CN110982126A - Composition containing hyaluronate and preparation method thereof - Google Patents
Composition containing hyaluronate and preparation method thereofDownload PDFInfo
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- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
- 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
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- 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
- 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
- C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
- C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
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Abstract
Description
Technical Field
The invention relates to the technical field of hyaluronic acid, in particular to a hyaluronic acid double-gel system and a preparation method and application thereof.
Background
Hyaluronic acid is a straight-chain macromolecular acidic mucopolysaccharide composed of D-glucuronic acid and N-acetylglucosamine disaccharide units, is called hyaluronic acid, is widely present in human bodies, animals and microorganisms, has no species specificity, is non-toxic and non-irritant, has good biocompatibility and has high safety. Various parts of human body, such as articular cartilage, crystalline lens, dermal layer of skin, etc., contain hyaluronic acid, wherein the hyaluronic acid contained in the skin accounts for more than half of the total hyaluronic acid in human body. As people age, the content of hyaluronic acid in human skin gradually decreases, and the decrease of hyaluronic acid causes the skin to gradually lose elasticity and become rough and dull. The physical properties of hyaluronic acid make it possible to retain water in excess of 500 times its weight, thus maintaining the bulk and elasticity of the skin, and since it is species-specific, the absence of endogenous hyaluronic acid can be supplemented by exogenous hyaluronic acid. At present, the application of the non-crosslinked hyaluronic acid in treating skin wrinkles, skin laxity, dryness and the like is very wide, for example, internationally
HMW、Cytocare502、Cytocare516、Cytocare532、
Products such as Hydro, NCTF 135HA and the like are applied to the neck, face, hands, chest and the like.
Disclosure of Invention
Due to the existence of hyaluronidase in the body, the non-crosslinked hyaluronic acid injected into the human body is easily decomposed by the hyaluronidase, and the retention time in the body is short.
The cross-linked hyaluronic acid is formed by processing hyaluronic acid through a cross-linking process to change a linear macromolecular hyaluronic acid structure into a net structure, due to the specificity of enzyme, the cross-linked hyaluronic acid has the advantages that the capability of resisting the decomposition of hyaluronidase is enhanced due to the structural change, the cross-linked hyaluronic acid can be injected into a human body to prolong the retention time in the body, in addition, the cross-linked hyaluronic acid has better viscoelasticity and stronger shaping capability, can achieve the aim of supplementing volume and contour plasticity, and has irreplaceable effects on improving the deep and large wrinkles such as nasolabial folds, face lifting, depression volume supplementing and nose and lower jaw contour improvement. However, the cross-linked hyaluronic acid gel has the problems of high viscosity, low fluidity, large pushing force and the like when being filled in a pre-filled syringe, and is easy to have skin hills, nodules and other symptoms when being injected to the thin parts of the skin, such as the neck and the superficial layer of the dermis of the skin.
Based on the disadvantages of the prior art of cross-linked hyaluronic acid and non-cross-linked hyaluronic acid, it is an object of the present application to provide a composition comprising a hyaluronic acid salt and a method for the preparation thereof.
In order to achieve the purpose, the following technical scheme is adopted in the application:
a composition containing hyaluronate comprises a dual gel system consisting of crosslinked hyaluronate gel and non-crosslinked hyaluronate gel, wherein the dual gel system has a pH value of 6.0-8.0, an average particle size of 100-300 μm, and a kinematic viscosity of 10-80mm2And/s, the extrusion force is 5-30N.
In a particular aspect, the two-gel system described herein has an average particle size of 150-280 μm.
In a particular aspect, the kinematic viscosity of the dual gel system described herein is from 30 to 60mm2/s。
In a particular aspect, the dual gel system described herein has a push force of 8 to 20N.
In a specific aspect, the mass ratio of the cross-linked hyaluronate gel to the non-cross-linked hyaluronate gel described herein is 1 (1-10).
In a specific aspect, the mass ratio of the cross-linked hyaluronate gel to the non-cross-linked hyaluronate gel described herein is 1 (2-5).
In a specific aspect, the composition further comprises any one or more of glycerol, potassium chloride, sodium chloride, phosphate, amino acid, sodium acetate, and vitamin.
The present application also provides a method for preparing a composition comprising a hyaluronic acid salt, the method comprising:
preparing a cross-linked hyaluronate gel using a first hyaluronic acid or hyaluronate;
preparing a non-crosslinked hyaluronate gel using a second hyaluronic acid or hyaluronate;
and mixing the cross-linked hyaluronate gel and the non-cross-linked hyaluronate gel, and performing high-temperature treatment to obtain a double-gel system.
In a particular aspect, the first hyaluronic acid or hyaluronate salt described herein has a molecular weight of between 100 and 250 kilodaltons, preferably between 120 and 180 kilodaltons.
