A method for preparing thiolized hyaluronic acid and its application

Abstract

The application discloses a preparation method and application of thiolated hyaluronic acid, and the technical scheme of the application is that a thiol-modified hyaluronic acid derivative is synthesized through a polycyclic compound, the grafting rate of the thiolated hyaluronic acid can be accurately adjusted through the regulation of a feeding ratio, and the product is purified through dialysis, freeze drying and the like. The product has low content of toxic substances, mild reaction conditions, high grafting efficiency and better biocompatibility. The compound, the preparation method and the product of the application have wide application in the preparation of various clinical devices and materials.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for preparing thiolized hyaluronic acid and its application. Background Technology

[0002] Hyaluronic acid (HA) is a high-molecular-weight biopolysaccharide first isolated from the vitreous humor of bovine eyes by Professor Karl Mayer and his assistant John Palmer in 1934. Hyaluronic acid is a naturally occurring biopolymer with important biological functions in bacteria and higher animals (including humans). HA is mostly found in connective tissues, particularly in synovial fluid, vitreous humor of the eye, umbilical cord, and rooster combs. HA is synthesized by a class of intact membrane proteins called hyaluronidases and can be degraded by a series of hyaluronidases. Hyaluronic acid can be obtained through two routes: one is extraction from animal tissues (bovine eyes, rooster combs, etc.). However, hyaluronic acid derived from animal tissues contains exogenous proteins, thus its purity is insufficient for biomedical applications. Therefore, another method for preparing hyaluronic acid using microbial fermentation has been developed to address the purity issue.

[0003] Hyaluronic acid (HA) has numerous applications in clinical practice, tissue engineering repair, and gene and drug delivery. Cross-linked HA hydrogels can serve as biomaterials for cartilage repair. In gene delivery, HA-DNA microspheres and DNA-HA matrices can be used for controlled release and targeted release of DNA at specific sites. However, certain liver receptors can specifically recognize high molecular weight HA and rapidly clear it from systemic circulation. Therefore, the application of cross-linked HA can significantly prolong the duration of HA in vivo and extend its therapeutic effect. Commercially available cross-linked HA products are mainly prepared by reacting two small molecule cross-linking agents with the hydroxyl groups in HA (butanediol diglycidyl ether and divinyl sulfone). Both cross-linking agents can cause protein denaturation in the human body, thereby triggering allergic reactions. Therefore, the development of novel hyaluronic acid derivatives and cross-linking technologies has broad application prospects and commercial value. Summary of the Invention

[0004] To address the technical problems in the prior art, the present invention provides the following technical solution:

[0005] This invention provides compounds represented by any of the following chemical structural formulas or pharmaceutically acceptable salts thereof.

[0006]

[0007] Where n independently represents an integer greater than or equal to 1.

[0008] Hyaluronic acid of different molecular weights (in the form of solutions of varying viscosities, gels of varying viscoelasticity, sponges, films, or diaphragms) is used in human medical treatment and surgery. For example, it is used as a substitute for synovial fluid, an anti-adhesion agent for tissues, a substitute for vitreous fluid, artificial tears, and in vivo tissue remodeling agents (such as an extracellular matrix for osteoblast transplantation and subsequent calcification of bone zones (plates); an extracellular matrix for fibroblast transplantation and subsequent connective-skin tissue formation). It is also used as a material for preparing artificial skin in burn treatment or morphological surgery; a biocompatible vascular repair coating; and a carrier of active pharmaceutical ingredients in controlled-release formulations, etc.

[0009] The term "medicinal salt" as used in this invention refers to salts that are non-toxic at the amount and concentration in which they are administered. Such salts can be prepared for pharmacological applications by altering the physical properties of the compound without preventing them from exerting their physiological effects.

[0010] Furthermore, the pharmaceutically usable salts include acid addition salts and alkali addition salts.

[0011] Furthermore, the acid addition salts include, but are not limited to, any one or a combination of at least two of the following: hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate, salicylate, malonate, adipate, hexanoate, arginine, fumarate, nicotinate, phthalate, or oxalate of FTY720.

[0012] Furthermore, the alkali addition salts include, but are not limited to, lithium, sodium, potassium, barium, calcium, magnesium, aluminum, iron, ferrous, copper, and zinc salts of FTY720, or salts composed of FTY720 with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine, or histidine.

[0013] Furthermore, the pharmaceutically usable salt is obtained from an acid, which includes organic or inorganic acids.

[0014] Furthermore, the inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.

[0015] Furthermore, the organic acids include formic acid, acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, α-hydroxy acids such as citric acid or tartaric acid, amino acids, aromatic acids, and sulfonic acids.

