Lysine cross-linked hyaluronic acid gel as well as preparation method and application thereof

Abstract

The invention discloses lysine cross-linked hyaluronic acid gel as well as a preparation method and application thereof. The preparation method of the lysine cross-linked hyaluronic acid gel comprises the following steps: mixing a hyaluronic acid substance, DMTMM and lysine, and carrying out a cross-linking reaction to prepare the lysine cross-linked hyaluronic acid gel, the hyaluronic acid substance is added in the form of a hyaluronic acid substance solution, and the solution comprises a hyaluronic acid substance with the weight-average molecular weight of 70WDa-180WDa and a buffer solution; the hyaluronic acid substance accounts for 3-5% of the hyaluronic acid substance solution in percentage by mass; the mass ratio of the DMTMM to the hyaluronic acid substance is (0.74-1.1): 1. The cross-linked hyaluronic acid gel is prepared by taking lysine as a cross-linking agent, so that the prepared gel is ensured to have ideal biocompatibility and use safety. The gel prepared by the method also has ideal mechanical properties, and the degradation speed meets the requirements of injection filling materials.

Description

Technical Field

[0001] The present invention relates to the field of medical aesthetics and medicine, and in particular to a lysine-crosslinked hyaluronic acid gel, and a preparation method and application thereof. Background Art

[0002] With age and external stimuli such as ultraviolet rays, the content of components such as hyaluronic acid and collagen in the skin layer decreases, and the skin shows aging conditions such as wrinkles and water loss. Aging and damaged skin can be treated by injectable filling materials. Hyaluronic acid is a polysaccharide composed of repeated disaccharide units composed of D-glucuronic acid and N-acetyl-D-glucosamine. It is an endogenous natural mucopolysaccharide in the human body. For example, it is distributed in the interstitial matrix of human cells, vitreous body, synovial fluid and skin layer. It has good biocompatibility, high hydrophilicity and cell repair effects. It is one of the widely used injectable filling materials in the field of medical beauty. However, hyaluronic acid is affected by factors such as hyaluronidase and free radicals in the body. It degrades quickly in the body, has a short retention time, poor viscoelasticity, and an unsatisfactory filling effect.

[0003] At present, BDDE cross-linking method is used to modify hyaluronic acid. The physical and chemical properties of the cross-linked hyaluronic acid gel obtained are significantly improved compared with natural hyaluronic acid, overcoming the defects of hyaluronic acid such as rapid degradation in the body and unsatisfactory filling effect. However, there is BDDE residue in the cross-linked hyaluronic acid gel obtained by this method. The epoxy group in BDDE has high reactivity and is listed as a group that needs to be regulated by the Food and Drug Administration. The residual epoxy group causes the cross-linked hyaluronic acid gel to be potentially toxic.

[0004] Therefore, there is an urgent need in the art to develop a cross-linked hyaluronic acid gel that is highly safe to use and has a filling effect and degradation rate that meet the requirements of injectable filling products. Summary of the invention

[0005] The technical problem to be solved by the present application is to overcome the defects of poor viscoelasticity of hyaluronic acid, fast degradation rate in vivo, poor safety of BDDE cross-linked hyaluronic acid gel in the prior art, and provide a lysine cross-linked hyaluronic acid gel, and its preparation method and application. The present application uses lysine as a cross-linking agent to prepare cross-linked hyaluronic acid gel. Since lysine is an endogenous substance in the human body, it is ensured that the obtained lysine cross-linked hyaluronic acid gel has ideal biocompatibility and safety in use. The lysine cross-linked hyaluronic acid gel obtained by the method also has ideal mechanical properties, and the degradation rate meets the requirements of injection filling materials, wherein the elastic modulus and viscosity modulus are both high, and on the premise of ensuring good support, it also has ideal viscosity, and when filling the bridge of the nose or nasolabial groove and other parts, it is not easy to slide down, and the filling effect is ideal.

[0006] This application solves the above technical problems through the following technical solutions.

