CN113929792A - Aldehyde modified hyaluronic acid (sodium) and synthesis method and application thereof - Google Patents
Aldehyde modified hyaluronic acid (sodium) and synthesis method and application thereof Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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- A61L26/008—Hydrogels or hydrocolloids
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- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/91—Injection
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
Abstract
The invention relates to novel aldehyde modified hyaluronic acid (sodium) and a preparation method and application thereof. The hyaluronic acid (sodium) modified by aldehyde group is prepared by performing aldehyde group functionalization on carboxyl of hyaluronic acid (sodium).
Description
Technical Field
The invention belongs to the fields of biomedicine and medical cosmetology, and particularly relates to novel aldehyde modified hyaluronic acid (sodium) and a preparation method and application thereof.
Background
Hyaluronic acid, hydraronic acid, abbreviated as HA, is a high molecular weight biopolysaccharide that was first isolated in 1934 by professor kari meier and his assistant john paler in the vitreous of bovine eyes. Hyaluronic acid is a naturally occurring biopolymer with important biological functions in bacteria and higher animals including humans. HA is in most cases present in connective tissue, and is present in high amounts in particular in synovial fluid, vitreous humor of the eyeball, umbilical cord and cockscomb. HA is synthesized by a class of intact membrane proteins called hyaluronan synthase and can be degraded by a range of hyaluronidases. Hyaluronic acid is available in two ways: one approach is via animal tissue extraction, such as: hyaluronic acid extracted from animal tissues of bulls-eyes, cockscombs and the like often contains exogenous proteins, so that the purity of the hyaluronic acid cannot meet the application in the biomedical field. Therefore, another method for preparing hyaluronic acid using microbial fermentation was developed to solve the problem of purity.
HA HAs many applications in clinical, tissue engineering repair, and gene and drug delivery. Hyaluronic acid HAs good biocompatibility, so that the hyaluronic acid plays an important role in biological development and body injury repair processes, but HA HAs the defects of excessively strong water solubility, short metabolic cycle and the like in a human body, and can be crosslinked to overcome the defects. Crosslinking HA can greatly prolong the in vivo maintenance time of HA and prolong the action effect. Commercial cross-linked HA products are mainly prepared by reacting two small-molecule cross-linking agents, namely butanediol diglycidyl ether and divinyl sulfone, with hydroxyl groups in HA. Both crosslinking agents can cause denaturation of proteins in the human body, thereby causing allergic reactions. Therefore, the development of a new hyaluronic acid crosslinking technology and the development of a new hyaluronic acid derivative and a crosslinking product thereof have wide application prospects and commercial values.
In the prior art, the hydrogel is generally prepared by crosslinking hyaluronic acid with a small-molecule crosslinking agent, and after the crosslinking reaction is completed in vitro and the small-molecule crosslinking agent is purified and removed, the hydrogel can be applied to organisms, so that the hydrogel has obvious disadvantages, including: 1. hydrogels are too viscous to or difficult to inject through standard bore injection needles, and replacing large bore injection needles can increase patient pain and increase the risk of inflammation and trauma. 2. The physical and chemical properties of the hydrogel are relatively fixed, and the specific requirements in the fields of biological medicine and medical cosmetology cannot be well matched. 3. The small-molecule cross-linking agent often remains in the hydrogel, because part of the small-molecule cross-linking agent is locked in the three-dimensional space structure of the hydrogel in the preparation process and cannot be completely removed in the purification process of the hydrogel, and the residues of the small-molecule cross-linking agent such as butanediol diglycidyl ether and divinyl sulfone bring safety risks and adverse reaction events, thereby restricting the application of the hydrogel in the fields of biomedicine and medical cosmetology.
Researchers in the related art have made many efforts to develop in situ cross-linked hydrogel compositions suitable for various application requirements, which are injected into organisms in a liquid form rather than a hydrogel form, and the cross-linking reaction thereof to form the hydrogel is completed in the organisms. Because it is in a flowable liquid form prior to injection, when injected into a target site in a living being, a hydrogel is formed that conforms exactly to the shape of the target site. The injectable property not only makes the operation process simple and convenient, but also can avoid the pain of the patient caused by the implantation operation, and greatly reduces the traumatic property of the operation. For example, WO 95/15168 reports the synthesis of HA hydrazide derivatives by chemical coupling using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, EDC, hydrazide-HA can be crosslinked with homo-or hetero-bifunctional or Traut's crosslinkers to form hydrogels for drug delivery. WO 00/016818 discloses in situ formation of hydrogels by crosslinking aldehyde or amine functionalized derivatives of HA (e.g., adipic dihydrazide-HA) with homo-or hetero-bifunctional crosslinkers (e.g., bifunctional N-hydroxysuccinimide ester crosslinkers such as (SPA) 1-PEG). Dahlmann et al (Biomaterials,2013,34: 940-.
