A high-viscoelastic cross-linked biopolysaccharide gel and its preparation method and application

Abstract

The present invention relates to the technical field of polysaccharide material modification, and in particular to a high-viscoelastic cross-linked biopolysaccharide gel and a preparation method and application thereof, wherein a biopolysaccharide containing a carboxyl structure and a diaminodiketopiperazine compound cross-linking agent are dissolved and mixed uniformly, and then a cross-linking reaction is generated under the action of a condensing agent to obtain a high-viscoelastic cross-linked biopolysaccharide gel; the obtained high-viscoelastic cross-linked biopolysaccharide gel can meet application conditions after wet heat sterilization; it is used to prepare an internal injection preparation, an internal filling preparation, or an external drug carrier preparation; the cross-linked biopolysaccharide gel prepared by the present invention has a large downward exploration space in terms of viscoelasticity data for wet heat sterilization, which can ensure that the product is sterile and has good viscoelastic properties, and has low cytotoxicity, high product purity and a long service life.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide material modification, and in particular to a high-viscoelastic cross-linked biopolysaccharide gel and a preparation method and application thereof. Background Art

[0002] In recent years, the number of osteoarthritis patients has increased year by year, posing a huge threat to the health of the general public. The viscoelastic supplementation treatment program, which is consistent with the physiological characteristics of the knee joint, has been widely recognized by clinical professionals in a variety of mainstream knee osteoarthritis treatment programs for its advantages in improving joint function and solving pain. In recent years, the application of sodium hyaluronate (also known as hyaluronic acid, HA) for injection viscoelastic supplementation treatment has become a mature treatment option for early and middle-stage arthritis. However, natural sodium hyaluronate is easily biodegraded and has a very short residence time in the body, which makes the efficacy of sodium hyaluronate injection preparations short-lived. Generally, the product requires 5 injections, and the efficacy is generally 3-6 months, which increases the discomfort of patients taking the medicine. In addition, as an endogenous component of the body, sodium hyaluronate has a wide range of applications in the field of medical beauty and surgical adhesion prevention, but it also faces the problem of rapid degradation in the body.

[0003] Cross-linked sodium hyaluronate uses one or more combined cross-linking agents to react with the carboxyl or hydroxyl groups on sodium hyaluronate to form a network structure between sodium hyaluronate molecules, thereby improving its mechanical strength and in vitro and in vivo stability. In recent years, cross-linked sodium hyaluronate products have made great progress in clinical applications. At present, most domestic and foreign products use divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol diglycidyl ether (PEGDE) as cross-linking agents. These cross-linking agents contain chemically active structural units such as epoxy or double bonds, which can covalently bind to functional proteins and nucleic acids in the body, thereby affecting their normal functions. They have high potential cytotoxicity and poor biocompatibility. In addition, the residues of these cross-linking agents themselves or their degradation products can also cause inflammatory reactions in the body. For example, data show that the incidence of adverse reactions such as hypersensitivity, thrombocytopenia, and systemic adverse events caused by DVS cross-linked products is 7.2%, which is 12 times that of uncross-linked products (such as Alzhi, with an incidence of 0.52%), which greatly limits its development and application. Compared with BDDE or DVS, lysine derivatives are safer, but these cross-linking processes either require the reaction with organic salts of sodium hyaluronate in organic solvents to prepare, or it is difficult to avoid further polymerization of polylysine itself and introduce new polymer impurities into the sodium hyaluronate gel. In addition, existing products often have insufficient viscoelasticity after wet heat sterilization, which leads to a short service life (i.e., short efficacy). Therefore, the development of a cross-linking agent with safe materials, efficient cross-linking, and long degradation cycle has become the development direction of cross-linked hyaluronic acid. Summary of the invention

[0004] In order to solve the technical problems of high impurities and short efficacy of cross-linked biopolysaccharide gel in the prior art, a high viscoelastic cross-linked biopolysaccharide gel and its preparation method and application are provided. The diaminodiketopiperazine compound of the present invention is used as a cross-linking agent to cross-link biopolysaccharides. The product of the present invention has the advantages of superior physical and chemical properties, clear cross-linking process, high safety, strong anti-degradation ability, high stability, uniform granulation, etc., and has the advantages of small dosage per course of treatment, long maintenance time of efficacy and good effect in clinical use, and has broad application prospects in the fields of tissue filling, postoperative adhesion prevention and osteoarthritis treatment.

[0005] In order to achieve the above objectives, the present invention is implemented by the following technical solutions:

[0006] A method for preparing a highly viscoelastic cross-linked biopolysaccharide gel comprises the following steps:

[0007] After the biopolysaccharide containing a carboxyl structure and the diaminodiketopiperazine compound cross-linking agent are dissolved and mixed evenly, a condensation agent is added to form a reaction solution, and a cross-linking reaction is generated to obtain a high-viscoelastic cross-linked biopolysaccharide gel.