[0016] Furthermore, the medicinal salts include alkali addition salts, specifically alkali metal salts (such as sodium salts, potassium salts, etc.) and alkaline earth metal salts (such as calcium salts, magnesium salts, etc.).

[0017] This invention provides a method for preparing thiolized hyaluronic acid, the method comprising:

[0018] Hyaluronic acid is dissolved in water and added dropwise to a solution containing side-chain modified groups. The reaction is then carried out by adding DTT reducing agent solution and stirring. The reaction solution is transferred to a dialysis bag for deoxygenation and dialysis. The purified solution is then freeze-dried to obtain a thiol-modified hyaluronic acid derivative.

[0019] The side chain modification groups include BDDE and / or DSDE.

[0020] The term "BDDE" used in this invention refers to 1,4-butanediol diglycidyl ether, PubChem CID:17046, English name 1,4-Butanediol diglycidyl ether.

[0021] In this invention, the term "DSDE" refers to 2-[2-[2-(2-(Oxiran-2-ylmethoxy)ethyldisulfanyl]ethoxymethyl]oxirane, PubChem CID: 102416550, English name 2-[2-[2-(Oxiran-2-ylmethoxy)ethyldisulfanyl]ethoxymethyl]oxirane.

[0022] Furthermore, after reacting with the solution containing side-chain modified groups, small molecule impurities are removed by dialysis.

[0023] Furthermore, after the reaction with the solution containing the side-chain modified group is added dropwise and then dialyzed, the product can be freeze-dried, and the freeze-dried product can be re-dissolved in water.

[0024] In some embodiments, the freeze-dried product is reconstituted in water to prepare a solution with a final concentration of 2.0% (w / v).

[0025] Furthermore, the reaction temperature for adding the solution containing the side-chain modified group is 25°C.

[0026] Furthermore, the reaction time for adding the solution containing the side-chain modified group is 2 hours.

[0027] Furthermore, the reaction temperature during which the DTT reducing agent solution is added dropwise and stirred is 25°C.

[0028] Furthermore, the stirring time for adding the DTT reducing agent solution dropwise is 12-24 hours.

[0029] In some embodiments, the stirring time for adding the DTT reducing agent solution dropwise is 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, or 24h.

[0030] In this invention, the term "DTT" refers to dithiothreitol.

[0031] Furthermore, the oxygen dedialysis process lasts for 3 days.

[0032] Furthermore, the temperature of the deoxygenated dialysis is 25°C.

[0033] Furthermore, the water is ultrapure water.

[0034] Furthermore, the final concentration of the hyaluronic acid dissolved in water is 1% w / v to 2% w / v.

[0035] In some embodiments, the final concentration of the hyaluronic acid dissolved in water is 1% w / v, 1.1% w / v, 1.2% w / v, 1.3% w / v, 1.4% w / v, 1.5% w / v, 1.6% w / v, 1.7% w / v, 1.8% w / v, 1.9% w / v, or 2% w / v.

[0036] Furthermore, the molecular weight of the hyaluronic acid is 800 Da-10000 kDa.

[0037] In some embodiments, the molecular weight of the hyaluronic acid is 800 Da, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 20 kDa, 30 kDa, 50 kDa, 60 kDa, 80 kDa, 100 kDa, 200 kDa, 500 kDa, 800 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 5000 kDa, or 10000 kDa. In some embodiments, thiolated hyaluronic acid can be prepared from hyaluronic acid of any molecular weight.

[0038] Furthermore, the pH value of the solution is adjusted after the hyaluronic acid is dissolved in water.

[0039] Furthermore, the pH value of the adjusted solution is 8.0-9.0.

[0040] In some implementations, the pH value of the adjusted solution is 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

[0041] Furthermore, the pH value of the adjusted solution is 8.5.

[0042] Furthermore, the reagent used to adjust the pH of the solution is a NaOH solution.

[0043] Furthermore, the concentration of the NaOH solution is 5M.

[0044] Furthermore, the molar ratio of hyaluronic acid to the side-chain modified group is 1:(0.5-10).

[0045] In some embodiments, the molar ratio of hyaluronic acid to the side-chain modified group is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, or 1:10.

[0046] Furthermore, the molar ratio of hyaluronic acid to the side-chain modification group is 1:0.5, 1:1, 1:2, 1:2.5, 1:5, and 1:10.

[0047] Furthermore, the specific steps of the deoxygenated dialysis are as follows: nitrogen gas is pre-passed into the pure water for dialysis for 1 hour to remove dissolved oxygen, and then the dialysis bag containing the sample is placed in it for dialysis for 3 days.

