CN115572396B - Sodium hyaluronate gel capable of being degraded in gradient manner and preparation method thereof - Google Patents
Sodium hyaluronate gel capable of being degraded in gradient manner and preparation method thereof Download PDFInfo
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- 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
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
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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
- C08J3/00—Processes of treating or compounding macromolecular substances
- 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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- 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 the technical field of biomedical materials, and provides a sodium hyaluronate gel capable of being degraded in a gradient manner and a preparation method thereof; the preparation method comprises the following steps: s1, dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide, uniformly stirring, adding a cross-linking agent, and obtaining cross-linked gel after reaction; neutralizing the crosslinked gel to be neutral, fully swelling to a proper concentration, and homogenizing to obtain crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 1; s2, dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide, adding a cross-linking agent, uniformly stirring, adding the particles 1 prepared in the step S1, uniformly stirring, performing secondary cross-linking reaction, and obtaining cross-linked gel after the reaction is finished; neutralizing the crosslinked gel to be neutral, swelling to a proper concentration, and homogenizing to obtain crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 2; and S3, repeating the step S2 at least once to obtain the multiple cross-linked sodium hyaluronate gel which has excellent gradient degradation performance and mechanical property.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a sodium hyaluronate gel capable of being degraded in a gradient manner and a preparation method thereof.
Background
Sodium hyaluronate is an inherent component in human body, is a kind of glucuronic acid, has no species specificity, is widely present in tissues and organs such as placenta, amniotic fluid, crystalline lens, articular cartilage, skin dermis and the like, has lubricating and nourishing effects on cells and cell organs contained in the tissues and organs, and is usually present in a sodium salt form. The sodium hyaluronate has wide application and can be used as bone joint cavity lubricating fluid, artificial tears, skin fillers, medical lubricants and the like. In the plastic and beauty industry, after the cross-linked hyaluronic acid gel is injected and filled, the cross-linked hyaluronic acid gel is attacked by hyaluronidase molecules expressed by a human body and gradually degraded and collapsed. Therefore, in order to improve the overall gel retention time and the gel-forming effect, it is necessary to suppress the enzymatic hydrolysis efficiency of hyaluronidase in human body. At present, the mode of improving the chemical crosslinking degree by using a crosslinking agent is basically used for improving the maintaining time of the hyaluronic acid gel, but the subsequent adverse reaction can be caused by the improvement of the content of the chemical crosslinking agent.
Chinese patent publication No. CN109758607A, for example, provides a cross-linked hyaluronic acid gel resistant to hydrolysis by hyaluronidase: comprises crosslinked hyaluronic acid gel and natural flavin coenzyme and derivatives thereof, including riboflavin, isoalloxazine, 7-M-8-C, 7-B-8-M, 7-M-8-B, 7-C-8-E, and 7,8-D. The invention provides a group of methods for reducing the activity of hyaluronidase by using natural flavin coenzyme and derivatives thereof and effectively enhancing the maintaining time and plasticity effect of cross-linked hyaluronic acid gel, and the catalytic efficiency of the hyaluronidase is reduced by more than 50% by using the methods in an in vitro cross-linked hyaluronic acid enzymolysis experiment. However, although the enzymolysis resistance of the crosslinked sodium hyaluronate gel is greatly improved, the effectiveness of the crosslinked sodium hyaluronate gel can slowly disappear in 3 to 6 months during actual clinical use, and the effect can be maintained only by repeated injection; moreover, once the crosslinked sodium hyaluronate gel is degraded, the mechanical properties thereof are drastically reduced, and the filling effect cannot be maintained. This brings great inconvenience to the patient, and the popularization thereof is limited.
Disclosure of Invention
The first object of the present invention is to provide a method for preparing a sodium hyaluronate gel, which not only realizes gradient degradation of the sodium hyaluronate gel, but also maintains mechanical properties after initial injection.
The second purpose of the invention is to provide a sodium hyaluronate gel which has excellent gradient degradation performance and mechanical property.
The embodiment of the invention is realized by the following technical scheme:
a preparation method of sodium hyaluronate gel comprises the following steps:
1) Dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide to prepare a solution with the concentration of 5-30%, preferably 10-25%, and fully stirring for 10-40min until the solution is uniform.