The aldehyde group functionalized hyaluronic acid is a hyaluronic acid derivative which is easy to crosslink, good in biocompatibility, good in stability and easy to store. In recent years, various documents report that the hydrogel formed by the hyaluronic acid derivative shows good application value in the fields of tissue engineering and regenerative medicine. However, the synthesis method of aldehyde modified hyaluronic acid reported at present is mainly obtained by oxidizing vicinal diol sites in HA disaccharide unit. The method inevitably hydrolyzes the glycosidic bond of the hyaluronic acid, thereby causing the molecular weight of the hyaluronic acid to be reduced, and further causing the mechanical property of the end product to be reduced. This problem greatly hinders the progress of the aldehyde-based hyaluronic acid derivatives in industrial applications.
Disclosure of Invention
The invention provides a method for preparing aldehyde modified hyaluronic acid (sodium) by aldehyde functionalization of carboxyl of hyaluronic acid (sodium), the aldehyde modified hyaluronic acid (sodium) method for carboxyl of hyaluronic acid (sodium) can not cause hydrolysis of glycosidic bond of hyaluronic acid (sodium), the reaction is mild and controllable, high temperature is not needed, and byproducts are easy to remove.
The aldehyde modified hyaluronic acid (sodium) can form hydrogel after crosslinking, and particularly, the hydrogel forming mode can be realized in a self-crosslinking mode without using a small-molecule crosslinking agent, so that in-situ crosslinking in a living body can be realized, various defects of the hydrogel using the small-molecule crosslinking agent (butanediol diglycidyl ether, divinyl sulfone and the like) in the prior art are avoided, and a better choice is provided for the application of the hydrogel in the fields of biomedicine and medical cosmetology.
In addition, the aldehyde hyaluronic acid (sodium) can also be subjected to Schiff base condensation reaction with a micromolecule cross-linking agent containing amino or hydrazide groups to form hydrogel, the cross-linking reaction is dynamic and reversible, and the obtained hydrogel has the characteristics of self-healing and shear thinning: the self-healing has important significance in solving the damage repair problem of the flexible biological material and realizing the intelligent and efficient development of the biological material; the shear thinning properties give the hydrogel good injectability. During the colloid injection process, the tangential shearing force can change the gel into a solution form, and when the colloid passes through the needle tube, the external shearing force disappears, and the solution form can be restored to the gel form. The performance can well prevent cells in the colloid from being damaged by high shearing force. The self-healing and shear thinning make the hydrogel of the invention have attractive application prospect in the field of injection filling molding cosmetic products.
According to one aspect of the present invention, the present invention relates to an aldehyde modified hyaluronic acid or sodium hyaluronate having the structure represented by the following formula:
it will be appreciated by those skilled in the art that hyaluronic acid or sodium hyaluronate of various molecular weights may be used in the present invention. In a preferred embodiment, the hyaluronic acid or sodium hyaluronate modified by hydroformylation has a molecular weight of 10-2200kDa, preferably 50-1000kDa, further preferably 90-110kDa, most preferably about 100 kDa.
In a preferred embodiment, the modification rate (or degree of substitution) of the aldehyde-modified hyaluronic acid or sodium hyaluronate is 10-90%, preferably 50-80%, most preferably 60-70%.
It will be appreciated by those skilled in the art that hyaluronic acid (sodium) from a variety of sources may be used in the present invention, either commercially available or self-made, as an extract, or prepared by biological fermentation or the like.
According to another aspect of the invention, the invention relates to a preparation method of the aldehyde modified hyaluronic acid or sodium hyaluronate, which comprises the following steps:
(1) dissolving hyaluronic acid (sodium) in water and adjusting the pH of the resulting solution to 4.5-5.0, preferably 4.7-4.8, adding EDCI (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and stirring to dissolve it, followed by adding the propylene glycol hydrazide derivative 2,3-dihydroxypropyl-3- ((3-hydrazino-3-oxopropyl) thio) -2-methylpropionate (2,3-dihydroxypropyl-3- ((3-hydrazino-3-oxopropyl) thio) -2-methylpropionate), reacting with stirring at 0 ℃ -90 ℃, preferably 20 ℃ -50 ℃, most preferably at room temperature for 0.5-12h, preferably 3-5h, most preferably about 4h, adjusting the pH of the reaction solution to 6.0-8.0, preferably 6.9-7.1 to terminate the reaction, dialyzing the reaction solution and freeze-drying to obtain propylene glycol modified hyaluronic acid (sodium);
preferably, the pH of the reaction solution is controlled to be 4.5-5.0, more preferably 4.7-4.8 during the reaction;
preferably, the molecular weight of the hyaluronic acid (sodium) is between 10 and 2200kDa, preferably between 50 and 1000kDa, further preferably between 90 and 110kDa, most preferably about 100 kDa;
preferably, the mass/volume concentration of the aqueous hyaluronic acid (sodium) solution is between 0.1% and 2.5%, more preferably between 0.5 and 1.5%, most preferably about 1.0%;
preferably, the mass ratio of EDCI to hyaluronic acid (sodium) is 1:2-4:1, more preferably 1:1-3:1, most preferably about 2: 1;
preferably, the EDCI is added and stirred to dissolve, followed by stirring for 2-60 minutes, more preferably 5-30 minutes, and most preferably about 15 minutes before the addition of the propylene glycol hydrazide derivative to activate the reactive sites to be modified;
preferably, the mass ratio of the propylene glycol hydrazide derivative to the hyaluronic acid (sodium) is 1:4 to 16:1, more preferably 1:1 to 8:1, most preferably about 4: 1;
one skilled in the art will appreciate that dialysis is performed to remove reaction residues and impurities. Preferably, a dialysis bag is used.
