CN118878717A - A preparation method and application of thiolated hyaluronic acid - Google Patents

Abstract

The invention discloses a preparation method and application of sulfhydrylation hyaluronic acid, the technical proposal of the invention obtains sulfhydrylation modified hyaluronic acid derivatives through synthesis of multiple epoxy compounds, the grafting rate of the thiolated hyaluronic acid can be accurately regulated by regulating the charging ratio, and the thiolated hyaluronic acid can be purified by dialysis, freeze drying and the like. The product of the invention 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 invention have wide application in preparing various clinical equipment and materials.

Description

A preparation method and application of thiolated hyaluronic acid

Technical Field

The invention belongs to the field of biotechnology and relates to a preparation method and application of thiolated hyaluronic acid.

Background Art

Hyaluronic acid (HA) is a high molecular weight biopolysaccharide that was first isolated in the vitreous of bovine eyes by Professor Karl Mayer and his assistant John Palmer in 1934. Hyaluronic acid is a naturally occurring biopolymer that has important biological functions in bacteria and higher animals (including humans). HA is found in connective tissues in most cases, especially in synovial fluid, vitreous humor of the eyeball, umbilical cord and rooster comb. HA is synthesized by a class of integral membrane proteins called hyaluronan synthases and can be degraded by a series of hyaluronidases. Hyaluronic acid can be obtained in two ways: one is through animal tissue extraction (bovine eyes, rooster combs, etc.), and hyaluronic acid extracted from animal tissues contains exogenous proteins, so its purity cannot meet the application in the biomedical field. Therefore, another method of preparing hyaluronic acid using microbial fermentation has been developed to solve the purity problem.

HA has many applications in clinical practice, tissue engineering repair, and gene and drug delivery. Cross-linked HA hydrogels can be used as biomaterials for cartilage repair. In terms of gene delivery, HA-DNA microspheres and DNA-HA matrices can be used for controlled release of DNA and targeted release at specific sites. However, certain liver receptors can specifically recognize high molecular weight HA and quickly remove it from the systemic circulation. Therefore, the application of cross-linked HA can greatly prolong the maintenance time of HA in the body and prolong the effect. Commercial cross-linked HA products are mainly prepared by reacting two small molecule cross-linkers with the hydroxyl groups in HA (butanediol diglycidyl ether and divinyl sulfone). Both cross-linkers can cause protein denaturation in the human body, thereby triggering allergic reactions. Therefore, the development of new hyaluronic acid derivatives and cross-linking technologies has broad application prospects and commercial value.

Summary of the invention

In order to solve the technical problems in the prior art, the present invention provides the following technical solutions:

The present invention provides a compound represented by any of the following chemical formulas or a pharmaceutically acceptable salt thereof:

wherein n independently represents an integer greater than or equal to 1.

Hyaluronic acid with different molecular weights (in the form of solutions with different viscosities, gels with different viscoelasticities, sponges, films or membranes) is used in human medical treatment and surgical operations, for example, it is used as a substitute for synovial fluid, an anti-adhesion agent for tissues, a substitute for vitreous humor, an artificial tear, a reconstructor of in vivo tissues (such as as an extracellular matrix for osteoblast migration and the subsequent formation of bone areas (sheets) after calcification; as an extracellular matrix for the formation of connective-skin tissue after fibroblast migration), a material for preparing artificial skin in burn treatment or allopathic surgery; a coating agent for vascular repair with physiological compatibility, a carrier of active pharmaceutical ingredients in controlled release formulations, and the like.

The term "pharmaceutically acceptable salt" used in the present invention is non-toxic in the amount and concentration used. The preparation of such salts can facilitate pharmacological applications by changing the physical properties of the compound without preventing it from exerting its physiological effect.

Furthermore, the pharmaceutically acceptable salts include acid addition salts and base addition salts.

Further, the acid addition salt includes, but is not limited to, any one of the hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, argininate, fumarate, nicotinate, phthalate or oxalate of FTY720, or a combination of at least two thereof.