2) Then adding a cross-linking agent 1, 4-butanediol diglycidyl ether for cross-linking reaction, wherein the content of the cross-linking agent is 0.1-10% (relative to the mass of the sodium hyaluronate), and preferably 0.5-5%. The reaction time is 18 to 36h;
3) After the reaction is finished, neutralizing the crosslinked gel to be neutral, and fully swelling the crosslinked gel until the content of the sodium hyaluronate is 3 mg/ml-50 mg/ml, preferably 5 mg/ml-30 mg/ml; homogenizing to obtain cross-linked sodium hyaluronate gel particles with proper particle size, and marking as particles 1;
4) Dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide, adding a cross-linking agent 1, 4-butanediol diglycidyl ether to prepare a solution with the concentration of 5-30%, preferably 10-25%, and the content of the cross-linking agent of 0.1-10% (relative to the mass of the sodium hyaluronate), preferably 0.5-5%, fully stirring for 10-40min, and adding the particles 1 after uniform stirring;
5) Then carrying out crosslinking reaction for 18 to 36h;
6) After the reaction is finished, neutralizing the crosslinked gel to be neutral, and fully swelling the crosslinked gel until the content of the sodium hyaluronate is 3 mg/ml-50 mg/ml, preferably 5 mg/ml-30 mg/ml. Homogenizing to obtain cross-linked sodium hyaluronate gel particles with proper particle size, and marking as particles 2;
7) Dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide, adding a cross-linking agent 1, 4-butanediol diglycidyl ether to prepare a solution with the concentration of 5-30%, preferably 10-25%, and the content of the cross-linking agent of 0.1-10% (relative to the mass of the sodium hyaluronate), preferably 0.5-5%, fully stirring for 10-40min, and adding the particles 2 after uniform stirring;
8) Then carrying out crosslinking reaction for 18 to 36h;
9) After the reaction is finished, neutralizing the crosslinked gel to be neutral, and fully swelling the crosslinked gel until the content of the sodium hyaluronate is 3 mg/ml-50 mg/ml, preferably 5 mg/ml-30 mg/ml. And then homogenized to form cross-linked sodium hyaluronate gel particles of appropriate size, labeled as particles 3.
The above steps are repeated until N times of crosslinked sodium hyaluronate gel is obtained, and the number of times of crosslinking reaction is preferably 3 to 10.
According to the invention, the cross-linking agent reacts with sodium hyaluronate and forms a cross-linking form of a layer-by-layer coating through multiple cross-linking, and in order to build independence and mechanical properties after each coating, the cross-linked gel is neutralized to be neutral and then swells during each cross-linking, so that the preparation time is shortened, the state after cross-linking is more independent and stable, a good attachment basis is provided for the next coating, the performance of the sodium hyaluronate gel in the inner layer is not influenced, and the cross-linking effect of the finished sodium hyaluronate gel is better.
The multi-crosslinking sodium hyaluronate gel provided by the invention is like wearing a plurality of layers of coats on the once crosslinked sodium hyaluronate gel, and the crosslinking degree of the multi-layer coats can be controlled and selected by controlling different crosslinking times and crosslinking amounts, so that the multi-layer crosslinked coats not only realize the gradient degradation of the sodium hyaluronate gel after application, but also do not influence the performance of the sodium hyaluronate gel in the inner layer after the sodium hyaluronate gel in the outermost layer is degraded, and can also maintain the mechanical property after initial injection, thereby greatly improving the satisfaction degree of patients.
The crosslinking agent is 1, 4-butanediol diglycidyl ether, and commercially available crosslinking agents such as divinyl sulfone (DVS) or 1,2,7, 8-diepoxyoctane, or a combination thereof may be used.
The invention also provides a cross-linked sodium hyaluronate gel prepared by the preparation method. The raw materials comprise: sodium hyaluronate, a crosslinking agent; wherein the sodium hyaluronate accounts for 90% or more of the total mass of the gel, the content of the cross-linking agent is 0.1-10% of the mass of the sodium hyaluronate, and the cross-linking reaction is carried out for 3 times or more.
