An injectable subcutaneous filling hydrogel based on gluco-galacto- polysaccharide, its preparation method and application

Abstract

The present application relates to a kind of injectable subcutaneous filling hydrogel based on gluco-galactomannan and its preparation method and application.Gluco-galactomannan polysaccharide is dissolved in NaOH aqueous solution, stirred to be uniform, then a proper amount of crosslinking agent is added and stirred uniformly, and finally the gluco-galactomannan hydrogel is obtained under appropriate conditions.The preparation condition is mild, the method is simple, the reagent is less and there is no violent reaction, the cost is low, the industrialization is easy to realize, the prepared polysaccharide hydrogel has excellent degradation resistance, excellent mechanical properties, good biocompatibility, long duration in vivo, and is suitable for subcutaneous filling, wrinkle removal and joint cavity lubricating fluid replacement.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and hydrogel technology, specifically relating to an injectable subcutaneous filling hydrogel based on glucosamine, its preparation method, and its application. Background Technology

[0002] Tumor resection, traumatic injury, fat atrophy, congenital defects, and the aging process can all lead to the loss of normal structures in soft tissues such as subcutaneous fat, collagen, and muscle. To repair these defects, many artificial fillers have been widely used in clinical soft tissue restoration.

[0003] Ideal soft tissue fillers should possess good biocompatibility, flexibility, and biostability. Polysaccharide-based hydrogels have the advantage of abundant natural sources. Hyaluronic acid is a crucial component of the natural extracellular matrix and dominates the health and cosmetics markets. However, due to the presence of hyaluronidase, hyaluronic acid is readily degraded in vivo. Existing commercially available hyaluronic acid hydrogels are made by combining hyaluronic acid and cross-linking agents under specific conditions. While this prolongs the degradation time, repeated injections are still required within a year, increasing patient discomfort.

[0004] The applicant disclosed a strain of *Agrobacterium* and a method for producing and preparing its anti-inflammatory extracellular polysaccharide in another patent publication, CN111286466A. Simultaneously, the applicant's published paper (Anti-tumor activity and immunogenicity of a succinoglycan ricin. *Carbohydr Polym.* 2021 Mar 1; 255:117370.) provides the structural analysis of the aforementioned extracellular polysaccharide. Glucosylgalactan is a polysaccharide obtained by treating the aforementioned polysaccharide using an alkaline heating method, resulting in the removal of succinyl groups. Summary of the Invention

[0005] The problem this invention aims to solve is to provide an injectable polysaccharide hydrogel with high safety, excellent mechanical properties, and good biocompatibility to meet the needs of subcutaneous filling.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing an injectable subcutaneous filling hydrogel based on glucosamine, comprising the following steps:

[0008] (1) Pure glucan was obtained by treating succino-Riclin by alkaline heat treatment.

[0009] (2) Dissolve glucosamine in NaOH aqueous solution and stir at room temperature to obtain a transparent solution, which is glucosamine solution;

[0010] (3) Add the required crosslinking agent to the glucogalactan solution, stir at room temperature, and obtain glucogalactan hydrogel after chemical crosslinking;

[0011] (4) Neutralize the glucosamine hydrogel with an acidic solution, and then wash it with neutral deionized water 8 to 15 times to obtain a neutral glucosamine hydrogel.

[0012] (5) The neutral glucosamine hydrogel was swollen to a certain volume and sterilized at high temperature to obtain a sterile, neutral glucosamine hydrogel.

[0013] As a further aspect of the present invention: in step (1), the concentration of succinyl polysaccharide Riclin solution is 0.5-2wt%, the concentration of NaOH is 0.1wt%, and the reaction conditions are 105℃ for 10 minutes.

[0014] As a further aspect of the present invention: in step (2), the concentration of the glucan solution is 8-10 wt%, and the concentration of the NaOH aqueous solution is 0.1-0.2 M.

[0015] As a further aspect of the present invention: in step (3), polyethylene glycol diglycidyl ether (PEGDE) is used as a crosslinking agent, and the volume ratio of glucosamine solution to crosslinking agent is 1:0.036-1:0.108.

[0016] As a further aspect of the present invention: in step (3), the crosslinking temperature is 30-60℃ and the reaction time is 8-16h.

