CN116396498A

CN116396498A - Cross-linked polyglutamic acid gel and preparation method and application thereof

Info

Publication number: CN116396498A
Application number: CN202310012576.9A
Authority: CN
Inventors: 王志伟; 陈雄伟
Original assignee: Imeik Technology Development Co ltd
Current assignee: Imeik Technology Development Co ltd
Priority date: 2022-01-05
Filing date: 2023-01-05
Publication date: 2023-07-07

Abstract

The invention discloses a cross-linked polyglutamic acid gel, a preparation method and application thereof, in particular to a cross-linked polyglutamic acid gel for injection filling and related products thereof. According to the invention, the cross-linking agent is adopted, the pH of the solution is regulated, the polyglutamate and the cross-linking agent form an amide bond under the action of the catalyst, and the cross-linked polyglutamate gel is obtained through further crushing, washing, drying, re-dissolving and wet heat sterilization, and is resistant to wet heat sterilization, particularly, the dosage of the cross-linking agent is low, the removal process of the cross-linking agent and the catalyst is increased, the biocompatibility of the gel is improved, and the safety risk of injection use is reduced. The prepared crosslinked polyglutamic acid gel is safe and nontoxic, has high elastic modulus, stable performance and good biocompatibility, can ensure the maintenance time on the filling effect when being particularly used in products like skin filling, and has important clinical significance and application value, and meanwhile, the stability of the performance and the higher elastic modulus provide better support and compression resistance for tissue filling.

Description

Cross-linked polyglutamic acid gel and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a crosslinked polyglutamic acid gel and a preparation method and application thereof, and especially relates to a crosslinked polyglutamic acid gel for injection filling and related products thereof.

Background

Polyglutamic acid (gamma-PGA) is a water-soluble polyamino acid produced by fermentation of Bacillus subtilis and has a structure in which glutamic acid units form a high molecular polymer of peptide bonds through alpha-amino groups and gamma-carboxyl groups. The gamma-PGA contains carboxyl with higher activity, is easy to react with some substances to generate stable complex, has strong hydrophilicity, has moisture retention capacity exceeding that of hyaluronic acid, can be completely degraded in nature or human body, can be degraded into endogenous substance glutamic acid in human body, does not generate toxic or side effect, is widely applied to the fields of cosmetics, medicines, health care products, sanitary products and the like, and is an ideal biodegradable medical high polymer material.

The hydrogel is a high molecular three-dimensional network polymer formed by crosslinking linear molecular chains, can absorb a large amount of water without dissolution, effectively improves the degradation period of the molecular chains, has the advantages of high water retention, excellent biocompatibility, endogenous glutamic acid as a degradation product and the like, and has the potential of being applied to facial skin injection, reducing wrinkles, improving the appearance and structure of the skin and preventing skin aging.

Less reports on the preparation method of cross-linked polyglutamic acid gel for injection are provided, chinese patent CN106730029B discloses cross-linked polyglutamic acid gel particles for injection filling, and a preparation method and application thereof, wherein 1, 4-butanediol diglycidyl ether is used as a cross-linking agent, and gel particles are obtained after premixing, homogenizing, pH adjusting, separating and sterilizing to increase the maintenance time for tissue filling. However, the elastic modulus of the gel prepared by the method is obviously reduced after the gel is subjected to wet heat sterilization, and the removal process of the cross-linking agent is lacked, so that the effect of tissue filling is directly affected by the reduction of the elastic modulus, the residual cross-linking agent or byproducts of the cross-linking reaction also have potential carcinogenicity, and particularly, in order to prolong the in-vivo maintenance time of the polyglutamic acid gel, the cross-linking degree is improved by increasing the adding amount of the cross-linking agent, so that the safety risk of the product is further improved. Chinese patent CN 109503864B discloses a method for preparing an injectable hydrogel with cohesive enhanced properties, which mixes polyamino and polycarboxy polymers to form ionic bonds, adds EDC/NHS or DMTMM as a catalyst to form part of the ionic bonds to form amide bonds, rearranges molecular chains through alkalization, sedimentation and re-dissolution processes, and increases dynamic viscosity of the hydrogel after wet heat sterilization, wherein the increase of dynamic viscosity is caused by breaking a crosslinked network structure to form more macromolecular chain structures, which results in loss of elastic modulus of the gel, and the increase of viscosity of the gel prepared by the method is caused by ionic bond bonding among polymers along with the rearrangement between molecular chains after the storage time is prolonged, but the viscosity increase is unstable, particularly the long-term storage, can affect the stability of product performance, and can generate risks for practical use. In addition, the method refers to the use of phosphate buffer solution as a solvent to control the pH value of a reaction system taking EDC/NHS as a catalyst, so that the catalytic efficiency is reduced, the effective use amount of the cross-linking agent is reduced, the same cross-linking effect is achieved, the use amount is increased, the economic cost is increased, the risk of the residual amount of the cross-linking agent is increased, and the safety risk of a product is increased.

In summary, no report has been made on a method for preparing a polyglutamic acid gel for injection filling with low addition amount of a crosslinking agent and high elastic modulus characteristics, and particularly, the polyglutamic acid gel for injection filling has a certain significance in that the gel can still maintain a higher elastic modulus after wet heat sterilization and still remain stable after a period of storage and can provide high compression resistance as a tissue filler, and therefore, the polyglutamic acid gel for injection tissue filling can be provided to meet clinical diversified demands.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a cross-linked polyglutamic acid gel, a preparation method and application thereof, and in particular relates to a cross-linked polyglutamic acid gel for injection filling and related products thereof.