[0048] In some embodiments, the method for preparing thiolated hyaluronic acid can significantly reduce the residual toxic substances in the preparation process of the prior art, including EDC, acylhydrazine, etc.

[0049] In some embodiments, the method for preparing thiolized hyaluronic acid can precisely control and adjust the grafting rate of thiol groups to obtain functionalized hyaluronic acid with an accurate grafting rate. In more specific embodiments, the grafting rate is adjusted by adjusting the feed ratio of HA, BDDE, and DTT. In more specific embodiments, the grafting rate is adjusted by adjusting the feed ratio of HA and DSDE.

[0050] In some embodiments, the method for preparing thiolized hyaluronic acid can introduce active functional groups through side chain modification without changing the disaccharide backbone of hyaluronic acid, thus avoiding the degradation of the HA backbone during the reaction process.

[0051] The present invention provides a product, which is a product prepared by the preparation method described above or a derivative thereof, including hydrogels, films, and sponges.

[0052] Furthermore, the grafting rate of the thiolated hyaluronic acid obtained from HA and DSDE in the product is 5%-80%.

[0053] In some embodiments, the grafting rate of thiolated hyaluronic acid obtained from HA and DSDE in the product is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%. %, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%.

[0054] Furthermore, the grafting rates of the thiolated hyaluronic acid obtained from HA and DSDE in the product are 17.5%, 33.3%, 47.6%, 62.3%, 69.7%, and 78.2%.

[0055] Furthermore, the grafting rate of the thiolated hyaluronic acid obtained from HA and BDDE in the product is 5%-60%.

[0056] In some implementations, the HA and BDDE crosslink after purification, making it impossible to accurately determine the grafting rate. Therefore, the grafting rate of the final thiol group after the addition of DTT is used as the grafting rate of the product. More specifically, the final grafting rate of thiol groups after adding DTT is between 5% and 60%, specifically including 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%. In some more specific embodiments, the grafting rates of the thiol groups ultimately formed by the BDDE are 11.2% (100 μL BDDE, 2.57 g DTT), 19.8% (200 μL BDDE, 5.14 g DTT), 24.1% (300 μL BDDE, 7.71 g DTT), 46.7% (600 μL BDDE, 15.42 g DTT), and 58.9% (1000 μL BDDE, 25.70 g DTT).

[0057] Furthermore, the product is in gel form.

[0058] Furthermore, the product is in solid or semi-solid form.

[0059] In some implementations, the optimal grafting ratio range is related to the specific application. For example, a high grafting ratio is needed to enhance material strength in scenarios where it is required; while a low grafting ratio is needed to extend the range of material flexibility in scenarios where it is required.

[0060] This invention provides the use of the compounds, preparation methods, or products described above in the preparation of synovial fluid, intraocular vitreous substitutes, drug-controlled release matrices, healing agents, anti-adhesion agents, vascular repairs, hybrid physiological organs, healing devices, ophthalmic and otological compositions, prostheses, implants, and medical devices.

[0061] In some embodiments, the thiolated hyaluronic acid and its derivatives can be used as substitutes for synovial fluid in joints for the treatment of osteoarthritis; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as substitutes for vitreous fluid for the treatment of ophthalmic symptoms and side effects; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as a matrix for artificial tear formulations for the treatment of dry eye; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as a matrix for controlled drug release, specifically for drugs including anti-inflammatory drugs, antibiotics, β-adrenergic agonists and antagonists, aldose reductase inhibitors, anti-acne drugs, anti-allergy drugs, anti-hair loss drugs, anti-tumor drugs, anti-glaucoma drugs, anti-itch drugs, anti-psoriasis drugs, and anti-skin diseases. Seborrhea, anti-ulcer drugs, antiviral agents, growth factors; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as preparations of various breathable membranes or sponges; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as known applications of hyaluronic acid in various fields, specifically including the production of various solid or semi-solid devices or malleable devices for the manufacture of vascular repairs (anti-adhesion dressings for blood vessels, artificial heart valves, etc.); biohybrid organs (artificial pancreas, liver); ophthalmic products (lens substitutes, contact lenses); otological products; anti-adhesion implants commonly used in abdominal, gynecological, plastic, orthopedic, neurological, ophthalmic, thoracic, otolaryngological and other surgical fields; Stenson molds, catheters, cannulas and other medical devices.

[0062] The application of thiolated hyaluronic acid in some other patent documents can also be applied to the thiolated hyaluronic acid provided in this invention.

