High-viscoelastic crosslinked biopolysaccharide gel, preparation method therefor, and application thereof

Abstract

The present invention relates to the technical field of polysaccharide material modification, and specifically relates to a high-viscoelastic crosslinked biopolysaccharide gel, a preparation method therefor, and an application thereof. After a carboxyl-group-containing biopolysaccharide and a diaminodiketopiperazine-based crosslinking agent are dissolved and mixed uniformly, a crosslinking reaction is generated under the action of a condensing agent to obtain a high-viscoelastic crosslinked biopolysaccharide gel; following moist heat sterilization, the resulting high-viscoelastic crosslinked biopolysaccharide gel meets conditions for the application thereof, and can be applied to the preparation of injectable formulations, internal filler formulations, or topical pharmaceutical carrier formulations. The crosslinked biopolysaccharide gel prepared according to the present invention exhibits large headroom, in terms of viscoelasticity data, to accommodate moist heat sterilization, which ensures product sterility while guaranteeing the product has good viscoelastic performance, with low cytotoxicity, high product purity, and long service life.

Description

A highly viscoelastic cross-linked biopolysaccharide gel, its preparation method and application Technical Field

[0001] This invention relates to the field of polysaccharide material modification technology, specifically to a highly viscoelastic cross-linked biopolysaccharide gel, its preparation method, and its applications. Background Technology

[0002] In recent years, the number of patients with osteoarthritis has been increasing year by year, posing a huge threat to the health of the general public. Viscoelasticity supplementation therapy, which is well-suited to the physiological characteristics of the knee joint, has gained widespread recognition among clinical professionals due to its advantages in improving joint function and relieving pain. In recent years, the application of sodium hyaluronate (also known as hyaluronic acid, HA) injections for viscoelasticity supplementation has become a mature treatment option for early to mid-stage arthritis. However, natural sodium hyaluronate is easily biodegradable and has a very short residence time in the body, resulting in a relatively short therapeutic effect for sodium hyaluronate injections. Generally, five injections are required, and the therapeutic effect typically lasts 3-6 months, increasing patient discomfort. Furthermore, as an endogenous component, sodium hyaluronate has wide applications in the medical aesthetics field and in preventing adhesions during surgery, but it also faces the problem of rapid degradation in the body.

[0003] Cross-linked sodium hyaluronate uses one or more cross-linking agents to react with the carboxyl or hydroxyl groups on sodium hyaluronate, forming 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 seen significant development in clinical applications. Currently, most domestic and international 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 reactive structural units such as epoxy or double bonds, which can covalently bind to functional proteins and nucleic acids in the body, thus affecting their normal function. They have high potential cytotoxicity and poor biocompatibility. Furthermore, the residues of these cross-linking agents themselves or their degradation products can also cause inflammatory reactions in vivo. For example, data shows 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 higher than that of non-cross-linked products (such as Alzheimer's, with an incidence of 0.52%). This significantly limits its development and application. Compared to BDDE or DVS, lysine derivatives offer higher safety. However, these crosslinking processes either require reaction with the organic salt of sodium hyaluronate in an organic solvent or inevitably lead to further polymerization of polylysine itself, introducing new polymer impurities into the sodium hyaluronate gel. Furthermore, existing products often lack sufficient viscoelasticity after moist heat sterilization, resulting in a short lifespan (i.e., short therapeutic effect). Therefore, developing a crosslinking agent that is materially safe, highly efficient in crosslinking, and has a long degradation cycle is the future direction for crosslinked hyaluronic acid. Summary of the Invention

[0004] To address the technical problems of high impurities and short therapeutic effects in existing cross-linked biopolysaccharide gels, this invention provides a highly viscoelastic cross-linked biopolysaccharide gel, its preparation method, and its applications. This invention uses diaminodikepiperazine compounds as cross-linking agents to cross-link biopolysaccharides. The product of this invention possesses advantages such as superior physicochemical properties, a well-defined cross-linking process, high safety, strong anti-degradation ability, high stability, and uniform particle size. In clinical use, it offers advantages such as low dosage per treatment course, long-lasting therapeutic effect, and good efficacy, showing broad application prospects in tissue filling, postoperative adhesion prevention, and osteoarthritis treatment.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

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

[0007] After dissolving and mixing carboxyl-containing biopolysaccharides with diaminodikepiperazine crosslinking agents, a condensing agent is added to form a reaction solution, resulting in a crosslinking reaction to obtain a highly viscoelastic crosslinked biopolysaccharide gel.

[0008] Furthermore, the specific steps include the following:

[0009] S1. Dissolve one of the following compounds in a buffer solution: a carboxyl-containing biopolysaccharide or a diaminodikepiperazine crosslinking agent. Then add the other compound and mix thoroughly to form a mixture. The other compound can be directly mixed with the former to form a mixture, or it can be mixed thoroughly with the buffer solution to form a mixture.