[0007] The present application provides a method for preparing a lysine-crosslinked hyaluronic acid gel, comprising the following steps: mixing a hyaluronic acid substance, DMTMM and lysine, and performing a crosslinking reaction to obtain the lysine-crosslinked hyaluronic acid gel;

[0008] The hyaluronic acid substance is added in the form of a hyaluronic acid substance solution, which includes a hyaluronic acid substance with a weight average molecular weight of 70WDa to 180WDa and a buffer solution; the mass percentage of the hyaluronic acid substance in the hyaluronic acid substance solution is 3% to 5%; the mass ratio of the DMTMM to the hyaluronic acid substance is (0.74 to 1.1):1.

[0009] In some embodiments, the hyaluronic acid-like substance is selected from hyaluronic acid and / or hyaluronate.

[0010] Wherein, the hyaluronate is selected from at least one of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate and zinc hyaluronate.

[0011] In some embodiments, the weight average molecular weight of the hyaluronic acid substance is 130 WDa to 180 WDa.

[0012] In some embodiments, the mass percentage of the hyaluronic acid substance in the hyaluronic acid substance solution is 3.5% to 5%.

[0013] In some embodiments, the buffer is selected from phosphate buffer and / or citrate buffer.

[0014] Preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate and water. More preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate, NaCl and water. Preferably, the citrate buffer comprises citric acid, dihydrogen phosphate and water. Wherein, the dihydrogen phosphate is preferably selected from potassium dihydrogen phosphate and / or sodium dihydrogen phosphate. The dihydrogen phosphate is preferably selected from dipotassium hydrogen phosphate and / or disodium hydrogen phosphate.

[0015] In a preferred embodiment, the phosphate buffer comprises 20-30 g / L of Na2HPO4, 5-9 g / L of NaH2PO4 and water; for example, the phosphate buffer comprises 26.7 g / L of Na2HPO4, 6.9 g / L of NaH2PO4 and water.

[0016] In some embodiments, the pH value of the buffer solution is 6.2-7.5, for example, 7.

[0017] In some embodiments, the osmotic pressure of the buffer solution is 150-300 mOsm / L, for example 250 mOsm / L.

[0018] In some embodiments, the method for preparing the hyaluronic acid solution comprises the following steps: mixing the hyaluronic acid with the buffer solution.

[0019] The mixing time may be conventional in the art, generally, it is sufficient to allow the hyaluronic acid substance to be completely dissolved in the buffer solution, for example, 3 to 4 hours.

[0020] In some embodiments, the mass ratio of the DMTMM to the hyaluronic acid is (0.75-0.9): 1. DMTMM is also known as 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride.

[0021] In some embodiments, the mass ratio of the lysine to the hyaluronic acid is 1:(6-16), preferably 1:(8-14), for example 1:10.

[0022] In some embodiments, the lysine can be added in the form of a lysine solution according to conventional practice in the art, wherein the solvent in the lysine solution is a buffer solution.

[0023] In the lysine solution, the mass ratio of the lysine to the buffer solution is (0.05-0.15):1, for example, 0.1:1.

[0024] In the lysine solution, the buffer is selected from phosphate buffer and / or citrate buffer.

[0025] Preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate and water. More preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate, NaCl and water. Preferably, the citrate buffer comprises citric acid, dihydrogen phosphate and water. Wherein, the dihydrogen phosphate is preferably selected from potassium dihydrogen phosphate and / or sodium dihydrogen phosphate. The dihydrogen phosphate is preferably selected from dipotassium hydrogen phosphate and / or disodium hydrogen phosphate.

[0026] In a preferred embodiment, the phosphate buffer comprises 20-30 g / L of Na2HPO4, 5-9 g / L of NaH2PO4 and water; for example, the phosphate buffer comprises 26.7 g / L of Na2HPO4, 6.9 g / L of NaH2PO4 and water.

[0027] In the lysine solution, the pH value of the buffer solution is 6.2-7.5, for example, 7.

[0028] In the lysine solution, the osmotic pressure of the buffer solution is 150-300 mOsm / L, for example, 250 mOsm / L.

[0029] In some embodiments, the cross-linking reaction is performed under static conditions.