[0063] This invention provides the application of the aforementioned compounds or the aforementioned preparation methods in products that reduce organic solvent residue, improve the biocompatibility of thiolated hyaluronic acid, reduce human rejection of thiolated hyaluronic acid, and / or reduce human inflammatory response to thiolated hyaluronic acid during the preparation of thiolated hyaluronic acid.

[0064] The term "thiolization" as used in this invention refers to a reaction that provides a thiol (-SH) group to a specific molecule.

[0065] Advantages and benefits of the present invention:

[0066] 1) Pioneering use of BDDE and DSDE as side-chain modifying groups;

[0067] 2) It avoids the use of toxic substances such as EDC and hydrazide in traditional thiolated hyaluronic acid;

[0068] 3) The reaction modification conditions are mild and the grafting efficiency is high;

[0069] 4) It avoids the use of organic solvents, fundamentally eliminating the problem of residual organic solvents in the product, resulting in better biocompatibility and a significant reduction in the probability of rejection and inflammatory reactions after implantation into the human body. Attached Figure Description

[0070] Figure 1 This is a diagram of the reaction equations for preparing thiolized hyaluronic acid using the BDDE modification method;

[0071] Figure 2 This is a flowchart illustrating the preparation process of thiolized hyaluronic acid using the BDDE modification method.

[0072] Figure 3 This is the 1H-NMR spectrum of thiolized hyaluronic acid prepared using the BDDE modification method;

[0073] Figure 4 This is a diagram of the reaction equations for preparing thiolized hyaluronic acid using the DSDE modification method;

[0074] Figure 5 This is a flowchart of the preparation process for thiolized hyaluronic acid using the DSDE modification method;

[0075] Figure 6 This is the 1H-NMR spectrum of thiolized hyaluronic acid prepared using the DSDE modification method;

[0076] Figure 7 This is a graph showing the results of the cytological toxicity evaluation;

[0077] Figure 8 This is a graph showing the results of an animal's evaluation of inflammatory response. Detailed Implementation

[0078] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0079] Example 1: Preparation of BDDE-modified hyaluronic acid

[0080] 1. Preparation steps

[0081] 1) Dissolve 0.5g (1.25mmol) of sodium hyaluronate with a molecular weight of 370kDa in 50ml of ultrapure water to prepare a 1.0% (w / v) solution. Then add 250μL of 5M NaOH solution to the solution and adjust the pH of the solution to 8.5.

[0082] 2) Then slowly add 600 μL of BDDE to it and react for 2 h;

[0083] 3) After the reaction was completed, the dialysis bag containing the reaction product was placed in pre-purified nitrogen-filled pure water for dialysis for 3 days, and then freeze-dried to obtain BDDE-modified hyaluronic acid with the following structure.

[0084] 2. Preparation process and results

[0085] The specific reaction equation for the preparation of BDDE-modified hyaluronic acid is as follows: Figure 1 As shown, BDDE-modified hyaluronic acid was finally obtained with the following chemical formula.

[0086]

[0087] Example 2: Preparation of thiolated hyaluronic acid

[0088] 1) Dissolve 0.5g of BDDE-modified hyaluronic acid obtained in Example 1 in 25ml of ultrapure water to prepare a 2.0% (w / v) solution. Dissolve 15.42g of DTT in 25ml of ultrapure water and stir until completely dissolved.

[0089] 2) Slowly add a 2.0% BDDE-modified hyaluronic acid solution dropwise into a DTT aqueous solution, and then react for 12 hours;

[0090] 3) After the reaction is completed, the resulting reaction solution is subjected to deoxygenated dialysis in a dialysis bag. The specific steps are as follows: nitrogen gas is introduced into the dialysis solution for 1 hour to remove dissolved oxygen, and then the dialysis bag is placed in it for deoxygenated dialysis for 3 days. The solution is then freeze-dried to obtain thiolized hyaluronic acid with the following structure.

[0091]

[0092] The reaction equation is shown below. Figure 1 See the detailed flowchart. Figure 2 The 1H-NMR spectrum of the thiolized hyaluronic acid obtained from the above reaction is shown in [reference needed]. Figure 3 The grafting rate of DTT is determined by the ratio of the integrated area of ​​the characteristic peaks of the methyl group of hyaluronic acid and DTT in the 1H NMR spectrum, i.e., the grafting rate. The grafting rate of the thiolized hyaluronic acid obtained in this example was measured to be 46.7%.