Preferably, lyophilization is carried out until the water content is below 10%.
(2) Dissolving the propylene glycol modified hyaluronic acid (sodium) obtained in the step (1) in water, and adding sodium periodate (NaIO)4) Sodium silicate (Na)2SiO3) And (3) stirring the aqueous solution for 2-72h, preferably 16-32h and most preferably about 24h at 0-90 ℃, preferably 20-50 ℃ and most preferably room temperature in the dark, adding ethylene glycol, stirring for 0.2-4 h, preferably 0.5-2h and most preferably about 1h to terminate the reaction, dialyzing the reaction solution and freeze-drying to obtain aldehyde modified hyaluronic acid (sodium).
Preferably, the mass/volume concentration of the aqueous solution of propylene glycol modified hyaluronic acid (sodium) is 0.1% to 2.5%, more preferably 0.5 to 1.5%, most preferably about 1.0%;
preferably, the mass ratio of sodium periodate to propylene glycol modified hyaluronic acid (sodium) is from 1:2 to 4:1, more preferably from 1:1 to 3:1, most preferably about 2: 1;
preferably, sodium periodate is sodium silicate (Na)2SiO3) A mass/volume concentration in the aqueous solution of 0.5% to 15%, more preferably 8% to 12%, most preferably about 10%;
preferably, sodium silicate (Na)2SiO3) The mass/volume concentration of sodium silicate in the aqueous solution is 0.2% to 5%, more preferably 0.5 to 1.5%, most preferably about 1.0%;
one skilled in the art will appreciate that dialysis is performed to remove reaction residues and impurities. Preferably, dialysis bags are used.
Preferably, lyophilization is carried out until the water content is below 10%.
It will be appreciated by those skilled in the art that various pH adjusting agents commonly used in the art may be used in the present invention. Preferably, the pH is adjusted using an aqueous hydrochloric acid solution and/or an aqueous sodium hydroxide solution, more preferably, the concentration of the aqueous hydrochloric acid solution is about 1M and the concentration of the aqueous sodium hydroxide solution is about 1M.
The specific reaction route of the above reaction is as follows:
the aldehyde modified hyaluronic acid or sodium hyaluronate can perform self-crosslinking reaction with a polymer containing amino or hydrazide groups without a small molecule crosslinking agent, so that in-situ crosslinking in a living body can be realized.
According to another aspect of the present invention, the present invention relates to a composition comprising the aldehydized modified hyaluronic acid or sodium hyaluronate of the present invention and an amino or hydrazide group-containing polymer. Preferably, the composition further comprises water. Preferably, the composition is free of cross-linking agents, including small molecule cross-linking agents. Preferably, the composition consists of the aldehydized modified hyaluronic acid or sodium hyaluronate of the present invention and a polymer containing an amino group or a hydrazide group. Preferably, the composition consists of the aldehydized modified hyaluronic acid or sodium hyaluronate of the present invention, an amino or hydrazide group-containing polymer and water.
According to another aspect of the present invention, the present invention relates to a kit comprising an aldehydized modified hyaluronic acid or sodium hyaluronate and an amino or hydrazide group-containing polymer, which are present independently of each other. Preferably, the kit further comprises water independently present. Preferably, the kit does not contain a crosslinking agent, including a small molecule crosslinking agent.
According to the present invention, in the above-mentioned composition and kit, an amino group-containing polymer such as chitosan, collagen, gelatin, etc. One skilled in the art can select the appropriate molecular weight of these amino group-containing polymers as desired. For example, the molecular weight is 10-2200kDa, preferably 50-1000kDa, more preferably 90-110kDa, and most preferably about 100 kDa.
According to the present invention, in the above-mentioned composition and kit, the hydrazide group-containing polymer such as hydrazide-modified biocompatible polymer compound includes, but is not limited to, hydrazide-modified hyaluronic acid or sodium hyaluronate, hydrazide-modified chondroitin sulfate, hydrazide-modified heparin, hydrazide-modified sodium alginate, hydrazide-modified cellulose, hydrazide-modified gelatin, hydrazide-modified polyethylene glycol, hydrazide-modified dendrimer polyamide, and the like. One skilled in the art can select the appropriate molecular weight of these hydrazide group-containing polymers as desired. For example, the molecular weight is 10-2200kDa, preferably 50-1000kDa, more preferably 90-110kDa, and most preferably about 100 kDa.
