CN104761735A - Preparation method of cross-linked sodium hyaluronate gel having hyaluronidase activity inhibiting effect - Google Patents
Preparation method of cross-linked sodium hyaluronate gel having hyaluronidase activity inhibiting effect Download PDFInfo
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Abstract
The invention relates to a preparation method of a cross-linked sodium hyaluronate gel having a hyaluronidase activity inhibiting effect. The method utilizes a fact that different groups in sodium hyaluronate molecules are activated under different pH conditions, then different pH conditions are controlled, a hyaluronidase inhibitor is introduced into the sodium hyaluronate molecules, and finally the cross-linked sodium hyaluronate gel having the hyaluronidase activity inhibiting effect is obtained. Compared with a gel prepared by a traditional process, the cross-linked sodium hyaluronate gel prepared by the method has the advantages of low viscosity, long in-vivo residual time and the like, also can inhibit the hyaluronidase activity, and has the highest inhibition rate up to 65%. The low viscosity of the cross-linked sodium hyaluronate gel prepared by the method ensures convenience of clinical use, and the cross-linked sodium hyaluronate gel inhibits the hyaluronidase activity, significantly increases the in-vivo residual time, and has good application prospects in the aspects of cosmetic injections, artificial vitreous bodies and degenerative arthritis supplementary treatment.
Description
Technical field
The present invention relates to a kind of preparation method of cross-linking sodium hyaluronate gel of hyaluronidase inhibitor, the present invention first by hyaluronic acid enzyme inhibitor by being covalently bonded on hyaluronic acid sodium gel molecule, then carry out secondary crosslinking and form gel, this gel can be used for Soft-tissue operation, artificial vitreous, joint cavity lubricating fluid supplement, slow releasing carrier of medication etc., belongs to medical field.
Technical background
The linear high score polysaccharide that hyaluronic acid is made up of glucuronic acid and N-acetyl-glucosamine disaccharide repeating unit, is distributed widely in the Organ and tissues such as mammiferous reticular tissue, vitreum, placenta.Hyaluronic acid has good biocompatibility and non-immunogenicity, is widely used in the fields such as medical treatment, beauty treatment, makeup.But owing to containing specific Unidasa in human body, natural sodium hyaluronate gel in vivo can by degraded, metabolism rapidly.Cross-linking hyaluronic acid sodium is that hyaluronate sodium modifies the high-molecular gel obtained by chemically crosslinked, compensate for the drawback that hyaluronate sodium retention time is short, and traditional cross-linking sodium hyaluronate gel mostly is particulate state or viscosity is excessive, pushing force is larger, be difficult to first-class by small-bore syringe needle, limit its further expansive approach.
Nowadays hyaluronic acid sodium gel product many employings divinylsulfone (DVS) market occurred, 1, 4-butyleneglycol bisglycidyl ethers (BDDE) etc. are as linking agent, in order to pursue longer degradation time in vivo, often increase the add-on of linking agent, improve the degree of crosslinking of product, but above-mentioned linking agent has toxicity or carinogenicity more, residual cross-linker enters in body, very large risk can be caused, simultaneously in existing preparation technology in dialysis procedure, because the hyaluronic acid sodium gel after crosslinked is three-dimensional netted three-dimensional arrangement, linking agent embedding wherein, be difficult to remove totally, therefore, in order to improve the biological safety of product, the add-on of linking agent need be reduced, but the degree of crosslinking of product can be affected so simultaneously, and the residence time in the final body affecting product.
Seem a difficult problem for contradiction for above-mentioned one, also do not occur that the patent of being correlated with is to set forth the problems referred to above at present, the present invention just for this to paradox, solve this problem from another one angle.Residence time after et al. Ke cross-linking sodium hyaluronate gel is the Degradation of Unidasa in body in essence, improving in body the residence time in traditional patent mainly extends the residence time by improving degree of crosslinking, and this patent looks for another way, hyaluronic acid enzyme inhibitor is covalently bound on hyaluronic acid sodium gel molecule, reaches the residence time in extension body by the activity of Unidasa in suppression body.