[0010] That is: dissolve the carboxyl-containing biopolysaccharide in a buffer solution, add a diaminodikepiperazine crosslinking agent, and mix thoroughly to form a mixture; or dissolve the diaminodikepiperazine compound in a buffer solution, then add the biopolysaccharide, and mix thoroughly to form a mixture.

[0011] S2. Dissolve the condensing agent in the buffer solution, and then add it to the mixture to form a reaction solution for cross-linking reaction. After the reaction is completed, a highly viscoelastic cross-linked biopolysaccharide gel is obtained.

[0012] Furthermore, the diaminodikepiperazine crosslinking agent has the following chemical structure: Formula I:

[0013] Where R is a normal alkyl or isoalkyl group with 10 or fewer carbon atoms.

[0014] Preferably, the crosslinking agent of the diaminodiketopiperazine compound is selected from one or more of 3,6-bis(4-aminobutyl)-2,5-diketopiperazine or its salts, 3,6-bis(3-aminopropyl)-2,5-diketopiperazine or its salts, and the salts of the compound are, for example, hydrochloride, sulfate, nitrate, etc.

[0015] Furthermore, the purity of the diaminodikepiperazine crosslinking agent is at least 99 wt%, and the amount of the diaminodikepiperazine crosslinking agent is 1%-50% of the molar amount of the repeating unit of the biopolysaccharide, preferably 5%-20%, more preferably 5%-15%.

[0016] Further, the molecular weight of the biopolysaccharide is 0.5 MDa-10 MDa, preferably 0.5 MDa-2.0 MDa; the biopolysaccharide is selected from one or more of hyaluronic acid or its salt, alginate 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%. Different concentrations of biopolysaccharide in the reaction solution result in different swelling ratios of the obtained cross-linked biopolysaccharide gel, which in turn affects the content of biopolysaccharide in the gel. That is, the swelling ratio of the present invention is controllable, and the content of biopolysaccharide in the final product can be controlled by adjusting the concentration of biopolysaccharide in the initial reaction solution.

[0017] The buffer solution is one of a phosphate buffer system or a phosphate buffer system / sodium chloride solution, and the pH of the buffer solution is in the range of 6.0-8.5, preferably in the range of 6.4-8.2.

[0018] 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-methylpyridine iodide, and the molar amount of the condensing agent is 100%-300% of the molar amount of the diaminodikepiperazine compound crosslinking agent.

[0019] Furthermore, it also includes swelling and rinsing the highly viscoelastic cross-linked biopolysaccharide gel with a buffered saline solution, followed by crushing, filling, and moist heat sterilization to obtain the finished product.

[0020] Furthermore, the temperature of the crosslinking reaction is in the range of 0℃-30℃, preferably 10℃-25℃; the crosslinking reaction time is in the range of 10h-80h, preferably 24h-72h.

[0021] The conditions for moist heat sterilization are: sterilization at 121℃ for 10-40 minutes;

[0022] The amount of the buffer solution 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 h to 96 h, preferably 8 h to 24 h.

[0023] In another aspect, the present invention provides a highly viscoelastic cross-linked biopolysaccharide gel obtained by the above preparation method, having an elastic modulus of 200 Pa-5000 Pa and a viscous modulus of 50 Pa-500 Pa before moist heat sterilization; an elastic modulus of 100 Pa-400 Pa and a viscous modulus of 30 Pa-90 Pa after moist heat sterilization; and a biopolysaccharide content of 5 mg / mL-25 mg / mL, preferably 10 mg / mL-20 mg / mL.

[0024] The final aspect of this invention provides the application of the highly viscoelastic cross-linked biopolysaccharide gel obtained by the above preparation method in the preparation of intra-injectable formulations, intra-filler formulations, or topical drug carrier formulations.

[0025] Beneficial technical effects:

[0026] This invention uses diaminodikepiperazine compounds as cross-linking agents to cross-link biological polysaccharides to a certain extent under the action of condensing agents. Compared with commercially available products, the high viscoelastic cross-linked biological polysaccharide gel of this invention has more uniform and delicate particles with smaller particle size. The product also exhibits low cytotoxicity, high safety, good tissue compatibility, strong anti-degradation ability, high stability, and high viscoelasticity. When used, it has the advantages of low dosage per treatment course, long duration of therapeutic effect, and good efficacy. Furthermore, the production process of this invention is easy to control, the product swelling ratio is controllable, the rinsing effect is good, and there are few impurities remaining. The production equipment required for this invention is simple, facilitating large-scale production. The product of this invention has broad application prospects in tissue filling, postoperative anti-adhesion, and osteoarthritis treatment.