The technical scheme of the embodiment of the invention at least has the following advantages and beneficial effects:
the invention provides a preparation method of multiple cross-linked sodium hyaluronate gel, which is characterized in that a cross-linking agent and sodium hyaluronate react and form a cross-linked form of a layer-by-layer coating through multiple cross-linking, so that gradient degradation of the sodium hyaluronate gel is realized, the performance of the sodium hyaluronate gel in the inner layer is not influenced after the sodium hyaluronate gel in the outermost layer is degraded, the mechanical property after initial injection can be maintained, and the satisfaction degree of a patient is greatly improved. In addition, the raw materials and the cross-linking agent used by the gel are safe and non-toxic, meet the requirements of environmental protection and sustainable development, and are more suitable for being applied to the field of biomedicine.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
A multiple cross-linked sodium hyaluronate gel is prepared by the following steps:
(1) Weighing 5g of sodium hyaluronate, dissolving the sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide to prepare a 15% solution, and fully stirring for 20min until the solution is uniform;
(2) Then adding a cross-linking agent 1, 4-butanediol diglycidyl ether for cross-linking reaction, wherein the content of the cross-linking agent is 1 percent (relative to the mass of the sodium hyaluronate), and reacting for 24 hours;
(3) After the reaction is finished, hydrochloric acid is added to neutralize the crosslinked gel to be neutral, the crosslinked gel is fully swelled to have the sodium hyaluronate content of 20mg/ml, and then the crosslinked gel is homogenized into crosslinked sodium hyaluronate gel particles with proper particle size, which are marked as particles 1.
(4) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether, wherein the content of the crosslinking agent is 2% (relative to the mass of the sodium hyaluronate), preparing a 15% solution, fully stirring for 20min until the solution is uniform, and adding the particles 1;
(5) Then carrying out crosslinking reaction for 24h;
(6) After the reaction is finished, adding hydrochloric acid to neutralize the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of sodium hyaluronate is 20mg/ml, and homogenizing the crosslinked gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the crosslinked gel particles are marked as particles 2;
(7) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the content of the crosslinking agent being 2% to prepare a 15% solution, fully stirring for 20min, and adding the particles 2 after the solution is uniform;
(8) Then carrying out crosslinking reaction for 24h;
(9) And after the reaction is finished, adding hydrochloric acid to neutralize the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of the sodium hyaluronate is 20mg/ml, and homogenizing the crosslinked gel to form crosslinked sodium hyaluronate gel particles with proper particle size, thereby obtaining the crosslinked sodium hyaluronate gel.
The in vitro enzymolysis time of the cross-linked sodium hyaluronate gel prepared by the embodiment is 8 hours; the initial form is stable, and the phenomenon of scattering or dilution does not occur in the enzymolysis process.
Example 2
A multiple cross-linked sodium hyaluronate gel is prepared by the following steps:
(1) Weighing 5g of sodium hyaluronate, dissolving in 10% sodium hydroxide-containing aqueous solution to prepare 15% solution, and preferably fully stirring for 20min until the solution is uniform;
(2) Then adding a cross-linking agent 1, 4-butanediol diglycidyl ether for cross-linking reaction, wherein the content of the cross-linking agent is 2 percent (relative to the mass of the sodium hyaluronate), and reacting for 24 hours;
(3) After the reaction is finished, adding hydrochloric acid to neutralize the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of the sodium hyaluronate is 20mg/ml, and homogenizing the crosslinked gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the crosslinked gel particles are marked as particles 1;
(4) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether, wherein the content of the crosslinking agent is 3%, and preparing a 15% solution; stirring for 20min, and adding the granules 1;
(5) Then carrying out crosslinking reaction for 24h;
(6) After the reaction is finished, adding hydrochloric acid to neutralize the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of sodium hyaluronate is 20mg/ml, and homogenizing the crosslinked gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the crosslinked gel particles are marked as particles 2;
(7) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 3% to prepare a 15% solution, fully stirring for 20min, and adding the particles 2 after the solution is uniform;
(8) Then carrying out crosslinking reaction for 24h;
(9) And after the reaction is finished, adding hydrochloric acid to neutralize the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of the sodium hyaluronate is 20mg/ml, and homogenizing the crosslinked gel to form crosslinked sodium hyaluronate gel particles with proper particle size, thereby obtaining the crosslinked sodium hyaluronate gel.
The in vitro enzymolysis time of the cross-linked sodium hyaluronate gel prepared by the embodiment is 13h; the initial form is stable, and the phenomenon of scattering or dilution does not occur in the enzymolysis process.
Example 3
A multiple cross-linked sodium hyaluronate gel is prepared by the following steps:
(1) Weighing 5g of sodium hyaluronate, dissolving in an aqueous solution containing 10% of sodium hydroxide to prepare a 15% solution, and fully stirring for 20min until the solution is uniform;
(2) Then adding a cross-linking agent 1, 4-butanediol diglycidyl ether for cross-linking reaction, wherein the content of the cross-linking agent is 3 percent (relative to the mass of the sodium hyaluronate), and reacting for 24 hours;
(3) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 1;
(4) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 1 after the solution is uniform;
(5) Then carrying out crosslinking reaction for 24h;
(6) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of sodium hyaluronate is 20mg/ml, and homogenizing the crosslinked gel particles to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the crosslinked gel particles are marked as particles 2;
(7) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 2 after the solution is uniform;
(8) Then carrying out crosslinking reaction for 24h;
(9) And after the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of the sodium hyaluronate is 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, thereby obtaining the crosslinked sodium hyaluronate gel.
