Sulfhydrylation modified natural polysaccharide derivative as well as application and preparation method thereof
By introducing thiol functional groups at the hydroxy end of the natural polysaccharide, disulfide bonds are formed to achieve in situ crosslinking, the problem of natural polysaccharide derivatives being easily degraded in the body is solved, the physiological activity of the carboxyl group is retained and the water solubility is improved, and the application of antioxidant and tissue engineering scaffolds is realized.
Patent Information
- Application Number
- CN202411757478.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-03
- Publication Date
- 2025-03-07
AI Technical Summary
The existing natural polysaccharides and their derivatives are prone to degradation in the body, have a short metabolic cycle, and their physiological activity and water solubility are limited after carboxy group modification.
By introducing thiol functional groups at the hydroxy end of the natural polysaccharide, disulfide bonds are formed to achieve in situ crosslinking, retaining electronegative groups such as carboxyl groups, reducing viscosity and improving water solubility.
The antioxidant effect of natural polysaccharide derivatives and the application of tissue engineering scaffolds are realized, while improving its application potential in the field of biomedicine.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the fields of natural polysaccharide modification and biomedicine technology, and in particular to a thiol-modified natural polysaccharide derivative and its application and preparation method. Background Art
[0002] Natural polysaccharides and their derivatives are naturally occurring biopolymers that are widely found in animals, plants, and bacteria. Moreover, most natural polysaccharide derivatives also play a relatively important biological function, making them widely used in the biomedical field. For example, hyaluronic acid (HA) is a high molecular weight natural glycosaminoglycan that is widely distributed in the extracellular matrix of animals and humans. It is an important component of the cell matrix and various tissues. It has the functions of regulating cell proliferation, migration and differentiation, lubricating joints and protecting cartilage, and promoting wound healing. Chondroitin sulfate (CS) is also an important glycosaminoglycan that exists in tissues such as cartilage in animals and humans. It can effectively relieve pain in patients with osteoarthritis, improve joint function, and promote cartilage repair. Gellan gum (GG) is obtained through bacterial fermentation. The main component is a linear polysaccharide composed of glucose, glucuronic acid, and rhamnose in a ratio of 2:1:1 as repeating structural units. It has good gelling ability, can simulate the natural extracellular matrix, and promote cell adhesion and tissue regeneration.
[0003] Natural polysaccharides and their derivatives are widely used, for example, in food, clinical, tissue engineering repair and drug delivery, and are considered safe by regulatory agencies. They have good biocompatibility and biodegradability, allowing for gradual absorption or metabolism in the body. In addition, natural polysaccharides and their derivatives generally have more active groups, and their hydrophilicity makes them suitable for applications such as hydrogels. However, natural polysaccharides and their derivatives generally have the problem of being easily degraded, resulting in a shorter metabolic cycle in the body. Therefore, it is usually necessary to functionalize the active groups on the polysaccharide structure to give it better rheological properties and resistance to enzymatic hydrolysis, thereby further effectively expanding its application in the biomedical field.
[0004] Common natural polysaccharides and their derivatives, such as hyaluronic acid, chondroitin sulfate, alginate, gellan gum, cellulose, etc., all have many active functional groups, such as hydroxyl, amino, carboxyl, etc., which makes these polysaccharides easy to be functionally modified. For example, thiol-modified natural polysaccharides do not require crosslinking agents, and can directly form disulfide bonds under the action of oxygen to obtain in situ crosslinked hydrogels, which not only have antioxidant effects, but also form a three-dimensional hydrogel structure that can be used as a tissue engineering scaffold. In addition, thiol-modified natural polysaccharides can also be quickly crosslinked in situ with double-bond modified biocompatible crosslinkers (such as polyethylene glycol methacrylate, etc.) or natural polymers (such as methacrylate-modified gelatin / hyaluronic acid, etc.) through a "thiol-ene" biological click reaction to form a three-dimensional hydrogel, which can be used for drug sustained release and in situ cell embedding, etc., and has important prospects in the fields of medical cosmetology, drug delivery, and tissue regeneration and repair.
