Injectable hydrogel based on gamma-polyglutamic acid and preparation method thereof

Abstract

The preparation method comprises the following steps: adding a cross-linking agent, a carboxyl activator, a catalyst and a viscosity modifier on the basis of gamma-polyglutamic acid to form a hydrogel solution with a three-cross-linked structure; compared with hydrogel with a single component, the injectable hydrogel based on gamma-polyglutamic acid has the advantages that the mechanical property of hydrogel with a three-crosslinking structure is obviously enhanced, and the injectable hydrogel has better thermal stability and biocompatibility.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to an injectable hydrogel based on gamma-polyglutamic acid and a preparation method thereof. Background Art

[0002] Injectable hydrogels are liquid at room temperature and can be delivered into the human body through a simple injection. They then solidify into a gel inside the body or at the injection site. Therefore, they have broad application potential in drug delivery, tissue engineering, minimally invasive surgery and other fields.

[0003] Chinese patent CN202010137448.3 discloses an injectable hydrogel with high mechanical strength and stability, and its preparation method and application. The patent comprises two components: a buffer solution containing a hydroxylamine-terminated polyethylene glycol block polymer and a buffer solution containing an aldehyde-terminated S4 arm polyethylene glycol. The two components are evenly mixed in a molar ratio of 1:1 between the hydroxylamine group and the aldehyde group, and then allowed to stand to ensure that they are completely gelled. The injectable hydrogel has good biocompatibility and can be used in the field of biomaterials. However, in actual applications, this type of injectable hydrogel may darken in color due to oxidation, affecting its stability, because its molecular structure contains functional groups that are easily oxidized.

[0004] Chinese patent CN201810553473.2 discloses a method for preparing an injectable self-healing hydrogel, which uses γ-polyglutamic acid and tyramine hydrochloride as the main components of the material, and also uses NHS and EDC as activators and reaction catalysts to prepare the product. Compared with traditional injectable hydrogels, the self-healing hydrogel can uniformly encapsulate drugs, and produce better reproducible experimental results and higher therapeutic activity of drugs and cells under in vitro physiological conditions. After injection into the body, the broken hydrogel fragments can regenerate a complete biodegradable hydrogel at the target location, avoiding the risk of catheter blockage caused by premature polymerization, and can achieve drug controlled release. However, the material has the problems of insufficient injectability and filling properties. Summary of the invention

[0005] The purpose of the present invention is to provide an injectable hydrogel based on gamma-polyglutamic acid and a preparation method thereof.

[0006] The purpose and innovation of the present invention are as follows: on the basis of gamma-polyglutamic acid, a cross-linking agent, a carboxyl activator, a catalyst and a viscosity regulator are added to form a hydrogel with a three-cross-linking structure; on the basis of gamma-polyglutamic acid, cross-linking with the cross-linking agent can form more chemical binding sites and groups; the viscosity regulator can form a hydration layer to regulate antioxidant; compared with a single-component hydrogel, the hydrogel with the three-cross-linking structure has significantly enhanced mechanical properties and better thermal stability and biocompatibility; an ultrafiltration method is used in the production process, the process is simple, the cost is low, the endotoxin of the material can be effectively reduced, and better colloid particle formation can be achieved.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0008] An injectable hydrogel based on γ-polyglutamic acid and a preparation method thereof, comprising the following steps:

[0009] Step S1: dissolving 3-9 parts by weight of γ-polyglutamic acid and 2-7 parts by weight of a cross-linking agent in 40-60 parts by weight of distilled water at room temperature and dissolving both completely to obtain a precursor solution of γ-polyglutamic acid and the cross-linking agent; the γ-polyglutamic acid is a γ-polyglutamic acid with a molecular weight of 500,000 to 1.2 million prepared by a microbial fermentation method;

[0010] Step S2: adding 2-9 parts by weight of a carboxyl activator to the precursor solution of step S1, and stirring to mix the two evenly to form a first solution;

[0011] Step S3: adding 1-5 parts by weight of a catalyst to the first solution, and rapidly stirring until the mixture is uniformly mixed to form a second solution;

[0012] Step S4: adding 0.1-0.5 parts by weight of a viscosity modifier to the second solution, and stirring at 37° C. using a magnetic stirrer or a water bath until the viscosity modifier is completely dissolved and mixed evenly to form an improved hydrogel solution with a three-crosslinked structure;

[0013] Step S5: ultrafiltration treatment is performed on the improved hydrogel solution with a three-crosslinked structure formed in step S4 to remove small molecules without crosslinking and reduce the content of endotoxin to obtain a solute;

[0014] Step S6: re-dissolving the solute obtained after ultrafiltration in step S5 with water to form an injectable hydrogel based on γ-polyglutamic acid.

[0015] Furthermore, in step S1, the cross-linking agent includes one or more of glutaraldehyde, polyethylene glycol, formaldehyde, and tyramine hydrochloride.