In a first aspect of the present invention, there is provided a method for preparing a crosslinked polyglutamic acid gel, comprising the steps of:

(1) Mixing polyglutamate, a crosslinking agent and water, and adjusting the pH of the obtained solution;

(2) And (3) adding a catalyst into the solution obtained in the step (1) to perform a reaction.

Specifically, the polyglutamate in step (1) is selected from: one or more of sodium polyglutamate, potassium polyglutamate, calcium polyglutamate, and magnesium polyglutamate, in particular sodium polyglutamate.

In particular, the molecular weight of the polyglutamate in step (1) is 500-2000kDa (e.g. 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000 kDa), in particular 800-1500kDa.

Specifically, the crosslinking agent in step (1) is selected from the group consisting of: one or more of an alkyl diamine, an aminopolysaccharide, and an amino acid ester compound, particularly one or more of an alkyl diamine and an amino acid ester compound, preferably a combination of an alkyl diamine and an amino acid ester compound.

Specifically, the alkyldiamine contains two amino groups (-NH) in the molecule ₂ ) It may be selected from: 1, 3-propanediamine, 1, 4-butanediamine, 1, 6-hexanediamine, bis-hexamethyltriamine (N- (6-aminohexyl) -1, 6-hexanediamine), in particular 1, 3-propanediamine or bis-hexamethyltriamine.

Specifically, aminopolysaccharide refers to amino group-containing polysaccharides such as cyclodextrin, chitosan, and the like.

Specifically, for amino acid ester compounds, the amino acid preferably has two amino groups (-NH) ₂ ) Such as lysine, asparagine, glutamine, arginine, in particular lysine; wherein the alcohol forming the amino acid ester can be C1-6 alcohol such as methanol, ethanol, i.e. the amino acid ester compound can be amino acid methyl ester, amino acid ethyl ester, especially L-lysine ethyl ester dihydrochloride or L-lysine methyl ester dihydrochloride.

In one embodiment of the invention, the crosslinking agent in step (1) is a combination of an alkyl diamine and an amino acid ester compound, wherein the molar ratio of amino groups of the two is 1:0.1-0.5 (e.g. 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5), in particular 1:0.1-0.3.

The method comprises the steps of combining alkyl diamine and amino acid ester compounds as a cross-linking agent, wherein the alkyl diamine with a simple molecular chain structure is subjected to cross-linking reaction with polyglutamate firstly, the amino acid ester with a longer molecular chain can be subjected to cross-linking reaction with carboxyl groups on the polyglutamate which are far away from the alkyl diamine and are not subjected to cross-linking reaction with the alkyl diamine, and the generated carboxyl groups and amino groups in the alkyl diamine form intermolecular hydrogen bonds along with the change of pH of a reaction system in the cross-linking process, so that the synergistic effect of the amino acid ester compounds and the alkyl diamine in the cross-linking process is realized, the final cross-linking efficiency is improved, and a more compact three-dimensional network structure is formed; in addition, the amino acid ester cross-linking agent is added, so that the cross-linked polyglutamic acid also contains a part of ester bonds, the part of ester bonds are subjected to hydrolytic cleavage after wet heat sterilization, and intermolecular hydrogen bonds are formed between generated carboxyl groups and free amino groups, thereby improving the stability of the gel after sterilization and prolonging the degradation time of the gel to a certain extent.

In one embodiment of the invention, the crosslinker in step (1) is a combination of 1, 3-propanediamine and L-lysine ethyl ester dihydrochloride or L-lysine methyl ester dihydrochloride, wherein the molar ratio of amino groups of the two is 1:0.1-0.5 (e.g. 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5), in particular 1:0.1-0.3.

In another embodiment of the present invention, the crosslinking agent in step (1) is a combination of bis-hexamethyltriamide and L-lysine ethyl ester dihydrochloride or L-lysine methyl ester dihydrochloride, wherein the molar ratio of amino groups of the two is 1:0.1-0.5 (e.g., 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5), especially 1:0.1-0.3.

Specifically, the mass ratio of polyglutamate to the crosslinking agent in step (1) is 1:0.01-0.09 (e.g., 1:0.01, 1:0.02, 1:0.04, 1:0.05, 1:0.06, 1:0.08, 1:0.09), particularly 1:0.02-0.08.

Specifically, the mass percentage of polyglutamate in the solution in the step (1) is 10-20% (e.g., 10%, 12%, 14%, 15%, 16%, 18%, 20%).

Specifically, the step of adjusting the pH in the step (1) is to adjust the pH to 5.5-6.5 (e.g., 5.5, 5.8, 6.0, 6.2, 6.5).

In particular, the reagents used to adjust the pH may be mineral acids (e.g., hydrochloric acid, sulfuric acid) and/or mineral bases (e.g., sodium hydroxide, potassium hydroxide).

In one embodiment of the invention, the catalyst in step (2) is a carbonium salt, e.g., O- (7-azabenzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HATU), O- (benzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HBTU).

In another embodiment of the present invention, the catalyst in step (2) is 1- (3-dimethylaminopropyl) -3-ylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS).

Specifically, the molar ratio of catalyst to crosslinker is 1-1.4:1 (e.g., 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1).

Specifically, the reaction temperature in step (2) is 25-40 ℃ (e.g., 25, 30, 35, 40 ℃).