The in vitro enzymolysis time of the cross-linked sodium hyaluronate gel prepared by the embodiment is 18h; the initial form is stable, and the phenomenon of scattering or dilution does not occur in the enzymolysis process.
Example 4
A multiple cross-linked sodium hyaluronate gel is prepared by the following steps:
(1) Weighing 5g of sodium hyaluronate, dissolving the sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide to prepare a 15% solution, and fully stirring for 20min until the solution is uniform;
(2) Then adding a cross-linking agent 1, 4-butanediol diglycidyl ether for cross-linking reaction, wherein the content of the cross-linking agent is 3 percent (relative to the mass of the sodium hyaluronate), and reacting for 24 hours;
(3) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 1;
(4) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 1 after the solution is uniform;
(5) Then carrying out crosslinking reaction for 24h;
(6) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 2;
(7) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the content of the crosslinking agent of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 2 after the solution is uniform;
(8) Then carrying out crosslinking reaction for 24h;
(9) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 3;
(10) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 3 after the solution is uniform;
(11) Then carrying out crosslinking reaction for 24h;
(12) And after the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of the sodium hyaluronate is 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, thereby obtaining the crosslinked sodium hyaluronate gel.
The in vitro enzymolysis time of the cross-linked sodium hyaluronate gel prepared by the embodiment is 26h; the initial form is stable, and the phenomenon of scattering or dilution does not occur in the enzymolysis process.
Example 5
A multiple cross-linked sodium hyaluronate gel is prepared by the following method:
(1) Weighing 5g of sodium hyaluronate, dissolving in an aqueous solution containing 10% of sodium hydroxide to prepare a 15% solution, and fully stirring for 20min until the solution is uniform;
(2) Then adding a cross-linking agent 1, 4-butanediol diglycidyl ether for cross-linking reaction, wherein the content of the cross-linking agent is 3 percent (relative to the mass of the sodium hyaluronate), and reacting for 24 hours;
(3) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 1;
(4) 1g of sodium hyaluronate is dissolved in an aqueous solution containing 10% of sodium hydroxide, and a crosslinking agent 1, 4-butanediol diglycidyl ether is added to prepare a 15% solution, wherein the crosslinking agent content is 5%. Stirring for 20min, and adding the granules 1;
(5) Then carrying out crosslinking reaction for 24h;
(6) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 2;
(7) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 2 after the solution is uniform;
(8) Then carrying out crosslinking reaction for 24h;
(9) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 3;
(10) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 3 after the solution is uniform;
(11) Then carrying out crosslinking reaction for 24h;
(12) After the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel to the content of sodium hyaluronate of 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, wherein the particles are marked as particles 4;
(13) Dissolving 1g of sodium hyaluronate in an aqueous solution containing 10% of sodium hydroxide, adding a crosslinking agent 1, 4-butanediol diglycidyl ether with the crosslinking agent content of 5% to prepare a 15% solution, fully stirring for 20min, and adding the particles 4 after the solution is uniform;
(14) Then carrying out crosslinking reaction for 24h;
(15) And after the reaction is finished, neutralizing the crosslinked gel to be neutral, fully swelling the crosslinked gel until the content of the sodium hyaluronate is 20mg/ml, and homogenizing the gel to form crosslinked sodium hyaluronate gel particles with proper particle size, thereby obtaining the crosslinked sodium hyaluronate gel.
The in vitro enzymolysis time of the cross-linked sodium hyaluronate gel prepared by the embodiment is 30 hours; the initial form is stable, and the phenomenon of scattering or dilution does not occur in the enzymolysis process.
Comparative example 1
(1) 5g of sodium hyaluronate are dissolved in an aqueous solution containing 10% sodium hydroxide to prepare a 15% solution, which is preferably stirred sufficiently for 20min to be uniform.
(2) Then adding a cross-linking agent 1, 4-butanediol diglycidyl ether for cross-linking reaction, wherein the content of the cross-linking agent is 1 percent (relative to the mass of the sodium hyaluronate), and reacting for 24 hours;
(3) After the reaction is finished, the cross-linked gel is neutralized to be neutral, fully swelled to reach the sodium hyaluronate content of 20mg/ml, and then homogenized to form the cross-linked sodium hyaluronate gel particles with proper particle size.