[0008] Furthermore, the method specifically includes the following steps:

[0009] S1, dissolving one of the carboxyl-containing biopolysaccharide or diaminodiketopiperazine cross-linking agent in a buffer solution, then adding the other compound, and mixing them evenly to form a mixed solution;

[0010] That is, the biopolysaccharide containing a carboxyl structure is dissolved in a buffer solution, a diaminodiketopiperazine compound cross-linking agent is added, and the mixture is mixed evenly to form a mixed solution; or the diaminodiketopiperazine compound is dissolved in a buffer solution, and then the biopolysaccharide is added, and the mixture is mixed evenly to form a mixed solution; the other compound can be directly mixed with the former to form a mixed solution, or can be mixed evenly with the buffer solution to form a mixed solution;

[0011] S2, dissolving the condensing agent in a buffer solution, then adding the condensing agent into the mixed solution to form a reaction solution, and performing a cross-linking reaction. After the reaction is completed, a highly viscoelastic cross-linked biopolysaccharide gel is obtained.

[0012] Furthermore, the diaminodiketopiperazine compound cross-linking agent has the following chemical structure:

[0013] ,

[0014] wherein R is a normal alkyl group or an isomeric alkyl group having 10 or less carbon atoms.

[0015] Preferably, the diaminodiketopiperazine compound crosslinking agent is selected from one or more of 3,6-di(4-aminobutyl)-2,5-diketopiperazine or its salt, 3,6-di(3-aminopropyl)-2,5-diketopiperazine or its salt, such as the hydrochloride, sulfate, nitrate and the like.

[0016] Furthermore, the purity of the diaminodiketopiperazine compound crosslinking agent is at least 99 wt %, and the amount of the diaminodiketopiperazine compound crosslinking agent is 1%-50%, preferably 5%-20%, and more preferably 5%-15% of the molar amount of the repeating unit of the biopolysaccharide;

[0017] Furthermore, the molecular weight of the biopolysaccharide is 0.5MDa-10MDa, preferably 0.5MDa-2.0MDa; the biopolysaccharide is selected from one or more of hyaluronic acid or its salt, alginic acid or its salt, carboxymethyl chitin or its salt, carboxymethyl chitosan or its salt, carboxymethyl cellulose or its salt, and chondroitin compounds; the mass fraction of the biopolysaccharide in the reaction solution is 0.5%-10%, preferably 2%-6%. The swelling ratio of the cross-linked biopolysaccharide gel obtained is different due to the different concentration of the biopolysaccharide in the reaction solution, which further affects the content of the biopolysaccharide in the gel, that is, the swelling ratio of the present invention is controllable, and the biopolysaccharide content in the final product can be controlled by adjusting the biopolysaccharide concentration of the initial reaction solution;

[0018] The buffer is one of a phosphate buffer system and a phosphate buffer system / sodium chloride solution, and the pH of the buffer is in the range of 6.0-8.5, preferably the pH of the buffer is in the range of 6.4-8.2;

[0019] The condensing agent is selected from one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine tetrafluoroborate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, and 2-chloro-1-methylpyridinium iodide, and the molar amount of the condensing agent is 100%-300% of the molar amount of the diaminodiketopiperazine compound cross-linking agent.

[0020] Furthermore, the method also includes swelling and rinsing the highly viscoelastic cross-linked biopolysaccharide gel with a buffered saline solution after obtaining the gel, followed by crushing, filling, and wet heat sterilization to obtain a finished product.

[0021] Furthermore, the cross-linking reaction temperature is in the range of 0°C-30°C, preferably 10°C-25°C; the cross-linking reaction time is in the range of 10h-80h, preferably 24h-72h;

[0022] The wet heat sterilization conditions are: sterilization at 121°C for 10min-40min;

[0023] The amount of the buffer used for swelling or rinsing is 3 to 10 times the weight of the reaction product obtained after the cross-linking reaction (i.e., the required weight of the entire system after the cross-linking reaction), and the rinsing time is 4 to 96 hours, preferably 8 to 24 hours.

[0024] On the other hand, the present invention provides a high-viscoelastic cross-linked biopolysaccharide gel obtained by the above preparation method, wherein the elastic modulus is 200Pa-5000Pa and the viscosity modulus is 50Pa-500Pa before moist heat sterilization; the elastic modulus is 100Pa-400Pa and the viscosity modulus is 30Pa-90Pa after moist heat sterilization; the biopolysaccharide content is 5mg / mL-25mg / mL, preferably 10 mg / mL-20mg / mL.

[0025] The last aspect of the present invention provides the use of the highly viscoelastic cross-linked biopolysaccharide gel obtained by the above preparation method in the preparation of internal injection preparations, internal filling preparations, or external drug carrier preparations.