[0030] In some embodiments, the cross-linking reaction time is 12 to 72 hours, such as 24 hours or 48 hours.

[0031] In some embodiments, the temperature of the cross-linking reaction is 2-8°C, for example 4°C.

[0032] In some embodiments, the cross-linking reaction may be further followed by at least one of dialysis, screening and sterilization.

[0033] Wherein, the dialysate used for the dialysis is selected from phosphate buffer and / or citrate buffer.

[0034] Preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate and water. More preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate, NaCl and water. Preferably, the citrate buffer comprises citric acid, dihydrogen phosphate and water. Wherein, the dihydrogen phosphate is preferably selected from potassium dihydrogen phosphate and / or sodium dihydrogen phosphate. The dihydrogen phosphate is preferably selected from dipotassium hydrogen phosphate and / or disodium hydrogen phosphate.

[0035] In a preferred embodiment, the phosphate buffer comprises 20-30 g / L of Na2HPO4, 5-9 g / L of NaH2PO4 and water; for example, the phosphate buffer comprises 26.7 g / L of Na2HPO4, 6.9 g / L of NaH2PO4 and water.

[0036] The pH value of the dialysate used in the dialysis is 6.2-7.5, for example 7.

[0037] The osmotic pressure of the dialysate used in the dialysis is 150-300 mOsm / L, for example, 250 mOsm / L.

[0038] Wherein, the dialysis time is 2 to 4 days, for example, 3 days.

[0039] The mesh size of the sieve used for the sieving is 200 to 500 meshes, such as 300 meshes or 400 meshes.

[0040] Wherein, the sterilization method is the moist heat sterilization method conventionally used in the art.

[0041] In a preferred embodiment, the preparation method of the lysine cross-linked hyaluronic acid gel comprises the following steps:

[0042] (1) The DMTMM and the hyaluronic acid substance are mixed and activated to obtain material A;

[0043] (2) The material A is mixed with the lysine, and subjected to the cross-linking reaction to obtain the lysine-cross-linked hyaluronic acid gel.

[0044] In step (1), the activation can be carried out under stirring conditions according to the conventional practice in the art.

[0045] In step (1), the activation time is 20 to 30 minutes.

[0046] In step (1), the activation temperature is room temperature.

[0047] In step (2), the mixing comprises the following steps: mixing the lysine and a buffer solution to obtain a lysine solution, and adding the lysine solution dropwise into the material A.

[0048] Wherein, the mass ratio of the lysine to the buffer is (0.05-0.15):1, for example, 0.1:1.

[0049] In step (2), the mixing time may be conventional in the art, preferably 10 to 30 minutes, for example 20 minutes.

[0050] The present application also provides a lysine-crosslinked hyaluronic acid gel, which is prepared by the preparation method of the lysine-crosslinked hyaluronic acid gel as described above.

[0051] The present application also provides a use of the lysine cross-linked hyaluronic acid gel as described above as a raw material in the preparation of an injectable filling material.

[0052] In some embodiments, the injectable filling material is selected from a nose bridge filling material, a nasolabial fold filling material, a neck wrinkle filling material, a forehead wrinkle filling material or a joint cavity filling material.

[0053] The present application also provides an injectable filling material, which includes the lysine cross-linked hyaluronic acid gel as described above.

[0054] In some embodiments, the injectable filling material further includes an anesthetic.

[0055] Wherein, the anesthetic is selected from at least one of lidocaine hydrochloride, lidocaine carbonate, tetracaine, prilocaine, procaine, mepivacaine and bupivacaine.

[0056] The amount of the anesthetic added may be conventional in the art.

[0057] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0058] The reagents and raw materials used in this application are commercially available.

[0059] The positive progress of the present application is that: the present application uses lysine as a cross-linking agent to prepare a cross-linked hyaluronic acid gel. Since lysine is an endogenous substance in the human body, the prepared lysine cross-linked hyaluronic acid gel is ensured to have ideal biocompatibility and safety in use. The lysine cross-linked hyaluronic acid gel prepared by this method also has ideal mechanical properties, and the degradation rate meets the requirements of injection filling materials, wherein the elastic modulus and the viscosity modulus are both high, and on the premise of ensuring good support, it also has ideal viscosity, and when filling the nasolabial groove behind the bridge of the nose and other parts, it is not easy to slide down, and the filling effect is ideal. DETAILED DESCRIPTION

[0060] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0061] The reagents and raw materials used in the following examples are all commercially available, and the purity of the reagents used is injection grade.

