modified hyaluronic acid product prepared by the invention has good biocompatibility, can be applied to the treatment of osteoarthritis, has the advantages of enzymolysis resistance and capability of realizing long-acting retention in vivo, gel is not formed in the modification process, and the modified hyaluronic acid still keeps good injectability.
Description
Technical Field
The invention belongs to the technical field of hyaluronic acid modification, and particularly relates to long-acting hyaluronic acid for treating osteoarthritis and a preparation method thereof.
Background
Hyaluronic acid is a macromolecular glycosaminoglycan with high viscoelasticity, which is polymerized by taking D-glucuronic acid and N-acetylglucosamine as disaccharide units, and has unique physicochemical properties and wide physiological functions, such as: has the effects of moisturizing, regulating inflammatory reaction, lubricating joints, protecting cartilage, promoting wound healing and the like, and is widely applied to the fields of medical cosmetology, orthopedic treatment, ophthalmic treatment and the like.
When applied in vivo, the hyaluronic acid is rapidly degraded by the action of hyaluronidase, and the retention time is short. Therefore, compared with the hyaluronic acid with small molecular weight, the hyaluronic acid with ultra-high molecular weight (Mw is more than 1000kDa) has unique advantages in application scenes such as minimally invasive fillers and osteoarthritis treatment due to relatively low degradation speed. However, since such high molecular weight hyaluronic acid has a chain-like open structure and is easily degraded by the action of an enzyme, the retention time in the body is still not preferable. To solve this problem, researchers have proposed cross-linking hyaluronic acid molecules to prepare polymer gels to compensate for the too short residence time in vivo. For example, patent CN111440334A discloses a method for preparing an injectable hyaluronic acid-based hydrogel, which forms a hyaluronic acid hydrogel material with controllable mechanical strength and degradation rate through schiff base reaction between modified hyaluronic acid containing amino groups and a cross-linking agent containing a plurality of aldehyde groups. Other commonly used hyaluronic acid cross-linking agents include: BDDE (butanediol glycidyl ether), GMA (glycidyl methacrylate ether), DVS (divinyl sulfone), and the like. However, such hyaluronic acid crosslinking agents tend to be highly toxic and cause a significant decrease in injectability after gelling, limiting their use. Therefore, there is an urgent need to develop a long-acting hyaluronic acid which can increase the in vivo retention time and is advantageous for in vivo application.
Disclosure of Invention
The invention aims to: in view of the problems of the prior art, the present invention aims to provide a long-acting hyaluronic acid for osteoarthritis treatment, which can improve the residence time of hyaluronic acid in vivo and is beneficial to in vivo application; a second object of the present invention is to provide a method for preparing long-acting hyaluronic acid for the treatment of osteoarthritis.
The technical scheme is as follows: the invention provides long-acting hyaluronic acid for treating osteoarthritis, which is prepared by blending and modifying branched macromolecules, thiolated hyaluronic acid and a solvent; the branched macromolecules are polyethylene glycol macromolecules with vinyl end groups, and the solvent is water or PBS buffer solution.
Furthermore, the polyethylene glycol polymer with the vinyl end group is one or more of dendritic polyethylene glycol ((mPEG)4- (PEG)2-MAL), two-Arm polyethylene glycol ((Propargyl-PEG)2-Allyl), three-Arm polyethylene glycol (3Arm (PEG-Allyl)3), four-Arm polyethylene glycol (4Arm (PEG-Allyl)4), six-Arm polyethylene glycol (6Arm-PEG-DA), eight-Arm polyethylene glycol maleimide (8Arm-PEG-MAL) and branched polyethylene glycol diacrylate (HB-PEGDA).
Furthermore, the sulfhydrylation hyaluronic acid is a functional polymer material obtained by grafting sulfhydryl onto a hyaluronic acid chain through a chemical method, the molecular weight is 5-3000 kDa, and the substitution rate of the sulfhydryl is 0.1-2.0 mmol/g.
The preparation method of the long-acting hyaluronic acid for treating osteoarthritis comprises the following specific preparation steps:
(1) adding the branched macromolecules into a solvent, stirring and dissolving to obtain a solution with the concentration range of 0.5-5% (w/v), wherein the preferable concentration is 4%;
(2) adding thiolated hyaluronic acid into a solvent, stirring and dissolving to obtain a solution with the concentration range of 0.1-1% (w/v);
(3) blending and modifying the solutions prepared in the step (1) and the step (2) in proportion to prepare a hyaluronic acid modified product;
(4) dialyzing the hyaluronic acid modified product prepared in the step (3) and separating and purifying unreacted branched macromolecules;
(5) and freeze-drying the separated hyaluronic acid modified product to obtain the long-acting hyaluronic acid for treating osteoarthritis.