[0026] Beneficial technical effects:

[0027] The present invention adopts diaminodiketopiperazine compounds as cross-linking agents, and cross-links the biopolysaccharide to a certain extent under the action of a condensing agent. Compared with commercially available products, the prepared cross-linked biopolysaccharide gel has more uniform and delicate particles and smaller particle size, and the product has low cytotoxicity, high safety, good tissue compatibility, strong anti-degradation ability, high stability, and high viscoelasticity. When used, it has the advantages of small dosage per course of treatment, long maintenance time of therapeutic effect and good effect; at the same time, the production process of the present invention is easy to control, the product swelling ratio is controllable, the rinsing effect is good, and less impurity residues; the production equipment required for the production of the present invention is simple, which is conducive to large-scale production; the product of the present invention has broad application prospects in the fields of tissue filling, postoperative adhesion prevention, and osteoarthritis treatment;

[0028] (1) The present invention is the first to use diamino-containing 2,5-diketopiperazine compounds and their acid salts as crosslinking agents with stable structure, high purity and high safety, which are safer than crosslinking agents such as DVS, BDDE and PEGDE;

[0029] (2) The prepared cross-linked biopolysaccharide is covalently bonded to the carboxyl groups of the biopolysaccharide through amide bonds at the two amino groups at both ends of the cross-linker. Since the reactivity of the amino groups on the cross-linker is much higher than the reactivity of the hydroxyl groups of the biopolysaccharide itself, ineffective cross-linking of the cross-linker and self-cross-linking of the biopolysaccharide can be avoided. At the same time, there is no risk of self-polymerization of the polylysine cross-linker, and the cross-linking process is clear, definite and controllable.

[0030] (3) In the preparation process of the cross-linked biopolysaccharide gel with high viscoelasticity of the present invention, all process steps including material dissolution, cross-linking reaction, product washing and purification, swelling, etc. are carried out in water. This is due to the good water solubility of materials such as cross-linking agents, condensation agents and their degradation products, which can be purified in water, making the entire preparation process green and environmentally friendly, and conducive to commercial production;

[0031] (4) The swelling ratio of the cross-linking process of the present invention is controllable, and the obtained cross-linked biopolysaccharide gel has a low impurity content, a high viscoelastic modulus, uniform particles, and high thermal stability. It can withstand sufficient terminal sterilization to ensure the sterility of the product, thereby improving the safety of the product. In addition, the cross-linked biopolysaccharide gel of the present invention has stronger resistance to enzymatic hydrolysis, and when used, it can effectively prolong its retention time in the body, thereby prolonging the efficacy time, thereby reducing the frequency of administration and improving patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The graph is a comparison of the particle sizes of the high-viscoelastic cross-linked biopolysaccharide gel of the present invention before and after sterilization and the commercially available product, butylene glycol cross-linked sodium hyaluronate injection. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention will be described clearly and completely below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0034] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. The technology and method known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology and method should be considered as a part of the specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments can have different values. In addition, being recorded as "within the scope" is usually considered as not including endpoint values.

[0035] The experimental methods in the following examples without specifying specific conditions are usually measured in accordance with national standards; if there is no corresponding national standard, they are carried out in accordance with general standard requirements or general methods.

[0036] Note: The molecular weight of a polymer compound usually refers to the number average molecular weight.

[0037] Example 1

[0038] This case is the preparation of the cross-linking agent 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride:

[0039] 100g of 2-amino-6-(trifluoroacetylamino)hexanoic acid (CAS 10009-20-8), 17.5g of phosphorus pentoxide, and 206mL of N-methylpyrrolidone were placed in a reaction vessel, reacted at 165°C for 2 hours for ring-closing reaction, and then the reaction solution was poured into purified water to precipitate solids, which were filtered and dried to obtain an intermediate. Subsequently, the intermediate was placed in 270mL of ammonia water (mass fraction 19%) and reacted at 50°C for 22 hours for detrifluoroacetyl reaction to obtain a crude product of 3,6-di(4-aminobutyl)-2,5-diketopiperazine. The crude product was dissolved in 60mL of 1M dilute hydrochloric acid, and 600mL of tetrahydrofuran was added dropwise for crystallization to obtain 30g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride (molecular weight 329.27g / mol), with an HPLC purity of 99%.

[0040] The product was tested by NMR carbon spectrum and hydrogen spectrum, and the results are as follows:

[0041] 13 CNMR (101 MHz, D2O) δ: 170.28, 53.98, 39.16, 31.76, 26.41, 20.17;

[0042] 1 HNMR (400 MHz, D2O) δ: 4.25(t, 2H), 3.01(t, 4H), 1.99-1.77(m, 4H), 1.74-1.62(m, 4H), 1.54-1.28(m, 4H).