[0005] In the prior art, thiol functional groups are generally introduced at the hydroxyl end or carboxyl end of natural polysaccharides. When thiol groups are introduced at the hydroxyl end, a large amount of auxiliary agents (such as tetrabutylammonium hydroxide, lutidine, acetone, dimethyl sulfoxide, etc.) are required; when thiol groups are introduced at the carboxyl end, an activator needs to be added before the thiol groups can be introduced through a series of reactions. However, carboxyl groups often play an important role in organisms, such as participating in the regulation of various cell activities and signal transduction. After modification, the carboxyl groups are reduced, and the role they play in the organism will also be weakened; in addition, the viscosity of natural polysaccharides is generally large, resulting in poor water solubility. All of these lead to certain restrictions on the application of carboxyl groups after thiol modification. Summary of the invention
[0006] The technical problem to be solved by the present invention is to provide a thiol-modified natural polysaccharide derivative and its application and preparation method, wherein the thiol-modified natural polysaccharide derivative is a natural polysaccharide derivative in which a modified group is introduced into the hydroxyl end, and electronegative groups such as carboxyl groups are retained, so that the physiological activity is retained, and the viscosity is also reduced, so that the water solubility is improved; the preparation method of the thiol-modified natural polysaccharide derivative is simple in process and suitable for industrial production.
[0007] In order to solve the above technical problems, the first aspect of the present invention is to provide a thiol-modified natural polysaccharide derivative having a structural unit of formula I:
[0008]
[0009] In formula I, L is selected from one of -COOH, -CH2OH or -CH3;
[0010] At least one of R1 and R2 is a thiol-containing functional group; preferably, R1 and R2 are independently selected from one of -OH, -NH-CH2-CH2-SH, -NH-NH2-CO-CH2-CH2-SH or -NH-NH2-CO-CH2-CH2-CH2-SH, and R1 and R2 are not -OH at the same time.
[0011] In the present invention, in the structural formula of formula I, "*" is other structural units in natural polysaccharides or their derivatives, wherein the structural units of formula I may also be included; R1 and R2 are adjacent hydroxyl groups on the six-membered ring of natural polysaccharides or their derivatives that are modified, and contain thiol groups and reduced hydroxyl groups; the thiol-modified natural polysaccharide derivatives having the structure of formula I can, for example, form in situ cross-linked hydrogels through the formation of disulfide bonds by thiol groups under the action of oxygen, which has antioxidant effects and can also be used as tissue engineering scaffolds; for example, they can also be formed through "thiol-ene" rapid in situ cross-linking with biocompatible unsaturated double-bond modified polymers or small molecule cross-linking agents to form hydrogels, which have controllable rapid response cross-linking gelation, and the concentration of free radicals generated during the reaction is low, which can minimize the chemical toxicity of the adverse damage to the reaction mixture; for example, they can also form multi-cross-linked network hydrogels through thiol-disulfide bond oxidation reaction cross-linking to enhance the anti-degradation ability. It should be noted that the thiol-modified natural polysaccharide of the present invention and the polymer material modified with unsaturated double bonds are rapidly cross-linked in situ through the "thiol-ene" click reaction. The reaction is fast and the conditions are mild, so rapid in situ gelation in vivo can be achieved, and various shortcomings of the prior art of first forming a gel and then injecting it into the organism can be avoided.
[0012] In the present invention, the six-membered ring is opened to reduce the viscosity of the natural polysaccharide, thereby increasing its solubility, which is beneficial to increasing the concentration of its solution. In addition, the present invention modifies the hydroxyl group of the natural polysaccharide or its derivative, retains its original electronegativity (such as retaining the carboxyl group, etc.), so that it maintains its original physiological activity, which is beneficial to cell regulation and signal transduction; the thiol-modified natural polysaccharide derivative of the present invention also has good biocompatibility.
[0013] In a specific embodiment, when both R1 and R2 are not -OH, R1 and R2 are the same.
[0014] In a specific embodiment, the thiol-modified natural polysaccharide derivatives include thiol-modified hyaluronic acid derivatives, thiol-modified chondroitin sulfate derivatives, thiol-modified alginic acid derivatives, thiol-modified gellan gum derivatives, and thiol-modified cellulose derivatives. The preferred thiol modification rate is 0.1%-10% or 4%-8% or 5%-7% or 6%-7%, among which 4%-8% or 5%-7% or 6%-7% are more preferred, 5%-7% or 6%-7% are further optimized, and 6%-7% is the most optimized.
[0015] In a specific embodiment, the thiol-modified natural polysaccharide derivative includes a compound with the following structure:
[0016]
[0017]
[0018]
[0019]
[0020] In formulae II to XIII, the value range of x is 1≤x≤400, the value range of y is 1≤y≤4500, and the value range of z is 1≤z≤400;
[0021] n in formula III, V, VII, IX, XI and XIII is 2 or 3.