According to the invention, in the composition and the kit, the mass ratio of the aldehyde modified hyaluronic acid or sodium hyaluronate to the amino or hydrazide group-containing polymer is 1:10-10:1, preferably 1:5-5:1, and most preferably about 1: 1.
According to the present invention, the mass/volume concentration of the aldehyde-modified hyaluronic acid or sodium hyaluronate is 0.1-2.5%, more preferably 0.5-1.5%, most preferably about 1.0%, and the mass/volume concentration of the amino or hydrazide group-containing polymer is 0.1-2.5%, more preferably 0.5-1.5%, most preferably about 1.0%.
It will be understood by those skilled in the art that the above compositions may be formulated prior to use, or that the components of the above kits, which are separate from each other, may be mixed and formulated into a composition prior to use. In general, the crosslinking reaction occurs immediately after the composition is formulated and is completed in a time period of about 2 minutes to 2 hours. It will be appreciated by those skilled in the art that the above composition may be injected into an organism at any time after the formulation is complete until the crosslinking reaction is complete.
According to another aspect of the present invention, the present invention relates to a hydrogel obtained by crosslinking the aldehyde-modified hyaluronic acid or sodium hyaluronate with the amino or hydrazide group-containing polymer.
According to the invention, the mass ratio of the aldehyde modified hyaluronic acid or sodium hyaluronate to the amino or hydrazide group-containing polymer is 1:10 to 10:1, preferably 1:5 to 5:1, and most preferably about 1: 1.
According to another aspect of the present invention, the present invention relates to a method for preparing the above hydrogel, comprising the steps of: preparing an aqueous solution containing the aldehyde modified hyaluronic acid or sodium hyaluronate and the amino or hydrazide group-containing polymer, and reacting at 0-90 deg.C, preferably 20-50 deg.C, most preferably room temperature.
According to the present invention, the mass/volume concentration of the aldehyde modified hyaluronic acid or sodium hyaluronate in the solution is 0.1% to 2.5%, more preferably 0.5% to 1.5%, most preferably about 1.0%, and the mass/volume concentration of the amino or hydrazide group-containing polymer is 0.1% to 2.5%, more preferably 0.5% to 1.5%, most preferably about 1.0%.
The hyaluronic acid or sodium hyaluronate modified by aldehyde group can be crosslinked under the action of a micromolecule crosslinking agent, or crosslinked with biocompatible high molecular compounds modified by acrylic ester.
According to another aspect of the invention, the invention relates to a hydrogel which is obtained by crosslinking the aldehyde modified hyaluronic acid or sodium hyaluronate with a small molecule crosslinking agent containing amino or hydrazide groups.
According to another aspect of the invention, the invention relates to a hydrogel, which is obtained by crosslinking the aldehyde modified hyaluronic acid or sodium hyaluronate, the acrylate modified biocompatible macromolecular compound and the amino or hydrazide group-containing small molecule crosslinking agent.
According to the present invention, the amino group-containing small molecule crosslinking agent is, for example, a small molecule compound containing two or more primary amines or a physiologically acceptable salt thereof, a small molecule compound containing two or more primary amines such as cystine, lysine, ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexylenediamine, p-phenylenediamine, m-phenylenediamine, p-phenylenediamine, biphenyldiamine, etc., a salt such as hydrochloride, sulfate, methanesulfonate, citrate, etc.
According to the present invention, the small molecule crosslinking agent containing a hydrazide group is, for example, a small molecule compound containing two or more hydrazides, such as oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, or the like.
According to another aspect of the present invention, the present invention relates to a method for preparing the above hydrogel, comprising the steps of: preparing an aqueous solution containing the aldehyde modified hyaluronic acid or sodium hyaluronate and an aqueous solution containing the amino or hydrazide group-containing small molecule cross-linking agent, dripping the aqueous solution containing the amino or hydrazide group-containing small molecule cross-linking agent into the aqueous solution containing the aldehyde modified hyaluronic acid or sodium hyaluronate, and stirring at 0-90 ℃, preferably 20-50 ℃ and most preferably at room temperature until the cross-linking reaction is completed.
Preferably, the aqueous solution containing the aldehyde-modified hyaluronic acid or sodium hyaluronate further contains an acrylate-modified biocompatible polymer compound.
The hydrogel can be applied to the fields of biomedicine and medical cosmetology, for example, the hydrogel can be prepared into external wound dressings, nose and lip filling for medical cosmetology, skeleton materials for replacing hyaluronic acid injection in the existing medical cosmetology and moderate-to-deep venous ulcer wound dressings, and the like.
The invention has the beneficial effects that:
1. the aldehyde functional group is introduced into the carboxyl position of hyaluronic acid (sodium) creatively;
2. the irreversible degradation reaction caused by introducing aldehyde group into the adjacent diol site of hyaluronic acid (sodium) in the prior art is avoided;
3. the first step of modification reaction has mild conditions and high modification efficiency;
4. the degree of the second oxidation reaction step is quantitative and controllable, and toxic byproducts are not generated;
5. the use of organic solvent is avoided, the residue problem of the organic solvent in the product is fundamentally eliminated, the biocompatibility is better, and the occurrence probability of rejection reaction and inflammatory reaction of implanted organisms such as human bodies can be greatly reduced.