In a particular aspect, the concentration of hyaluronate in the crosslinked hyaluronate gel described herein is between 0.5% and 2.5%, preferably between 0.8% and 2.0%.
In a particular aspect, the preparation of a non-crosslinked hyaluronate gel as described herein comprises: dissolving hyaluronic acid or hyaluronate in water, and filtering to obtain non-crosslinked hyaluronate gel.
In a particular aspect, the second hyaluronic acid or hyaluronate salt described herein is selected from at least one, preferably two or three, hyaluronic acid or hyaluronate salts having a molecular weight of from 5 kilodalton to 40 kilodalton, from 80 kilodalton to 150 kilodalton and from 180 kilodalton to 300 kilodalton.
In a particular aspect, the concentration of hyaluronate in the non-crosslinked hyaluronate gel described herein is between 0.5% and 3.0%, preferably between 1.0% and 2.0%.
In a specific aspect, the crosslinked hyaluronate gel and the non-crosslinked hyaluronate gel described herein are mixed in a mass ratio of 1 (1-10).
In a particular aspect, the cross-linking agent used to prepare the cross-linked hyaluronate gel described herein is an epoxy based cross-linking agent, preferably 1, 4-butanediol diglycidyl ether (BDDE).
In a specific aspect, the amount of the cross-linking agent is 2% to 12% by mass of the hyaluronic acid or hyaluronate salt.
In one particular aspect, the screens described herein are used with a mesh size of 120-200 mesh.
In a particular aspect, the high temperature treatment described herein is carried out at a temperature of 100 ℃ to 130 ℃ for a time of 8 to 60 minutes.
The present application also provides a composition comprising a hyaluronate salt, the composition comprising a dual gel system consisting of a cross-linked hyaluronate gel and a non-cross-linked hyaluronate gel, prepared using any of the preparation methods described above.
In a specific aspect, the pH of the dual gel system is 6.0-8.0, the average particle size is 100-300 μm, and the kinematic viscosity is 10-80mm2And/s, the extrusion force is 5-30N.
Specifically, the present application relates to the following aspects:
1. a composition containing hyaluronate is characterized by comprising a double-gel system consisting of crosslinked hyaluronate gel and non-crosslinked hyaluronate gel, wherein the pH value of the double-gel system is 6.0-8.0, the average particle size is 100-300 mu m, and the kinematic viscosity is 10-80mm2And/s, the extrusion force is 5-30N.
2. The composition according to item 1, wherein the average particle size of the two-gel system is 150-280 μm.
3. Composition according to item 1, characterized in that the kinematic viscosity of the double gel system is between 30 and 60mm2/s。
4. The composition according to item 1, wherein the dual gel system has a push force of 8 to 20N.
5. The composition according to item 1, wherein the mass ratio of the crosslinked hyaluronate gel to the non-crosslinked hyaluronate gel is 1 (1-10).
6. The composition according to item 1, wherein the mass ratio of the crosslinked hyaluronate gel to the non-crosslinked hyaluronate gel is 1 (2-5).
7. The composition according to claim 1, which further comprises one or more of glycerol, potassium chloride, sodium chloride, phosphate, amino acid, sodium acetate and vitamins.
8. A method for preparing a composition comprising a hyaluronic acid salt, comprising:
preparing a cross-linked hyaluronate gel using a first hyaluronic acid or hyaluronate;
preparing a non-crosslinked hyaluronate gel using a second hyaluronic acid or hyaluronate;
and mixing the cross-linked hyaluronate gel and the non-cross-linked hyaluronate gel, and performing high-temperature treatment to obtain a double-gel system.
9. The process according to claim 8, wherein the first hyaluronic acid or hyaluronate salt has a molecular weight of 100-250 kilodalton, preferably 120-180 kilodalton.
10. The method according to item 9, wherein the concentration of the hyaluronic acid salt in the crosslinked hyaluronic acid salt gel is 0.5% to 2.5%, preferably 0.8% to 2.0%.
11. The method of claim 8, wherein the preparing the non-crosslinked hyaluronate gel comprises: dissolving hyaluronic acid or hyaluronate in water, and filtering to obtain non-crosslinked hyaluronate gel.
12. The method according to claim 11, wherein the second hyaluronic acid or hyaluronate salt is at least one, preferably two or three, hyaluronic acid or hyaluronate salts having a molecular weight of 5-40 kilodalton, 80-150 kilodalton and 180-300 kilodalton.
13. The method according to item 12, wherein the concentration of the hyaluronic acid salt in the non-crosslinked hyaluronic acid salt gel is 0.5% to 3.0%, preferably 1.0% to 2.0%.