Further, the blending modification process in the step (3) is that double bonds in the branched macromolecules and mercapto functional groups on the hyaluronic acid generate Michael addition reaction at normal temperature and are grafted to the hyaluronic acid skeleton.
Further, the pH of the Michael addition reaction is 5 to 10, preferably 6 to 8, and more preferably 7.
Further, the proportion in the step (3) is that the branched macromolecules: the hyaluronic acid is 1: 1-10: 1(w/v), preferably 5: 1.
Further, the cut-off molecular weight of the dialysis bag used for dialysis purification in step (4) is 1000-.
Further, the time period for freeze-drying in step (5) is 1 to 5 days, preferably 2 to 4 days, and more preferably 4 days. .
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the steric hindrance of the branched polyethylene glycol macromolecules is utilized to inhibit the contact of hyaluronic acid and enzyme, so that the in-vivo retention time of hyaluronic acid can be obviously prolonged.
(2) The materials of all components of the reaction are nontoxic, have good biocompatibility and small damage to cells.
(3) Compared with the traditional crosslinking method, the strategy for modifying the chain polymer by using the branched polyethylene glycol macromolecules provided by the invention has the advantages that no gel is formed in the modification process, and the modified hyaluronic acid still keeps good injectability.
Drawings
FIG. 1 is a structural diagram of a long-acting hyaluronic acid molecule prepared according to the invention;
FIG. 2 is a structural diagram of an HA-SH molecule prepared in example 1 of the present invention;
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the long-acting hyaluronic acid prepared by the invention;
FIG. 4 is a graph of the cell activity of HB-PEGDA prepared in example 1 of the invention;
FIG. 5 is a graph of the cell activity of HA-SH prepared in example 1 of the present invention;
FIG. 6 is a graph of cell activity of long-acting hyaluronic acid prepared in example 2 of the present invention;
FIG. 7 is a diagram of the mechanism of the long-acting hyaluronic acid resistant to enzymatic hydrolysis prepared by the present invention;
FIG. 8 is a graph comparing the degradation of long-acting hyaluronic acid and pure hyaluronic acid prepared according to the invention;
FIG. 9 is a graph of safranin fast green staining of long-acting hyaluronic acid prepared in accordance with the present invention and control treated knee joints;
FIG. 10 is a graph comparing the effect of the long-acting hyaluronic acid prepared according to the invention on the treatment of knee joints with a control group.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
Example 1
(1) Synthesis of hyperbranched polyethylene glycol (HB-PEGDA) macromolecule: 11.5g of PEGDA monomer (average Mn. about.575 g/mol) was weighed out and added to 50ml of butanone and stirred to dissolve it completely. 0.1839g of Azobisisobutyronitrile (AIBN) and 0.2372g of tetraethylthiuram Disulfide (DS) were then added and the container sealed and the sealing film was wrapped to evacuate the air from the container. And then placing the mixture in an oil bath at 70 ℃, fully and uniformly mixing the mixture through magnetic stirring, stopping the reaction immediately after reacting for 6 hours, introducing air and cooling the mixture to room temperature. The product was purified by pouring a mixed solution of ether and n-hexane (v/v ═ 2:1) into a beaker, and then removing the residual solvent using a rotary evaporator. Adding a certain amount of HB-PEGDA into MEM complete culture medium, preparing a series of HB-PEGDA mixed liquor with concentration gradients of 0, 50, 100, 250, 500 and the like, co-culturing with l-929 cells, and quantitatively characterizing the growth state of the cells by using MTT detection agent after 24h, thereby characterizing the biocompatibility of the synthesized HB-PEGDA.
(2) Preparation of thiolated hyaluronic acid (HA-SH): 100mg of HA was weighed and dissolved in 10ml of MES buffer (pH 4.75, 0.1M), and stirred to dissolve completely; adding 96mg of EDCL, keeping the pH value of the reaction solution at 4.75, and reacting for 5 h; after the reaction, adjusting the pH of the reaction solution to 7.0 by using NaOH; 501mg of DTT were further added and the pH of the reaction solution was adjusted to 8.5 with NaOH; stirring and reacting for 24 hours at normal temperature, and adjusting the pH of the reaction solution to 3.5 by HCl after the reaction; transferring the acidified solution to a dialysis bag, dialyzing with 0.1M NaCl (pH 3.5) solution for 24h, and then dialyzing with deionized water (pH 3.5) for 24 h; and finally, freeze-drying the obtained liquid mixture to obtain the solid HA-SH. And adding a certain amount of solid HA-SH into MEM complete culture medium for full dissolution to prepare a series of HA-SH mixed solutions with concentration gradients, co-culturing with l-929 cells, and quantitatively characterizing the growth state of the cells by using an MTT (methyl thiazolyl tetrazolium) detection agent after 24 hours to characterize the biocompatibility of synthesized HA-SH.