Drawings
FIG. 1 is an infrared spectrum of hydrazide-modified sodium hyaluronate prepared in example 1, 1717cm-1The peak is an aldehyde group.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.
Preparation example 1 preparation of sodium hyaluronate modified by hydrazoylation
Dissolving 1.0g of sodium hyaluronate with the molecular weight of 100kDa in 100mL of purified water to obtain 1.0% sodium hyaluronate aqueous solution, adding 18.3g of adipic dihydrazide into the sodium hyaluronate aqueous solution, and adjusting the pH of the sodium hyaluronate aqueous solution to 4.75 by using 1M HCl aqueous solution; adding EDCI 1.7g, stirring at room temperature for reaction for 2 hours, adjusting the pH of the reaction solution to 7.0 by using 1M NaOH aqueous solution to terminate the reaction, dialyzing and purifying the reaction solution, and freeze-drying to obtain the sodium hyaluronate modified by hydrazidation, wherein the hydrazidation modification rate is 78% by detection.
Example 1 two-step Synthesis of sodium hyaluronate modified by hydroformylation
(1) Modification reaction: adding 50.0g of sodium hyaluronate with the molecular weight of 100kDa into 5000mL of ultrapure water, magnetically stirring to dissolve the sodium hyaluronate to obtain 1% (w/v) of clear transparent solution, and adjusting the pH of the clear transparent solution to 4.75 by using a proper amount of 1M HCl aqueous solution; adding 100.0g of EDCI, magnetically stirring to dissolve the EDCI, and continuously stirring for 15 minutes; adding 200.0g of propylene glycol hydrazide derivative 2,3-dihydroxypropyl-3- ((3-hydrazino-3-oxopropyl) thio) -2-methylpropionate, reacting for 4 hours at room temperature under magnetic stirring, and controlling the pH of the reaction solution to be between 4.7 and 4.8 by using 1M aqueous HCl solution during the reaction; adjusting the pH of the reaction solution to 7.0 by using 1M NaOH aqueous solution to terminate the coupling reaction; transferring the reaction solution into a dialysis bag (Shibipure, 8kDa molecular weight cut-off) for dialysis for 3 days, and freeze-drying the purified solution obtained after dialysis to obtain propylene glycol modified sodium hyaluronate, wherein the water content is below 10 percent, and the hyaluronic acid is stored in a nitrogen-filled sealed and light-proof manner.
(2) And (3) oxidation reaction: adding 10.0g of propylene glycol modified sodium hyaluronate obtained in the step (1) into 1000mL of ultrapure water, and magnetically stirring to dissolve the sodium hyaluronate to obtain 1% (w/v) clear transparent solution; adding a solution containing 20.0g NaIO4200mL of 1% sodium silicate (Na)2SiO3) Reacting the aqueous solution for 24 hours under the condition of keeping out of the sun and magnetic stirring at room temperature, adding a proper amount of glycol and magnetically stirring for 1 hour to terminate the reaction; transferring the reaction solution into a dialysis bag (Shibipure, 8kDa molecular weight cut-off) for dialysis for 3 days, and freeze-drying the purified solution obtained after dialysis to obtain the aldehyde modified sodium hyaluronate, wherein the water content is below 10%, the aldehyde modification rate is 63% by measurement, and the hyaluronic acid is stored in a nitrogen-filled sealed and dark place. The calculated yield was 87.1%.
Example 2 preparation of hydrogel from sodium hyaluronate modified by hydroformylation with Small molecule Cross-linker
Dissolving 1.0g of the aldehyde modified sodium hyaluronate prepared in example 1 in 20mL of distilled water to obtain a solution with the concentration of 5.0%; dissolving 120mg of small molecular cross-linking agent cystine hydrochloride into 2mL of distilled water to obtain a cross-linking agent solution; dropwise adding a cross-linking agent solution into the sodium hyaluronate solution subjected to hydroformylation modification, and magnetically stirring at room temperature to react for 2 hours to obtain a gel substance; soaking in distilled water, and shaking for 24 hr to obtain hydrogel.
Example 3 preparation of hydrogel by self-crosslinking of sodium hyaluronate modified by hydroformylation
Dissolving 1.0g of hydrazide modified sodium hyaluronate prepared in preparation example 1 in 50mL of purified water to obtain a solution with the concentration of 2.0%; in addition, 1.0g of the sodium hyaluronate subjected to hydroformylation modification prepared in example 1 was dissolved in 50mL of purified water to obtain a solution with a concentration of 2.0%. Mixing the two solutions, stirring and reacting at room temperature for 1 minute to form a viscous colloidal substance, wherein the viscosity of the viscous colloidal substance continuously increases with the continuous crosslinking reaction, and does not increase after about 20 minutes to obtain hydrogel, wherein the viscosity of the hydrogel is measured to be 2.152 +/-0.007.