[0022] In order to solve the above technical problems, the second aspect of the present invention is to provide an application of the aforementioned thiol-modified natural polysaccharide derivative in the preparation of biopharmaceutical products or medical cosmetic products. Biopharmaceutical products may be, for example, wound repair products or tissue repair products; medical cosmetic products may be, for example, facial masks or water-light products. Understandably, in the present invention, since the thiol-modified natural polysaccharide derivative has functional thiol groups and hydroxyl groups with good water solubility, it is more convenient for subsequent applications. For example, by forming disulfide bonds through thiol groups under the action of oxygen to form in-situ cross-linked hydrogels, it has an antioxidant effect and can also be used as a tissue engineering scaffold, suitable for preparing, for example, skin tissue repair products; for example, it can also be formed by rapid in-situ cross-linking with a biocompatible unsaturated double bond-modified polymer or a small molecule cross-linking agent through "thiol-ene" to form a hydrogel, suitable for preparing, for example, viscoelastic agents for ophthalmic intraocular lens implantation surgery; for example, it can also be cross-linked through "thiol-ene" and thiol-disulfide bond oxidation reactions to form a multi-cross-linked network hydrogel, enhancing the ability to resist degradation, suitable for preparing fillers for joint surgeries such as bone / cartilage repair.
[0023] In order to solve the above technical problems, the third aspect of the present invention is to provide a method for preparing the aforementioned thiol-modified natural polysaccharide derivative, comprising the following steps:
[0024] S1, dissolving a natural polysaccharide or a derivative thereof, adding an oxidant to react at room temperature in the dark, and then continuing to add an oxidant and a polyol compound to react after a period of reaction to obtain a product containing an aldehyde group;
[0025] S2, adjusting the pH of the product of step S1 to 3-8, and then reacting with a thiol-amino reagent to obtain a product containing an aldehyde side chain and a disulfide bond structure side chain;
[0026] S3, reacting the product of step S2 with a first reducing agent to reduce the imine bond in the form of a double bond to a carbon-nitrogen single bond;
[0027] S4, reacting the product of step S3 with a second reducing agent to reduce the disulfide bonds to thiol groups, and then post-treating to obtain a thiol-modified natural polysaccharide derivative.
[0028] In step S1 of the present invention, the main function of adding an oxidant is to oxidize and open part of the six-membered ring of the natural polysaccharide or its derivative, and the solubility increases after the ring opening, and an aldehyde group is formed at the ring opening position, and then a polyol compound is added to neutralize the excess oxidant to terminate the oxidation reaction; in step S2, the amino group of the thiol-amino reagent is added and the aldehyde group of the product of step S1 is reacted by Schiff base to generate a cystamine / cystamine salt or hydrazide-modified product, which has an imine bond in the form of an unstable double bond; in step S3, the purpose of the reaction of the product of step S2 with the first reducing agent is to reduce the unstable imine bond in the form of a double bond to a stable carbon-nitrogen single bond, thereby increasing its stability for subsequent reactions; in step S4, the product of step S3 continues to react with the second reducing agent to reduce the disulfide bond to a thiol group, and then the thiol-modified natural polysaccharide derivative is finally obtained through post-treatment, wherein the post-treatment is a purification treatment. The preparation method of the thiol-modified natural polysaccharide derivative of the present invention is simple, suitable for industrial production, and avoids the use of a large number of auxiliary agents.
[0029] In a specific scheme, in step S1, the mass / volume concentration of the natural polysaccharide or its derivative after dissolution is not more than 2% or between 0.5%-1% or 1%; the natural polysaccharide or its derivative is selected from one or more of hyaluronic acid, hyaluronate, modified derivatives of hyaluronate, chondroitin sulfate, chondroitin sulfate salt, modified derivatives of chondroitin sulfate salt, alginic acid, alginate, modified derivatives of alginate, gellan gum, modified derivatives of gellan gum, cellulose, and modified derivatives of cellulose with a molecular weight of 90-2200kDa or 100-200kDa or 150kDa-180kDa or 180kDa containing adjacent hydroxyl six-membered ring structure; the oxidant is sodium periodate; and the polyol compound is selected from one of ethylene glycol, propylene glycol or glycerol.