The present invention adopts BDDE or DVS as linking agent, first utilize in acid condition in hyaluronic acid sodium molecule by the carboxyl activated, enzyme inhibitor containing carboxyl is covalently cross-linking on hyaluronic acid sodium gel molecule, and then add natural sodium hyaluronate gel, be adjusted to alkalescence utilize alkaline condition under hydroxyl by the feature activated, hyaluronic acid sodium molecule containing enzyme inhibitors is cross-linked, pass through alcohol precipitation again, obtain the cross-linking hyaluronic acid sodium dry powder containing enzyme inhibitors, PBS redissolves the cross-linking sodium hyaluronate gel obtained containing Hyaluronidase inhibitor.
Containing Hyaluronidase inhibitor in cross-linking sodium hyaluronate gel prepared by the method, the activity suppressing Unidasa can effectively be fallen, and then the residence time in extension body.The preparation method of cross-linked hyaluronic acid gel is in the past (as patent CN 101036808, patent CN1950039 etc.) gel that obtains is water-fast solid gel, need by be prepared as there is certain diameter particulate after could use, each step of the method that patent of the present invention provides all is carried out under homogeneous aqueous environment, unreacted linking agent can be removed to greatest extent by alcohol precipitation, improve the biological safety of product.
Summary of the invention
Invention advantage
1. introduce hyaluronate sodium enzyme inhibitors in the present invention, after implanting, effectively can suppress the activity of Unidasa.
2. the present invention removes unreacted linking agent by alcohol precipitation and the method combined of dialysing.
3. the present invention carries out secondary crosslinking reaction respectively under acidity and alkaline condition, and the utilization ratio that improve linking agent reduces the risk after implanting for a long time in body, improves the biological safety of use in body.
For realizing above object, technical scheme provided by the invention is:
The first step: the configuration of sodium hyaluronate solution
Second step: acid cross-linked reacts
3rd step: alkaline crosslinking reaction
4th step: alcohol precipitation
5th step: the redissolution of precipitation dry powder
6th step: dialysis.
Accompanying drawing explanation
The cross-linking sodium hyaluronate gel of the different gamma-polyglutamic acid-content of Fig. 1 is on the impact of hyaluronidase activity.
Embodiment
Now in conjunction with the embodiments, the present invention is described in detail:
Embodiment one
Taking molecular weight is 1,200,000 daltonian hyaluronate sodium dry powder 10g, molecular weight is the gamma-polyglutamic acid-dry powder 5g of 1,000,000,100ml is adopted to contain 0.5g1, the 0.5%(w/w of 4-butanediol diglycidyl ether) hydrochloric acid soln by its solvent, then room temperature 25 DEG C reaction 24 hours, obtains acid cross-linked gel.Then in acid cross-linked gel, add the sodium hydroxide solution that 100ml concentration is 1%, after being uniformly dissolved, putting into the reaction of 5 DEG C of fixed temperature and humidity reaction cabinets after 7 days, obtain alkaline cross linked gel.Adopt 0.5% sodium hydroxide that secondary crosslinking gel is diluted to 1000ml, then adopt the alcohol settling of 95%, wash 2 times.Then adopt phosphate buffer soln that the dry powder obtained is dissolved into 400ml, be uniformly dissolved rear dress dialysis tubing and dialyse 3 days, obtain sample 1.