The in-vitro enzymolysis time of the sodium hyaluronate gel prepared by the comparative example is 3 hours; the initial form is stable, and the phenomenon of scattering or dilution does not occur in the enzymolysis process.
Comparative example 2
This comparative example differs from example 1 in that: the step (3) does not include neutralization operation, i.e., swelling is performed after the reaction is completed.
The in-vitro enzymolysis time of the sodium hyaluronate gel prepared by the comparative example is 3.5 hours; the initial form is unstable, and the phenomena of scattering and dilution appear in the enzymolysis process.
The properties of the sodium hyaluronate gel finally prepared in each embodiment show that the enzymolysis time is gradually prolonged along with the increase of the crosslinking times, and the sodium hyaluronate gel has excellent mechanical properties and can keep a good shape without scattering or dilution in the enzymolysis process. While comparative examples 1-2 all showed the problem of excessively fast enzymatic hydrolysis time, comparative example 2 showed the phenomenon that the morphology of the gel was not maintained during the enzymatic hydrolysis. As can be seen from examples 1 to 3, the more the crosslinking agent is added, the longer the enzymolysis time is, with the same number of crosslinks. As can be seen from examples 3 to 5, the enzymatic hydrolysis time increases with the increase in the number of crosslinking.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A preparation method of a sodium hyaluronate gel capable of being degraded in a gradient manner is characterized by comprising the following steps:
s1, dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide, uniformly stirring, adding a cross-linking agent for a cross-linking reaction, and obtaining a cross-linked gel after the reaction is finished; neutralizing the crosslinked gel to be neutral, fully swelling to a proper concentration, and homogenizing to obtain crosslinked sodium hyaluronate gel particles with a proper particle size;
s2, dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide, adding a cross-linking agent, uniformly stirring, adding the cross-linked sodium hyaluronate gel particles prepared in the previous step, uniformly stirring, carrying out secondary cross-linking reaction, and obtaining cross-linked gel after the reaction is finished; neutralizing the crosslinked gel to be neutral, swelling to a proper concentration, and homogenizing to obtain crosslinked sodium hyaluronate gel particles with proper particle size;
and S3, repeating the method for preparing the crosslinked sodium hyaluronate gel particles in the previous step at least once according to the step S2 to obtain the multiple crosslinked sodium hyaluronate gel.
2. The method for preparing a gradient-degradable sodium hyaluronate gel according to claim 1, wherein S3 comprises: dissolving sodium hyaluronate in an aqueous solution containing sodium hydroxide, adding a cross-linking agent, fully and uniformly stirring, adding the cross-linked sodium hyaluronate gel particles prepared in the step S2, uniformly stirring to perform secondary cross-linking reaction, neutralizing the cross-linked gel to be neutral after the reaction is finished, swelling to a proper concentration, and homogenizing to obtain the cross-linked sodium hyaluronate gel particles with proper particle size.
3. The method for preparing gradient degradable sodium hyaluronate gel according to claim 1 or 2, wherein sodium hyaluronate is dissolved in an aqueous solution containing sodium hydroxide to prepare a solution of 5% to 30% in each crosslinking reaction.
4. The preparation method of the gradient degradable sodium hyaluronate gel according to claim 1 or 2, wherein the addition amount of the cross-linking agent in each cross-linking reaction is 0.1-10% of the mass of sodium hyaluronate, and the reaction time of the cross-linking reaction is 18-36h.
5. The method for preparing gradient degradable sodium hyaluronate gel according to claim 1 or 2, wherein swelling is carried out until the content of sodium hyaluronate is 3 mg/mL-50 mg/mL in each crosslinking reaction.
6. The method for preparing a gradient-degradable sodium hyaluronate gel according to claim 1, wherein the mass ratio of the sodium hyaluronate in S2 to the sodium hyaluronate in S1 is 1 to 3-10.
7. The method of claim 6, wherein the amount of sodium hyaluronate in S3 is equal to the amount of sodium hyaluronate in S2.
8. The method for preparing a gradient-degradable sodium hyaluronate gel according to claim 1, wherein the crosslinking agent is one or more of 1, 4-butanediol diglycidyl ether, divinyl sulfone or 1,2,7, 8-diepoxyoctane.
9. A gradient-degradable sodium hyaluronate gel, characterized by being prepared by the preparation method of any one of claims 1 to 8.
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