[0030] In a specific embodiment, the molar ratio of the total amount of the oxidant used to the natural polysaccharide or its derivative is 0.8-1.2:1 or 1:1, and the molar ratio of the polyol compound to the oxidant is 0.5-2:1 or 1:1. In the present invention, the degree of oxidation (i.e., the degree of six-membered ring opening) is directly related to the viscosity and mechanical properties of the final product (i.e., the thiol-modified natural polysaccharide derivative), so the amount of the oxidant and the oxidation time can be determined according to the requirements of the final product, wherein the purpose of adding the oxidant in step S1 is to open part of the six-membered rings in the natural polysaccharide or its derivative and form an aldehyde group at the opening position, and the polyol compound is added after the opening to neutralize the excess oxidant and terminate the oxidation reaction. The reaction time of adding the oxidant is 0.1-24h or 0.5-2h or 1-2h or 2h, among which 0.5-2h or 1-2h or 2h is a more preferred reaction time, 1-2h or 2h is a further preferred reaction time, and 2h is the best reaction time; the reaction time after continuing to add the polyol compound is 0.5-3h or 1-2h or 2h, among which 1-2h or 2h is a more preferred reaction time, and 2h is the best reaction time.
[0031] In a specific scheme, in step S2, the thiol-amino reagent is a diamine containing a disulfide bond or a dithiodihydrazide compound. Preferably, the diamine containing a disulfide bond is cystamine and / or cystamine salt; the dithiodihydrazide compound is 3,3'-dithiodipropionyl hydrazide or 4,4'-dithiodibutyryl hydrazide. Preferably, the molar ratio of the thiol-amino reagent to the aldehyde group is 0.5-2:1 or 0.5-1.2:1 or 1:1, wherein 0.5-1.2:1 or 1:1 is a more optimal molar ratio, and 1:1 is the best molar ratio.
[0032] In the present invention, the reaction between the thiol-amino reagent and the product of step S1 is a Schiff base reaction, which is a reversible reaction that depends on the pH of the solution. Under acidic pH conditions, the degree of substitution may increase when the Schiff base reaction is mainly a forward reaction, but the present invention finds that too low a pH will reduce the grafting degree of the thiol group, so the preferred pH value is 3-8 or 5-7 or 5-6, wherein 5-7 or 5-6 is a more preferred pH value, and 5-6 is the most preferred pH value, at which time the grafting degree of the thiol group is the maximum, and when the pH is 7-8, the grafting degree of the thiol group will be reduced. The reaction time of step S2 is 0.1-24h or 0.5-2h or 2h, wherein 0.5-2h or 2h is a preferred reaction time, and 2h is the optimal reaction time.
[0033] In a specific embodiment, in step S3, the first reducing agent is borohydride or cyanoborohydride. Preferably, the molar ratio of the first reducing agent to the natural polysaccharide or its derivative is 1-3:1 or 2:1, wherein 2:1 is a more preferred molar ratio.
[0034] In a specific scheme, in step S3, the pH of the reaction system is maintained at 5-7 or 6-7 or 6.5, wherein 6-7 or 6.5 are preferred pH values, and 6.5 is a more preferred pH value; the reaction time is 2-48h or 2-24 or 18-24h, wherein 2-24 or 18-24h are preferred reaction times, and 18-24h is a more preferred reaction time.
[0035] In a specific embodiment, in step S4, the second reducing agent is dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride. Preferably, the molar ratio of the second reducing agent to the aldehyde group is 1-1.5:1 or 1:1, wherein 1:1 is a more preferred molar ratio.
[0036] In a specific scheme, in step S4, the pH of the reaction system is maintained at 5-7 or 6-7 or 6.5, wherein 6-7 or 6.5 are preferred pH values and 6.5 is a more preferred pH value; the reaction time is 1-24h or 1-3 or 3h, wherein 1-3 or 3h are preferred reaction times and 3h is a more preferred reaction time. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0038] Figure 1a is the infrared spectrum of the sodium hyaluronate raw material in Example 1;
[0039] Figure 1b is the infrared spectrum of the product of step S1 in Example 1;
[0040] Figure 1c is the infrared spectrum of the final product in Example 1;
[0041] Figure 2 is the effect of oxidation time in step S1 on aldehyde grafting rate and viscosity;
[0042] Figure 3 It is the influence of the pH value of the reaction system in step S2 on the thiol grafting rate of the final product. DETAILED DESCRIPTION
[0043] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0044] In the present invention, a natural polysaccharide containing adjacent hydroxyl six-membered ring structural units or a derivative thereof is used as the reactant. When the thiol-forming agent is a diamine agent containing a disulfide bond, namely cystamine or cystamine salt, the reaction formula is as follows:
[0045]
[0046] Wherein, L is a residue such as -COOH, -CH2OH or -CH3; 400≥x≥1, 4500≥y≥1, 400≥z≥1.