Embodiment two
Taking molecular weight is 1,200,000 daltonian hyaluronate sodium dry powder 12g, molecular weight is the gamma-polyglutamic acid-dry powder 5g of 1,000,000,100ml is adopted to contain 0.5g1, the 0.5%(w/w of 4-butanediol diglycidyl ether) hydrochloric acid soln by its solvent, then room temperature 25 DEG C reaction 48 hours, obtains acid cross-linked gel.Then in acid cross-linked gel, add the potassium hydroxide solution that 100ml concentration is 1%, after being uniformly dissolved, putting into the reaction of 5 DEG C of fixed temperature and humidity reaction cabinets after 7 days, obtain alkaline cross linked gel.Adopt 0.5% potassium hydroxide that secondary crosslinking gel is diluted to 1000ml, then adopt the alcohol settling of 95%, wash 2 times.Then adopt phosphate buffer soln that the dry powder obtained is dissolved into 400ml, be uniformly dissolved rear dress dialysis tubing and dialyse 5 days, obtain sample 2.
Embodiment three
Taking molecular weight is 1,200,000 daltonian hyaluronate sodium dry powder 12g, molecular weight is the gamma-polyglutamic acid-dry powder 5g of 1,000,000,100ml is adopted to contain 0.5g1, the 0.5%(w/w of 4-butanediol diglycidyl ether) hydrochloric acid soln by its solvent, then room temperature 25 DEG C reaction 48 hours, obtains acid cross-linked gel.Then in acid cross-linked gel, add the potassium hydroxide solution that 100ml concentration is 1%, after being uniformly dissolved, putting into the reaction of 5 DEG C of fixed temperature and humidity reaction cabinets after 5 days, obtain alkaline cross linked gel.Adopt 0.5% potassium hydroxide that secondary crosslinking gel is diluted to 1000ml, then adopt the alcohol settling of 95%, wash 2 times.Then adopt phosphate buffer soln that the dry powder obtained is dissolved into 400ml, be uniformly dissolved rear dress dialysis tubing and dialyse 5 days, obtain sample 3.
Embodiment four
Taking molecular weight is 1,200,000 daltonian hyaluronate sodium dry powder 10g, molecular weight is the gamma-polyglutamic acid-dry powder 5g of 1,000,000,100ml is adopted to contain 0.5g1, the 0.5%(w/w of 4-butanediol diglycidyl ether) hydrochloric acid soln by its solvent, then room temperature 25 DEG C reaction 24 hours, obtains acid cross-linked gel.Then in acid cross-linked gel, add the sodium hydroxide solution that 100ml concentration is 1%, after being uniformly dissolved, putting into the reaction of 5 DEG C of fixed temperature and humidity reaction cabinets after 5 days, obtain alkaline cross linked gel.Adopt 0.5% sodium hydroxide that secondary crosslinking gel is diluted to 1000ml, then adopt the alcohol settling of 95%, wash 2 times.Then adopt phosphate buffer soln that the dry powder obtained is dissolved into 400ml, be uniformly dissolved rear dress dialysis tubing and dialyse 3 days, obtain sample 4.
Comparative example one
Taking molecular weight is 1,200,000 daltonian hyaluronate sodium dry powder 6.4g, after adopting 100ml distilled water to dissolve, add concentration be 25% the alkalization of sodium carbonate solution 25ml low temperature spend the night.Then add divinylsulfone 0.35g, put into 40 DEG C of fixed temperature and humidity reaction cabinets reaction 4 hours after stirring, be cut into small pieces dialysis after 6 days, pulverize and obtain comparative sample 1.
Comparative example two
Taking molecular weight is 1,200,000 daltonian hyaluronate sodium dry powder 10g, adopt 0.3g 1, the 50ml concentration of 4-butanediol diglycidyl ether be 1.5% sodium hydroxide solution be uniformly dissolved, put into 5 DEG C of fixed temperature and humidity reaction cabinets reaction 7 days, be cut into small pieces dialysis to 500ml, then pulverizing, obtain cross-linking sodium hyaluronate gel particulate, is comparative sample 2.