Furthermore, the base addition salt includes but is not limited to lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt of FTY720, or a salt of FTY720 with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

Furthermore, the pharmaceutically acceptable salt is obtained from an acid, which includes an organic acid or an inorganic acid.

Furthermore, the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.

Furthermore, the organic acid may be 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 acid such as citric acid or tartaric acid, amino acid, aromatic acid, or sulfonic acid.

Furthermore, the pharmaceutically acceptable salts include base 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.).

The present invention provides a method for preparing thiolated hyaluronic acid, the preparation method comprising:

The hyaluronic acid is dissolved in water, and a solution containing a side chain modification group is added dropwise to react, and then added dropwise to a DTT reducing agent solution with stirring. The reaction solution is transferred into a dialysis bag for deoxygenation and dialysis, and the obtained purified solution is freeze-dried to obtain a thiol-modified hyaluronic acid derivative;

Wherein, the side chain modification groups include BDDE and/or DSDE.

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

The term "DSDE" used in the present invention refers to 2-[2-[2-(Oxiran-2-ylmethoxy)ethyldisulfanyl]ethoxymethyl]oxirane, PubChem CID: 102416550, English name 2-[2-[2-(Oxiran-2-ylmethoxy)ethyldisulfanyl]ethoxymethyl]oxirane.

Furthermore, after the solution containing the side chain modification group is added dropwise for reaction, dialysis is performed to remove small molecule impurities.

Furthermore, the solution containing the side chain modifying group is added dropwise for reaction and then dialyzed, and then freeze-dried, and the freeze-dried product is redissolved in water.

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

Furthermore, the reaction temperature of the dropwise addition of the solution containing the side chain modification group is 25°C.

Furthermore, the reaction time of dropwise adding the solution containing the side chain modification group is 2 hours.

Furthermore, the reaction temperature of the dropwise addition into the DTT reducing agent solution and stirring is 25°C.

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

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.

The term "DTT" as used herein refers to dithiothreitol.

Furthermore, the deoxygenation dialysis time is 3 days.

Furthermore, the temperature of the deoxygenation dialysis is 25°C.

Furthermore, the water is ultrapure water.

Furthermore, the final concentration of the hyaluronic acid dissolved in water is 1% w/v-2% w/v.

In some embodiments, the final concentration of 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.

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

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

Furthermore, the hyaluronic acid is dissolved in water and the pH value of the solution is adjusted.

Furthermore, the pH value of the solution is adjusted to 8.0-9.0.

In some embodiments, the pH 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.

Furthermore, the pH value of the solution is adjusted to 8.5.

Furthermore, the reagent for adjusting the pH value of the solution is a NaOH solution.

Furthermore, the concentration of the NaOH solution is 5M.

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

In some embodiments, the molar ratio of hyaluronic acid to side chain modifying groups 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, 1:10.

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.

Furthermore, the specific steps of the deoxygenation dialysis are to pre-introduce nitrogen into the pure water for dialysis for 1 hour to remove dissolved oxygen, and then place the dialysis bag containing the sample into the pure water for dialysis for 3 days.

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, and the toxic substances include EDC, hydrazide, etc.

In some embodiments, the preparation method of the thiolated hyaluronic acid can accurately control and adjust the grafting rate of the thiol group to obtain a functionalized hyaluronic acid with an accurate grafting rate. In some more specific embodiments, the grafting rate is adjusted by adjusting the feed ratio of HA, BDDE and DTT. In some more specific embodiments, the grafting rate is adjusted by adjusting the feed ratio of HA and DSDE.

In some embodiments, the method for preparing thiolated hyaluronic acid can introduce active functional groups through side chain modification without changing the disaccharide backbone of hyaluronic acid, thereby avoiding degradation of the HA main chain during the reaction.

The present invention provides a product, which is a product prepared by the above-mentioned preparation method or a derivative of the product prepared by the above-mentioned preparation method, and the derivative includes a hydrogel, a membrane, and a sponge.

Furthermore, the grafting rate of the thiolated hyaluronic acid obtained from HA and DSDE as raw materials in the product is 5%-80%.

In some embodiments, the grafting rate of thiolated hyaluronic acid obtained from HA and DSDE as raw materials 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, %, 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%.