[0027] (1) This invention is the first to use 2,5-dikepiperazine compounds containing diamino groups and their acid salts as crosslinking agents, which are structurally stable, highly pure and safe, and are safer than crosslinking agents such as DVS, BDDE and PEGDE.

[0028] (2) The cross-linked biopolysaccharide prepared is obtained by covalently bonding the two amino groups at both ends of the cross-linking agent with the carboxyl groups of the biopolysaccharide through amide bonds. Since the reactivity of the amino groups on the cross-linking agent is much higher than that of the hydroxyl groups of the biopolysaccharide itself, the ineffective cross-linking of the cross-linking agent and the self-cross-linking of the biopolysaccharide can be avoided. At the same time, there is no risk of self-polymerization of polylysine cross-linking agents. The cross-linking process is clear, specific and controllable.

[0029] (3) The present invention has a high viscoelastic cross-linked biopolysaccharide gel preparation process. 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 agent, condensing agent and their degradation products, which can be purified in water, making the whole preparation process green and environmentally friendly, and conducive to commercial production.

[0030] (4) The swelling ratio of the cross-linking process of the present invention is controllable, and the resulting cross-linked biopolysaccharide gel has low impurity content, high viscoelastic modulus, uniform particles, and high thermal stability. It can withstand sufficient terminal sterilization to ensure product sterility and improve product safety. In addition, the cross-linked biopolysaccharide gel of the present invention has stronger resistance to enzymatic hydrolysis and can effectively prolong its retention time in the body during application, thereby prolonging the duration of drug efficacy. Therefore, it can reduce the frequency of administration and improve patient compliance. Attached Figure Description

[0031] Figure 1 is a comparison of particle size staining of the high viscoelastic cross-linked biopolysaccharide gel before and after sterilization of the present invention, and the commercially available product, butyric acid cross-linked sodium hyaluronate injection. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. Techniques and methods known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques and methods should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. Furthermore, the expression "within a range" is generally considered to exclude endpoint values.

[0034] Experimental methods not specified in the following examples are generally performed according to national standards; if there is no corresponding national standard, they are performed according to general standard requirements or general methods.

[0035] Note: The molecular weight of polymers is usually expressed as number-average molecular weight.

[0036] Example 1

[0037] This case study details the preparation of the crosslinking agent 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride.

[0038] 100 g of 2-amino-6-(trifluoroacetamido)hexanoic acid (CAS10009-20-8), 17.5 g of phosphorus pentoxide, and 206 mL of N-methylpyrrolidone were placed in a reaction vessel and reacted at 165 °C for 2 hours to carry out a ring-closure reaction. The reaction solution was then poured into purified water to precipitate a solid, which was filtered and dried to obtain an intermediate. The intermediate was then placed in 270 mL of ammonia water (19% by mass) and reacted at 50 °C for 22 hours to carry out a detrifluoroacetylation reaction, yielding crude 3,6-di(4-aminobutyl)-2,5-diketopiramate. This crude product was dissolved in 60 mL of 1M dilute hydrochloric acid, and 600 mL of tetrahydrofuran was added dropwise for crystallization, yielding 30 g of 3,6-di(4-aminobutyl)-2,5-diketopiramate hydrochloride (molecular weight 329.27 g / mol), with an HPLC purity of 99%.

[0039] The product was subjected to carbon and proton NMR spectroscopy, and the results are as follows:

[0040] 13 CNMR(101MHz,D2O)δ:170.28,53.98,39.16,31.76,26.41,20.17;

[0041] 1 HNMR(400MHz,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).

[0042] Example 2

[0043] This case study focuses on the preparation of the crosslinking agent 3,6-bis(3-aminopropyl)-2,5-diketopiramate hydrochloride.

[0044] 100 g of 2-amino-5-(trifluoroacetamido)valerate (CAS 5123-49-9), 17.2 g of phosphorus pentoxide, and 200 mL of N-methylpyrrolidone were placed in a reaction vessel and reacted at 165 °C for 2 hours to carry out a ring-closure reaction. The reaction solution was then poured into purified water to precipitate a solid, which was filtered and dried to obtain an intermediate. The intermediate was then placed in 250 mL of ammonia water (19% by mass) and reacted at 50 °C for 20 hours to carry out a detrifluoroacetylation reaction to obtain crude 3,6-di(3-aminopropyl)-2,5-diketopiperazine. This crude product was dissolved in 60 mL of 1M dilute hydrochloric acid, and 600 mL of tetrahydrofuran was added dropwise for crystallization to obtain 25 g of 3,6-di(3-aminopropyl)-2,5-diketopiperazine hydrochloride (molecular weight 301.21 g / mol), with an HPLC purity of 99%.

[0045] The product was subjected to proton NMR spectroscopy, and the results are as follows:

[0046] 1 HNMR(400MHz,D2O)δ:4.17(t,2H),3.05(t,4H),1.96-1.71(m,4H),1.78-1.65(m,4H).