Specifically, the reaction time in step (2) is 12 to 18 hours (e.g., 12, 14, 16, 18 hours).

Specifically, the reaction in step (2) is carried out under closed conditions, for example in a closed forced air drying oven.

More specifically, the preparation method further comprises the step (3): swelling the gel obtained by the reaction in the step (2), crushing, washing, separating and drying.

In particular, the solvent used for swelling is water, in particular purified water.

In particular, the multipurpose solvent for washing is water, in particular purified water, and the number of washing may be one or more.

Specifically, the separation step includes an alcohol precipitation step; more specifically, the alcohol is ethanol.

Specifically, the drying method is vacuum drying; more specifically, the vacuum degree of vacuum drying is-0.10 MPa to 0MPa (e.g., -0.09, -0.08, -0.06, -0.04, -0.02 MPa); more specifically, the temperature of the vacuum drying is 30-50deg.C (e.g., 30, 35, 40, 45, 50deg.C); more specifically, the time for vacuum drying is 6-36 hours (e.g., 6, 12, 18, 24, 30, 36 hours).

More specifically, the preparation method further comprises the step (4): and (3) redissolving the dried product obtained in the step (3) and sterilizing.

Specifically, the pH of the resulting solution is 7.2.+ -. 2 (e.g., pH 7.2.+ -. 1, pH 7.2.+ -. 0.5, pH 7.2.+ -. 0.2), for example pH 7.0.

Specifically, the resulting solution is reconstituted to an osmotic pressure of 280-350mOsm/L (e.g., 280, 300, 320, 340, 350 mOsm/L).

In particular, the solvent used for reconstitution may be a pH neutral buffer, such as a pH 7.0 phosphate buffer.

Specifically, the sterilization mode is damp-heat sterilization; more specifically, the sterilization temperature is 120-130 ℃ (e.g., 120, 121, 122, 124, 125, 126, 128, 130 ℃); more specifically, the sterilization time is 1-30 minutes (e.g., 1,5, 10, 15, 20, 25, 30 minutes).

In one embodiment of the invention, the sterilizing step comprises: the re-dissolved solution is filled in a pre-filled syringe for moist heat sterilization (the cross-linked polyglutamic acid gel for injection filling is obtained, and can be injected and filled in skin tissues for achieving the purposes of eliminating the looseness of skin due to aging, repairing skin defects and the like).

In a second aspect of the present invention, there is provided a crosslinked polyglutamic acid gel prepared by the method of the first aspect.

Specifically, the crosslinked polyglutamic acid gel is a crosslinked polyglutamic acid gel for injection filling.

In particular, the crosslinked polyglutamic acid gel is sterile.

In a third aspect of the present invention there is provided a gel composition comprising the cross-linked polyglutamic acid gel of the second aspect, and one or more adjuvants.

Specifically, the composition may further comprise one or more of moisturizers, osmotic pressure regulator, bioactive agent, cosmetic active agent, cell attachment agent, and the like.

Specifically, humectants include, but are not limited to, glycerin, propylene glycol, butylene glycol, lactic acid, sodium lactate, urea, erythritol, xylitol, rhamnose, mannose, sorbitol, trehalose, raffinose, and the like; osmotic pressure regulators include, but are not limited to, sodium chloride, dextrose, mannitol, potassium chloride, calcium chloride, sorbitol, and the like; bioactive agents include, but are not limited to, cell growth factors, peptides, peptidomimetics, antibodies, nucleic acids, polysaccharides, and the like; cosmetic actives include, but are not limited to, anti-aging agents, anti-free radical agents, antioxidants, hydrating agents, whitening agents, sunscreens, muscle relaxants, and the like.

In a fourth aspect of the present invention, there is provided the use of the crosslinked polyglutamic acid gel of the second aspect in the preparation of tissue fillers, tissue engineering scaffolds, wound repair materials, drug carriers, cosmetics (external use), food stabilizers, sewage treatment agents, water-retaining agents and the like, in particular in the preparation of tissue fillers.

In a fifth aspect of the present invention, there is provided a tissue-filling product comprising a syringe and the crosslinked polyglutamic acid gel of the second aspect, wherein the crosslinked polyglutamic acid gel is encapsulated in the syringe.

Specifically, the syringe includes a syringe barrel having a distal end and a proximal end, a cross-linked polyglutamic acid gel contained therein, a plunger stop slidably positioned within the syringe barrel and providing a seal against the cross-linked polyglutamic acid gel at the proximal end within the syringe barrel, and a closure device coupled to the distal end of the syringe barrel, the closure device having an outlet engagement portion that sealingly engages and closes the distal open outlet end of the syringe barrel. More specifically, the syringe may further comprise a needle.

In particular, the crosslinked polyglutamic acid gel is sterile.

The invention has the beneficial effects that:

(1) According to the invention, one or more of alkyl diamine, aminopolysaccharide and amino acid ester compounds are adopted as a cross-linking agent, the concentration of polyglutamate is adjusted to be 10-20%, the pH of the solution is 5.5-6.5, and under the action of a catalyst, even when a small amount of cross-linking agent is used (the mass ratio of polyglutamate to the cross-linking agent is 1:0.01-0.09), the reaction efficiency can reach more than 80%, the consumption of the cross-linking agent is effectively reduced, and the residue of the cross-linking agent is reduced; and under the condition of using a small amount of cross-linking agent, the polyglutamic acid gel with low loss of elastic modulus and good stability after wet heat sterilization can be obtained.