[0017] As a further aspect of the present invention: in step (4), the acidic solution is a 0.1-0.2M HCl solution.

[0018] As a further aspect of the present invention: in step (4), the glucosamine content in the obtained neutral glucosamine hydrogel is 20 mg / mL.

[0019] As a further aspect of the present invention: in step (5), the high-temperature sterilization conditions are 121°C for 20 minutes.

[0020] Another objective of this invention is to provide an injectable subcutaneous filler of glucosamine hydrogel that can be maintained subcutaneously for a long time.

[0021] Another object of the present invention is to provide the application of the above-mentioned galactomannan hydrogel in the preparation of subcutaneous soft tissue filling materials or in the preparation of joint cavity lubricating fluid.

[0022] The beneficial effects of this invention are:

[0023] Polyethylene glycol (PEG) is a non-toxic, FDA-approved amphiphilic polyether compound. Low molecular weight (Mw < 1000) PEG can be degraded and eliminated from the body. This invention prepares a novel polysaccharide-based hydrogel under alkaline conditions via a crosslinking reaction between polyethylene glycol diglycidyl ether (PEGDE) and glucosan. Results show that the glucosan hydrogel is a promising soft tissue reinforcing biomaterial.

[0024] (1) The present invention provides a novel injectable hydrogel with chemical cross-linking of microbial polysaccharides. The microbial polysaccharide hydrogel has good biocompatibility, excellent mechanical properties, and long duration in vivo, showing great potential in cosmetic medicine.

[0025] (2) The preparation process disclosed in this invention is simple, the reaction conditions are mild, it is easy to synthesize, and it is suitable for widespread use. Attached Figure Description

[0026] Figure 1 This is a morphological diagram of the gelation of glucogalactan hydrogel.

[0027] Figure 2 This is a schematic diagram illustrating the injectability of glucosamine hydrogel, in which... Figure 2 Figure (a) shows the shear rate scan curve of the glucogalactan hydrogel and the injection force diagram of the glucogalactan hydrogel injected through 25-gauge and 27-gauge needles. Figure 2 Figure (b) shows a schematic diagram of the injection of R-0.5 hydrogel, R-1.0 hydrogel and R-1.5 hydrogel through a 27-gauge needle;

[0028] Figure 3 The image shows the effect of injecting glucosamine hydrogel into the back of a mouse. Figure 3 Figure (a) shows macroscopic photographs of the glucan hydrogel after the skin on the back of the mouse was dissected at weeks 1, 6, and 16 post-surgery. Figure 3 Figure (b) shows the degradation trend of the hydrogel in mice;

[0029] Figure 4 The diagram shows the biocompatibility of the glucogalactan hydrogel. Figure 4 Figure (a) shows the survival rate of L929 cells cultured for 24 hours with different concentrations of glucan hydrogel extract. Figure 4 Figure (b) shows the histological evaluation of major organs (heart, liver, spleen, lung, and kidney) one week after subcutaneous injection of glucosamine hydrogel. Detailed Implementation

[0030] Unless otherwise specified, all technical and scientific terms used in this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise specified, the reagents or materials used in the following examples can be commercially available or synthesized by referring to existing methods.

[0031] The preferred embodiments of the present invention have been described in detail above. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

[0032] Example 1

[0033] Preparation of glucan-galactan:

[0034] (1) Dissolve succinyl polysaccharide Riclin powder in pure water to obtain a Riclin polysaccharide solution with a concentration of 0.5 wt%.

[0035] (2) Add 0.1 wt% NaOH to the Riclin polysaccharide solution, stir and mix well, and react at 105℃ for 10 minutes.

[0036] (3) Filter the reacted polysaccharide solution through a 1μm fiber membrane, adjust the pH of the filtrate to neutral, and add alcohol to obtain polysaccharide precipitate, which is glucogalactan; the structural formula of glucogalactan is:

[0037]

[0038] Where n is between 1000 and 2000.

[0039] Example 2

[0040] Preparation of injectable subcutaneous filler hydrogel containing glucosan:

[0041] (1) Dissolve the glucosamine polysaccharide powder prepared in Example 1 in a 0.1M NaOH aqueous solution and homogenize it at room temperature to obtain a transparent solution with a sugar concentration of 8wt%, which is a glucosamine solution;

[0042] (2) A certain amount of polyethylene glycol diglycidyl ether crosslinking agent was added to the galactomannan solution so that the volume ratio of the polysaccharide solution to the crosslinking agent was 1:0.036, 1:0.072, and 1:0.108. The mixture was stirred evenly at room temperature and then subjected to a chemical crosslinking reaction at 40°C for 8 hours to obtain galactomannan hydrogel.