[0016] Furthermore, in step S3, the catalyst includes one or more of N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and ferrous sulfate.

[0017] Furthermore, in step S4, the viscosity modifier includes one or more of polyvinyl alcohol, methyl cellulose, sodium hyaluronate, and chitosan.

[0018] The beneficial effects of the present invention are:

[0019] First, the present invention forms more chemical binding sites and groups by cross-linking with a cross-linking agent on the basis of γ-polyglutamic acid, thereby increasing its filling stability, and uses a viscosity modifier to form a hydration layer to regulate antioxidant properties. This is also the first time that a three-crosslinked chemical structure has been applied to this field. The three crosslinks are: self-crosslinking of γ-polyglutamic acid, where γ-polyglutamic acid can form a polymer network through intermolecular and intramolecular reactions; chemical crosslinking of γ-polyglutamic acid and tyramine hydrochloride, where γ-polyglutamic acid and tyramine hydrochloride (which can also be glutaraldehyde, polyethylene glycol, or formaldehyde) can be induced to undergo an amidation reaction by chemical crosslinking agents such as EDC and NHS to form an amide bond, thereby achieving chemical crosslinking; physical and chemical crosslinking of a viscosity modifier, sodium hyaluronate (which can also be polyvinyl alcohol, methylcellulose, or chitosan), where sodium hyaluronate reacts with the amino group of γ-polyglutamic acid or the amino group of tyramine hydrochloride through its carboxyl group, and this reaction can also involve physical adsorption and physical crosslinking of non-covalent bonds such as hydrogen bonds; compared with a single-component hydrogel, the hydrogel with the three-crosslinked structure has significantly enhanced mechanical properties and better thermal stability and biocompatibility.

[0020] Second: Reduce endotoxins and small biological molecules through ultrafiltration and re-dissolution, use 500,000-1.2 million molecular weight γ-polyglutamic acid prepared by microbial fermentation as the basis, and then use a series of cross-linking agents to form stable covalent bonds, thereby promoting cross-linking, and then use one or more carboxyl activators to make the carboxyl group more easily react with the amino group and further improve the reactivity of the carboxyl group, thereby improving the cross-linking efficiency. This process forms a preliminary cross-linked network structure, enhances the mechanical stability of the hydrogel, and brings it the desired physical and chemical properties.

[0021] Third: The preparation process of this application is simple, the preparation cost is low, and it is more suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention provides a physical picture of various stages of an injectable hydrogel based on γ-polyglutamic acid.

[0023] Figure 2This is an XPS graph of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention.

[0024] Figure 3 This is a TEM comparison diagram of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention and the Hi-body brand sodium hyaluronate composite solution for injection.

[0025] Figure 4 This is a DLS comparison chart of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention and the Hi-body brand sodium hyaluronate composite solution for injection.

[0026] Figure 5 This is a graph showing the viscosity test results of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention.

[0027] Figure 6 This is a comparison chart of endotoxin testing performed on the injectable hydrogel based on γ-polyglutamic acid provided by the present invention.

[0028] Figure 7 This is a graph showing the results of a CCK8 cytotoxicity experiment conducted on the injectable hydrogel based on γ-polyglutamic acid provided by the present invention.

[0029] Figure 8 This is a physical picture of the injection experiment of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention.

[0030] Fig. 9 The present invention provides an absorbance spectrum diagram of the injectable hydrogel based on γ-polyglutamic acid and the hemolysis rate of mouse blood.

[0031] Fig.10 It is a series of characterization pictures of the staining of mouse viscera and skin in the subcutaneous experiment of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings.

[0033] An injectable hydrogel based on γ-polyglutamic acid and a preparation method thereof, comprising the following steps:

[0034] Step S1: dissolving 3-9 parts by weight of γ-polyglutamic acid and 2-7 parts by weight of a cross-linking agent in 40-60 parts by weight of distilled water at room temperature and dissolving both completely to obtain a precursor solution of γ-polyglutamic acid and the cross-linking agent; the γ-polyglutamic acid is a γ-polyglutamic acid with a molecular weight of 500,000 to 1.2 million prepared by a microbial fermentation method;

[0035] Step S2: adding 2-9 parts by weight of a carboxyl activator to the precursor solution of step S1, and stirring to mix the two evenly to form a first solution;

[0036] Step S3: adding 1-5 parts by weight of a catalyst to the first solution, and rapidly stirring until the mixture is uniformly mixed to form a second solution;

[0037] Step S4: adding 0.1-0.5 parts by weight of a viscosity modifier to the second solution, and stirring at 37° C. using a magnetic stirrer or a water bath until the viscosity modifier is completely dissolved and mixed evenly to form an improved hydrogel solution with a three-crosslinked structure;