(2) According to the invention, the amino acid ester compound, such as L-lysine ethyl ester dihydrochloride or L-lysine methyl ester dihydrochloride, and the alkyl diamine form a compound crosslinking agent, wherein the alkyl diamine with a simple molecular chain structure is subjected to crosslinking reaction with polyglutamate, the amino acid ester with a longer molecular chain can be subjected to crosslinking reaction with carboxyl on the polyglutamate which is far away from the alkyl diamine and is not subjected to crosslinking reaction, and the ester bond in the amino acid ester can be partially hydrolyzed along with the change of pH of a reaction system in the crosslinking process, so that the generated carboxyl and amino in the alkyl diamine form intermolecular hydrogen bonds, the synergistic effect of the amino acid ester compound and the alkyl diamine in the crosslinking process is realized, the final crosslinking efficiency is improved, and a more compact three-dimensional network structure is formed; in addition, the amino acid ester cross-linking agent is added, so that the cross-linked polyglutamic acid also contains a part of ester bonds, the part of ester bonds are subjected to hydrolytic cleavage in the wet heat sterilization, and intermolecular hydrogen bonds are formed between generated carboxyl groups and free amino groups, thereby improving the stability of the gel after sterilization and prolonging the degradation time of the gel to a certain extent.

(3) The polyglutamic acid gel obtained by the preparation process disclosed by the invention is safe, nontoxic, resistant to damp and heat sterilization, high in elastic modulus, good in long-term stability, capable of being injected and filled in skin tissues, capable of eliminating the problems of relaxation, skin defect repair and the like of skin caused by aging, and long in degradation time after filling.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

Various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental methods for which specific conditions are not specified in the examples are generally as described in conventional conditions and handbooks, or as suggested by the manufacturer; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

Example 1

4.00g of sodium polyglutamate (molecular weight: 800 kDa) was weighed, then 0.08g (molar number: 365. Mu. Mol) of L-lysine ethyl ester dihydrochloride was added, dissolved in 36mL of water, the pH of the system was adjusted to 6.0 with 6mol/L of sodium hydroxide solution, 0.068g (molar number: 365. Mu. Mol) of EDC and 0.014g of NHS were weighed, added to the solution, stirred uniformly, and placed in a blast drying oven at 40℃for reaction for 12 hours in a sealed manner. After the reaction, 400mL of water is added to fully swell the gel, the gel is homogenized and crushed, the gel is separated from free water, the gel is continuously washed for 5 times by 400mL of purified water, then equal amount of absolute ethyl alcohol is added into the gel, the sediment is separated, and the sediment is placed into a vacuum oven and dried for 24 hours at 40 ℃ under the vacuum degree of minus 0.09 MPa. After complete drying, 1.0g of the dried product is taken, 50mL of phosphate buffer with the pH of 7.0 and the concentration of 10mg/mL is added, after the gel is completely swelled, the gel is filled into a prefilled syringe, and moist heat sterilization is carried out at 121 ℃ for 1,5min, thus obtaining the final product gel.

Example 2

4.00g of potassium polyglutamate (molecular weight: 1,500 kDa) was weighed, 0.32g (molar number: 1373. Mu. Mol) of L-lysine methyl ester dihydrochloride was further added, 16mL of purified water was added, and the mixture was stirred to dissolve completely, the pH of the system was adjusted to 5.5 with 6mol/L of sodium hydroxide solution, 0.3672g (molar number: 1922. Mu. Mol) of EDC and 0.0734g of NHS were weighed, added to the solution, stirred uniformly, and the mixture was placed in a blow drying oven at 25℃for reaction for 18 hours in a sealed manner. The other steps were the same as in example 1.

Example 3

Weighing 4.00g of magnesium polyglutamate (molecular weight 2,000 kDa), adding 0.08g (mol number 1081 mu mol) of 1, 3-propanediamine, adding 26mL of purified water, stirring to dissolve completely, adjusting the pH value of the system to 5.7 by using 6mol/L hydrochloric acid solution, adding 1297 mu mol of HATU into the solution, stirring uniformly, sealing and placing in a blast drying oven at 30 ℃ for reaction for 14h. The other steps were the same as in example 1.

Example 4

Weighing 4.00g of calcium polyglutamate (molecular weight 500 kDa), adding 0.32g (mol number 1116 mu mol) of bis (hexamethyltriamide), adding 26mL of purified water, stirring to dissolve completely, adjusting the pH value of the system to 6.5 by using 6mol/L hydrochloric acid solution, adding 1451 mu mol of HATU into the solution, stirring uniformly, and sealing and placing in a blast drying oven at 40 ℃ for reaction for 16h. The other steps were the same as in example 1.

Example 5

The cross-linking agent is 1, 3-propylene diamine and L-lysine methyl ester dihydrochloride, which are mixed according to the amino molar ratio of 1:0.1, and the mole number of the cross-linking agent is 1081 mu mol. The other steps were the same as in example 3.

Example 6

The cross-linking agent is 1, 3-bis (hexamethyltriamide) and L-lysine ethyl ester dihydrochloride, which are mixed according to the amino mol ratio of 1:0.3, and the mole number of the cross-linking agent is 1116 mu mol. The other steps were the same as in example 4.