Embodiment five
Take sample 1, sample 2, sample 3, sample 4, comparative sample 1 and each 0.32g of comparative sample 2 respectively, adding respectively containing hyaluronidase concentration is the phosphate buffered saline buffer 8.0ml of 50U/ml, at 37 DEG C of enzymes, 24h falls, then glucuronic acid content in enzymolysis solution is detected, calculate the 24h degradation rate of 4 samples, be respectively:
| Sample name | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Comparative sample 1 | Comparative sample 2 |
| Degraded percentage | 65.14% | 59.31% | 69.11% | 63.43% | 92.70% | 86.39% |
As seen from the above table, sample 1,2, the degraded percentage of 3,4 far below comparative sample 1,2.
Embodiment six
Adopt rotational rheometer to detect the viscosity taking sample 1, sample 2, sample 3, sample 4, comparative sample 1 and comparative sample 2 respectively, the viscosity results of getting 0.25Hz place is as follows:
| Sample name | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Comparative sample 1 | Comparative sample 2 |
| Viscosity mPas | 52631 | 76627 | 45268 | 67489 | 263010 | 507600 |
As seen from the above table, sample 1, sample 2, sample 3, the viscosity of sample 4 is well below control sample 1 and control sample 2, and sample prepared by the method has lower viscosity.
Embodiment seven
Take appropriate amount of sample 1,2,3,4 respectively, utilize Kjeldahl nitrogen determination total nitrogen content, then deduct hyaluronate sodium nitrogen content inherently, obtain the percentage composition that 4 samples are different, result is as follows:
| Sample name | Sample 1 | Sample 2 | Sample 3 | Sample 4 |
| The percentage composition (%) of polyglutamic acid | 3.68 | 4.63 | 3.19 | 4.50 |
As seen from the above table, sample 1,2,3,4 have different polyglutamic acid content, and the result of 5 can be found out in conjunction with the embodiments, and polyglutamic acid content is higher, higher to the inhibit activities of Unidasa.
Embodiment eight
Respectively by sample 1,2,3,4 are diluted to the gel that concentration is 1%, add 400U/ml Unidasa, then at 37 DEG C with hyaluronic acid enzyme reaction 20min.In reaction soln, add the hyaluronate sodium of 0.4mg/ml and react 20min at 37 DEG C, then adding 0.4NNaOH termination reaction.In reaction soln, add the p-dimethylaminobenzaldehyde of 3ml1% and react 20min at 37 DEG C, finally under absorbancy 585nm, utilizing the enzymic activity of Morgan-Ai Ademilson method (Moegan-Elson) assaying reaction solution.Calculation sample 1,2, the hyaluronic acid enzyme level percentage ratio (the results are shown in Figure of description 1) of 3,4.
Claims (7)
1. the preparation method of the cross-linking sodium hyaluronate gel of a hyaluronidase inhibitor, it is characterized in that controlling the different activating group of hyaluronic acid sodium molecule and different crosslinking times, in cross-linking hyaluronic acid sodium molecule, introduce Hyaluronidase inhibitor; First under acidic conditions, Hyaluronidase inhibitor is introduced hyaluronic acid sodium molecule, and then in the basic conditions the hyaluronic acid sodium molecule introducing Hyaluronidase inhibitor is carried out crosslinking reaction, obtain the cross-linking hyaluronic acid sodium dry powder containing Hyaluronidase inhibitor finally by alcohol precipitation, use phosphoric acid buffer (PBS) again to dissolve subsequently and obtain cross-linking sodium hyaluronate gel.
2. the Hyaluronidase inhibitor described in claim 1 refers to the hyaluronic acid enzyme inhibitor containing carboxyl such as gamma-polyglutamic acid-, heparin sodium.
3. the linking agent described in claim 1 refers to BDO bisglycidyl ether (BDDE) and divinylsulfone (DVS).
4. the activating group that hyaluronic acid sodium molecule according to claim 1 is different refers to that carboxyl under acid and alkaline condition in hyaluronic acid sodium molecule and hydroxyl are activated respectively.