[0047] Example 3

[0048] This case study describes the cytotoxicity assay of the products from Examples 1 and 2.

[0049] 3,6-Di(4-aminobutyl)-2,5-diketopiperazine hydrochloride (hereinafter referred to as compound a) and 3,6-di(3-aminopropyl)-2,5-diketopiperazine hydrochloride (hereinafter referred to as compound b) were prepared into samples of different concentrations as shown in Table 1 below (using phosphate buffer solution as the solvent). These samples were co-incubated with human primary chondrocytes for a certain period of time, followed by chondrocyte viability assay. Specifically: After the human primary chondrocytes reached the exponential growth phase and were in good condition, viable cell counts were performed using a cell counter. The cell suspension was adjusted to a suitable cell concentration using the appropriate culture medium. 100 μL of cell suspension was added to each well of a 96-well cell culture plate, 5000 cells / well, 3 replicates, and incubated overnight (approximately 24 h) at 37°C in a 5% CO2 incubator. The next day, after cell attachment, the culture supernatant was gently aspirated, and 120 μL of the corresponding concentration of the test compound prepared in the culture medium was added to each group. After incubation for a certain period of time, cell viability was measured.

[0050] Cell viability was determined by measuring ATP content using a chemiluminescence method to reflect the number of live cells. Following the kit instructions, pre-melted and equilibrated reagents (equilibration time approximately 30 min) were added to each well. The cells were then shaken and mixed at room temperature for 2 minutes to fully induce cell lysis. The fluorescence signal value was then measured using a multi-functional microplate reader.

[0051] Cell viability calculation: Cell viability = (RLU value per well) / (average RLU value of blank control group) × 100%.

[0052] The results are shown in Table 1.

[0053] Table 1. Cytotoxicity results of the cross-linking agent compounds

[0054] As shown in Table 1, under experimental conditions with concentrations far exceeding those of the crosslinking agent in the gel formulation, neither of the two crosslinking agents showed a significant inhibitory effect on chondrocyte proliferation. This indicates that the crosslinking agent used in this invention has the advantages of low cytotoxicity and high safety.

[0055] Example 4

[0056] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0057] S1. Weigh 4.0125g of sodium hyaluronate (molecular weight 0.8MDa), add 80.14g of phosphate buffer system / sodium chloride solution (pH=6.81), and stir for 24h.

[0058] Weigh 0.1653 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add it to a phosphate buffer / sodium chloride solution (pH = 6.81) until the total weight is 10.0313 g, and dissolve it thoroughly.

[0059] Stir and mix the two for 30 minutes to form a mixture;

[0060] S2. Weigh 0.4972 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add it to a phosphate buffer system / sodium chloride solution (pH=6.81) until the total weight is 10.0231 g, and dissolve it evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture and stir for about 30 min to form a reaction solution. Then, stir at 20 °C for 24 h to carry out the cross-linking reaction to obtain the reaction product, a highly viscoelastic cross-linked biopolysaccharide gel.

[0061] S3. Take 56.30g of the reaction product after the S2 crosslinking reaction is completed, add phosphate buffer system / sodium chloride solution (pH=6.81) to it until the total weight is 530.48g, stir and wash for 8h; filter through a sieve, drain the retained gel, crush it to obtain the product, and detect the elastic modulus G' and viscous modulus G” of the product at a frequency of 2.5Hz to calculate the swelling ratio and the final concentration of the product.

[0062] Example 5

[0063] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0064] S1. Weigh 2.0087g of sodium hyaluronate (molecular weight 0.6MDa), add 78.00g of phosphate buffer system / sodium chloride solution (pH=6.73), and stir for 24h.

[0065] Weigh 0.3140 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add it to a phosphate buffer / sodium chloride solution (pH=6.73) until the total weight is 10.0106 g, and dissolve it evenly;

[0066] Stir and mix the two for 30 minutes to form a mixture;

[0067] S2. Weigh 0.9958 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add it to a phosphate buffer system / sodium chloride solution (pH=6.73) until the total weight is 10.0592 g, and dissolve it evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture and stir for about 30 min to form a reaction solution. Then, stir at 15 °C for 48 h to carry out the cross-linking reaction to obtain the reaction product, a highly viscoelastic cross-linked biopolysaccharide gel.

[0068] S3. Take 42.22g of the reaction product after the S2 crosslinking reaction is completed, add phosphate buffer system / sodium chloride solution (pH=6.73) to it until the total weight is 248.94g, stir and wash for 24h; filter through a sieve, drain the retained gel, crush it to obtain the product, and detect the elastic modulus G' and viscous modulus G” of the product at a frequency of 2.5Hz to calculate the swelling ratio and the final concentration of the product.