[0062] The configuration of the phosphate buffer used in the following examples and comparative examples is as follows: 26.7 g of disodium hydrogen phosphate (Na2HPO4) and 6.9 g of sodium dihydrogen phosphate (NaH2PO4) are added to every 1 L of water for injection; the pH value of the phosphate buffer is 7, and the osmotic pressure is 250 mOsm / L.

[0063] Example 1

[0064] (1) Sodium hyaluronate with a weight average molecular weight of 70 WDa was mixed with phosphate buffer and stirred for 3 hours to prepare a sodium hyaluronate solution with a sodium hyaluronate mass percentage of 3.5%; DMTMM was added to the sodium hyaluronate solution, with a mass ratio of DMTMM to sodium hyaluronate of 0.75:1, and the mixture was stirred and activated for 20 minutes to obtain material A;

[0065] (2) Lysine and phosphate buffer were mixed at a mass ratio of 0.1:1 to prepare a lysine solution, and the lysine solution was added dropwise to material A at a mass ratio of lysine to sodium hyaluronate of 1:10, and stirred for 20 minutes. The material was allowed to stand for cross-linking at 4°C for 24 hours. The cross-linked material was dialyzed in phosphate buffer for 3 days, and the dialysate was replaced twice during the dialysis process. The dialyzed material was weighed to 20 mg / g (i.e., 20 mg of sodium hyaluronate per gram of the system), passed through a 300-mesh sieve twice, and then sterilized by wet heat at 121°C for 12 minutes to obtain a lysine-cross-linked hyaluronic acid gel.

[0066] Examples 2 to 5 and Comparative Examples 1 to 4

[0067] Compared with Example 1, the only difference is the weight average molecular weight of sodium hyaluronate, the concentration of sodium hyaluronate solution or the mass ratio of DMTMM to sodium hyaluronate. Other conditions and parameters are the same as those in Example 1. Please refer to Table 1 for details.

[0068] Table 1

[0069]

[0070]

[0071] Comparative Example 5 (Compared with Example 2, the difference is that the mass percentage of sodium hyaluronate in the sodium hyaluronate solution is 2%)

[0072] (1) Sodium hyaluronate with a weight average molecular weight of 130 WDa was mixed with phosphate buffer and stirred for 3 hours to prepare a sodium hyaluronate solution with a sodium hyaluronate mass percentage of 2%; DMTMM was added to the sodium hyaluronate solution, with a mass ratio of DMTMM to sodium hyaluronate of 0.75:1, and the mixture was stirred and activated for 20 minutes to obtain material A;

[0073] (2) Lysine and phosphate buffer were mixed at a mass ratio of 0.1:1 to prepare a lysine solution, and the lysine solution was added dropwise to material A at a mass ratio of lysine to sodium hyaluronate of 1:10, and stirred for 20 minutes. The cross-linked material was allowed to stand for 24 hours at 4°C. The cross-linked material was dialyzed in phosphate buffer for 3 days, and the dialysate was replaced twice during the dialysis process. The dialyzed material was passed through a 300-mesh sieve twice, and then sterilized by wet heat at 121°C for 12 minutes to obtain a lysine-cross-linked hyaluronic acid gel.

[0074] Effect Example 1 Mechanical Properties Test

[0075] The sample states of the lysine cross-linked hyaluronic acid gels prepared in the above examples and comparative examples were observed, and the elastic modulus G', viscous modulus G" and complex viscosity η* were tested. The results are shown in Table 2 below.

[0076] Test method: Use a rheometer to perform dynamic frequency scanning, set the test temperature to 25°C, and the frequency range is 0.1-10Hz. Compare the elastic modulus G', viscous modulus G" and complex viscosity η* at 1Hz.