5. the acidic conditions in claim 1 refers to that by adjust ph such as acidic solution example hydrochloric acid, acetic acid and phosphoric acid be 3-5.
6. the alkaline condition in claim 1 refers to basic solution as sodium hydroxide, sodium carbonate or potassium hydroxide etc. regulate pH to be 9-13.
7. the different crosslinking time described in claim 1 refers to the time in cross-linking hyaluronic acid sodium reaction under sour environment and alkaline environment, and the reaction times under sour environment is 1-2 days, and the reaction times under alkaline condition is 5-7 days.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111246919A (en) * | 2017-07-26 | 2020-06-05 | 多肽治疗解决公司 | Cross-linked polymers composed of polysaccharides and polyamino acids and use thereof |
| CN112442199A (en) * | 2019-08-29 | 2021-03-05 | 上海其胜生物制剂有限公司 | Flexible high-stability gel and preparation method thereof |
| CN113087935A (en) * | 2021-05-19 | 2021-07-09 | 青岛琛蓝海洋生物工程有限公司 | Composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis and preparation method thereof |
| WO2022222907A1 (en) | 2021-04-20 | 2022-10-27 | 中国海洋大学 | Thiolated polysaccharide derivative hydrogel, preparation method therefor, and application thereof |
| CN118063833A (en) * | 2024-04-19 | 2024-05-24 | 四川兴泰普乐医疗科技有限公司 | Micro-crosslinking sodium hyaluronate gel and preparation method thereof |
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| CN101821294A (en) * | 2007-09-28 | 2010-09-01 | 株式会社资生堂 | Swellable crosslinked hyaluronic acid powder and method for producing the same |
| CN102757572A (en) * | 2011-04-28 | 2012-10-31 | 上海其胜生物制剂有限公司 | Preparation method for agranular crosslinking sodium hyaluronate with high-temperature-resistant and enzymatic-hydrolysis-resistant characteristics |
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| CN1433432A (en) * | 2000-02-03 | 2003-07-30 | 电气化学工业株式会社 | Hyaluronic acid gel, process for producing the same and medical material containing the same |
| CN101821294A (en) * | 2007-09-28 | 2010-09-01 | 株式会社资生堂 | Swellable crosslinked hyaluronic acid powder and method for producing the same |
| CN102757572A (en) * | 2011-04-28 | 2012-10-31 | 上海其胜生物制剂有限公司 | Preparation method for agranular crosslinking sodium hyaluronate with high-temperature-resistant and enzymatic-hydrolysis-resistant characteristics |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111246919A (en) * | 2017-07-26 | 2020-06-05 | 多肽治疗解决公司 | Cross-linked polymers composed of polysaccharides and polyamino acids and use thereof |
| CN111246919B (en) * | 2017-07-26 | 2022-03-08 | 多肽治疗解决公司 | Cross-linked polymers composed of polysaccharides and polyamino acids and use thereof |
| CN112442199A (en) * | 2019-08-29 | 2021-03-05 | 上海其胜生物制剂有限公司 | Flexible high-stability gel and preparation method thereof |
| CN112442199B (en) * | 2019-08-29 | 2023-01-10 | 上海其胜生物制剂有限公司 | Flexible high-stability gel and preparation method thereof |
| WO2022222907A1 (en) | 2021-04-20 | 2022-10-27 | 中国海洋大学 | Thiolated polysaccharide derivative hydrogel, preparation method therefor, and application thereof |
| CN113087935A (en) * | 2021-05-19 | 2021-07-09 | 青岛琛蓝海洋生物工程有限公司 | Composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis and preparation method thereof |
| CN113087935B (en) * | 2021-05-19 | 2022-05-27 | 青岛琛蓝海洋生物工程有限公司 | Composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis and preparation method thereof |
| CN118063833A (en) * | 2024-04-19 | 2024-05-24 | 四川兴泰普乐医疗科技有限公司 | Micro-crosslinking sodium hyaluronate gel and preparation method thereof |
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