14. The method according to item 8, wherein the crosslinked hyaluronate gel and the non-crosslinked hyaluronate gel are mixed at a mass ratio of 1 (1-10).
15. The method according to item 8, wherein the crosslinking agent used for the preparation of the crosslinked hyaluronate gel is an epoxy compound-based crosslinking agent, preferably 1, 4-butanediol diglycidyl ether (BDDE).
16. The method according to claim 15, wherein the amount of the crosslinking agent is 2 to 12% by mass of the hyaluronic acid or the hyaluronic acid salt.
17. The production method according to item 8, wherein the sieve is used with a mesh size of 120-200 mesh.
18. The method according to item 8, wherein the high-temperature treatment is carried out at a temperature of 100 ℃ to 130 ℃ for 8 to 60 minutes.
19. A composition comprising a hyaluronic acid salt, comprising a dual gel system consisting of a cross-linked hyaluronic acid salt gel and a non-cross-linked hyaluronic acid salt gel, which is prepared by the preparation method according to any of the claims 8-18.
20. The composition comprising a hyaluronic acid salt according to item 19, wherein the pH of the dual gel system is 6.0-8.0, the average particle size is 100-300 μm, and the kinematic viscosity is 10-80mm2And/s, the extrusion force is 5-30N.
Effects of the invention
Compared with the traditional hyaluronic acid water-optical product, the composition containing the hyaluronate has the advantages of enhanced hyaluronidase resistance, longer in-vivo action time, low gel viscosity, high smoothness, enhanced fluidity, easier extrusion operation in clinical use and capability of reducing the occurrence of side reactions such as skin humps and red swelling.
Detailed Description
The present application will be described in detail below.
Compositions containing hyaluronate salts provided herein compriseA double-gel system consisting of cross-linked hyaluronate gel and non-cross-linked hyaluronate gel, wherein the pH value of the double-gel system is 6.0-8.0, the average particle size is 100-300 mu m, and the kinematic viscosity is 10-80mm2And/s, the extrusion force is 5-30N.
Wherein the average particle size of the double gel system is determined by: the particle size and particle size distribution of the sample was measured using a Winner2308A laser particle size distribution instrument and the average particle size of the sample was finally recorded.
The kinematic viscosity of the two-gel system was determined by: after diluting 4g of the sample with purified water by 4 times, the sample is stirred to mix it uniformly, and then the kinematic viscosity of the diluent is measured at a predetermined temperature by using a proper type of a Brookfield viscometer (for example,. phi.1.0 mm,. phi.1.5 mm, or. phi.2.0 mm).
The push force of the two-gel system was determined by: filling 2.5mL of sample subjected to high-temperature treatment into a 3.0mL pre-filled syringe, installing a matched push rod and a 30G injection needle, discharging a small amount of air at the front end of the syringe, then installing the syringe on a universal material testing machine (Jinan Zhengzhong WDW-01E type), setting testing parameters of the universal material testing machine, starting testing, and reading the maximum force, the minimum force and the average force of the full range.
Further, the average particle size of the double gel system is 150-280 μm. For example, it may be 160. mu.m, 170. mu.m, 180. mu.m, 190. mu.m, 200. mu.m, 210. mu.m, 220. mu.m, 230. mu.m, 240. mu.m, 250. mu.m, 260. mu.m, 270. mu.m. Further, the kinematic viscosity of the double-gel system is 30-60mm2And s. For example, it may be 30mm2/s、35mm2/s、40mm2/s、45mm2/s、50mm2/s、55mm2/s。
Further, the extrusion force of the double-gel system is 8-20N. For example, 8N, 9N, 10N, 11N, 12N, 13N, 14N, 15N, 16N, 17N, 18N, 19N, 20N may be used.
Specifically, the mass ratio of the cross-linked hyaluronate gel to the non-cross-linked hyaluronate gel is 1 (1-10). For example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 may be used.
In the present application, the crosslinked hyaluronate gel is a hydrogel having a network structure and a granular appearance, which is obtained by crosslinking hyaluronic acid or a hyaluronate salt with a crosslinking agent.
The non-crosslinked hyaluronate gel refers to a hydrogel obtained by dissolving hyaluronic acid or hyaluronate of one, two or three molecular weights in water.
Further, the mass ratio of the cross-linked hyaluronate gel to the non-cross-linked hyaluronate gel is 1 (2-5).
Specifically, the composition of the invention further comprises one or more of glycerol, potassium chloride, sodium chloride, phosphate, amino acid, sodium acetate and vitamin.