[0069] Example 6

[0070] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0071] S1. Weigh 4.0126g of sodium hyaluronate (molecular weight 0.8MDa), add 156.00g of phosphate buffer system / sodium chloride solution (pH=7.21), and stir for 24h.

[0072] Weigh 0.1599 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add it to a phosphate buffer / sodium chloride solution (pH=7.21) until the total weight is 20.0344 g, and dissolve it evenly;

[0073] Stir and mix the two for 30 minutes to form a mixture;

[0074] S2. Weigh 0.4981 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add it to a phosphate buffer system / sodium chloride solution (pH=7.21) to a total weight of 20.0238 g, and dissolve it evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture and stir for about 30 min to form a reaction solution. Then, stir at 25 °C for 24 h to carry out the cross-linking reaction to obtain the reaction product, a highly viscoelastic cross-linked biopolysaccharide gel.

[0075] S3. The reaction product after the crosslinking reaction in S2 was added to the phosphate buffer system / sodium chloride solution (pH=7.21) for dilution. No obvious blocky gel was filtered out, indicating that the combination of sodium hyaluronate concentration and crosslinking agent concentration in this case could not produce a washable and filterable gel product.

[0076] Example 7

[0077] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0078] S1. Weigh 3.0035g of sodium hyaluronate (molecular weight 1.0MDa), add 80.47g of phosphate buffer system / sodium chloride solution (pH=7.70), and stir for 24h.

[0079] Weigh 0.2462 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add it to a phosphate buffer / sodium chloride solution (pH=7.70) until the total weight is 10.0171 g, and dissolve it evenly;

[0080] Stir and mix the two for 30 minutes to form a mixture;

[0081] S2. Weigh 0.7482 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add it to a phosphate buffer system / sodium chloride solution (pH=7.70) to a total weight of 10.0008 g, and dissolve it evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture and stir for about 30 min to form a reaction solution. Then, stir at 5 °C for 72 h to carry out the cross-linking reaction to obtain the reaction product, a highly viscoelastic cross-linked biopolysaccharide gel.

[0082] S3. Take 53.78g of the reaction product after the S2 crosslinking reaction is completed, add phosphate buffer system / sodium chloride solution (pH=7.70) to it until the total weight is 253.34g, stir and wash for 16h; filter through a sieve, drain the retained gel, crush it to obtain the product, and detect the elastic modulus G' and viscous modulus G” of the product at a frequency of 2.5Hz to calculate the swelling ratio and the final concentration of the product.

[0083] Example 8

[0084] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0085] S1. Weigh 5.0058g of sodium hyaluronate (molecular weight 0.8MDa), add 80.08g of phosphate buffer system / sodium chloride solution (pH=7.96), and stir for 24h.

[0086] Weigh 0.2069 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add it to a phosphate buffer / sodium chloride solution (pH=7.96) until the total weight is 10.0155 g, and dissolve it evenly;

[0087] Stir and mix the two for 30 minutes to form a mixture;

[0088] S2. Weigh 0.3593g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 7084-11-9), add it to a phosphate buffer system / sodium chloride solution (pH=7.96) until the total weight is 10.0438g, and dissolve it evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture and stir for about 30min to form a reaction solution. Then, stir at 25℃ for 72h to carry out the cross-linking reaction to obtain the reaction product, a highly viscoelastic cross-linked biopolysaccharide gel.

[0089] S3. Take 52.71g of the reaction product after the S2 crosslinking reaction is completed, add phosphate buffer system / sodium chloride solution (pH=7.96) to it until the total weight is 250.39g, stir and wash for 4h; filter through a sieve, drain the retained gel, crush it to obtain the product, and detect the elastic modulus G' and viscous modulus G” of the product at a frequency of 2.5Hz to calculate the swelling ratio and the final concentration of the product.

[0090] Example 9

[0091] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0092] S1. Weigh 8.0172g of sodium hyaluronate (molecular weight 0.8MDa), add 171.74g of phosphate buffer system / sodium chloride solution (pH=6.74), and stir for 24h.

[0093] Weigh 0.6598 g of 3,6-bis(3-aminopropyl)-2,5-diketopiramate hydrochloride, add it to a phosphate buffer / sodium chloride solution (pH=6.74) until the total weight is 10.0056 g, and dissolve it evenly;

[0094] Stir and mix the two for 30 minutes to form a mixture;

[0095] S2. Weigh 1.4864 g of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, add it to a phosphate buffer system / sodium chloride solution (pH=6.74) until the total weight is 10.0539 g, and dissolve it evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture and stir for about 30 min to form a reaction solution. Then, stir at 25 °C for 24 h to carry out the cross-linking reaction to obtain the reaction product, a highly viscoelastic cross-linked biopolysaccharide gel.