[0047] Taking a natural polysaccharide or its derivative containing adjacent hydroxyl six-membered ring structural units as its reactant, when the thiol-reducing agent is a dithiodihydrazide compound, the reaction formula is as follows:
[0048]
[0049] Wherein, L is a residue such as -COOH, -CH2OH or -CH3; 400≥x≥1, 4500≥y≥1, 400≥z≥1.
[0050] Embodiment 1
[0051] S1. Dissolve 5 g (0.0125 mol) of sodium hyaluronate (molecular weight: 180 KDa) in purified water and stir until completely dissolved to form a 1 w / v% homogeneous transparent solution. Add 25 mL of 0.5 M sodium periodate solution dropwise at room temperature and in the dark. Stir and react for 5 min. Then add 5 mL of ethylene glycol and continue stirring and reacting for 2 h. Then, dialyze with a 12-14 kDa cutoff dialysis bag to remove sodium periodate and ethylene glycol to obtain the following product;
[0052]
[0053] Among them, 400≥x≥1, 4500≥y≥1, the aldehyde grafting rate is 12%, and the viscosity is 6000 Pa.s.
[0054] S2, the product of step S1 was transferred to a beaker, 2.82 cystamine dihydrochloride (0.0125 mol) was added, and the reaction was stirred for 30 min. During the reaction, the pH value of the reaction system was maintained in the range of 4-5 with glacial acetic acid / sodium hydroxide (5 M). After the reaction was completed, the excess cystamine and glacial acetic acid were removed by dialysis with a 12-14 kDa cut-off dialysis bag for 1 d (1 day) to obtain a product with the following structure:
[0055]
[0056] Among them, 400≥x≥1, 4500≥y≥1.
[0057] S3, the product of step S2 was transferred to a beaker, placed at room temperature, 1.57g (0.025mol) of sodium cyanoborohydride was added, and the reaction was stirred for 48h. During the reaction, the pH value of the reaction system was maintained in the range of 6-7 with glacial acetic acid / sodium hydroxide (5M). After the reaction was completed, the excess cystamine and glacial acetic acid were removed by dialysis with a 12-14kDa cut-off dialysis bag for 1d to obtain a product with the following structure:
[0058]
[0059] Among them, 400≥x≥1, 4500≥y≥1.
[0060] S4, the product of step S3 was transferred to a beaker, placed at room temperature, 3.58 (0.0125 mol) TCEP.HCL reducing agent was added, and the reaction was carried out for 3 hours. During the reaction, the pH value of the reaction system was maintained in the range of 6-7 with glacial acetic acid / sodium hydroxide (5M). After the reaction was completed, the excess TCEP.HCL was removed by dialyzing with a 12-14 kDa cut-off dialysis bag for 4 days, and the mixture was frozen overnight at -80°C and freeze-dried to obtain a product with the following structure (Formula II):
[0061]
[0062] Wherein, 400≥x≥1, 4500≥y≥1, 400≥z≥1, the thiol grafting rate is 0.81%, and the infrared spectra of the raw material hyaluronic acid, the product of step S1 and the final product are as follows: Figure 1a-1c shown.
[0063] Embodiment 2
[0064] S1. On the basis of step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 1 h, and the other conditions remained unchanged. The aldehyde grafting rate of the obtained product was 22% and the viscosity was 2000 Pa.s;
[0065] S2, based on step S2 of embodiment 1, this step does not perform interception processing;
[0066] S3, based on step S3 of embodiment 1, this step does not perform interception processing;
[0067] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 2.5%.
[0068] Embodiment 3
[0069] S1. On the basis of step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 1 h, and the other conditions remained unchanged. The aldehyde grafting rate of the obtained product was 22% and the viscosity was 2000 Pa.s;
[0070] S2. Based on step S2 of Example 1, the reaction time with cystamine dihydrochloride was extended to 120 min, and during the reaction, the pH of the reaction system was maintained in the range of 5-6, and the other conditions were the same as those of Example 1;
[0071] S3, perform the same operation as step S3 in embodiment 1;
[0072] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 3.3%.
[0073] Embodiment 4
[0074] S1. On the basis of step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 1 h, and the other conditions remained unchanged. The aldehyde grafting rate of the obtained product was 22% and the viscosity was 2000 Pa.s;
[0075] S2. Based on step S2 of Example 1, the reaction time with cystamine dihydrochloride was extended to 120 min, and during the reaction, the pH of the reaction system was maintained in the range of 5-6, and the other conditions were the same as those of Example 1;
[0076] S3, based on step S3 of embodiment 1, this step does not perform interception processing;
[0077] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 5.6%.