Example 4 physicochemical Properties of sodium hyaluronate modified by hydroformylation
1.0g of the aldehyde-modified sodium hyaluronate prepared in example 1 and 1.0g of the commercially available sodium hyaluronate having a molecular weight equivalent thereto were dissolved in 100mL of ultrapure water to obtain a solution having a concentration of 1% (w/v), and the dynamic viscosity of the obtained solution was measured at 25 ℃ with a rotational viscometer, and the results are shown in Table 1.
Meanwhile, 1.0g of the aldehyde-modified sodium hyaluronate prepared in example 1 and 1.0g of commercially available sodium hyaluronate having a molecular weight equivalent thereto were taken, and the stability including appearance, water content, viscosity and the like was examined at room temperature and under normal pressure, and the results are shown in table 1.
Table 1 viscosity and stability observations of sodium hyaluronate modified by hydroformylation
Example 5 physicochemical Properties of hydrogels made from sodium hyaluronate modified by hydroformylation
The aldehyde-modified sodium hyaluronate hydrogels prepared in examples 2 and 3 and commercially available sodium hyaluronate hydrogels were prepared into 20mL solutions with a concentration of 0.5% w/v, and the dynamic viscosity was measured at 25 ℃ using a rotational viscometer, and the results are shown in Table 2. Meanwhile, the stability including appearance, water content, viscosity, etc. was examined at room temperature and under normal pressure, and the results are shown in table 2.
Table 2 viscosity and stability investigation results of aldehyde modified sodium hyaluronate hydrogel
Example 6 MTT assay of aldehyde-modified sodium hyaluronate and hydrogel thereof
The aldehyde-modified sodium hyaluronate prepared in example 1 and the hydrogel prepared in examples 2 and 3 were prepared into a series of samples (0, 500, 1000, 2500mg/mL) with DMEM cell preparation solutions, respectively, and were subjected to filtration sterilization. The sample cytotoxicity test was performed on L929 fibroblasts using the MTT method. One group at a concentration of 0 was a blank control group, and the others were experimental groups. L929 cells grown in log phase were seeded into 96-well cell culture plates (cell concentration 8.0X 10)3One/well), after the cells adhere to the wall, adding the blank control group solution and the experimental group solution respectively, wherein each well is 100 mu L, and 3 multiple wells are arranged. After 24h of culture, MTTThe method measures the absorbance at 570nm and calculates the relative survival rate of the cells.
The calculation formula is as follows: cell viability (%). gtoreq (OD mean of experimental group/OD mean of blank control group) × 100%
The results show that the cell survival rate of each experimental group is over 95%. The results show that neither the aldehydized modified sodium hyaluronate prepared in example 1 nor the hydrogels prepared in examples 2 and 3 have significant toxicity.
In this patent, the concentration 1M of the aqueous HCl or NaOH solution is 1mol/L, as it is used throughout.
Claims (10)
1. An aldehyde modified hyaluronic acid or sodium hyaluronate having the structure shown by the following formula:
preferably, the hyaluronic acid or sodium hyaluronate modified by hydroformylation has a molecular weight of 10-2200kDa, more preferably 50-1000kDa, more preferably 90-110kDa, most preferably about 100 kDa.
Preferably, the modification rate of the aldehyde modified hyaluronic acid or sodium hyaluronate is 10-90%, more preferably 50-80%, and most preferably 60-70%.
2. The method for preparing hyaluronic acid or sodium hyaluronate according to claim 1, wherein the method comprises the following steps:
(1) dissolving hyaluronic acid or sodium hyaluronate in water, adjusting the pH of the obtained solution to 4.5-5.0, preferably 4.7-4.8, adding EDCI, stirring to dissolve, subsequently adding the propylene glycol hydrazide derivative 2,3-dihydroxypropyl-3- ((3-hydrazino-3-oxopropyl) thio) -2-methylpropionate, stirring to react at 0-90 ℃, preferably 20-50 ℃, most preferably room temperature for 0.5-12h, preferably 3-5h, most preferably about 4h, adjusting the pH of the reaction solution to 6.0-8.0, preferably 6.9-7.1 to terminate the reaction, dialyzing the reaction solution and freeze-drying to obtain propylene glycol modified hyaluronic acid or sodium hyaluronate;
preferably, the pH of the reaction solution is controlled to be 4.5-5.0, more preferably 4.7-4.8 during the reaction;
preferably, the molecular weight of the hyaluronic acid or sodium hyaluronate is between 10 and 2200kDa, further preferably between 50 and 1000kDa, more preferably between 90 and 110kDa, most preferably about 100 kDa;
preferably, the mass/volume concentration of the aqueous solution of hyaluronic acid or sodium hyaluronate is between 0.1% and 2.5%, more preferably between 0.5 and 1.5%, most preferably about 1.0%;
preferably, the mass ratio of EDCI to hyaluronic acid or sodium hyaluronate is from 1:2 to 4:1, more preferably from 1:1 to 3:1, most preferably about 2: 1;
preferably, the EDCI is added and stirred to dissolve, followed by stirring for 2 to 60 minutes, more preferably 5 to 30 minutes, and most preferably about 15 minutes before the addition of the propylene glycol hydrazide derivative;
preferably, the mass ratio of the propylene glycol hydrazide derivative to the hyaluronic acid or sodium hyaluronate is from 1:4 to 16:1, more preferably from 1:1 to 8:1, most preferably about 4: 1;
preferably, lyophilization is carried out until the water content is below 10%.