The present invention also provides a method for preparing a composition comprising a hyaluronic acid salt, the method comprising:
preparing a cross-linked hyaluronate gel using a first hyaluronic acid or hyaluronate;
preparing a non-crosslinked hyaluronate gel using a second hyaluronic acid or hyaluronate;
and mixing the cross-linked hyaluronate gel and the non-cross-linked hyaluronate gel, and performing high-temperature treatment to obtain a double-gel system.
Specifically, the preparation of the cross-linked hyaluronate gel comprises the following steps:
dissolving hyaluronic acid or hyaluronate in an alkaline solution containing a cross-linking agent, and reacting for 5-30h at 20-35 ℃;
adding water to swell the gel mass, dialyzing, removing unreacted cross-linking agent and base.
In particular, the first hyaluronic acid or hyaluronate salt has a molecular weight of 100-250 kilodaltons, e.g. may be 100-kilodaltons, 110-kilodaltons, 120-kilodaltons, 130-kilodaltons, 140-kilodaltons, 150-kilodaltons, 160-kilodaltons, 170-kilodaltons, 180-kilodaltons, 190-kilodaltons, 200-kilodaltons, 210-kilodaltons, 220-kilodaltons, 230-kilodaltons, 240-kilodaltons, 250-kilodaltons. Preferably from 120 kilodaltons to 180 kilodaltons.
Further, the concentration of the hyaluronic acid salt in the crosslinked hyaluronic acid salt gel is 0.5% to 2.5%, for example, may be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%. Preferably 0.8% to 2.0%.
Specifically, the crosslinking agent is an epoxy compound crosslinking agent, and preferably 1, 4-butanediol diglycidyl ether (BDDE).
Specifically, the dosage of the cross-linking agent is 2-12% of the mass of the hyaluronic acid or the hyaluronate.
Specifically, the alkali is any one of sodium hydroxide and sodium carbonate, and sodium hydroxide is preferred. The amount of base used is 1% to 5% by mass of the hyaluronic acid or hyaluronate, for example, 1%, 2%, 3%, 4%, 5%. Preferably 2% to 3%.
Specifically, the solution used for swelling the gel block by adding water may be either or both of a phosphate buffer solution and a dilute hydrochloric acid solution having a pH of 2 to 4.
Specifically, the crosslinked hyaluronate gel and the non-crosslinked hyaluronate gel are mixed in a mass ratio of 1 (1-10).
Specifically, the preparation of the non-crosslinked hyaluronate gel comprises the following steps: dissolving hyaluronic acid or hyaluronate in water, and filtering to obtain non-crosslinked hyaluronate gel.
In particular, the second hyaluronic acid or hyaluronate salt is selected from at least one, preferably two or three, hyaluronic acid or hyaluronate salts having a molecular weight of 5-40 kilodalton, 80-150 kilodalton and 180-300 kilodalton. From 5 kilodalton to 40 kilodalton, and may be, for example, 5 kilodalton, 10 kilodalton, 20 kilodalton, 30 kilodalton, 40 kilodalton. From 80 kilodaltons to 150 kilodaltons, for example, 80 kilodaltons, 90 kilodaltons, 100 kilodaltons, 110 kilodaltons, 120 kilodaltons, 130 kilodaltons, 140 kilodaltons, 150 kilodaltons may be present. 180 kilodaltons to 300 kilodaltons, for example, may be 180 kilodaltons, 190 kilodaltons, 200 kilodaltons, 210 kilodaltons, 220 kilodaltons, 230 kilodaltons, 240 kilodaltons, 250 kilodaltons, 260 kilodaltons, 270 kilodaltons, 280 kilodaltons, 290 kilodaltons, 300 kilodaltons.
Specifically, the concentration of the hyaluronic acid salt in the non-crosslinked hyaluronic acid salt gel is 0.5% to 3.0%, and for example, may be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%. Preferably 1.0% to 2.0%.
Specifically, the screen used for the screening has a size of 120-200 meshes, for example, 120 meshes, 130 meshes, 140 meshes, 150 meshes, 160 meshes, 170 meshes, 180 meshes, 190 meshes, 200 meshes.
Specifically, the high temperature treatment is performed at 100-130 deg.C, such as 100 deg.C, 110 deg.C, 121 deg.C, 130 deg.C, for 8-60min, such as 8min, 10min, 15min, 20min, 30min, 40min, 50min, 60 min.
Has the advantages that: compared with the traditional water-light product, the composition has the advantages that the capability of resisting the decomposition of hyaluronidase is enhanced, the acting time in vivo can be prolonged, the gel viscosity is low, the smoothness is high, the fluidity is enhanced, and the pushing operation is easier during clinical use; when the gel is used for cosmetic injection filling of a shallower part of the skin, compared with the traditional cross-linked sodium hyaluronate gel, the gel can reduce the occurrence of side reactions such as skin humps, red swelling and the like.