[0096] S3. Take 100.80g of the reaction product after the S2 crosslinking reaction, add phosphate buffer system / sodium chloride solution (pH=6.74) to it until the total weight is 611.42g, stir and wash for 24h; filter through a sieve, drain the retained gel, crush it to obtain the product, and detect the elastic modulus G' and viscous modulus G” of the product at a frequency of 2.5Hz to calculate the swelling ratio and the final concentration of the product;

[0097] The obtained product was placed into multiple vials, capped, and sterilized at 121°C for 15 min, 20 min, and 30 min, respectively. The elastic modulus G' and viscous modulus G of the product at a frequency of 2.5 Hz after sterilization were measured.

[0098] Example 10

[0099] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0100] S1. Weigh 10.0011g of sodium hyaluronate (molecular weight 1.2MDa), add 169.21g of phosphate buffer system / sodium chloride solution (pH=6.74), and stir for 24h.

[0101] Weigh 0.6598 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add it to a phosphate buffer / sodium chloride solution (pH=6.74) until the total weight is 10.0080 g, and dissolve it evenly;

[0102] Stir and mix the two for 30 minutes to form a mixture;

[0103] S2. Weigh 1.2452 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add it to a phosphate buffer system / sodium chloride solution (pH=6.74) until the total weight is 10.1350 g, and dissolve it evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture and stir for about 30 min to form a reaction solution. Then, stir at 10 °C for 24 h to carry out the cross-linking reaction to obtain the reaction product, a highly viscoelastic cross-linked biopolysaccharide gel.

[0104] S3. Take 100.86g of the reaction product after the S2 crosslinking reaction, add phosphate buffer system / sodium chloride solution (pH=6.74) to it until the total weight is 707.25g, stir and wash for 96h; filter through a sieve, drain the retained gel, crush it to obtain the product, and detect the elastic modulus G' and viscous modulus G” of the product at a frequency of 2.5Hz to calculate the swelling ratio and the final concentration of the product;

[0105] The obtained product was placed into multiple vials, capped, and sterilized at 121°C for 15 min, 20 min, and 30 min, respectively. The elastic modulus G' and viscous modulus G of the product at a frequency of 2.5 Hz after sterilization were measured.

[0106] Example 11

[0107] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0108] S1. Weigh 1.0283g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add 400.14g of phosphate buffer / sodium chloride solution (pH=6.86), stir for 30min, then add 25.0369g of sodium hyaluronate (molecular weight 0.8MDa) while stirring, and stir for 21.5h to form a mixture;

[0109] S2. Weigh 3.1787 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add 68.20 g of phosphate buffer / sodium chloride solution (pH=6.86), and dissolve evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture, stir for about 30 min to form a reaction solution, and then let it stand at 25℃ for 24 h for cross-linking reaction to obtain the reaction product, high viscoelastic cross-linked biopolysaccharide gel;

[0110] S3. Take 454.15g of the reaction product after the S2 crosslinking reaction, add 2502.47g of phosphate buffer system / sodium chloride solution (pH=6.86) to it, stir and wash for 12h; filter through a sieve, drain the retained gel to obtain 1206.98g of gel product; filter the gel product through 0.8mm, 0.4mm, 0.2mm and 0.15mm pore sizes respectively and pulverize it into gel particle products. Detect the elastic modulus G' and viscous modulus G” of the product at a frequency of 2.5Hz, and calculate the swelling ratio and final concentration of the product.

[0111] The obtained product was placed into multiple vials, capped, and sterilized at 121℃ for 30 min and 40 min respectively. The elastic modulus G' and viscous modulus G of the product at 2.5 Hz after sterilization were measured.

[0112] Another sample was taken, and HPLC was used to detect the presence of six impurities in the product: 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). The results of the impurity content determination are shown in Table 2.

[0113] Table 2 Impurity content in cross-linked biopolysaccharide gels

[0114] As can be seen from the test data in Table 2, the cross-linked biopolysaccharide gel prepared by this invention has good product quality, few impurities, and will have higher safety in application.

[0115] Example 12

[0116] The elastic modulus G', viscous modulus G”, swelling ratio, and final product concentration of the cross-linked biopolysaccharide gels prepared in Examples 4-11 before sterilization were detected and analyzed. The results are shown in Table 3.

[0117] Table 3 Properties of the cross-linked biopolysaccharide gels obtained in Examples 4-11 before sterilization

[0118] (Note: Commercially available product I is Hyruan ONE, manufactured by LG Chem, Ltd., batch number SIH22068; commercially available product II is Synvisc, manufactured by Genzyme Corporation, batch number CRSP006B; commercially available product III is Haimei, manufactured by Shanghai Qisheng Biological Reagents Co., Ltd., batch number Q2305QS11; sodium hyaluronate concentration indicates the mass fraction of sodium hyaluronate in the reaction solution; crosslinking agent dosage refers to the percentage of the molar amount of crosslinking agent to the molar amount of sodium hyaluronate repeating unit; swelling ratio is the ratio of the weight increase of the gel after swelling to the original weight; the final concentration of the product is expressed as sodium hyaluronate content.)