As shown in FIG. 4-FIG. 5, the cell activities were all above 80% in the concentration range of the experiment, which indicates that the synthesized HB-PEGDA HAs good biocompatibility with the modified HA-SH.
Example 2
(1) Synthesis of hyperbranched polyethylene glycol (HB-PEGDA) macromolecule: the synthesis was carried out as described in step (1) of example 1.
(2) Preparation of thiolated hyaluronic acid (HA-SH): the synthesis was performed according to the method described in step (2) of example 1.
(3) Preparing long-acting hyaluronic acid: adding a proper amount of HB-PEGDA into the PBS solution to prepare 4% HB-PEGDA solution, adding a proper amount of solid HA-SH into the PBS solution to prepare 0.5% HA-SH solution, and blending the 4% HB-PEGDA solution and the 0.5% HA-SH solution to make the branched macromolecule: hyaluronic acid 8:1, controlling pH to 7-8, performing Michael addition crosslinking reaction to form polymer mucus, dialyzing with deionized water, and freeze drying for storage.
(4) Adding a certain amount of freeze-dried long-acting hyaluronic acid into MEM complete culture medium, fully dissolving, preparing into HB-PEGDA/HA-SH mixed solution with a series of concentration gradients of 0, 50, 100, 250, 500 and the like, co-culturing with l-929 cells, and quantitatively representing the growth state of the cells by using MTT detection agent after 24h, thereby representing the biocompatibility of the synthesized high polymer material.
As shown in the cell activity chart of FIG. 6, the cell activities were all above 80% in the concentration range of the experiment, which indicates that the finally synthesized HB-PEGDA/HA-SH HAs good biocompatibility.
Example 3
(1) Synthesis of hyperbranched polyethylene glycol (HB-PEGDA) macromolecule: the synthesis was carried out as described in step (1) of example 1.
(2) Preparation of thiolated hyaluronic acid (HA-SH): the synthesis was carried out according to the method described in step (2) of example 1.
(3) Preparing long-acting hyaluronic acid: adding a proper amount of HB-PEGDA into a PBS solution to prepare a 4% HB-PEGDA solution, adding a proper amount of solid HA-SH into the PBS solution to prepare a 0.8% HA-SH solution, and blending the 4% HB-PEGDA solution and the 0.8% HA-SH solution to ensure that the branched macromolecules: controlling the pH value to be 6-7, carrying out Michael addition crosslinking reaction to obtain high-molecular mucus, dialyzing with deionized water, and freeze-drying for storage.
(4) And (3) adding a certain amount of the freeze-dried solid into the PBS solution to prepare mucus with certain viscosity.
(5) 50U/mL hyaluronidase was added to the mucus and the degradation was characterized by changes in viscosity of the mucus over time using a Haake rheometer.
As shown in the degradation-to-degradation curve of FIG. 8, the pure hyaluronic acid is degraded completely in 6 hours, while the modified hyaluronic acid is degraded completely in 48 hours, which shows that the modified hyaluronic acid polymer has better enzymolysis resistance, and the specific enzymolysis resistance mechanism diagram is shown in FIG. 7.
Example 4
(1) Synthesis of hyperbranched polyethylene glycol (HB-PEGDA) macromolecule: the synthesis was carried out as described in step (1) of example 1.
(2) Preparation of thiolated hyaluronic acid (HA-SH): the synthesis was performed according to the method described in step (2) of example 1.
(3) Preparing long-acting hyaluronic acid: adding a proper amount of HB-PEGDA into the PBS solution to prepare 4% HB-PEGDA solution, adding a proper amount of solid HA-SH into the PBS solution to prepare 1% HA-SH solution, and blending 4% HB-PEGDA and 1% HA-SH solution to make the branched macromolecules: controlling the pH value to be 7-8, carrying out Michael addition crosslinking reaction to obtain high-molecular mucus, dialyzing with deionized water, and freeze-drying for storage.
(4) An appropriate amount of the freeze-dried solid was added to physiological saline (0.9% NaCl solution) to prepare a 1.5% HB-PEGDA/HA-SH solution.