The following examples of the present invention are merely illustrative of specific embodiments for carrying out the present invention and are not to be construed as limiting the invention. Other changes, modifications, substitutions, combinations, and simplifications which may be made without departing from the spirit and principles of the invention are intended to be equivalents thereof and to fall within the scope of the invention.
Examples
The experimental methods used in the following examples are all conventional methods, unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Dissolving 20g of sodium hyaluronate with the molecular weight of 180 ten thousand daltons in 100mL of solution containing 0.4g of BDDE and 0.4g of sodium hydroxide, and reacting for 24 hours at 25 ℃; cutting the gel to about 1cm3Adding phosphate buffer solution (with concentration of 0.3mg/mL and pH value of 6.0-8.0) into the granules to swell the gel block, dialyzing, removing unreacted cross-linking agent and alkali, controlling the concentration of hyaluronic acid to be 1.5%, controlling the pH value of the gel to be 6.0-8.0, and sieving with a 120-mesh stainless steel sieve to obtain the cross-linked sodium hyaluronate gel.
Taking 1.0g of sodium hyaluronate with the molecular weight of 80 ten thousand daltons, adding the sodium hyaluronate into 100mL of 0.5% glycerol aqueous solution, stirring until the sodium hyaluronate is dissolved, and filtering through a 5-micron filter element to obtain non-crosslinked sodium hyaluronate gel with the pH of 6.0-8.0.
Mixing the sodium hyaluronate gel and the non-crosslinked sodium hyaluronate gel in a mass ratio of 1:10, uniformly mixing, and performing heat treatment at 100 ℃ for 60 minutes to obtain a hyaluronate double-gel system.
And testing the pH value, the average particle size, the kinematic viscosity, the extruding force and the enzymolysis resistance of the obtained double-gel system.
Wherein the average particle size of the double gel system is determined by: the particle size distribution of the sample was measured using a Winner2308A laser particle size analyzer and the average particle size of the sample was finally recorded.
Kinematic viscosity is determined by: taking 4g of sample, diluting the sample to 4 times with purified water, stirring the sample to be uniformly mixed, and then selecting a proper type of a plat's viscometer to test the kinematic viscosity of the diluent at a specified temperature.
The squeeze force was determined by: filling 2.5mL of the sample subjected to high-temperature treatment into a 3mL pre-filled and sealed injector, installing a matched push rod and an injection needle, discharging a small amount of air at the front end of the injector, then installing the injector on a universal material testing machine (Jinan Zhongzheng WDW-01E type), setting testing parameters of the universal material testing machine, starting testing, and reading the full-scale maximum force, the minimum force and the average force. The test conditions were as follows: the type of the needle head: 30G; and (3) testing temperature: room temperature; extrusion speed: the extrusion speed was set at 10 mm/min.
And (3) enzyme resistance test: the test principle is as follows: the hyaluronic acid is degraded by hyaluronic acid microbial enzyme to become hyaluronic acid disaccharide containing carbon-carbon double bonds, the hyaluronic acid disaccharide has stronger absorption at the ultraviolet absorption wavelength of 232nm, and the concentration of the hyaluronic acid disaccharide in the solution is in direct proportion to the absorbance at the 232nm, so the enzymolysis resistance of the hyaluronic acid can be evaluated by testing the absorbance at the 232nm wavelength.
The test method comprises the following steps: putting 2g of the hyaluronate double gel system into a 50mL volumetric flask, adding a proper amount of phosphate buffer (5mM), fixing the volume, shaking up, then adding 250 mu L of hyaluronic acid microbial enzyme with the enzyme activity of 8356IU/mL, and shaking up to be used as a sample; in addition, a blank control sample is prepared by the same method by replacing the hyaluronic acid microbial enzyme with inactivated hyaluronic acid microbial enzyme, wherein the enzyme activity of the hyaluronic acid microbial enzyme is 8356 IU/mL. And (3) placing the sample to be tested and the blank control sample in a water bath at 42 ℃ for enzymolysis reaction, sampling for 0h, 0.5h, 1h, 2h, 4h, 6h and 8h respectively, immediately boiling the sample for 2 minutes after sampling to inactivate the enzyme, and then testing the absorbance at 232nm by using an ultraviolet-visible spectrophotometer. After blank deduction, the absorbance of the test sample at different time points can reflect the enzymolysis resistance of different test samples.