[0119] Table 3 shows that the cross-linked biopolysaccharide gels obtained under different reaction conditions exhibit a wide range of modulus data before sterilization: elastic modulus 567 Pa-2009 Pa, and viscous modulus 54 Pa-464 Pa, demonstrating good viscoelastic properties. During moist heat sterilization, the viscoelastic data of the cross-linked biopolysaccharide gel decreases significantly. Referring to the elastic and viscous modulus data of commercially available butyric acid cross-linked sodium hyaluronate and Xinweike measured under the same conditions, the cross-linked biopolysaccharide gel prepared in this invention has a large downward potential in viscoelastic data for moist heat sterilization. This ensures both product sterility and good viscoelastic properties. Furthermore, the cross-linked biopolysaccharide gel prepared in this invention exhibits a suitable swelling ratio after post-treatment, thereby controlling the sodium hyaluronate content in the gel to 10-20 mg / mL, consistent with commercially available products.

[0120] Example 13

[0121] The cross-linked biopolysaccharide gels prepared in Examples 9, 10 and 11 were subjected to terminal moist heat sterilization, and their tolerance was investigated. The data are shown in Table 4.

[0122] Table 4. Performance of cross-linked biopolysaccharide gels after moist heat sterilization in Examples 9-11.

[0123] As can be seen from the results in Table 4, the cross-linked biopolysaccharide gel prepared by this invention can withstand moist heat sterilization at 121°C for 15 minutes or even longer while ensuring the viscoelastic properties of the product, thereby ensuring the application safety of the end product.

[0124] Example 14

[0125] This case study tests the enzyme degradation stability of a product.

[0126] The anti-enzyme degradation ability of the cross-linked biopolysaccharide gel product (sample 1) sterilized for 40 min in Example 11 and the commercially available product Qisheng Haimei (sample 2) was compared.

[0127] The enzyme degradation assay protocol is as follows:

[0128] Weigh out approximately 8 mg of HA from the gel and place it in a stoppered tube. Add 15 mL of hyaluronidase solution (80 U / mL), vortex for 30-60 seconds, and then incubate at 37°C for enzymatic hydrolysis. Samples were taken out at 6 h, 24 h, and 48 h, respectively. The supernatant was centrifuged and the glucuronic acid content was determined and the enzyme degradation rate was calculated. The enzyme degradation data are shown in Table 5.

[0129] Table 5 Enzyme degradation data

[0130] As shown in Table 5, the cross-linked biopolysaccharide gel prepared by this invention has good anti-enzymatic degradation properties and a long duration of action in vivo.

[0131] Example 15

[0132] The cross-linked biopolysaccharide gel prepared in Example 11 before sterilization and after sterilization for 40 minutes were compared with commercially available butyric acid cross-linked sodium hyaluronate (commercially available I) in a staining experiment. The results are shown in Figure 1. As can be seen from Figure 1, compared with the commercially available butyric acid cross-linked sodium hyaluronate injection, the cross-linked sodium hyaluronate product prepared in this invention has more uniform and delicate particles with a smaller particle size. Therefore, the resistance during syringe injection is relatively lower, which is more conducive to injection.

[0133] Example 16

[0134] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0135] S1. Weigh 0.1270 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add 18.03 g of phosphate buffer / 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 mixture;

[0136] S2. Weigh 0.3869 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add 1.98 g of phosphate buffer / sodium chloride solution (pH=6.86), and dissolve evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture, stir for about 30 min to form a reaction solution, and then let it stand at 25℃ for 24 h for cross-linking reaction to obtain the reaction product, high viscoelastic cross-linked biopolysaccharide gel;

[0137] S3. Take 20.83g of the reaction product after the S2 crosslinking reaction, add 207.61g of phosphate buffer / sodium chloride solution (pH=6.82), stir and wash for 12h; filter through a sieve, drain the retained gel to obtain 63.34g of gel product; filter the gel product through a 0.15mm pore size filter and pulverize it into gel particles. The calculated swelling ratio is 204.08%, and the final product concentration is 16.4mg / mL; the elastic modulus G' of the product is 412.8Pa and the viscous modulus G” is 104.4Pa when measured at a frequency of 2.5Hz.

[0138] The obtained product was placed into vials, capped, and sterilized at 121℃ for 8 minutes. The elastic modulus G' of the product after sterilization at a frequency of 2.5Hz was 109.4Pa and the viscous modulus G” was 15.5Pa.