[0038] Step S5: ultrafiltration treatment is performed on the hydrogel solution with a three-crosslinked structure formed in step S4 to obtain solutes, remove small molecules without crosslinking, and reduce the content of endotoxins; the specific method is to add the prepared hydrogel solution with a three-crosslinked structure into an ultrafiltration centrifuge tube; put the ultrafiltration centrifuge tube with a lid into a centrifuge rotor and balance it with a similar ultrafiltration tube; use a swing bucket rotor for centrifugation at a maximum of 4000×g for 15-60 minutes; insert a pipette (gun tip) into the inner tube of the ultrafiltration centrifuge tube, move it left and right to absorb the sample, recover the concentrated product, ensure complete recovery, and the filtrate can be stored in the outer tube of the centrifuge;

[0039] Step S6: re-dissolving the solute obtained after ultrafiltration in step S5 with water to form an injectable hydrogel based on γ-polyglutamic acid.

[0040] Furthermore, the microbial fermentation method for preparing γ-polyglutamic acid with a molecular weight of 500,000 to 1.2 million is a prior art and will not be described in detail here.

[0041] like Figure 1 As shown, (a) is the actual precursor solution of γ-polyglutamic acid and a cross-linking agent obtained in step S1, (b) is the actual hydrogel solution with a three-cross-linked structure obtained in step S4 after improvement, and (c) is the actual injectable hydrogel based on γ-polyglutamic acid obtained after ultrafiltration and redissolution in steps S5 and S6.

[0042] Furthermore, in step S1, the cross-linking agent includes one or more of glutaraldehyde, polyethylene glycol, formaldehyde, and tyramine hydrochloride.

[0043] Furthermore, in step S3, the catalyst includes one or more of N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and ferrous sulfate.

[0044] Furthermore, in step S4, the viscosity modifier includes one or more of polyvinyl alcohol, methyl cellulose, sodium hyaluronate, and chitosan.

[0045] Further, in order to facilitate storage and transportation, the injectable hydrogel based on γ-polyglutamic acid of the present application can also be prepared into a lyophilized powder. The specific method is to mix and stir the injectable hydrogel based on γ-polyglutamic acid prepared in step S6 with a plasticizer, and then quickly freeze it at low temperature. The commonly used method is to use the pre-cooling function of liquid nitrogen or a freeze dryer, first freeze it at minus 80 degrees for one night, then open the container, wrap the tin foil, put it back in the refrigerator, and then prepare the sample in the freeze dryer. The freeze-dried powder needs to be collected in a sterile and dry environment to avoid contamination or moisture absorption. The freeze-dried powder is sealed in a moisture-proof and light-proof packaging material and stored in a dry and cool environment to avoid temperature and humidity changes to maintain the stability of the product. The method for preparing freeze-dried powder is prior art and will not be repeated here.

[0046] Figure 2 (a) is an XPS graph of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention; (b) is a graph of the binding energy peak of the Na element, and (c) is a graph of the binding energy peak of the Cl element. The binding energy peaks of the Cl and Na elements can be seen from the graph, indicating that tyramine hydrochloride and sodium hyaluronate have been successfully combined into the material system.

[0047] Figure 3 TEM comparison diagram of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention and the composite solution of sodium hyaluronate for injection of Hibody brand, wherein:

[0048] (a) is the TEM image of γ-polyglutamic acid hydrogel;

[0049] (b) is the TEM image of the composite solution of sodium hyaluronate for injection of HiTi brand;

[0050] (c) TEM image of the improved hydrogel solution with a triple cross-linked structure;

[0051] (d) is a TEM image of the injectable hydrogel based on γ-polyglutamic acid obtained after ultrafiltration and re-dissolution;

[0052] (e) is a single particle image of the injectable hydrogel based on γ-polyglutamic acid obtained after ultrafiltration and re-dissolution;

[0053] (f) is an enlarged image of a single particle of the injectable hydrogel based on γ-polyglutamic acid obtained after ultrafiltration and re-dissolution;

[0054] By comparison, we can see that the injectable hydrogel based on γ-polyglutamic acid is well formed.

[0055] Figure 4 This is a DLS comparison chart of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention and the composite solution of sodium hyaluronate for injection of Hibody brand, wherein:

[0056] (a) is the DLS graph of HiTi brand sodium hyaluronate complex solution for injection (3 tests);

[0057] (b) is the DLS graph of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention (3 tests); by comparison, it is known that the injectable hydrogel based on γ-polyglutamic acid of the present application has a better DLS distribution.

[0058] Figure 5 A viscosity test was conducted on the injectable hydrogel based on γ-polyglutamic acid provided in the present application; the test results showed that the viscosity of the hydrogel sample in the laboratory (viscosity value 32) can reach the industrial index, and the viscosity can be adjusted by adding the content of injection viscosity.