Example 2
20g of sodium hyaluronate with a molecular weight of 120 ten thousand daltons was taken, dissolved in 100mL of a solution containing 1.0g of BDDE and 0.6g of sodium hydroxide, and reacted at 25 ℃ for 24 hours. Cutting the gel to about 1cm3Adding phosphate buffer solution (with concentration of 0.3mg/mL and pH value of 6.5) to swell the gel block, dialyzing for 3-6 times to remove unreacted cross-linking agent and alkali, controlling the concentration of hyaluronic acid to be 1.5% and the pH value of the gel to be 6.0-8.0, and sieving with a 120-mesh stainless steel sieve to obtain the cross-linked sodium hyaluronate gel.
Adding 1.0g of sodium hyaluronate with the molecular weight of 10 ten thousand daltons and 0.5g of sodium hyaluronate with the molecular weight of 100 ten thousand daltons into 100mL of 0.5% glycerol aqueous solution, stirring until the sodium hyaluronate is dissolved, and filtering by a 5-micrometer filter element to obtain non-crosslinked sodium hyaluronate gel with the pH of 6.0-8.0.
Mixing the sodium hyaluronate gel and the non-crosslinked sodium hyaluronate gel in a mass ratio of 1:8, uniformly mixing, and performing heat treatment at 121 ℃ for 15 minutes to obtain a hyaluronate double-gel system.
And testing the pH value, the average particle size, the kinematic viscosity, the extruding force and the enzymolysis resistance of the obtained double-gel system. The test method was the same as in example 1.
Example 3
20g of sodium hyaluronate with a molecular weight of 130 kilodaltons was dissolved in 100mL of a solution containing 2.0g of BDDE and 0.5g of sodium hydroxide, and reacted at 25 ℃ for 24 hours. Cutting the gel to about 1cm3Adding phosphate buffer solution (with concentration of 0.3mg/mL and pH value of 6.0) into the granules to swell the gel blocks, dialyzing for 3-6 times to remove unreacted cross-linking agent and alkali, controlling the concentration of hyaluronic acid to be 1.5% and the pH value of the gel to be 6.0-8.0, and sieving with a 120-mesh stainless steel sieve to obtain the cross-linked sodium hyaluronate gel.
Adding 1.0g of sodium hyaluronate with the molecular weight of 30 ten thousand daltons and 0.5g of sodium hyaluronate with the molecular weight of 90 ten thousand daltons into 100mL of 0.5% glycerol aqueous solution, stirring until the mixture is dissolved, and filtering through a 5-micrometer filter element to obtain non-crosslinked sodium hyaluronate gel with the pH of 6.0-8.0.
Mixing the sodium hyaluronate gel and the non-crosslinked sodium hyaluronate gel in a mass ratio of 1:2, uniformly mixing, and performing heat treatment at 125 ℃ for 20 minutes to obtain a hyaluronate double-gel system.
And testing the pH value, the average particle size, the kinematic viscosity, the extruding force and the enzymolysis resistance of the obtained double-gel system. The test method was the same as in example 1.
Example 4
20g of sodium hyaluronate with a molecular weight of 140 kilodaltons was dissolved in 100mL of a solution containing 0.8g of BDDE and 0.55g of sodium hydroxide, and reacted at 25 ℃ for 24 hours. Cutting the gel to about 1cm3Adding phosphate buffer solution (concentration of 0.3mg/mL, pH 6.0) to swell the gel mass, dialyzing for 3-6 times to remove unreacted cross-linking agent and alkaliControlling the concentration of hyaluronic acid to be 1.5 percent and the pH value of the gel to be 6.0-8.0, and sieving the gel with a 120-mesh stainless steel sieve to obtain the cross-linked sodium hyaluronate gel.
Adding 1.0g of sodium hyaluronate with the molecular weight of 120 ten thousand daltons and 0.5g of sodium hyaluronate with the molecular weight of 250 ten thousand daltons into 100mL of glycerol aqueous solution containing 0.5 percent, stirring until the mixture is dissolved, and filtering through a 5-micron filter element to obtain non-crosslinked sodium hyaluronate gel with the pH of 6.0-8.0.
Mixing the sodium hyaluronate gel and the non-crosslinked sodium hyaluronate gel in a mass ratio of 1:1, uniformly mixing, and performing heat treatment at 130 ℃ for 8 minutes to obtain a hyaluronate double-gel system.
And testing the pH value, the average particle size, the kinematic viscosity, the extruding force and the enzymolysis resistance of the obtained double-gel system. The test method was the same as in example 1.
Example 5
20g of sodium hyaluronate with a molecular weight of 150 kilodaltons was dissolved in 100mL of a solution containing 1.5g of BDDE and 0.45g of sodium hydroxide, and reacted at 25 ℃ for 24 hours. Cutting the gel to about 1cm3Adding phosphate buffer solution (with concentration of 0.3mg/mL and pH value of 6.0) into the granules to swell the gel blocks, dialyzing for 3-6 times to remove unreacted cross-linking agent and alkali, controlling the concentration of hyaluronic acid to be 1.5% and the pH value of the gel to be 6.0-8.0, and sieving with a 120-mesh stainless steel sieve to obtain the cross-linked sodium hyaluronate gel.