Example 7

The cross-linking agent is 1, 3-propylene diamine and L-lysine methyl ester dihydrochloride, which are mixed according to the amino molar ratio of 1:0.5, and the mole number of the cross-linking agent is 1081 mu mol. The other steps were the same as in example 3.

Example 8

The cross-linking agent was chitosan (degree of deacetylation 95%, weight average molecular weight 5,000 Da), and 0.05g of chitosan was added, and the procedure was the same as in example 1.

Comparative example 1

4.00g of sodium polyglutamate (molecular weight: 300 kDa) was weighed, 0.5g (molar number: 2281. Mu. Mol) of L-lysine ethyl ester dihydrochloride was further added, 56mL of purified water was added, and the mixture was stirred to dissolve completely, the pH of the system was adjusted to 5.0 with 6mol/L of sodium hydroxide solution, 0.421g (molar number: 2281. Mu. Mol) of EDC and 0.087g of NHS were weighed, added to the solution, stirred uniformly, and the mixture was placed in a blast drying oven at 40℃for reaction for 12 hours in a sealed manner. The other steps were the same as in example 1.

Comparative example 2

4.00g of magnesium polyglutamate (molecular weight: 2,000 kDa) was weighed, 0.08g (mol: 1081. Mu. Mol) of 1, 3-propanediamine was added to the magnesium polyglutamate solution, 26mL of purified water was added thereto, and stirring was conducted to dissolve it completely, the pH of the system was adjusted to 7.0 with 6mol/L of hydrochloric acid solution, 1297. Mu. Mol of HATU was added to the solution, stirring was conducted uniformly, and the mixture was sealed and placed in a blast drying oven at 30℃for 14 hours of reaction. The other steps were the same as in example 1.

Comparative example 3

4.00g of potassium polyglutamate (molecular weight: 1,500 kDa) was weighed, 0.32g (molar number: 1373. Mu. Mol) of L-lysine methyl ester dihydrochloride was added to the sodium polyglutamate solution, 56mL of purified water was added thereto, and stirring was conducted to dissolve it completely, the pH value of the system was adjusted to 5.5 with 6mol/L of sodium hydroxide solution, 0.3672g (molar number: 1922. Mu. Mol) of EDC and 0.0734g of NHS were weighed, stirred uniformly in the solution, and the mixture was sealed and placed in a blow drying oven at 25℃for 18 hours of reaction. The other steps were the same as in example 1. .

Comparative example 4 (crosslinking method with reference to CN 106730029B)

1.4g of 1, 4-butanediol diglycidyl ether is added into 110g of deionized water, and after being stirred uniformly, 25g of sodium polyglutamate with the weight average molecular weight of 170 ten thousand daltons is added, and the mixture is stirred uniformly and is kept at 100 ℃ for 0.5h; 1.0L of 0.9% NaCl aqueous solution was added to homogenize, and about 0.074g NaH was added to the homogenate ₂ PO ₄ And 0.040g Na ₂ HPO ₄ Adjusting the pH to 6.5; further adding NaCl of about 0.65g to regulate osmotic pressure to 288 mOsm/kg.H ₂ O; centrifuging at 5000r/min for 10min, and collecting gel particles of the precipitation layer; packaging, and sterilizing with steam at 157 deg.C for 3 s.

Comparative example 5 (crosslinking method with reference to CN 109503864B)

6.0g of polylysine was weighed out and dissolved in 100g of 0.2M PBS solution, 8g of polyglutamic acid (molecular weight 800 kDa) was added and stirred and mixed well at 37 ℃. Then adding 6.8g DMTMM, stirring at 37 ℃ for reaction for 1 hour, and then transferring to 2-8 ℃ for reaction for 23 hours. 300g of 0.1M NaOH solution was then added to the mixture to alkalize the mixture, and the mixture was stirred at room temperature for 0.5 hour and at 2 to 8℃for 18 hours. Sedimentation was then performed with 1600mL ethanol. The resulting powder was washed thoroughly with 400mL of ethanol and the washing step was repeated 3 times. After the powder was drained, the powder was reconstituted with 400g of 0.9% NaCl solution, and after thoroughly stirring the solution, the solution was dialyzed in a dialysis bag to give 3000g of 0.9% NaCl solution as an external dialysis solution. Dialysis was performed for a total of 3 days with 2 changes of dialysate per day. After the dialysis was completed, the solution was filled into a glass syringe, and the procedure of example 1 was used for sterilization.

Comparative example 6

The crosslinking agent was 1, 3-propanediamine and L-lysine methyl ester dihydrochloride which were mixed in a 0.1:1 molar ratio of amino groups, the molar number of the crosslinking agent was 1081. Mu. Mol, and the pH was adjusted to 5.7, and the other steps were the same as in example 3.

Comparative example 7

The cross-linking agent is 1, 3-propylene diamine and L-lysine methyl ester dihydrochloride, which are mixed according to the amino molar ratio of 1:0.08, and the mole number of the cross-linking agent is 1081 mu mol. The other steps were the same as in example 3.

Comparative example 8

The cross-linking agent is 1, 3-propylene diamine and L-lysine methyl ester dihydrochloride, which are mixed according to the amino molar ratio of 1:0.6, and the mole number of the cross-linking agent is 1081 mu mol. The other steps were the same as in example 3.

Application example 1: detection of shear viscosity and elastic modulus of crosslinked polyglutamic acid gel

The crosslinked polyglutamic acid gels obtained in examples 1 to 8 and comparative examples 1 to 8 were examined for elastic modulus (G') and shear viscosity (Pa.s) before and after sterilization, respectively, using a TA DHR-2 type flat rheometer.