[0077] Table 2

[0078]

[0079] According to the results in Table 2, it can be seen that the weight average molecular weight of sodium hyaluronate, the concentration of sodium hyaluronate solution, and the mass ratio of DMTMM to sodium hyaluronate have a great influence on whether a gel can be prepared and the viscoelasticity of the prepared gel.

[0080] According to the results of Comparative Example 1, when the weight-average molecular weight of sodium hyaluronate is low, the obtained lysine cross-linked hyaluronic acid gel has poor thermal stability, insufficient strength after sterilization, small elastic modulus, poor deformation resistance and supporting capacity, and is not easy to rebound after being subjected to external force; and the viscous modulus and complex viscosity are both small, the adhesion ability is poor after injection, and it is easy to shift.

[0081] According to the results of Comparative Example 2, when the weight average molecular weight of sodium hyaluronate is too high, it can form a gel after cross-linking, but the gel state is destroyed after sterilization and becomes fluid, and has poor thermal stability and cannot be used as a filling material.

[0082] According to the results of Comparative Example 3, when the concentration of the sodium hyaluronate solution is high, stirring is difficult during the preparation process, and the sodium hyaluronate cannot be evenly dissolved in the phosphate buffer solution, resulting in inconsistent physical and chemical properties of various parts of the prepared lysine cross-linked hyaluronic acid gel.

[0083] According to the results of Comparative Example 4, when the amount of DMTMM added is high, it is not conducive to the cross-linking of hyaluronic acid, which will lead to a decrease in the elastic modulus of the prepared lysine cross-linked hyaluronic acid gel, poor deformation resistance and supporting capacity, and difficulty in rebounding after being subjected to external force.

[0084] In Comparative Example 5, the concentration of sodium hyaluronate is low. When the cross-linked gel is sterilized, the viscoelasticity drops suddenly, resulting in the elastic modulus of the obtained lysine cross-linked hyaluronic acid gel being only 52.97 Pa, with poor deformation resistance and supporting capacity, and it is not easy to rebound after being subjected to external force.

[0085] Effect Example 2 Anti-degradation Performance Test

[0086] The in vitro anti-enzymatic performance of the lysine cross-linked hyaluronic acid gel prepared in the above Example 1 and Comparative Example 1 was tested, and the results are shown in Table 3.

[0087] Test method: Soak the lysine cross-linked hyaluronic acid gel in a hyaluronidase solution, add hyaluronidase at a rate of 20 U / g of lysine cross-linked hyaluronic acid gel, treat in a water bath at 37°C, and test the shear viscosity at different time points.

[0088] Shear viscosity η test method: Use a rheometer to scan the shear rate, set the detection temperature to 25°C, and the shear rate range to 0.1s -1 ~100s -1 , take the shear rate as 1s -1 Shear viscosity value under certain conditions.

[0089] Table 3

[0090]

[0091] According to the results in Table 3, after hyaluronidase treatment, the shear viscosity of the lysine-crosslinked hyaluronic acid gel prepared in Example 1 changes significantly slower than that of Comparative Example 1 over time. It can be seen that the lysine-crosslinked hyaluronic acid gel prepared in Example 1 has ideal anti-degradation ability and maintains a longer time in the body.

[0092] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0093] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A method for preparing a lysine-crosslinked hyaluronic acid gel, characterized in that: The method comprises the following steps: mixing hyaluronic acid substances, DMTMM and lysine, and subjecting the mixture to a cross-linking reaction to obtain the lysine-cross-linked hyaluronic acid gel; The hyaluronic acid substance is added in the form of a hyaluronic acid substance solution, which includes a hyaluronic acid substance with a weight average molecular weight of 70WDa to 180WDa and a buffer solution; the mass percentage of the hyaluronic acid substance in the hyaluronic acid substance solution is 3% to 5%; the mass ratio of the DMTMM to the hyaluronic acid substance is (0.74 to 1.1):

1.