[0043] Example 2

[0044] This case is the preparation of the cross-linking agent 3,6-bis(3-aminopropyl)-2,5-diketopiperazine hydrochloride:

[0045] 100g of 2-amino-5-(trifluoroacetylamino)pentanoic acid (CAS 5123-49-9), 17.2g of phosphorus pentoxide, and 200mL of N-methylpyrrolidone were placed in a reaction vessel, reacted at 165°C for 2 hours for ring-closing reaction, and then the reaction solution was poured into purified water to precipitate solids, which were filtered and dried to obtain an intermediate. Subsequently, the intermediate was placed in 250mL of ammonia water (mass fraction 19%) and reacted at 50°C for 20 hours for detrifluoroacetyl reaction to obtain a crude product of 3,6-di(3-aminopropyl)-2,5-diketopiperazine. The crude product was dissolved in 60mL of 1M dilute hydrochloric acid, and 600mL of tetrahydrofuran was added dropwise for crystallization to obtain 25g of 3,6-di(3-aminopropyl)-2,5-diketopiperazine hydrochloride (molecular weight 301.21g / mol), with an HPLC purity of 99%.

[0046] The product was tested by nuclear magnetic resonance hydrogen spectrum, and the results were as follows:

[0047] 1 HNMR (400 MHz, D2O) δ: 4.17(t, 2H), 3.05(t, 4H), 1.96-1.71(m, 4H), 1.78-1.65(m, 4H).

[0048] Example 3

[0049] This case is a cytotoxicity test on the products of Example 1 and Example 2.

[0050] 3,6-bis(4-aminobutyl)-2,5-diketopiperazine hydrochloride (hereinafter referred to as compound a) and 3,6-bis(3-aminopropyl)-2,5-diketopiperazine hydrochloride (hereinafter referred to as compound b) were respectively prepared into samples of different concentrations as shown in Table 1 below (the solvent was phosphate buffer solution), and incubated with human primary chondrocytes for a certain period of time, and then the chondrocyte viability was detected. Specifically: When the human primary chondrocytes grow to a good exponential growth phase, the viable cells are counted using a cell counter. The cell suspension is adjusted to an appropriate cell concentration using the corresponding culture medium. 100 μL of cell suspension is added to each well of a 96-well cell culture plate, 5000 cells / well, 3 replicates, and cultured overnight (about 24 hours) in a 37°C, 5% CO2 incubator. The next day, after the cells adhered to the wall, the culture medium supernatant was gently aspirated, and 120 μL of the test compound of different concentrations prepared with the culture medium was added to each group. After incubation for a certain period of time, the cell viability was detected.

[0051] Cell viability was determined by chemiluminescence to measure the ATP content to reflect the number of living cells. According to the operating instructions of the kit, each well was added with a reagent that had been melted and equilibrated to room temperature (equilibrium time was about 30 min). The mixture was shaken at room temperature for 2 minutes to fully induce cell lysis, and the fluorescence signal value was measured with a multifunctional microplate reader.

[0052] Calculation of cell survival rate: Cell survival rate = each experimental well (RLU value) / blank control group (average RLU value) × 100%.

[0053] The results are shown in Table 1.

[0054] Table 1 Cytotoxicity results of cross-linker compounds

[0055]

[0056] As shown in Table 1, under the experimental conditions far exceeding the crosslinker concentration in the gel preparation, both crosslinkers showed no significant inhibitory effect on chondrocyte proliferation, that is, the crosslinker used in the present invention has the advantages of low cytotoxicity and high safety.

[0057] Example 4

[0058] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0059] S1. Weigh 4.0125 g of sodium hyaluronate (molecular weight 0.8 MDa), add 80.14 g of phosphate buffer system / sodium chloride solution (pH = 6.81), and stir for 24 hours;

[0060] Weigh 0.1653 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add phosphate buffer system / sodium chloride solution (pH= 6.81) to a total weight of 10.0313 g, and dissolve evenly;

[0061] The two were stirred and mixed for 30 min to form a mixed solution;

[0062] S2. Weigh 0.4972 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add phosphate buffer system / sodium chloride solution (pH= 6.81) to a total weight of 10.0231 g, and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to form a reaction solution, and then stir at 20° C. for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0063] S3. Take 56.30 g of the reaction product after the cross-linking reaction of S2 is completed, add phosphate buffer system / sodium chloride solution (pH= 6.81) to it to a total weight of 530.48 g, and stir and wash for 8 hours; filter through a sieve, drain and crush the retained gel to obtain the product, detect the elastic modulus G' and viscous modulus G'' of the product at a frequency of 2.5 Hz, and calculate the swelling ratio and the final concentration of the product.

[0064] Example 5

[0065] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0066] S1. Weigh 2.0087 g of sodium hyaluronate (molecular weight 0.6 MDa), add 78.00 g of phosphate buffer system / sodium chloride solution (pH = 6.73), and stir for 24 hours;

[0067] Weigh 0.3140 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add phosphate buffer system / sodium chloride solution (pH= 6.73) to a total weight of 10.0106 g, and dissolve evenly;

[0068] The two were stirred and mixed for 30 min to form a mixed solution;

[0069] S2. Weigh 0.9958 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add phosphate buffer system / sodium chloride solution (pH= 6.73) to a total weight of 10.0592 g, and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to form a reaction solution, and then stir at 15° C. for a cross-linking reaction for 48 hours to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0070] S3. Take 42.22 g of the reaction product after the cross-linking reaction of S2 is completed, add phosphate buffer system / sodium chloride solution (pH= 6.73) to it to a total weight of 248.94 g, and stir and wash for 24 hours; filter through a sieve, drain and crush the retained gel to obtain the product, detect the elastic modulus G' and viscous modulus G'' of the product at a frequency of 2.5 Hz, and calculate the swelling ratio and the final concentration of the product.