[0078] Embodiment 5
[0079] S1. On the basis of step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 1 h, and the other conditions remained unchanged. The aldehyde grafting rate of the obtained product was 22% and the viscosity was 2000 Pa.s;
[0080] S2, based on step S2 of Example 1, the reaction time with cystamine dihydrochloride was extended to 120 min, and during the reaction, the pH of the reaction system was maintained in the range of 3-4, and no interception treatment was performed, and the other conditions were the same as in Example 1;
[0081] S3, based on step S3 of embodiment 1, this step does not perform interception processing;
[0082] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 1.7%.
[0083] Embodiment 6
[0084] S1. On the basis of step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 1 h, and the other conditions remained unchanged. The aldehyde grafting rate of the obtained product was 22% and the viscosity was 2000 Pa.s;
[0085] S2, based on step S2 of Example 1, the reaction time with cystamine dihydrochloride was extended to 120 min, and during the reaction, the pH of the reaction system was maintained in the range of 6-7, and no interception treatment was performed, and the other conditions were the same as in Example 1;
[0086] S3, based on step S3 of embodiment 1, this step does not perform interception processing;
[0087] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 6.2%.
[0088] Embodiment 7
[0089] S1. On the basis of step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 1 h, and the other conditions remained unchanged. The aldehyde grafting rate of the obtained product was 22% and the viscosity was 2000 Pa.s;
[0090] S2, based on step S2 of Example 1, the reaction time with cystamine dihydrochloride was extended to 120 min, and during the reaction, the pH of the reaction system was maintained in the range of 7-8, and no interception treatment was performed, and the other conditions were the same as in Example 1;
[0091] S3, based on step S3 of embodiment 1, this step does not perform interception processing;
[0092] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 4.96%.
[0093] Embodiment 8
[0094] S1. Based on step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 2 h, and the other conditions remained unchanged. The obtained product had an aldehyde grafting rate of 30% and a viscosity of 800 Pa.s;
[0095] S2, based on step S2 of Example 1, the reaction time with cystamine dihydrochloride was extended to 120 min, and during the reaction, the pH of the reaction system was maintained in the range of 6-7, and no interception treatment was performed, and the other conditions were the same as in Example 1;
[0096] S3, based on step S3 of embodiment 1, this step does not perform interception processing;
[0097] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 7.96%.
[0098] Embodiment 9
[0099] S1. On the basis of step S1 of Example 1, the reaction time of adding sodium periodate solution was extended to 24 h, and the other conditions remained unchanged. The aldehyde grafting rate of the obtained product was 70%, and the viscosity was less than 100 Pa.s;
[0100] S2, based on step S2 of Example 1, the reaction time with cystamine dihydrochloride was extended to 120 min, and during the reaction, the pH of the reaction system was maintained in the range of 6-7, and no interception treatment was performed, and the other conditions were the same as in Example 1;
[0101] S3, based on step S3 of embodiment 1, this step does not perform interception processing;
[0102] S4. Perform the same operation as step S4 in Example 1 to obtain a product with a thiol grafting rate of 3.56%.
[0103] In Examples 1 to 9, the effects of different oxidation times on the aldehyde grafting rate and viscosity of the product of step S1 are as follows: Figure 2 As shown, it means that with the extension of oxidation time, the grafting rate gradually increases, while the viscosity gradually decreases. The main reason is that prolonging the oxidation time is conducive to the opening of more six-membered rings containing adjacent hydroxyl groups, thereby producing more aldehyde groups and reducing the viscosity.
[0104] In Examples 1 to 9, the pH value of the reaction with the thiol-amino reagent in step 2 affects the thiol grafting rate of the final product as follows: Figure 3 As shown in the figure, it indicates that the increase of acidic internal pH value is beneficial to the improvement of thiol grafting rate. If the pH value continues to increase to 7.5 or above, the thiol grafting rate will decrease.
[0105] Embodiment 10
[0106] S1. Dissolve 4 g (0.0185 mol) of sodium alginate (molecular weight: 180 KDa) in purified water and stir until completely dissolved to form a 1 w / v% homogeneous transparent solution. Add 37 mL of 0.5 M sodium periodate solution dropwise at room temperature and in the dark. Stir and react for 1 h. Then add 5 mL of ethylene glycol and continue stirring and reacting for 2 h. Then, dialyze with a 12-14 kDa cutoff dialysis bag to remove sodium periodate and ethylene glycol to obtain the following product;
[0107]
[0108] Among them, 400≥x≥1, 4500≥y≥1, the aldehyde grafting rate is 35%, and the viscosity is 1000 Pa.s.