[0139] Example 17

[0140] This case study describes a method for preparing highly viscoelastic cross-linked biopolysaccharide gels, which includes the following steps:

[0141] S1. Weigh 0.1268 g of 3,6-bis(4-aminobutyl)-2,5-diketopiramate hydrochloride, add 18.03 g of phosphate buffer / 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 mixture;

[0142] S2. Weigh 0.3870 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, add 2.00 g of phosphate buffer / sodium chloride solution (pH=6.82), and dissolve evenly to form a condensing agent solution; filter the condensing agent solution and add it to the mixture, stir for about 30 min to form a reaction solution, and then let it stand at 25℃ for 24 h for cross-linking reaction to obtain the reaction product, high viscoelastic cross-linked biopolysaccharide gel;

[0143] S3. Add 199.47g of phosphate buffer / sodium chloride solution (pH=6.82) to the reaction product after the S2 crosslinking reaction, stir and wash for 12h; filter through a sieve, drain the retained gel to obtain 28.35g of gel product; pulverize the gel product into gel particles by filtering through a 0.15mm pore size filter. The calculated swelling ratio is 36.10%, and the final product concentration is 36.8mg / mL; the elastic modulus G' of the product was measured at a frequency of 2.5Hz and was 5238Pa, and the viscous modulus G” was 266.9Pa.

[0144] The obtained product was placed into vials, capped, and sterilized at 121℃ for 30 minutes. The elastic modulus G' of the product after sterilization at a frequency of 2.5Hz was 2992Pa and the viscous modulus G” was 80.76Pa.

[0145] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, characterized in that, Includes the following steps: After dissolving and mixing carboxyl-containing biopolysaccharides with diaminodikepiperazine crosslinking agents, a condensing agent is added to form a reaction solution, resulting in a crosslinking reaction to obtain a highly viscoelastic crosslinked biopolysaccharide gel. The diaminodikepiperazine crosslinking agent has the following chemical structure (Formula I): Where R is a normal alkyl or isoalkyl group with 10 or fewer carbon atoms.

2. The method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 1, characterized in that, Specifically, the steps include the following: S1. Dissolve one of the following compounds in a buffer solution: a carboxyl-containing biopolysaccharide or a diaminodionepiperazine crosslinking agent; then add the other compound and mix thoroughly to form a mixture. S2. Dissolve the condensing agent in the buffer solution, and then add it to the mixture to form a reaction solution for 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 crosslinking agent of the diaminodikepiperazine compound is selected from one or more of 3,6-di(4-aminobutyl)-2,5-dikepiperazine or its salts, 3,6-di(3-aminopropyl)-2,5-dikepiperazine or its salts.

4. A method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 1 or 2, characterized in that, The purity of the diaminodikepiperazine crosslinking agent is at least 99 wt%, and the amount of the diaminodikepiperazine crosslinking agent 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 the following: hyaluronic acid or its salt, alginate or its salt, carboxymethyl chitin or its salt, carboxymethyl chitosan or its salt, carboxymethyl cellulose or its salt, and chondroitin compounds. The biopolysaccharide has a mass fraction of 0.5%-10% in the reaction solution; The buffer solution is one of a phosphate buffer system or a phosphate buffer system / sodium chloride solution, and the pH of the buffer solution 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-methylpyridine iodide, and the molar amount of the condensing agent is 100%-300% of the molar amount of the diaminodikepiperazine compound crosslinking agent.

6. A method for preparing a highly viscoelastic cross-linked biopolysaccharide gel according to claim 1 or 2, characterized in that, It also includes swelling and rinsing the highly viscoelastic cross-linked biopolysaccharide gel with buffer solution after obtaining it, followed by crushing, filling and moist heat sterilization to obtain the 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 crosslinking reaction is in the range of 0℃-30℃; the time of the crosslinking reaction is in the range of 10h-80h. The conditions for moist heat sterilization are: sterilization at 121℃ for 10-40 minutes; The amount of the buffer solution used for swelling or rinsing is 3 to 10 times the weight of the reaction product obtained after the crosslinking 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 1-7, characterized in that, Before moist heat sterilization, the highly viscoelastic cross-linked biopolysaccharide gel has an elastic modulus of 200 Pa to 5000 Pa and a viscous modulus of 50 Pa to 500 Pa; after moist heat sterilization, the highly viscoelastic cross-linked biopolysaccharide gel has an elastic modulus of 100 Pa to 400 Pa and a viscous modulus of 30 Pa to 90 Pa. The biopolysaccharide content in the highly viscoelastic cross-linked biopolysaccharide gel is 5 mg / mL-25 mg / mL.

9. The use of the highly viscoelastic cross-linked biopolysaccharide gel prepared by the preparation method according to any one of claims 1-7 in the preparation of intra-injection formulations, intra-filler formulations, or topical drug carrier formulations.

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