The effect of long-acting hyaluronic acid on treatment of osteoarthritis in mice was evaluated by histological evaluation. At 2 weeks after DMM surgery, the knee joint of the mice was injected with a commercial osteoarthritis Alzhen injection (clinical HA), a synthetic HB-PEGDA/HA-SH solution (long-acting HA), and physiological saline, respectively. The long-acting HA is injected once, and the clinical HA and the normal saline are injected once a week for four times. After 4 weeks of intra-articular injection, the knee joints were harvested for histological examination, and fig. 9 shows representative histological images of articular cartilage, and safranin fast green staining showed severe cartilage damage, rough and uneven articular cartilage surface, cartilage layer defect, cartilage four-layer disorder (cartilage surface layer, transitional layer, radiation layer, calcified layer), significantly reduced chondrocyte number, significant fibrosis and hyperplasia, multiple tide lines and uneven staining in untreated mice. However, after clinical HA and long-acting HA treatment, the shape and the structure of the articular cartilage are greatly improved, the four-layer structure is clear and distinguishable, no crack exists, the arrangement of chondrocytes is tight, the tide line is complete, the cell nucleus is clear, the staining is uniform, and the treatment effects are not observed when normal saline is injected. Meanwhile, the extent of degeneration of the articular cartilage surface of osteoarthritic mice was evaluated and graded using the OARSI scoring system. The standard score is based on the evaluation of four parameters: cartilage structure, chondrocytes, safranin O staining and tide lines were scored by three independent observers, taking the mean of the three scores as the final score. As shown in fig. 10, independent sample assay analysis was used within the group; n-3, p <0.05, p <0.01, p <0.001, ns: the OARSI score result shows that the mouse score of the operation group is 8.7, however, the osteoarthritis mouse injected with long-acting HA is greatly reduced to only 3.7, and HAs no significant difference with clinical HA, thereby showing that the synthetic natural polymer material HAs the potential in treating osteoarthritis.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. The long-acting hyaluronic acid for treating osteoarthritis is characterized by being prepared by blending and modifying branched macromolecules, thiolated hyaluronic acid and a solvent; the branched macromolecule is a polyethylene glycol macromolecule with a vinyl end group.
2. The long-acting hyaluronic acid for osteoarthritis treatment according to claim 1, wherein the vinyl-terminated polyethylene glycol polymer is one or more of dendrimer ((mPEG)4- (PEG)2-MAL), two-armed polyethylene glycol ((Propargyl-PEG)2-Allyl), three-armed polyethylene glycol (3Arm (PEG-Allyl)3), four-armed polyethylene glycol (4Arm (PEG-Allyl)4), six-armed polyethylene glycol (6Arm-PEG-DA), eight-armed polyethylene glycol maleimide (8Arm-PEG-MAL), and branched polyethylene glycol diacrylate (HB-PEGDA).
3. The long-acting hyaluronic acid for osteoarthritis treatment according to claim 1, wherein the thiolated hyaluronic acid is a functional polymer material obtained by chemically grafting a thiol group onto a hyaluronic acid chain, and has a molecular weight of 5-3000 kDa and a thiol group substitution rate of 0.1-2.0 mmol/g.
4. The long-acting hyaluronic acid for the treatment of osteoarthritis according to claim 1, wherein the solvent is water or PBS buffer.
5. The method of preparing long-acting hyaluronic acid for the treatment of osteoarthritis according to any of claims 1-4, comprising the steps of:
(1) adding the branched macromolecules into a solvent, stirring and dissolving to obtain a solution with the concentration range of 0.4-4% (w/v);
(2) adding the thiolated hyaluronic acid into a solvent, stirring and dissolving to obtain a solution with the concentration range of 0.1-1% (w/v);
(3) blending and modifying the solutions prepared in the steps (1) and (2) in proportion to prepare a hyaluronic acid modified product;
(4) dialyzing the hyaluronic acid modified product prepared in the step (3) and separating and purifying unreacted branched macromolecules;
(5) and freeze-drying the separated hyaluronic acid modified product to obtain the long-acting hyaluronic acid for treating osteoarthritis.
6. The method of claim 5, wherein the blending modification process comprises grafting double bonds in the branched macromolecules and thiol functional groups on the hyaluronic acid onto the hyaluronic acid skeleton by Michael addition reaction at room temperature.
7. The method of producing long-acting hyaluronic acid for the treatment of osteoarthritis according to claim 6, wherein the pH of the Michael addition reaction is 5-10.
8. The method for preparing long-acting hyaluronic acid for osteoarthritis treatment according to claim 5, wherein the ratio in step (3) is branched macromolecules: hyaluronic acid is 1: 1-10: 1 (w/v).
9. The method for preparing long-acting hyaluronic acid for the treatment of osteoarthritis as claimed in claim 5, wherein the molecular weight cut-off of the dialysis bag used in the step (4) is 1000-100000 Da.
10. The method of preparing long-acting hyaluronic acid for the treatment of osteoarthritis according to claim 5, wherein the time for lyophilization in step (5) is 1-5 days.
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