[0043] (3) Neutralize the glucosamine hydrogel with 0.1M HCl solution, and then wash it with neutral deionized water 8 to 15 times to obtain a neutral glucosamine hydrogel with a glucosamine content of 20 mg / mL.

[0044] (4) The neutral glucosamine hydrogel was sterilized at 121°C for 20 min under high temperature and high pressure to obtain a sterile, neutral glucosamine hydrogel.

[0045] According to the above preparation method, 8 wt% of glucosamine polysaccharide solution was cross-linked with polyethylene glycol diglycidyl ether at volume ratios of 1:0.036, 1:0.072, and 1:0.108, respectively. After swelling, R-0.5 hydrogel, R-1.0 hydrogel and R-1.5 hydrogel with glucosamine polysaccharide content of 20 mg / mL were obtained, respectively.

[0046] like Figure 1 The figures show the morphologies of R-0.5, R-1.0, and R-1.5 hydrogels formed by cross-linking of glucosamine polysaccharide.

[0047] Example 3

[0048] Determination of injectability of injectable subcutaneous filler hydrogels based on glucan:

[0049] The R-0.5, R-1.0, and R-1.5 hydrogels were tested sequentially, and the injectability of the dexgalactan hydrogels was characterized using a rheometer. In the composite viscosity test, the viscosity of the hydrogels as a function of shear rate (0.1-1000 L / s) was measured. Then, using 25 and 27 gauge needles, the R-0.5, R-1.0, and R-1.5 hydrogels were respectively loaded into 1 mL syringes for injectability testing.

[0050] like Figure 2 As shown in Figure (a), the viscosity of the R-0.5 hydrogel, R-1.0 hydrogel, and R-1.5 hydrogel decreased rapidly by three orders of magnitude with increasing shear rate. Injectability was verified at a macroscopic level.

[0051] like Figure 2 As shown in Figure (b), all three hydrogels can be successfully extruded through needles No. 25 and No. 27 and integrated together to form a viscous whole gel, further visually verifying the injectability of the glucan hydrogel.

[0052] Example 4

[0053] In vivo mouse studies of injectable subcutaneous hydrogel based on glucosan:

[0054] During the modeling process, mice were randomly divided into four groups: R-0.5, R-1.0, R-1.5, and a control group. Hair was removed from the backs of the mice, and either dextrogalactan hydrogel or commercially available hyaluronic acid hydrogel (Bloomage Biotech, Runbaiyan, Modified Sodium Hyaluronate Gel for Injection, Production License No.: Lu Food and Drug Administration Medical Device Production Permit 20120123) was injected into the dorsal side of the mice. Mice were sacrificed at weeks 1, 6, and 16 after hydrogel injection. Skin tissue and hydrogel from the hydrogel area were excised, and the morphology, size, and mass changes of the hydrogel were observed and measured.

[0055] like Figure 3 As shown in Figure (a), the control group consisted of commercially available hyaluronic acid hydrogel. With the passage of time after implantation, the morphology and size of the control group hydrogel changed significantly due to the presence of hyaluronidase in the body. The morphology and size of the glucan hydrogel also changed. The R-0.5 hydrogel was thin and soft, possibly due to its weak cross-linking strength, making it difficult to maintain its mechanical properties subcutaneously for a long time. In contrast, the R-1.0 and R-1.5 hydrogels showed better retention.

[0056] like Figure 3 As shown in Figure (b), commercial hyaluronic acid hydrogels degrade to about 50% of their original weight after more than 16 weeks in vivo. In contrast, R-1.0 and R-1.5 hydrogels retain more than 80% of their original weight and are not easily degraded in vivo. R-0.5 hydrogels, due to their weaker cross-linking strength, degrade to about 60% of their original weight.