[0071] Example 6

[0072] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0073] S1. Weigh 4.0126 g of sodium hyaluronate (molecular weight 0.8 MDa), add 156.00 g of phosphate buffer system / sodium chloride solution (pH=7.21), and stir for 24 hours;

[0074] Weigh 0.1599 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add phosphate buffer system / sodium chloride solution (pH= 7.21) to a total weight of 20.0344 g, and dissolve evenly;

[0075] The two were stirred and mixed for 30 min to form a mixed solution;

[0076] S2, weighing 0.4981 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, adding a phosphate buffer system / sodium chloride solution (pH=7.21) to a total weight of 20.0238 g, and dissolving evenly to form a condensing agent solution; filtering the condensing agent solution, adding it to the mixed solution, and stirring for about 30 minutes to mix to form a reaction solution, and then stirring at 25° C. for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0077] S3. The reaction product after the cross-linking reaction in S2 was added to a phosphate buffer system / sodium chloride solution (pH=7.21) for dilution. No obvious block gel was filtered out, indicating that the combination of sodium hyaluronate concentration and cross-linking agent concentration in this case could not produce a gel product that could be rinsed and filtered.

[0078] Example 7

[0079] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0080] S1. Weigh 3.0035 g of sodium hyaluronate (molecular weight 1.0 MDa), add 80.47 g of phosphate buffer system / sodium chloride solution (pH = 7.70), and stir and mix for 24 hours;

[0081] Weigh 0.2462 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add phosphate buffer system / sodium chloride solution (pH= 7.70) to a total weight of 10.0171 g, and dissolve evenly;

[0082] The two were stirred and mixed for 30 min to form a mixed solution;

[0083] S2. Weigh 0.7482 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add phosphate buffer system / sodium chloride solution (pH= 7.70) to a total weight of 10.0008 g, and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to form a reaction solution, and then stir at 5°C for a cross-linking reaction for 72 hours to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0084] S3. Take 53.78 g of the reaction product after the cross-linking reaction of S2 is completed, add phosphate buffer system / sodium chloride solution (pH= 7.70) to it to a total weight of 253.34 g, and stir and wash for 16 hours; filter through a sieve, drain and crush the retained gel to obtain the product, detect the elastic modulus G' and viscous modulus G'' of the product at a frequency of 2.5 Hz, and calculate the swelling ratio and the final concentration of the product.

[0085] Example 8

[0086] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0087] S1. Weigh 5.0058 g of sodium hyaluronate (molecular weight 0.8 MDa), add 80.08 g of phosphate buffer system / sodium chloride solution (pH = 7.96), and stir for 24 hours;

[0088] Weigh 0.2069 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add phosphate buffer system / sodium chloride solution (pH= 7.96) to a total weight of 10.0155 g, and dissolve evenly;

[0089] The two were stirred and mixed for 30 min to form a mixed solution;

[0090] S2. Weigh 0.3593 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 7084-11-9), add phosphate buffer system / sodium chloride solution (pH=7.96) to a total weight of 10.0438 g, and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to form a reaction solution, and then stir at 25° C. for a cross-linking reaction for 72 hours to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0091] S3. Take 52.71 g of the reaction product after the cross-linking reaction of S2 is completed, add phosphate buffer system / sodium chloride solution (pH=7.96) to it to make the total weight 250.39 g, stir and wash for 4 hours; filter through a sieve, drain and crush the retained gel to obtain the product, detect the elastic modulus G' and viscous modulus G'' of the product at a frequency of 2.5 Hz, and calculate the swelling ratio and the final concentration of the product.

[0092] Example 9

[0093] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0094] S1. Weigh 8.0172 g of sodium hyaluronate (molecular weight 0.8 MDa), add 171.74 g of phosphate buffer system / sodium chloride solution (pH = 6.74), and stir for 24 hours;

[0095] Weigh 0.6598 g of 3,6-bis(3-aminopropyl)-2,5-diketopiperazine hydrochloride, add phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.0056 g, and dissolve evenly;

[0096] The two were stirred and mixed for 30 min to form a mixed solution;

[0097] S2, weighing 1.4864 g of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, adding a phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.0539 g, and dissolving evenly to form a condensing agent solution; filtering the condensing agent solution, adding it to the mixed solution, and stirring for about 30 minutes to form a reaction solution, and then stirring at 25°C for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0098] S3, take 100.80g of the reaction product after the cross-linking reaction of S2, add phosphate buffer system / sodium chloride solution (pH=6.74) to it to a total weight of 611.42g, stir and wash for 24h; filter through a mesh, drain and crush the trapped gel to obtain the product, detect the elastic modulus G' and viscous modulus G'' of the product at a frequency of 2.5Hz, and calculate the swelling ratio and the final concentration of the product;

[0099] The obtained product was packed into a number of vials, stoppered and capped, and sterilized by wet heat at 121°C for 15 min, 20 min, and 30 min, respectively. The elastic modulus G' and viscous modulus G'' of the product after sterilization were tested at a frequency of 2.5 Hz.