[0109] S2, the product of step S1 was transferred to a beaker, 4.17 g of cystamine dihydrochloride (0.0185 mol) was added, and the reaction was stirred for 120 min. During the reaction, the pH value of the reaction system was maintained in the range of 6-7 with glacial acetic acid / sodium hydroxide (5 M);
[0110] S3, after the reaction of step S2 is completed, 2.33 g (0.037 mol) of sodium cyanoborohydride is added to the solution, and the reaction is stirred for 24 hours. During the reaction, the pH value of the reaction system is maintained in the range of 6-7 using glacial acetic acid / sodium hydroxide (5 M);
[0111] S4, the product of step S3 was transferred to a beaker, placed at room temperature, 5.30 g (0.0185 mol) of TCEP.HCL reducing agent was added, and the reaction was carried out for 3 hours. During the reaction, the pH value of the reaction system was maintained in the range of 6-7 with glacial acetic acid / sodium hydroxide (5 M). After the reaction was completed, the excess TCEP.HCL was removed by dialysis with a 12-14 kDa cut-off dialysis bag for 4 days, and the mixture was frozen overnight at -80°C and freeze-dried to obtain a product with the following structure (Formula VI):
[0112]
[0113] Among them, 400≥x≥1, 4500≥y≥1, 400≥z≥1, and the thiol grafting rate is 8.2%.
[0114] Example 11 Biocompatibility Test
[0115] (1) Mouse fibroblasts (chondrocytes) were evenly distributed in a culture dish and cultured in a cell culture incubator at 37°C and 0.5% CO2. After the cells filled the culture dish, the cells were collected and prepared into a cell suspension with a concentration of 1×104 cells / mL.
[0116] (2) The intermediate of aldehyde-modified hyaluronic acid and the final product of thiol-modified hyaluronic acid in Examples 1-9 were respectively dissolved in 0.5 mM PBS and adjusted to pH 7.0-7.4 with 3 M NaOH solution. Then, an appropriate amount of culture medium was added and shaken in a shaker at 37° C. for 72 h. After adding the above cell suspension and culturing in a cell culture incubator at 37° C. and 0.5% CO2 for 24 h, the proportion of live cells detected by MTT method was higher than 70%, indicating that the intermediates and final products of Examples 1-9 have good biocompatibility.
[0117] Embodiment 12
[0118] The final product of Example 6 (thiol-modified sodium hyaluronate with a thiol grafting rate of 6.2%) was dissolved in purified water at 37° C. to prepare a 4% uniform solution. The pH was adjusted to 7.0-7.2 with hydrochloric acid / sodium hydroxide, and then 8% double-bond-modified gelatin (pH between 7.0-7.4) was added in a volume ratio of 1:1 / 2:1 or 4:1. After mixing evenly, it was placed at 37° C. and directly solidified to form a hydrogel after 24 hours.
[0119] 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
1. A thiol-modified natural polysaccharide derivative, characterized in that: Containing structural units of formula I: In formula I, L is selected from one of -COOH, -CH2OH or -CH3; At least one of R1 and R2 is a thiol-containing functional group.
2. The thiol-modified natural polysaccharide derivative according to claim 1, characterized in that: R1 and R2 are independently selected from one of -OH, -NH-CH2-CH2-SH, -NH-NH2-CO-CH2-CH2-SH or -NH-NH2-CO-CH2-CH2-CH2-SH, and R1 and R2 are not -OH at the same time.
3. The thiol-modified natural polysaccharide derivative according to claim 2, characterized in that: When both R1 and R2 are not -OH, R1 and R2 are the same.
4. The thiol-modified natural polysaccharide derivative according to claim 1, characterized in that: The thiolated natural polysaccharide derivatives include thiolated hyaluronic acid derivatives, thiolated chondroitin sulfate derivatives, thiolated alginic acid derivatives, thiolated gellan gum derivatives, and thiolated cellulose derivatives.
5. The thiol-modified natural polysaccharide derivative according to claim 1, characterized in that: The thiol modification rate of the thiol-modified natural polysaccharide derivative is 0.1%-10% or 4%-8% or 5%-7% or 6%-7%.
6. The thiol-modified natural polysaccharide derivative according to claim 1, characterized in that: Thiol-modified natural polysaccharide derivatives include compounds with the following structures: In formulae II to XIII, the value range of x is 1≤x≤400, the value range of y is 1≤y≤4500, and the value range of z is 1≤z≤400; n in formula III, V, VII, IX, XI and XIII is 2 or 3.