Adding 0.8g of sodium hyaluronate with the molecular weight of 20 ten thousand daltons, 0.4g of sodium hyaluronate with the molecular weight of 110 ten thousand daltons and 0.3g of sodium hyaluronate with the molecular weight of 200 ten thousand daltons into 100mL of 0.5% glycerin aqueous solution, stirring until the mixture is dissolved, and filtering through a 5-micrometer filter element to obtain non-crosslinked sodium hyaluronate gel with the pH of 6.0-8.0.
Mixing the sodium hyaluronate gel and the non-crosslinked sodium hyaluronate gel in a mass ratio of 1:5, uniformly mixing, and performing heat treatment at 130 ℃ for 8 minutes to obtain a hyaluronate double-gel system.
And testing the pH value, the average particle size, the kinematic viscosity, the extruding force and the enzymolysis resistance of the obtained double-gel system. The test method was the same as in example 1.
Specific reaction conditions for examples 1-5 are shown in Table 1:
TABLE 1 reaction conditions of examples and comparative examples
The sodium hyaluronate used for preparing the cross-linked sodium hyaluronate gel is first sodium hyaluronate, and the sodium hyaluronate used for preparing the non-cross-linked sodium hyaluronate gel is second sodium hyaluronate.
Comparative example 1
A cross-linked sodium hyaluronate gel was prepared by the procedure of preparing the cross-linked sodium hyaluronate gel in example 5.
And testing the pH value, the average particle size, the kinematic viscosity, the extruding force and the enzymolysis resistance of the obtained double-gel system. The test method was the same as in example 1.
Comparative example 2
A non-crosslinked sodium hyaluronate gel was prepared by the procedure of preparing a non-crosslinked sodium hyaluronate gel in example 5.
And testing the pH value, the average particle size, the kinematic viscosity, the extruding force and the enzymolysis resistance of the obtained double-gel system. The test method was the same as in example 1.
For the above examples and comparative examples, the test results are shown in tables 2 to 6 below. Wherein, table 2 is the pH value of each example and comparative example gel system, table 3 is the average particle size of each example and comparative example gel system, table 4 is the kinematic viscosity of each example and comparative example gel system, table 5 is the extrusion force of each example and comparative example gel system, and table 6 is the enzymolysis performance test result of each example and comparative example gel system.
TABLE 2 pH values of gel systems of the examples and comparative examples
| Item | pH value |
| Example 1 | 6.82 |
| Example 2 | 6.95 |
| Example 3 | 7.03 |
| Example 4 | 7.10 |
| Example 5 | 6.99 |
| Comparative example 1 | 7.25 |
| Comparative example 2 | 6.58 |
TABLE 3 average particle size of gel systems of the examples and comparative examples
| Item | Average particle diameter (μm) |
| Example 1 | 225 |
| Example 2 | 236 |
| Example 3 | 215 |
| Example 4 | 240 |
| Example 5 | 247 |
| Comparative example 1 | 165 |
| Comparative example 2 | 0 |
TABLE 4 kinematic viscosity of the gel systems of the examples and comparative examples
| Item | Kinematic viscosity/mm2/s |
| Example 1 | 56.07 |
| Example 2 | 35.13 |
| Example 3 | 48.87 |
| Example 4 | 68.12 |
| Example 5 | 54.44 |
| Comparative example 1 | 166.74 |
| Comparative example 2 | 75.81 |
TABLE 5 extrusion force of the gel systems of the examples and comparative examples
| Item | Average pushing force/N |
| Example 1 | 13.15 |
| Example 2 | 10.77 |
| Example 3 | 11.85 |
| Example 4 | 15.56 |
| Example 5 | 12.03 |
| Comparative example 1 | 35.12 |
| Comparative example 2 | 22.45 |
TABLE 6 test results of the enzymolysis resistance of the gel systems of the examples and comparative examples
| Time/sample | 0h | 0.5h | 1h | 2h | 4h | 6h | 8h |
| Example 1 | 0.0025 | 0.2650 | 0.4523 | 0.5912 | 0.6585 | 0.6965 | 0.7078 |
| Example 2 | 0.0036 | 0.2455 | 0.4315 | 0.5862 | 0.6512 | 0.6932 | 0.7110 |
| Example 3 | 0.0015 | 0.2394 | 0.4165 | 0.5412 | 0.6054 | 0.6610 | 0.7055 |
| Example 4 | 0.0012 | 0.2294 | 0.4110 | 0.5355 | 0.6120 | 0.6600 | 0.7105 |
| Example 5 | 0.0021 | 0.2355 | 0.4200 | 0.5215 | 0.6052 | 0.6560 | 0.7085 |
| Comparative example 1 | 0.0020 | 0.1223 | 0.2312 | 0.3856 | 0.5341 | 0.6245 | 0.6912 |
| Comparative example 2 | 0.0015 | 0.3190 | 0.5110 | 0.6641 | 0.7012 | 0.7100 | 0.7122 |
And (4) analyzing results: from the test results of pH, average particle diameter, kinematic viscosity, extrusion force and enzymatic hydrolysis resistance of each example and comparative example measured in tables 2 to 6, it was found that: in examples 1 to 5 and comparative examples 1 to 2, although the pH of the gels was 6.5 to 7.5, the average particle diameter, kinematic viscosity, extrusion force, and enzymatic resistance of the gels were considerably different from those of comparative example 1 (crosslinked gel) and comparative example 2 (non-crosslinked gel) in examples 1 to 5 (two-gel system).