[0100] Example 10

[0101] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0102] S1. Weigh 10.0011 g of sodium hyaluronate (molecular weight 1.2 MDa), add 169.21 g of phosphate buffer system / sodium chloride solution (pH = 6.74), and stir for 24 hours;

[0103] Weigh 0.6598 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.0080 g, and dissolve evenly;

[0104] The two were stirred and mixed for 30 min to form a mixed solution;

[0105] S2. Weigh 1.2452 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add a phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.1350 g, and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to mix to form a reaction solution, and then stir at 10° C. for a cross-linking reaction for 24 hours to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0106] S3, take 100.86g of the reaction product after the cross-linking reaction of S2, add phosphate buffer system / sodium chloride solution (pH=6.74) to it to a total weight of 707.25g, and stir and wash for 96h; filter through a mesh, drain and crush the trapped gel to obtain the product, detect the elastic modulus G' and viscous modulus G'' of the product at a frequency of 2.5Hz, and calculate the swelling ratio and the final concentration of the product;

[0107] The obtained product was packed into a number of vials, stoppered and capped, and sterilized by wet heat at 121°C for 15 min, 20 min, and 30 min, respectively. The elastic modulus G' and viscous modulus G'' of the product after sterilization were tested at a frequency of 2.5 Hz.

[0108] Embodiment 11

[0109] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0110] S1. Weigh 1.0283 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add 400.14 g of phosphate buffer system / sodium chloride solution (pH=6.86), stir for 30 min, then add 25.0369 g of sodium hyaluronate (molecular weight 0.8 MDa) while stirring, and stir for 21.5 h to form a mixed solution;

[0111] S2. Weigh 3.1787 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add 68.20 g of phosphate buffer system / sodium chloride solution (pH=6.86), and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to mix well to form a reaction solution, and then stand at 25°C for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0112] S3. Take 454.15 g of the reaction product after the cross-linking reaction of S2, add 2502.47 g of phosphate buffer system / sodium chloride solution (pH=6.86) thereto, stir and wash for 12 hours; filter through a mesh, drain the trapped gel to obtain 1206.98 g of a gel product; filter the gel product through 0.8 mm, 0.4 mm, 0.2 mm, and 0.15 mm pore sizes, respectively, and crush it into a gel particle product, detect the elastic modulus G' and the viscous modulus G'' of the product at a frequency of 2.5 Hz, and calculate the swelling ratio and the final concentration of the product;

[0113] The obtained product was packed into a plurality of vials, stoppered and capped, and sterilized by wet heat sterilization at 121° C. for 30 min and 40 min, respectively. The elastic modulus G' and viscous modulus G'' of the product after sterilization were tested at a frequency of 2.5 Hz.

[0114] Another sample was taken and HPLC was used to detect whether six impurities, including lysine (Lys), 3,6-di(4-aminobutyl)-2,5-diketopiperazine (Las), 4,6-dimethoxy-1,3,5-triazine-2(1H)-one (DMTO), 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)morpholine (DMTM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), were present in the product. The results of the impurity content determination are shown in Table 2.

[0115] Table 2 Impurity content in cross-linked biopolysaccharide gel

[0116]

[0117] It can be seen from the test data in Table 2 that the cross-linked biopolysaccharide gel prepared by the present invention has better product quality, less product impurities, and higher safety in application.

[0118] Example 12

[0119] The elastic modulus G' and viscous modulus G'', swelling ratio and final concentration of the cross-linked biopolysaccharide gel prepared in Example 4-11 before sterilization were tested and analyzed. The results are shown in Table 3.

[0120] Table 3 Properties of the cross-linked biopolysaccharide gels prepared in Examples 4-11 before sterilization

[0121]

[0122] (Note: The commercially available I is Hyruan ONE, manufactured by LG Chem, Ltd., with a batch number of SIH22068; the commercially available II is Synvisc, manufactured by Genzyme Corporation, with a batch number of CRSP006B; the commercially available III is Haimei, manufactured by Shanghai Qisheng Biological Products Co., Ltd., with a batch number of Q2305QS11; the sodium hyaluronate concentration indicates the mass fraction of sodium hyaluronate in the reaction solution; the amount of cross-linking agent refers to the percentage of the molar amount of the cross-linking agent to the molar amount of the sodium hyaluronate repeating unit; the swelling ratio is the ratio of the increased weight of the gel after swelling to the original weight; the final concentration of the product is calculated based on the sodium hyaluronate content)