(2) Dissolving the propylene glycol modified hyaluronic acid or sodium hyaluronate obtained in the step (1) in water, adding sodium silicate aqueous solution containing sodium periodate, stirring and reacting for 2-72h, preferably 16-32h, most preferably about 24h at the temperature of 0-90 ℃ in the dark, preferably 20-50 ℃ and most preferably at room temperature, adding ethylene glycol, stirring for 0.2-4 h, preferably 0.5-2h, most preferably about 1h to terminate the reaction, dialyzing the reaction solution and freeze-drying to obtain aldehyde modified hyaluronic acid or sodium hyaluronate.
Preferably, the mass/volume concentration of the aqueous solution of propylene glycol modified hyaluronic acid or sodium hyaluronate is 0.1% to 2.5%, more preferably 0.5 to 1.5%, most preferably about 1.0%;
preferably, the mass ratio of sodium periodate to the propylene glycol modified hyaluronic acid or sodium hyaluronate is from 1:2 to 4:1, more preferably from 1:1 to 3:1, most preferably about 2: 1;
preferably, the mass/volume concentration of sodium periodate in the aqueous sodium silicate solution is between 0.5% and 15%, more preferably between 8% and 12%, most preferably about 10%;
preferably, the mass/volume concentration of sodium silicate in the aqueous sodium silicate solution is from 0.2% to 5%, more preferably from 0.5% to 1.5%, most preferably about 1.0%;
preferably, lyophilization is carried out until the water content is below 10%.
3. A composition comprising the aldehydized modified hyaluronic acid or sodium hyaluronate according to claim 1 and an amino or hydrazide group-containing polymer.
Preferably, the composition further comprises water.
Preferably, the composition is free of cross-linking agents, including small molecule cross-linking agents.
Preferably, the composition consists of the aldehydized modified hyaluronic acid or sodium hyaluronate according to claim 1 and a polymer containing amino or hydrazide groups.
Preferably, the composition consists of the aldehydized modified hyaluronic acid or sodium hyaluronate according to claim 1, an amino or hydrazide group-containing polymer and water.
Preferably, the mass/volume concentration of the aldehyde modified hyaluronic acid or sodium hyaluronate is 0.1% -2.5%, more preferably 0.5-1.5%, most preferably about 1.0%; the mass/volume concentration of the amino or hydrazide group-containing polymer is from 0.1% to 2.5%, more preferably from 0.5 to 1.5%, most preferably about 1.0%.
Preferably, the mass ratio of the aldehyde modified hyaluronic acid or sodium hyaluronate and the amino or hydrazide group-containing polymer is 1:10 to 10:1, preferably 1:5 to 5:1, and most preferably about 1: 1.
Preferably, the amino-containing polymer is selected from chitosan, collagen, gelatin; preferably, the molecular weight is between 10 and 2200kDa, more preferably between 50 and 1000kDa, more preferably between 90 and 110kDa, most preferably about 100 kDa.
The polymer containing hydrazide groups is selected from hydrazide modified hyaluronic acid or sodium hyaluronate, hydrazide modified chondroitin sulfate, hydrazide modified heparin, hydrazide modified sodium alginate, hydrazide modified cellulose, hydrazide modified gelatin, hydrazide modified polyethylene glycol and hydrazide modified dendritic polyamide; preferably, the molecular weight is between 10 and 2200kDa, more preferably between 50 and 1000kDa, more preferably between 90 and 110kDa, most preferably about 100 kDa.
4. A kit comprising the aldehydized modified hyaluronic acid or sodium hyaluronate according to claim 1 and an amino or hydrazide group-containing polymer independently present from each other.
Preferably, the kit further comprises water independently present.
Preferably, the kit does not contain a crosslinking agent, including a small molecule crosslinking agent.
Preferably, the mass ratio of the aldehyde modified hyaluronic acid or sodium hyaluronate and the amino or hydrazide group-containing polymer is 1:10 to 10:1, preferably 1:5 to 5:1, and most preferably about 1: 1.
Preferably, the amino-containing polymer is selected from chitosan, collagen, gelatin; preferably, the molecular weight is between 10 and 2200kDa, more preferably between 50 and 1000kDa, more preferably between 90 and 110kDa, most preferably about 100 kDa.