[0057] Example 5

[0058] Safety evaluation of injectable subcutaneous filler hydrogels based on glucan:

[0059] (1) Cytotoxicity test:

[0060] This experiment used the MTT assay and live / dead imaging method to evaluate the in vitro cytotoxicity of glucosan and glucosan hydrogel. L929 cells were seeded in 96-well plates and cultured for 24 hours. 1 mL of the hydrogel was incubated in DMEM for 48 hours to prepare the glucosan hydrogel extract at 37°C. The cells were then replaced with standard DMEM medium containing different concentrations of the glucosan hydrogel extract and cultured for another 24 hours. Cell viability was measured at 570 nm after MTT treatment.

[0061] (2) Visceral safety assessment:

[0062] This experiment evaluated the in vivo degradation and biocompatibility of dextrogalactan hydrogel by subcutaneous implantation in the back of Balb / c mice. 200 μL of sterile hydrogel was injected subcutaneously into the dorsal side of the mouse using a 27-gauge needle. The mice were euthanized 7 days after implantation. Major organs (heart, liver, spleen, lungs, and kidneys) were harvested for H&E and Masson staining to analyze the toxicity of the dextrogalactan hydrogel to the mouse's internal organs.

[0063] like Figure 4 As shown in Figure (a), cell viability exceeded 90% in all ranges, indicating that the glucogalactan hydrogel has good in vitro biocompatibility.

[0064] like Figure 4 As shown in Figure (b), no obvious inflammatory response or tissue necrosis was observed in the H&E stained images. This further confirms the low in vivo toxicity and good biocompatibility of the dextrogalactan hydrogel.

[0065] As shown in the above examples, this invention dissolves glucosamine polysaccharide in NaOH aqueous solution, stirs until homogeneous, adds an appropriate amount of crosslinking agent and stirs until homogeneous, reacts under appropriate conditions, and finally obtains glucosamine hydrogel. This preparation method is mild, simple, requires few reagents and involves no violent reactions, is low-cost, and easily industrialized. The prepared polysaccharide hydrogel exhibits excellent degradation resistance, superior mechanical properties, good biocompatibility, and long duration of action in vivo. It is suitable for applications such as subcutaneous filling, wrinkle removal, and replacement of joint lubricating fluid.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing an injectable subcutaneous filling hydrogel based on glucan, characterized in that, Includes the following steps: (1) Dissolve glucosamine in NaOH aqueous solution and stir at room temperature to obtain a transparent sugar solution, which is a glucosamine solution; the concentration of the glucosamine solution is 8wt%, and the structural formula of glucosamine is: ； Where n is between 500 and 10000; (2) Add the required crosslinking agent to the glucogalactan solution, stir at room temperature, and obtain glucogalactan hydrogel after chemical crosslinking. The volume ratio of the glucogalactan solution to the crosslinking agent is 1:0.072–1:0.108, and the crosslinking agent is polyethylene glycol diglycidyl ether. (3) Neutralize the glucosamine hydrogel with an acidic solution, and then wash it with neutral deionized water 8 to 15 times to obtain a neutral glucosamine hydrogel. (4) The neutral glucosamine hydrogel was swollen to a certain volume and sterilized at high temperature to obtain a sterile, neutral glucosamine hydrogel.

2. The method for preparing the injectable subcutaneous filling hydrogel based on glucan according to claim 1, characterized in that: In step (1), the concentration of the NaOH aqueous solution is 0.1-0.2 M.

3. The method for preparing the injectable subcutaneous filling hydrogel based on glucosamine according to claim 1, characterized in that: In step (2), the crosslinking temperature is 30-60 ℃ and the reaction time is 8-16 h.

4. The method for preparing the injectable subcutaneous filling hydrogel based on glucan according to claim 1, characterized in that: In step (3), the acidic solution is a 0.1-0.2 M HCl solution.

5. The method for preparing the injectable subcutaneous filling hydrogel based on glucosamine according to claim 1, characterized in that: In step (3), the glucosamine content in the obtained neutral glucosamine hydrogel is 20 mg / mL.

6. The method for preparing the injectable subcutaneous filling hydrogel based on glucan according to claim 1, characterized in that: In step (4), the high-temperature sterilization conditions are 121 °C for 20 min.

7. The glucan hydrogel obtained by the preparation method according to any one of claims 1 to 6.

8. The use of the glucan hydrogel according to claim 7 in the preparation of subcutaneous soft tissue filling materials or in the preparation of joint lubricating fluid.

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