Elastic modulus detection setting parameters: operating gap: 1000mm, loading gap: 45000 μm, operating temperature: 37 ℃, deformation amount: 1%, frequency: 0.9Hz, run time: 60s.

Detecting shear viscosity (pa·s) detection setting parameters: operating gap: 1000 μm, test mode: flowpeak hold mode, test temperature: 25 ℃, equilibrium time after reaching the specified temperature: 60s, test time: 60s, test rotational speed: 100s ^-1 。

The test results of the samples are shown in Table 1:

TABLE 1 detection results of elastic modulus and shear viscosity of gel samples

As can be seen from Table 1, examples 1 to 8 have very high elastic modulus after sterilization, the variation of the elastic modulus is within 10%, and the elastic modulus and the shear viscosity of the sample remain stable after sterilization for 30 days, wherein the variation of the elastic modulus of examples 5 to 7 after sterilization is about 2%, and the elastic modulus and the shear viscosity of the sample remain stable after sterilization for 30 days, which means that the L-lysine ethyl ester dihydrochloride or L-lysine methyl ester dihydrochloride and the alkyl diamine form a compound cross-linking agent, wherein the ester bond in the L-lysine ethyl ester dihydrochloride or L-lysine methyl ester dihydrochloride can be partially hydrolyzed along with the pH change of the reaction system during cross-linking, and the generated carboxyl and amino in the alkyl diamine form intermolecular hydrogen bonds, so that the synergistic effect of the amino acid ester compound and the alkyl diamine during cross-linking is realized, the final cross-linking efficiency is improved, a more three-dimensional structure is formed, and therefore, the thermal network elasticity modulus is low, the thermal stability is high, and the thermal stability is lost after sterilization is high; the decrease in elastic modulus after sterilization is remarkable in comparative example 1, comparative example 2 and comparative example 3, which is probably due to the fact that the pH value of the reaction system and the polyglutamic acid concentration are not in the proper reaction conditions, so that the crosslinking efficiency is low, and the finally obtained gel has low crosslinking degree, so that the elastic modulus after sterilization is greatly changed. Comparative example 4 shows a large change in elastic modulus after sterilization, indicating poor heat resistance of the final gel; comparative example 5 the elastic modulus of the gel changed by more than 50% before and after sterilization, and the shear viscosity increased after 30 days of storage after sterilization. The loss of the elastic modulus after the wet heat sterilization is 9 percent and 7.5 percent of the elastic modulus after the wet heat sterilization is far higher than the loss rate of 2 percent in the embodiment 5, which is probably due to the fact that the amount of the L-lysine methyl ester dihydrochloride in the 1, 3-propane diamine and L-lysine methyl ester dihydrochloride compound cross-linking agent is excessive, the cross-linking gel is mainly formed by connecting carboxyl groups on polyglutamate molecules through the L-lysine methyl ester dihydrochloride, a small amount of the 1, 3-propane diamine is connected with the carboxyl groups on polyglutamate structural units to form amide bonds, so that amino groups in the 1, 3-propane diamine can not form hydrogen bonds with the carboxyl groups after the ester group hydrolysis in the L-lysine methyl ester dihydrochloride, the cross-linked polyglutamate skeleton structure is relatively loose, and the loss rate of the elastic modulus after the wet heat sterilization is higher. In addition, in comparative example 7, the loss of elastic modulus after the wet heat sterilization was also significantly higher than that of example 5, probably because the amount of L-lysine methyl ester dihydrochloride in the composite crosslinking agent was low, and it was difficult to form an effective synergistic effect with 1, 3-propanediamine, so that the loss rate of elastic modulus after the wet heat sterilization was high. Further, the compound cross-linking agent is formed by alkyl diamine and amino acid ester compound, and the proportion of the compound cross-linking agent and the amino acid ester compound is set in a proper range, so that the synergistic effect of the compound cross-linking agent and the amino acid ester compound can be effectively improved, and a gel product with high cross-linking efficiency and more stable structure is formed.

Application example 2: detection of crosslinking efficiency

Preparation of test solution:

taking 2.0g of each of the gel of the example and the gel of the comparative example, adding 10mL of 0.5mol/L sulfuric acid solution into each sample, placing the mixture in a blast drying oven at 90+/-10 ℃ for 60-70 min, adding 1mol/L sodium hydroxide solution for neutralization, and fixing the volume to 50mL for later use.

In addition, a polyglutamic acid solution of 20mg/mL is prepared, 2.0mL is taken and treated in the same way, and the polyglutamic acid solution is used as a blank of a test sample for standby.

Preparing a standard substance solution:

an appropriate amount of the crosslinking agent used in examples and comparative examples was weighed and dissolved in distilled water to a concentration of 50. Mu.g/mL.

And (3) detection: diluting the standard solution to a series of concentrations of 0.5-50 mug/mL, taking 1mL of the standard solution, adding 0.2mol/L citric acid buffer solution (pH 5.0) to 1mL of the solution for thorough mixing, then adding 1mL of KCN-glycol methyl ether-ninhydrin solution (1.25 g of recrystallized ninhydrin is dissolved in 25mL of redistilled glycol methyl ether to form a 5% solution, 1.5mL of 10mmol/L KCN solution is diluted to 125mL of solution for thorough mixing, then 125mL of KCN-glycol methyl ether is dissolved in 25mL of ninhydrin-glycol methyl ether solution for thorough mixing, heating in a boiling water bath for 15min, taking out and adding 3mL of 60% ethanol for dilution, then cooling, measuring absorbance at a wavelength of 570nm, detecting the sample in the same way, drawing an absorbance-concentration standard curve, obtaining the content c (mug/mL) of a crosslinking agent in the sample from the standard curve, calculating the actual crosslinking molar ratio m according to the formula (I), and then calculating the crosslinking efficiency e according to the formula (II).