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

Furthermore, the grafting rate of the thiolated hyaluronic acid obtained from HA and BDDE as raw materials in the product is 5%-60%.

In some embodiments, the HA and BDDE will be cross-linked after purification and the grafting rate cannot be accurately determined. Therefore, the final grafting rate of thiol groups after the addition of DTT is used as the grafting rate of the product. More specifically, after adding DTT, the final thiol grafting rate 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 rate of the thiol group finally formed by the BDDE is 11.2% (BDDE feed 100 μL, DTT 2.57 g), 19.8% (BDDE feed 200 μL, DTT 5.14 g), 24.1% (BDDE feed 300 μL, DTT 7.71 g), 46.7% (BDDE feed 600 μL, DTT 15.42 g), and 58.9% (BDDE feed 1000 μL, DTT 25.70 g).

Further, the product is in the form of a gel.

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

In some embodiments, a good range of grafting rate is related to practical applications. For example, a high grafting rate is required to enhance material strength in order to achieve material strength; a low grafting rate is required to expand the range of material flexibility in order to achieve material flexibility.

The present invention provides the use of the aforementioned compound, the aforementioned preparation method, or the aforementioned product in the preparation of synovial fluid, intraocular vitreous substitute, drug controlled release matrix, healing agent, anti-adhesion agent, vascular repair material, hybrid physiological organ, healing device, ophthalmic and otological composition, prosthesis, in vivo implant and medical device.

In some embodiments, the thiolated hyaluronic acid and its derivatives can be used as substitutes for synovial fluid in joints for the treatment of bone and joint diseases; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as substitutes for vitreous humor for the treatment of symptoms and side effects related to ophthalmic surgery; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as a matrix for artificial tear formulations, suitable for the treatment of dry eye; in some embodiments, the thiolated hyaluronic acid and its derivatives can be used as a matrix for controlled release of drugs, and specific drug types include anti-inflammatory drugs, antibiotics, β-adrenergic agonists and antagonists, aldose reductase inhibitors, anti-acne, anti-allergy, anti-hair loss, anti-tumor, anti-glaucoma, anti-itch, anti-psoriasis, and anti-dermatitis. Liporrhea, 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 hyaluronic acid in various applications, specifically including the production of various solid or semi-solid devices or plastic devices for the manufacture of vascular repair products (anti-adhesion bandages of blood vessels, artificial heart valves, etc.); biohybrid organs (artificial pancreas, liver); ophthalmic products (lens substitutes, contact lenses); otolaryngological 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 equipment.

The thiolated hyaluronic acid provided by the present invention can be used in all the applications of thiolated hyaluronic acid in some other patent documents.

The present invention provides the use of the aforementioned compound or the aforementioned preparation method in a product for reducing organic solvent residues during the preparation of thiolated hyaluronic acid, improving the biocompatibility of thiolated hyaluronic acid, reducing human body's rejection reaction to thiolated hyaluronic acid, and/or reducing human body's inflammatory reaction to thiolated hyaluronic acid.

The term "thiolation" as used herein refers to a reaction that provides a thiol (-SH) group to a specific molecule.

Advantages and beneficial effects of the present invention:

1) Pioneering use of BDDE and DSDE as side chain modification groups;

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

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

4) It avoids the use of organic solvents and fundamentally eliminates the problem of residual organic solvents in the product. It has better biocompatibility and can greatly reduce the probability of rejection and inflammatory reactions after implantation in the human body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a reaction equation diagram for preparing thiolated hyaluronic acid using a BDDE modification method;

FIG2 is a flow chart of the preparation of thiolated hyaluronic acid using the BDDE modification method;

FIG3 is a 1H-NMR spectrum result diagram of thiolated hyaluronic acid prepared by the BDDE modification method;

FIG4 is a reaction equation diagram of preparing thiolated hyaluronic acid using the DSDE modification method;

FIG5 is a flow chart of the preparation of thiolated hyaluronic acid using the DSDE modification method;

FIG6 is a 1H-NMR spectrum result diagram of thiolated hyaluronic acid prepared by the DSDE modification method;

FIG7 is a diagram showing the results of cytological toxicity evaluation;

FIG. 8 is a graph showing the results of animal inflammatory evaluation.