[0123] As shown in Table 3, the modulus data range of the cross-linked biopolysaccharide gel obtained under different reaction conditions before sterilization is relatively wide: elastic modulus 567Pa-2009Pa, viscous modulus 54Pa-464Pa, with good viscoelastic properties. The viscoelastic data of the cross-linked biopolysaccharide gel during moist heat sterilization will be greatly reduced. Referring to the elastic modulus and viscous modulus data measured under the same conditions by commercially available butyl cross-linked sodium hyaluronate and Xinwei, the cross-linked biopolysaccharide gel prepared by the present invention has a large downward exploration space in terms of viscoelastic data for moist heat sterilization, which can ensure that the product is sterile and has good viscoelastic properties. In addition, the cross-linked biopolysaccharide gel prepared by the present invention has a suitable swelling ratio after post-treatment, so that the sodium hyaluronate content in the gel is controlled at 10-20mg / mL, which is consistent with commercially available products.

[0124] Example 13

[0125] The cross-linked biopolysaccharide gels prepared in Example 9, Example 10 and Example 11 were subjected to terminal moist heat sterilization, and their tolerance was investigated. The data are shown in Table 4.

[0126] Table 4 Performance of cross-linked biopolysaccharide gels after wet heat sterilization in Examples 9-11

[0127]

[0128] It can be seen from the results in Table 4 that the cross-linked biopolysaccharide gel prepared by the present invention can withstand wet heat sterilization at 121° C. for 15 min or even longer while ensuring the viscoelastic properties of the product, thereby ensuring the application safety of the terminal product.

[0129] Embodiment 14

[0130] This case is to test the enzymatic degradation stability of the product.

[0131] The ability of the cross-linked biopolysaccharide gel product sterilized for 40 minutes in Example 11 (sample 1) and the commercially available product Qisheng Haimei (sample 2) to resist enzyme degradation was compared respectively.

[0132] The enzymatic degradation test protocol is as follows:

[0133] Weigh the gel so that the total amount of HA taken is about 8 mg and place it in a stoppered tube. Add 15 mL of hyaluronidase solution (80 U / mL), vortex oscillate for 30s-60s and then place it at 37°C for constant temperature enzymolysis. Take out the samples after 6h, 24h, and 48h, respectively, centrifuge and take the supernatant to determine the glucuronic acid content and calculate the enzyme degradation rate. The enzyme degradation data are shown in Table 5.

[0134] Table 5 Enzyme degradation data

[0135]

[0136] It can be seen from Table 5 that the cross-linked biopolysaccharide gel prepared by the present invention has good resistance to enzyme degradation and has a longer action time in the body.

[0137] Embodiment 15

[0138] The cross-linked biopolysaccharide gel prepared in Example 11 before sterilization and after sterilization for 40 minutes were compared with the commercially available butylated cross-linked sodium hyaluronate (commercially available I) by staining test. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that compared with the commercially available product, butylene glycol cross-linked sodium hyaluronate injection, the particles of the cross-linked sodium hyaluronate product prepared by the present invention are more uniform and delicate, and the particle size is smaller, so the resistance during syringe push injection will be relatively lower, which is more conducive to injection.

[0139] Example 16

[0140] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0141] S1. Weigh 0.1270 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add 18.03 g of phosphate buffer system / sodium chloride solution (pH=6.82), stir for 30 min, then add 1.0008 g of carboxymethyl chitin (molecular weight 0.7 MDa) while stirring, and stir for 22 h to form a mixed solution;

[0142] S2. Weigh 0.3869 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add 1.98 g of phosphate buffer system / sodium chloride solution (pH=6.86), and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to mix well to form a reaction solution, and then stand at 25°C for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0143] S3, take 20.83g of the reaction product after the cross-linking reaction of S2, add 207.61g of phosphate buffer system / sodium chloride solution (pH=6.82), stir and wash for 12h; filter through a mesh, drain the trapped gel to obtain 63.34g of gel product; filter the gel product through a 0.15mm pore size filter to crush it into a gel particle product. The swelling ratio is calculated to be 204.08%, and the final concentration of the product is 16.4mg / mL; the elastic modulus G' of the product is 412.8Pa and the viscous modulus G'' is 104.4Pa at a frequency of 2.5Hz.

[0144] The obtained product was put into a vial, stoppered and capped, and sterilized by wet heat sterilization at 121° C. for 8 min. The elastic modulus G' of the product after sterilization at a frequency of 2.5 Hz was 109.4 Pa, and the viscous modulus G'' was 15.5 Pa.