In formula (I):

m is the actual crosslinking mole ratio;

n ₁ the unit is mole (mol) of the crosslinking agent added;

c is the content of amino in the test sample, and the unit is micrograms per milliliter (mug/ml);

v is the dilution volume of the test sample in milliliters (ml);

m is the molecular weight of the cross-linking agent;

n ₃ the unit is mole (mol) of carboxyl in polyglutamate in the crosslinking reaction.

In formula (II):

e is the crosslinking efficiency;

m is the actual crosslinking mole ratio;

n is the theoretical crosslinking mole ratio, n=n ₁ /n ₃ *100。

The test results of the samples are shown in Table 2:

TABLE 2 detection results of gel sample crosslinking efficiency

Examples	Theoretical crosslinking molar ratio	Actual crosslinking molar ratio	Crosslinking efficiency
				Example 1	1.34％	1.13％	84％
Example 2	5.05％	4.29％	85％
				Example 3	3.97％	3.65％	92％
Example 4	4.10％	3.81％	93％
				Example 5	3.97％	3.75％	94.5％
Example 6	3.97％	3.78％	95.1％
				Example 7	3.97％	3.70％	93.1％
Example 8	1.06％	0.83％	78.3％
				Comparative example 1	7.36％	2.84％	39％
Comparative example 2	3.97％	1.19％	30％
				Comparative example 3	5.05％	2.02％	40％
Comparative example 4 ^*	22.36％	10.06％	45％
				Comparative example 5	78.75％	15.75％	20％
Comparative example 6	3.97％	3.38％	85％
				Comparative example 7	3.97％	3.45％	87％
Comparative example 8	3.97％	3.34％	84％

* The method for detecting the crosslinking efficiency of this sample is different from other examples and comparative examples, and the specific method is as follows:

sample preparation: taking 4.0g of a sample of comparative example 4 before sterilization, precisely weighing, freeze-drying in a freeze dryer, adding 10ml of acetone, shaking, performing ultrasonic treatment for 1h (the control temperature is not more than 25 ℃), taking a proper amount of supernatant, filtering a membrane, taking a proper amount of subsequent filtrate as a sample solution, taking a proper amount of 1, 4-butanediol diglycidyl ether (BDDE) reference substance, precisely weighing, adding acetone to prepare a solution containing 0.8-50 mug of acetone in each 1ml as a reference substance solution.

Chromatographic conditions: chromatographic column: DB-17 column (30 m x 0.32mm,0.50 μm); column temperature: heating to a temperature of 150 ℃ at 30 ℃ per minute to 260 ℃ for 10 minutes; the detector is a hydrogen Flame Ionization Detector (FID) with a detector temperature of 300 ℃; the temperature of the sample inlet is 260 ℃; split sample introduction, wherein the split ratio is 1:1; the carrier gas is nitrogen, the flow rate of the column is 5ml/min, and the purging flow rate of the spacer is 10ml/min; the sample volume was 1. Mu.l.

The detection method comprises the following steps: precisely measuring 1 μl of each of the sample solution and the reference solution, injecting into gas chromatograph, recording chromatogram, drawing standard curve of peak area-concentration, and finding the content c of the crosslinking agent in the sample from the standard curve ₁ (μg/ml) the actual molar crosslinking ratio m is calculated by the following formula (III):

in formula (III):

m is the actual crosslinking mole ratio;

n1 is the mole number of the adding amount of the cross-linking agent, and the unit is mole (mol);

c ₁ for the BDDE content of the test sample, the unit is micrograms per milliliter (mug/ml);

m is the molecular weight of the crosslinking agent BDDE;

As can be seen from Table 2, examples 1 to 8 have a much higher crosslinking efficiency than comparative examples 1 to 8, which is a key to keeping the elastic modulus of the gel stable before and after sterilization, while comparative example 5 has a low crosslinking efficiency, indicating that a large amount of free amino groups are present, which may be caused by a low degree of reaction of polylysine on the one hand, and by cleavage of the molecular weight of polyglutamic acid by addition of sodium hydroxide solution on the other hand. In comparative examples 1 to 3, however, the pH value and polyglutamic acid concentration of the reaction system were not within the appropriate reaction conditions, resulting in low crosslinking efficiency; in comparative examples 6 to 8, since the ratio of the alkyl diamine to the L-lysine methyl ester dihydrochloride in the compound crosslinking agent is not set in a proper range, the synergistic effect of the alkyl diamine and the L-lysine methyl ester dihydrochloride is difficult to achieve, the crosslinking efficiency is obviously lower than that of example 5, and the crosslinking agent is further proved to have an obvious positive effect on improving the crosslinking efficiency by reasonably controlling the reaction condition of the system.