[0093] Example 3: Preparation of thiolized hyaluronic acid using DSDE

[0094] 1) Dissolve 0.5g (1.25mmol) of sodium hyaluronate with a molecular weight of 370kDa in 50ml of ultrapure water to prepare a 1.0% (w / v) solution. Then add 250μL of 5M NaOH solution to the solution and adjust the pH of the solution to 8.5.

[0095] 2) Then slowly add 600 μL of DSDE dropwise and react for 2 h;

[0096] 3) Add 3.085g of DTT reducing agent to the reaction solution and react for 24 hours;

[0097] After the reaction was completed, the resulting reaction solution was subjected to deoxygenated dialysis in a dialysis bag. The specific steps were as follows: nitrogen gas was introduced into the pure water for dialysis for 1 hour to remove dissolved oxygen, and then the dialysis bag was placed in it for deoxygenated dialysis for 3 days. After freeze drying, thiolized hyaluronic acid with the following structure was obtained.

[0098] The reaction equation is shown below. Figure 4 See the detailed flowchart. Figure 5 The 1H-NMR spectrum of the DSDE-modified hyaluronic acid obtained from the above reaction is shown in [reference needed]. Figure 6 The grafting rate of the thiol group was determined by the ratio of the integrated areas of the characteristic peaks associated with the methyl group and DSDE in the 1H NMR spectrum of hyaluronic acid, i.e., the grafting rate. The grafting rate of the thiolized hyaluronic acid obtained in this example was measured to be 62.3%.

[0099]

[0100] Example 4: Preparation of thiolized hyaluronic acid with different grafting rates

[0101] Thiol-modified hyaluronic acid with different grafting rates was prepared using the same method as in Example 3, according to the conditions given in Table 1.

[0102] Table 1

[0103]

[0104] Since HA and BDDE crosslink after purification, the grafting rate cannot be accurately measured. Therefore, the final grafting rate of thiol groups after the addition of DTT is used for statistical analysis, as follows:

[0105] Grafting rate 11.2% (BDDE 100μL, DTT 2.57g);

[0106] Grafting rate 19.8% (BDDE feed 200μL, DTT 5.14g);

[0107] Grafting rate 24.1% (BDDE feed 300μL, DTT 7.71g);

[0108] Grafting rate 46.7% (BDDE feed 600μL, DTT 15.42g);

[0109] Grafting rate 58.9% (BDDE 1000μL, DTT 25.70g).

[0110] Example 5: Cellular toxicity of new materials

[0111] The thiolated hyaluronic acid with different grafting rates from Example 4 was dissolved in injectable saline at a certain concentration and co-cultured with normal human skin fibroblasts for 3 days before cytological toxicity evaluation. The evaluation results are detailed in [link to relevant documentation]. Figure 7 .

[0112] Example 6: Inflammatory Effects of the New Material in Animals

[0113] The thiolated hyaluronic acid with different grafting rates from Example 4 was dissolved in physiological saline at a concentration of 10 mg / mL and injected into the subcutaneous tissue of the back of SD rats. Blood samples were then collected at different time points for inflammatory evaluation; the evaluation results are detailed below. Figure 8 .

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1.A method for preparing a thiolated hyaluronic acid, the method comprising: dissolving hyaluronic acid in water, dropwise adding a solution containing a side chain modification group, a reaction temperature of 25℃, a reaction time of 2h, removing small molecular impurities by dialysis, freeze-drying, re-dissolving the freeze-dried product in water, stirring in a DTT reducing agent solution, a stirring time of 12-24h, a reaction temperature of 25℃, transferring the reaction solution into a dialysis bag to remove oxygen and dialysis, a time of 3 days, a temperature of 25℃, and obtaining a thiol-modified hyaluronic acid derivative after freeze-drying the purified solution; wherein the side chain modification group is DSDE; the water is ultrapure water; the final concentration of the hyaluronic acid dissolved in water is 1% w / v-2% w / v; adjusting the pH value of the solution to 8.5 after dissolving the hyaluronic acid in water; and a molar ratio of hyaluronic acid to side chain modification group is 1: (2.5-10). 2.The method of claim 1, wherein the reagent for adjusting the pH value of the solution is a NaOH solution. 3.The method of claim 2, wherein the concentration of the NaOH solution is 5M. 4.The method of claim 1, wherein the oxygen removal dialysis is specifically performed by pre-bubbling nitrogen gas into the pure water used for dialysis for 1h to remove dissolved oxygen, and then placing the dialysis bag containing the sample into the water for dialysis for 3 days. 5.A thiolated hyaluronic acid prepared by the method of any one of claims 1-4. 6.A derivative comprising the thiolated hyaluronic acid of claim 5, wherein the derivative is a hydrogel, a film or a sponge. ​

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