DETAILED DESCRIPTION

The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

Example 1 Preparation of BDDE-modified hyaluronic acid

1. Preparation steps

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

2) Then slowly add 600 μL of BDDE and react for 2 h;

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

2. Preparation process and results

The specific reaction equation for the preparation of BDDE-modified hyaluronic acid is shown in FIG1 , and finally BDDE-modified hyaluronic acid with the following chemical formula is obtained.

Example 2 Preparation of thiolated hyaluronic acid

1) Dissolve 0.5 g of BDDE-modified hyaluronic acid obtained in Example 1 in 25 ml of ultrapure water to prepare a 2.0% (w/v) solution, and dissolve 15.42 g of DTT in 25 ml of ultrapure water, stirring until completely dissolved;

2) Slowly dripping 2.0% BDDE-modified hyaluronic acid solution into DTT aqueous solution, followed by reaction for 12 h;

3) After the reaction is completed, the reaction solution is dialyzed in a dialysis bag for deoxygenation. The specific steps are to introduce nitrogen into the dialysis solution for 1 hour to remove dissolved oxygen, then place the dialysis bag therein for deoxygenation dialysis for 3 days, and freeze-dry to obtain a thiolated hyaluronic acid having the following structure.

The reaction equation is shown in FIG1 , the specific flow chart is shown in FIG2 , and the 1H-NMR spectrum of the thiolated hyaluronic acid obtained by the above reaction is shown in FIG3 . The grafting rate of DTT is determined by the ratio of the integral area of the characteristic peaks of the methyl group of hyaluronic acid and DTT in the hydrogen nuclear magnetic resonance spectrum, i.e., the grafting rate. After measurement, the grafting rate of the thiolated hyaluronic acid obtained in this example is 46.7%.

Example 3 Preparation of Thiolated Hyaluronic Acid by DSDE

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

2) Then 600 μL of DSDE was slowly added dropwise and reacted for 2 h;

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

After the reaction is completed, the reaction solution is dialyzed in a dialysis bag for deoxygenation. The specific steps are to pass nitrogen into the pure water for dialysis for 1 hour to remove dissolved oxygen, then put the dialysis bag into the water for deoxygenation dialysis for 3 days, and freeze-dry to obtain thiolated hyaluronic acid having the following structure.

The reaction equation is shown in FIG4 , the specific flow chart is shown in FIG5 , and the 1H-NMR spectrum of the DSDE-modified hyaluronic acid obtained by the above reaction is shown in FIG6 . The grafting rate of the thiol group is determined by the ratio of the integral area of the characteristic peaks related to the methyl group of the hyaluronic acid and DSDE in the hydrogen nuclear magnetic resonance spectrum, i.e., the grafting rate. After measurement, the grafting rate of the thiolated hyaluronic acid obtained in this example is 62.3%.

Example 4 Preparation of thiolated hyaluronic acid with different grafting rates

The same method as in Example 3 was used to prepare thiolated hyaluronic acids with different grafting rates according to the conditions given in Table 1.

Table 1

HA and BDDE will cross-link after purification, so the grafting rate cannot be accurately determined. Therefore, the final grafting rate of thiol groups after adding DTT is statistically analyzed, which are:

Grafting rate 11.2% (BDDE feed 100 μL, DTT 2.57 g);

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

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

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

The grafting rate was 58.9% (BDDE feed 1000 μL, DTT 25.70 g).

Example 5 Cytotoxicity of New Materials

The thiolated hyaluronic acids with different grafting ratios in Example 4 were dissolved in a certain concentration in physiological saline for injection, and co-cultured with normal human skin fibroblasts for 3 days, and then the cytotoxicity was evaluated. The evaluation results are shown in FIG7 .