Application example 3: in vitro degradation characterization

Taking appropriate amounts of samples of examples 1-8 and comparative examples 1-8 respectively, freeze-drying in a vacuum freeze dryer, weighing 0.2g of the freeze-dried samples, precisely weighing, marking as m1, soaking in 50ml of PBS solution, taking out the samples in a shaking table at 37 ℃ on the 7 th day, 14 th day, 21 th day, 28 th day, 35 th day, 42 th day and 49 th day respectively, soaking in deionized water until the residual PBS solution is completely washed out, weighing after freeze-drying, marking as m2, calculating degradation rate according to the formula (m 1-m 2)/m 1 x 100%, preparing 3 parts of each sample in parallel, and calculating the average value of the degradation rate. The detection results are shown in FIG. 1.

As can be seen from fig. 1 of in vitro degradation, the degradation time of the example is longer than that of the sample of the comparative example, the degradation rate is lower within the same time range, and in particular, in examples 5 to 7, only about 60% of degradation occurs in the whole degradation period, which is probably due to the fact that after the combination of the alkyl diamine and the amino acid ester cross-linking agent, the alkyl diamine cross-linking agent with a simple molecular structure is connected with the adjacent polyglutamic acid molecular chain, and meanwhile, the L-lysine methyl ester dihydrochloride or L-lysine ethyl ester dihydrochloride with a larger molecular structure is combined with the structural units which are far apart, and hydrogen bond interaction exists between the alkyl diamine and the amino acid ester, so that a more compact cross-linked network structure is formed, and the longer in vitro degradation time is more favorably maintained.

In summary, the gel disclosed by the invention is used as a skin filling product, the gel simulates polypeptide components of natural extracellular matrix, promotes cell adhesion and growth, reduces the addition amount of a cross-linking agent, increases the removal process of the cross-linking agent and a catalyst, improves the biocompatibility of the gel, reduces the safety risk of injection use, ensures that the gel maintains the particle state of the cross-linked gel through moist heat sterilization, still has a certain elastic modulus, ensures the maintenance time of filling effect, and simultaneously provides better support and compression resistance for tissue filling due to the stability of performance and higher elastic modulus.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.

Claims

1. A method for preparing a crosslinked polyglutamic acid gel, comprising the steps of:

(2) Adding a catalyst into the solution obtained in the step (1) to perform a reaction;

preferably, the cross-linking agent in step (1) is selected from: one or more of alkyl diamine, aminopolysaccharide and amino acid ester compounds;

preferably, the mass ratio of polyglutamate to the cross-linking agent in the step (1) is 1:0.01-0.09;

preferably, the mass percentage of polyglutamate in the solution in the step (1) is 10-20%;

preferably, the step of adjusting the pH in the step (1) is to adjust the pH to 5.5-6.5;

preferably, the reaction temperature in step (2) is 25-40 ℃.

2. The method of claim 1, wherein the polyglutamate in step (1) is selected from the group consisting of: one or more of sodium polyglutamate, potassium polyglutamate, calcium polyglutamate, and magnesium polyglutamate;

preferably, the molecular weight of the polyglutamate is 500-2000kDa.

3. The method of claim 1, wherein the cross-linking agent in step (1) is selected from the group consisting of: one or more of an alkyl diamine and an amino acid ester compound, preferably a combination of an alkyl diamine and an amino acid ester compound;

preferably, the alkyl diamine is selected from: 1, 3-propanediamine, 1, 4-butanediamine, 1, 6-hexanediamine, bis-hexamethyltriamine;

preferably, the amino acid ester compound is L-lysine ethyl ester dihydrochloride or L-lysine methyl ester dihydrochloride;

more preferably, the crosslinking agent in step (1) is a combination of an alkyl diamine and an amino acid ester compound, wherein the molar ratio of the amino groups of the alkyl diamine and the amino acid ester compound is 1:0.1-0.5, preferably 1:0.1-0.3.

4. The process according to claim 1, wherein in step (2) the catalyst is a carbonium salt, preferably O- (7-azabenzotriazol-1-yl) -di (dimethylamino) carbonium Hexafluorophosphate (HATU), O- (benzotriazol-1-yl) -di (dimethylamino) carbonium Hexafluorophosphate (HBTU); or alternatively, the first and second heat exchangers may be,

the catalyst in the step (2) is 1- (3-dimethylaminopropyl) -3-ylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS).

5. The method of claim 1, wherein the molar ratio of catalyst to crosslinker is 1-1.4:1.

6. The method of manufacture of claim 1, further comprising step (3): swelling the gel obtained by the reaction in the step (2), crushing, washing, separating and drying.

7. The method of manufacture of claim 1, further comprising step (4): redissolving the dried product obtained in the step (3), and sterilizing;

preferably, the pH value of the solution obtained by the redissolution is 7.2+/-2;

preferably, the osmotic pressure of the solution obtained by the reconstitution is 280-350mOsm/L;

preferably, the sterilization step comprises: and filling the re-dissolved solution into a pre-filled syringe for damp-heat sterilization.

8. A crosslinked polyglutamic acid gel prepared by the method of any one of claims 1-7.

9. A tissue-filling product comprising a syringe and the crosslinked polyglutamic acid gel of claim 8, wherein the crosslinked polyglutamic acid gel is potted in the syringe.

10. The use of the crosslinked polyglutamic acid gel according to claim 8 for preparing tissue fillers, tissue engineering scaffolds, wound repair materials, drug carriers, cosmetics, food stabilizers, sewage treatment agents and water-retaining agents;

preferably, the use is the use of the crosslinked polyglutamic acid gel in the preparation of a tissue filler.