7. Use of the thiol-modified natural polysaccharide derivative according to any one of claims 1 to 6 in the preparation of biopharmaceutical products or medical cosmetic products.
8. The use according to claim 7, characterized in that Biopharmaceutical products include: hard-to-heal wound repair materials, tissue regeneration and repair fillers, and eye drops; Medical beauty products include: wound repair patches / masks, and hydrating water.
9. The method for preparing the thiol-modified natural polysaccharide derivative according to any one of claims 1 to 6, characterized in that: The steps include: S1, dissolving a natural polysaccharide or a derivative thereof, adding an oxidant to react at room temperature in the dark, and then adding a polyol compound to terminate the reaction after a period of reaction to obtain a product containing an aldehyde group; S2, adjusting the pH of the product of step S1 to 3-8, and then reacting with a thiol-amino reagent to obtain a product containing an aldehyde side chain and a disulfide bond structure side chain; S3, reacting the product of step S2 with a first reducing agent to reduce the imine bond in the form of a double bond to a carbon-nitrogen single bond; S4, reacting the product of step S3 with a second reducing agent to reduce the disulfide bonds to thiol groups, and then post-treating to obtain a thiol-modified natural polysaccharide derivative.
10. The preparation method according to claim 9, characterized in that: In step S1, the mass / volume concentration of the natural polysaccharide or its derivative after dissolution is no more than 2% or between 0.5% and 1% or 1%; The natural polysaccharide or its derivative is selected from one or more of hyaluronic acid, hyaluronate, hyaluronate derivatives, chondroitin sulfate, chondroitin sulfate salt, chondroitin sulfate salt derivatives, alginic acid, alginate, alginate derivatives, gellan gum, gellan gum derivatives, cellulose, cellulose derivatives, and 180kDa hyaluronic acid, hyaluronate, hyaluronate derivatives, chondroitin sulfate, chondroitin sulfate salt, chondroitin sulfate salt derivatives, alginic acid, alginate, alginate derivatives, gellan gum, gellan gum derivatives, cellulose, cellulose derivatives, and cellulose derivatives. The oxidizing agent is sodium periodate; The polyol compound is selected from one of ethylene glycol, propylene glycol or glycerol.
11. The preparation method according to claim 9, characterized in that: In step S1, the molar ratio of the total amount of the oxidant used to the natural polysaccharide or its derivative is 0.8-1.2:1 or 1:1; The molar ratio of the polyol compound to the oxidant is 0.5-2:1 or 1:
1.
12. The preparation method according to claim 9, characterized in that: In step S1, the reaction time of the oxidant is 5 min-24 h or 0.5-2 h or 1-2 h or 2 h; the reaction time after continuing to add the polyol compound is 0.5-3 h or 1-2 h or 2 h.
13. The preparation method according to claim 9, characterized in that: In step S2, the thiol-amino reagent is a diamine or dithiodihydrazide compound containing a disulfide bond.
14. The preparation method according to claim 13, characterized in that: The diamine containing a disulfide bond is cystamine and / or cystamine salt; the dithiodihydrazide compound is 3,3'-dithiodipropionohydrazide or 4,4'-dithiodibutyrohydrazide.
15. The preparation method according to claim 9, characterized in that: In step S2, the molar ratio of the thiol-amino reagent to the aldehyde group is 0.5-2:1 or 0.5-1.2:1 or 1:
1.
16. The preparation method according to claim 9, characterized in that: In step S2, the pH of the product of step S1 is adjusted to 3-8 or 5-7 or 5-6; The reaction time is 0.1-24h or 0.5-2h or 2h.
17. The preparation method according to claim 9, characterized in that: In step S3, the first reducing agent is borohydride or cyanoborocyanide.
18. The preparation method according to claim 9, characterized in that: In step S3, the molar ratio of the first reducing agent to the natural polysaccharide or its derivative is 1-3:1 or 2:
1.
19. The preparation method according to claim 9, characterized in that: In step S3, the pH of the reaction system is maintained at 5-7 or 6-7 or 6.5; The reaction time is 2-48h or 2-24 or 18-24h.
20. The preparation method according to claim 9, characterized in that: In step S4, the second reducing agent is dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride.
21. The preparation method according to claim 9, characterized in that: In step S4, the molar ratio of the second reducing agent to the aldehyde group is 1-1.5:1 or 1:
1.
22. The preparation method according to claim 9, characterized in that: In step S4, the pH of the reaction system is maintained at 5-7 or 6-7 or 6.5; The reaction time is 1-24h or 1-3 or 3h.