[0059] Figure 6 A comparison chart of endotoxin tests performed on the injectable hydrogel based on γ-polyglutamic acid provided by the present invention, wherein:

[0060] (a) is the test sample without endotoxin (blank control group);

[0061] (b) is the positive control group of the test article with an endotoxin content of 2λ;

[0062] (c) is the positive control group with endotoxin of 2λ;

[0063] (d) is the negative control group without endotoxin;

[0064] Endotoxin test is performed using the horseshoe crab reagent method. If the negative control group (d) and the blank control group (a) do not coagulate (negative), and the positive control group (c) and the test sample positive control group (b) coagulate (positive), it can be considered that the endotoxin content is below 0.5eu / ml. It can be seen from the figure that the endotoxin test sample interference experiment is qualified, and the endotoxin content meets the requirements of industrial production.

[0065] Figure 7 This is a result diagram of the CCK8 cytotoxicity experiment conducted on the injectable hydrogel based on γ-polyglutamic acid provided by the present invention; the results show that the survival rate of fibroblasts is above 80%, among which the cell survival rate of 125 μg / mL reaches 95%.

[0066] Figure 8 The following are the actual pictures of the injection experiment of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention. Among them, (a) and (b) show that the injectable hydrogel based on γ-polyglutamic acid provided by the present invention has good biocompatibility and subcutaneous filling effect; (c) shows the excellent elasticity and shaping ability of the injectable hydrogel based on γ-polyglutamic acid provided by the present invention; (d) is a real picture of the imaging of a small animal injected with the injectable hydrogel based on γ-polyglutamic acid provided by the present invention.

[0067] Fig. 9 The invention provides an absorption spectrum diagram of the hemolysis rate of the injectable hydrogel based on gamma-polyglutamic acid and mouse blood. The hemolysis rate can be calculated to be less than 5% by observing the absorption value of the material at 545nm.

[0068] Fig.10 It is a series of characterization diagrams of the injectable hydrogel based on γ-polyglutamic acid provided by the present application and the staining of mouse viscera and skin in subcutaneous experiments in mice, wherein:

[0069] (a)-(e) show that the injected materials did not affect or damage the internal organs of the mice;

[0070] (f)-(g) are H&E staining images of injected skin;

[0071] (h)-(i) are Masson staining images of injected skin;

[0072] It is shown that the injectable hydrogel based on γ-polyglutamic acid provided by the present application does not cause damage to the internal organs of mice, causes inflammation to the skin and is conducive to the generation of new tissues.

[0073] In addition, the Hi-Body brand sodium hyaluronate composite solution for injection used in some comparative experiments in this application is a relatively representative cosmetic filling hydrogel product on the market.

[0074] The described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. An injectable hydrogel based on γ-polyglutamic acid and a preparation method thereof, characterized in that: The following steps are included: Step S1: dissolving 3-9 parts by weight of γ-polyglutamic acid and 2-7 parts by weight of a cross-linking agent in 40-60 parts by weight of distilled water at room temperature and dissolving both completely to obtain a precursor solution of γ-polyglutamic acid and the cross-linking agent; the γ-polyglutamic acid is a γ-polyglutamic acid with a molecular weight of 500,000 to 1.2 million prepared by a microbial fermentation method; Step S2: adding 2-9 parts by weight of a carboxyl activator to the precursor solution of step S1, and stirring to mix the two evenly to form a first solution; Step S3: adding 1-5 parts by weight of a catalyst to the first solution, and rapidly stirring until the mixture is uniformly mixed to form a second solution; Step S4: adding 0.1-0.5 parts by weight of a viscosity modifier to the second solution, and stirring at 37° C. using a magnetic stirrer or a water bath until the viscosity modifier is completely dissolved and mixed evenly to form an improved hydrogel solution with a three-crosslinked structure; Step S5: ultrafiltration treatment is performed on the improved hydrogel solution with a three-crosslinked structure formed in step S4 to remove small molecules without crosslinking and reduce the content of endotoxin to obtain a solute; Step S6: re-dissolving the solute obtained after ultrafiltration in step S5 with water to form an injectable hydrogel based on γ-polyglutamic acid.

2. The injectable hydrogel based on γ-polyglutamic acid and the preparation method thereof according to claim 1, characterized in that: In the step S1, the cross-linking agent includes one or more of glutaraldehyde, polyethylene glycol, formaldehyde, and tyramine hydrochloride.

3. The injectable hydrogel based on γ-polyglutamic acid and the preparation method thereof according to claim 1, characterized in that: In step S3, the catalyst includes one or more of N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and ferrous sulfate.

4. The injectable hydrogel based on γ-polyglutamic acid and the preparation method thereof according to claim 1, characterized in that: In step S4, the viscosity modifier includes one or more of polyvinyl alcohol, methyl cellulose, sodium hyaluronate, and chitosan.

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