Example 6 Inflammatory properties of new materials in animals

The thiolated hyaluronic acid with different grafting ratios in Example 4 was dissolved in a 10 mg/mL concentration of physiological saline for injection and injected into the subcutaneous tissue of the back of SD rats. Blood samples were then collected at different time points for inflammatory evaluation, and the evaluation results are shown in Figure 8.

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 in the scope of protection of the present invention.

Claims (10)

1. A compound represented by any of the following chemical formulas or a pharmaceutically acceptable salt thereof, wherein n independently represents an integer greater than or equal to 1. 2. A method for preparing thiolated hyaluronic acid, the method comprising: The hyaluronic acid is dissolved in water, and a solution containing a side chain modification group is added dropwise to react, and then added dropwise to a DTT reducing agent solution with stirring. The reaction solution is transferred into a dialysis bag for deoxygenation and dialysis, and the obtained purified solution is freeze-dried to obtain a thiol-modified hyaluronic acid derivative; Wherein, the side chain modification groups include BDDE and/or DSDE. 3. The preparation method according to claim 2, wherein the solution containing the side chain modifying group is added dropwise and then dialyzed to remove small molecule impurities; Preferably, the solution containing the side chain modifying group is added dropwise for reaction and then dialyzed, and then freeze-dried, and the freeze-dried product is redissolved in water; Preferably, the reaction temperature of the dropwise addition of the solution containing the side chain modifying group is 25°C; Preferably, the reaction time of dropwise adding the solution containing the side chain modifying group is 2 hours; Preferably, the reaction temperature of the dropwise addition into the DTT reducing agent solution and stirring is 25°C; Preferably, the stirring time of adding dropwise into the DTT reducing agent solution is 12-24 hours; Preferably, the deoxygenation dialysis time is 3 days; Preferably, the temperature of the deoxygenation dialysis is 25°C. 4. The preparation method according to claim 2, wherein the water is ultrapure water; Preferably, the final concentration of the hyaluronic acid dissolved in water is 1% w/v-2% w/v. 5. The preparation method according to claim 2, wherein the pH value of the solution is adjusted after the hyaluronic acid is dissolved in water; Preferably, the pH value of the adjusted solution is 8.0-9.0; Preferably, the pH value of the adjusted solution is 8.5; Preferably, the reagent for adjusting the pH value of the solution is NaOH solution; Preferably, the concentration of the NaOH solution is 5M; Preferably, the molar ratio of hyaluronic acid to side chain modification group is 1:(0.5-10); Preferably, 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, or 1:10. 6. The preparation method according to claim 2, wherein the deoxygenation dialysis comprises the following steps: introducing nitrogen into the pure water for dialysis for 1 hour to remove dissolved oxygen, and then placing the dialysis bag containing the sample therein for dialysis for 3 days. 7. A product, wherein the product is prepared by the preparation method according to any one of claims 2 to 6 or comprises a derivative of the product prepared by the preparation method according to any one of claims 2 to 6, wherein the derivative comprises a hydrogel, a membrane, or a sponge. 8. The product according to claim 7, wherein the grafting rate of the thiolated hyaluronic acid obtained from HA and DSDE as raw materials is 5%-80%; Preferably, the grafting rate of the thiolated hyaluronic acid obtained from HA and BDDE as raw materials in the product is 5%-60%; Preferably, the product is in the form of a gel; Preferably, the product is in solid or semi-solid form. 9. Use of the compound of claim 1, the preparation method of any one of claims 2 to 6, or the product of claim 7 or 8 in the preparation of synovial fluid, intraocular vitreous substitutes, drug controlled release matrices, healing agents, anti-adhesion agents, vascular repair materials, hybrid physiological organs, healing devices, ophthalmic and otological compositions, prostheses, in vivo implants and medical devices. 10. Use of the compound according to claim 1 or the preparation method according to any one of claims 2 to 6 in a product for reducing residual organic solvents, reducing residual toxic small molecules, improving the biocompatibility of thiolated hyaluronic acid and its derivatives, reducing human body's rejection reaction to thiolated hyaluronic acid and its derivatives, and/or reducing human body's inflammatory reaction to thiolated hyaluronic acid and its derivatives during the preparation process of thiolated hyaluronic acid.