The method comprises the following specific steps:
average particle size: comparative example 1 and examples 1-5 were screened using a screen of the same pore size during the preparation process, but the unexpected results were: the average particle size of the resulting gels varied. The reason why example 5 HAs a larger average particle diameter than comparative example 1 is probably that the crosslinked gel particles in the two-gel system of example 5 are surrounded by a network of non-crosslinked HA of different molecular weights, and are more stable during heat treatment, and the particles are not easily broken.
Kinematic viscosity: as can be seen from Table 4, the kinematic viscosities of examples 1 to 5 are all 10 to 80mm2In the/s range, but it is unexpected that the kinematic viscosities of example 5 are all comparable to those of water aloneThe kinematic viscosity of each gel, i.e., the crosslinked gel (comparative example 1) and the non-crosslinked gel (comparative example 2), was low, i.e., the dual gel system was more fluid than either the crosslinked gel or the non-crosslinked gel alone, and when applied as a hydrogel product to a relatively shallow area of the skin, it was less likely to cause adverse reactions such as skin hills, redness, and swelling.
Extrusion force: as can be seen from Table 5, the extrusion forces of examples 1 to 5 are all in the range of 8 to 20N, and the extrusion force of example 5 is lower than that of comparative examples 1 and 2, which indicates that the double-gel system is more favorable for being pushed out from the injector in the using process, the force is less, and the control and the operation are more convenient.
Resistance to enzymatic hydrolysis: as can be seen from table 6, the enzyme resistance of the dual gel system is improved by 1 time compared to the enzyme resistance of the single non-crosslinked gel, and the time for the decomposition of hyaluronic acid is prolonged, so that the effect time on the skin is longer and the efficacy is better when the dual gel system is used as an aqueous photoproduct.
Claims (10)
1. A composition containing hyaluronate is characterized by comprising a double-gel system consisting of crosslinked hyaluronate gel and non-crosslinked hyaluronate gel, wherein the pH value of the double-gel system is 6.0-8.0, the average particle size is 100-300 mu m, and the kinematic viscosity is 10-80mm2And/s, the extrusion force is 5-30N.
2. The composition as claimed in claim 1, wherein the average particle size of the dual gel system is 150-280 μm.
3. Composition according to claim 1, characterized in that the kinematic viscosity of the double gel system is between 30 and 60mm2/s。
4. The composition of claim 1, wherein the dual gel system has a push force of 8 to 20N.
5. The composition according to claim 1, wherein the mass ratio of the cross-linked hyaluronate gel to the non-cross-linked hyaluronate gel is 1 (1-10).
6. The composition according to claim 1, wherein the mass ratio of the cross-linked hyaluronate gel to the non-cross-linked hyaluronate gel is 1 (2-5).
7. The composition according to claim 1, further comprising any one or more of glycerol, potassium chloride, sodium chloride, phosphate, amino acid, sodium acetate, and vitamins.
8. A method for preparing a composition comprising a hyaluronic acid salt, comprising:
preparing a cross-linked hyaluronate gel using a first hyaluronic acid or hyaluronate;
preparing a non-crosslinked hyaluronate gel using a second hyaluronic acid or hyaluronate;
and mixing the cross-linked hyaluronate gel and the non-cross-linked hyaluronate gel, and performing high-temperature treatment to obtain a double-gel system.
9. The method of claim 8, wherein the first hyaluronic acid or hyaluronate salt has a molecular weight of 100-250 kilodalton, preferably 120-180 kilodalton.
10. The method according to claim 9, wherein the concentration of the hyaluronic acid salt in the cross-linked hyaluronic acid salt gel is between 0.5% and 2.5%, preferably between 0.8% and 2.0%.
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