[0145] Embodiment 17

[0146] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0147] S1. Weigh 0.1268 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride, add 18.03 g of phosphate buffer system / sodium chloride solution (pH=6.86), stir for 30 min, then add 1.0006 g of carboxymethyl chitosan (molecular weight 0.5 MDa) while stirring, and stir for 22 h to form a mixed solution;

[0148] S2. Weigh 0.3870 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add 2.00 g of phosphate buffer system / sodium chloride solution (pH=6.82), and dissolve evenly to form a condensing agent solution; filter the condensing agent solution, add it to the mixed solution, and stir for about 30 minutes to mix well to form a reaction solution, and then stand at 25°C for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0149] S3. Add 199.47g of phosphate buffer system / sodium chloride solution (pH=6.82) to the reaction product after the cross-linking reaction of S2, stir and wash for 12h; filter through a mesh, drain the trapped gel to obtain 28.35g of gel product; filter the gel product through a 0.15mm pore size filter to crush it into a gel particle product. The swelling ratio is calculated to be 36.10%, and the final concentration of the product is 36.8mg / mL; the elastic modulus G' of the product is 5238Pa and the viscous modulus G'' is 266.9Pa at a frequency of 2.5Hz.

[0150] The obtained product was put into a vial, stoppered and capped, and sterilized by wet heat sterilization at 121° C. for 30 min. The elastic modulus G' of the product after sterilization at a frequency of 2.5 Hz was 2992 Pa, and the viscous modulus G'' was 80.76 Pa.

[0151] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, characterized in that: The steps include: After the carboxyl-containing biopolysaccharide and the diaminodiketopiperazine compound cross-linking agent are dissolved and mixed evenly, a condensation agent is added to form a reaction solution to generate a cross-linking reaction to obtain a high-viscoelastic cross-linked biopolysaccharide gel; The diaminodiketopiperazine compound cross-linking agent has the following chemical structure: wherein R is a normal alkyl group or an isomeric alkyl group having 10 or less carbon atoms.

2. The method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 1, characterized in that: The specific steps include: S1, dissolving one of the carboxyl-containing biopolysaccharide or diaminodiketopiperazine cross-linking agent in a buffer solution, then adding the other compound, and mixing them evenly to form a mixed solution; S2, dissolving the condensing agent in a buffer solution, then adding the condensing agent into the mixed solution to form a reaction solution, and performing a cross-linking reaction. After the reaction is completed, a highly viscoelastic cross-linked biopolysaccharide gel is obtained.

3. The method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 1 or 2, characterized in that: The diaminodiketopiperazine compound cross-linking agent is selected from one or more of 3,6-di(4-aminobutyl)-2,5-diketopiperazine or its salt, 3,6-di(3-aminopropyl)-2,5-diketopiperazine or its salt.

4. The method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 1 or 2, characterized in that: The purity of the diaminodiketopiperazine compound cross-linking agent is at least 99 wt %, and the amount of the diaminodiketopiperazine compound cross-linking agent used is 1%-50% of the molar amount of the repeating unit of the biopolysaccharide.

5. The method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 2, characterized in that: The molecular weight of the biopolysaccharide is 0.5 MDa-10 MDa; The biopolysaccharide is selected from one or more of hyaluronic acid or its salt, alginic acid or its salt, carboxymethyl chitin or its salt, carboxymethyl chitosan or its salt, carboxymethyl cellulose or its salt, and chondroitin compounds; The mass fraction of the biopolysaccharide in the reaction solution is 0.5%-10%; The buffer is one of a phosphate buffer system and a phosphate buffer system / sodium chloride solution, and the pH of the buffer is in the range of 6.0-8.5; The condensing agent is selected from one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine tetrafluoroborate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, and 2-chloro-1-methylpyridinium iodide, and the molar amount of the condensing agent is 100%-300% of the molar amount of the diaminodiketopiperazine compound cross-linking agent.

6. The method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 1 or 2, characterized in that: The method also includes swelling and rinsing the highly viscoelastic cross-linked biopolysaccharide gel with a buffer solution after obtaining the gel, and subsequently crushing, filling, and wet heat sterilizing the gel to obtain a finished product.

7. The method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 6, characterized in that: The temperature of the cross-linking reaction is in the range of 0°C-30°C; the cross-linking reaction time is in the range of 10h-80h; The wet heat sterilization conditions are: sterilization at 121°C for 10min-40min; The amount of the buffer used for swelling or rinsing is 3 to 10 times the weight of the reaction product obtained after the cross-linking reaction is completed, and the rinsing time is 4 to 96 hours.

8. The highly viscoelastic cross-linked biopolysaccharide gel prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The highly viscoelastic cross-linked biopolysaccharide gel has an elastic modulus of 200Pa-5000Pa and a viscosity modulus of 50Pa-500Pa before moist heat sterilization; and an elastic modulus of 100Pa-400Pa and a viscosity modulus of 30Pa-90Pa after moist heat sterilization. The biopolysaccharide content in the highly viscoelastic cross-linked biopolysaccharide gel is 5 mg / mL-25 mg / mL.

9. Use of the highly viscoelastic cross-linked biopolysaccharide gel prepared by the preparation method according to any one of claims 1 to 7 in the preparation of internal injection preparations, internal filling preparations, or external drug carrier preparations.

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