The polymer containing hydrazide groups is selected from hydrazide modified hyaluronic acid or sodium hyaluronate, hydrazide modified chondroitin sulfate, hydrazide modified heparin, hydrazide modified sodium alginate, hydrazide modified cellulose, hydrazide modified gelatin, hydrazide modified polyethylene glycol and hydrazide modified dendritic polyamide; preferably, the molecular weight is between 10 and 2200kDa, more preferably between 50 and 1000kDa, more preferably between 90 and 110kDa, most preferably about 100 kDa.
5. A hydrogel obtained by crosslinking the aldehyde-modified hyaluronic acid or sodium hyaluronate according to claim 1 with a polymer having an amino group or a hydrazide group.
Preferably, the mass ratio of the aldehyde modified hyaluronic acid or sodium hyaluronate and the amino or hydrazide group-containing polymer is 1:10 to 10:1, preferably 1:5 to 5:1, and most preferably about 1: 1.
Preferably, the amino-containing polymer is selected from chitosan, collagen, gelatin; preferably, the molecular weight is between 10 and 2200kDa, more preferably between 50 and 1000kDa, more preferably between 90 and 110kDa, most preferably about 100 kDa.
The polymer containing hydrazide groups is selected from hydrazide modified hyaluronic acid or sodium hyaluronate, hydrazide modified chondroitin sulfate, hydrazide modified heparin, hydrazide modified sodium alginate, hydrazide modified cellulose, hydrazide modified gelatin, hydrazide modified polyethylene glycol and hydrazide modified dendritic polyamide; preferably, the molecular weight is between 10 and 2200kDa, more preferably between 50 and 1000kDa, more preferably between 90 and 110kDa, most preferably about 100 kDa.
6. The method of making a hydrogel according to claim 5 comprising the steps of: preparing an aqueous solution containing the aldehydized modified hyaluronic acid or sodium hyaluronate according to claim 1 and the amino or hydrazide group-containing polymer, and reacting at 0 ℃ to 90 ℃, preferably 20 ℃ to 50 ℃, most preferably at room temperature.
Preferably, the mass/volume concentration of the aldehyde modified hyaluronic acid or sodium hyaluronate in the solution is 0.1% to 2.5%, more preferably 0.5% to 1.5%, most preferably about 1.0%, and the mass/volume concentration of the amino or hydrazide group-containing polymer is 0.1% to 2.5%, more preferably 0.5% to 1.5%, most preferably about 1.0%.
7. A hydrogel obtained by crosslinking the aldehyde-modified hyaluronic acid or sodium hyaluronate of claim 1 with a small molecule crosslinking agent containing an amino group or a hydrazide group.
Preferably, the amino-containing small molecule cross-linking agent is selected from small molecule compounds containing two or more primary amines or physiologically acceptable salts thereof; the small molecule compound containing two or more primary amines is selected from cystine, lysine, ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexylenediamine, p-phenylenediamine, m-phenylenediamine, p-phenylenediamine, and biphenyldiamine; the salt is preferably hydrochloride, sulfate, methanesulfonate, citrate.
Preferably, the hydrazide group-containing small molecule crosslinking agent is selected from small molecule compounds containing two or more hydrazides; preferred are oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide and terephthalic acid dihydrazide.
8. A hydrogel, which is obtained by crosslinking the aldehyde modified hyaluronic acid or sodium hyaluronate or acrylate modified biocompatible polymer compound of claim 1 with a small molecule crosslinking agent containing amino or hydrazide groups.
Preferably, the amino-containing small molecule cross-linking agent is selected from small molecule compounds containing two or more primary amines or physiologically acceptable salts thereof; the small molecule compound containing two or more primary amines is selected from cystine, lysine, ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexylenediamine, p-phenylenediamine, m-phenylenediamine, p-phenylenediamine, and biphenyldiamine; the salt is preferably hydrochloride, sulfate, methanesulfonate, citrate.
Preferably, the hydrazide group-containing small molecule crosslinking agent is selected from small molecule compounds containing two or more hydrazides; preferred are oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide and terephthalic acid dihydrazide.
9. A process for the preparation of a hydrogel according to claim 7 or 8, comprising the steps of: preparing an aqueous solution containing the aldehydized modified hyaluronic acid or sodium hyaluronate according to claim 1 and an aqueous solution containing a small molecule cross-linking agent containing an amino group or a hydrazide group, dropwise adding the aqueous solution containing the small molecule cross-linking agent containing an amino group or a hydrazide group to the aqueous solution containing the aldehydized modified hyaluronic acid or sodium hyaluronate, and stirring at 0 ℃ to 90 ℃, preferably 20 ℃ to 50 ℃, and most preferably at room temperature until the cross-linking reaction is completed.
Preferably, the aqueous solution containing the aldehyde-modified hyaluronic acid or sodium hyaluronate further contains an acrylate-modified biocompatible polymer compound.
10. Use of the aldehyde modified hyaluronic acid or sodium hyaluronate according to claim 1 or of the hydrogel according to claims 5, 7 or 8 in the biomedical and cosmetology field.
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