2. The method for preparing the lysine-crosslinked hyaluronic acid gel according to claim 1, characterized in that: The preparation method satisfies at least one of the following conditions (1) to (8): (1) The hyaluronic acid substance is selected from hyaluronic acid and / or hyaluronate; preferably, the hyaluronate is selected from at least one of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate and zinc hyaluronate; (2) The weight average molecular weight of the hyaluronic acid substance is 130 WDa to 180 WDa; (3) The mass percentage of the hyaluronic acid substance in the hyaluronic acid substance solution is 3.5% to 5%; (4) The buffer is selected from phosphate buffer and / or citrate buffer; preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate and water; more preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate, NaCl and water; preferably, the citrate buffer comprises citric acid, dihydrogen phosphate and water; wherein the dihydrogen phosphate is preferably selected from potassium dihydrogen phosphate and / or sodium dihydrogen phosphate; the dihydrogen phosphate is preferably selected from dipotassium hydrogen phosphate and / or disodium hydrogen phosphate; (5) The pH value of the buffer solution is 6.2 to 7.5; (6) The osmotic pressure of the buffer solution is 150 to 300 mOsm / L; (7) The mass ratio of the DMTMM to the hyaluronic acid substance is (0.75-0.9):1; (8) The mass ratio of the lysine to the hyaluronic acid is 1:(6-16), preferably 1:(8-14).

3. The method for preparing the lysine cross-linked hyaluronic acid gel according to claim 1 or 2, characterized in that: The lysine is added in the form of a lysine solution, wherein the solvent in the lysine solution is a buffer solution; Preferably, the mass ratio of the lysine to the buffer is (0.05-0.15):1; Preferably, the buffer is selected from phosphate buffer and / or citrate buffer; more preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate and water; further preferably, the phosphate buffer comprises dihydrogen phosphate, dihydrogen phosphate, NaCl and water; more preferably, the citrate buffer comprises citric acid, dihydrogen phosphate and water; wherein the dihydrogen phosphate is preferably selected from potassium dihydrogen phosphate and / or sodium dihydrogen phosphate; the dihydrogen phosphate is preferably selected from dipotassium hydrogen phosphate and / or disodium hydrogen phosphate; Preferably, the pH value of the buffer is 6.2 to 7.5; Preferably, the osmotic pressure of the buffer solution is 150-300 mOsm / L.

4. The method for preparing the lysine-crosslinked hyaluronic acid gel according to any one of claims 1 to 3, characterized in that: The preparation method satisfies at least one of the following conditions (1) to (4): (1) the cross-linking reaction is carried out under static conditions; (2) The cross-linking reaction time is 12 to 72 hours; (3) The temperature of the cross-linking reaction is 2 to 8°C; (4) The cross-linking reaction may be followed by at least one of dialysis, sieving and sterilization.

5. The method for preparing the lysine-crosslinked hyaluronic acid gel according to claim 4, characterized in that: The preparation method satisfies at least one of the following conditions (1) to (6): (1) The dialysate used in the dialysis is selected from phosphate buffer and / or citrate buffer; preferably, the phosphate buffer comprises dihydrogen phosphate, disodium hydrogen phosphate and water; more preferably, the phosphate buffer comprises dihydrogen phosphate, disodium hydrogen phosphate, NaCl and water; preferably, the citrate buffer comprises citric acid, disodium hydrogen phosphate and water; wherein the dihydrogen phosphate is preferably selected from potassium dihydrogen phosphate and / or sodium dihydrogen phosphate; the disodium hydrogen phosphate is preferably selected from dipotassium hydrogen phosphate and / or disodium hydrogen phosphate; (2) The pH value of the dialysate used in the dialysis is 6.2 to 7.5; (3) The osmotic pressure of the dialysate used in the dialysis is 150 to 300 mOsm / L; (4) The dialysis duration is 2 to 4 days; (5) The mesh size of the sieve used for the sieving is 200 to 500 meshes; (6) The sterilization method is moist heat sterilization.

6. The method for preparing the lysine-crosslinked hyaluronic acid gel according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) The DMTMM and the hyaluronic acid substance are mixed and activated to obtain material A; (2) The material A is mixed with the lysine, and subjected to the cross-linking reaction to obtain the lysine-cross-linked hyaluronic acid gel.

7. The method for preparing the lysine-crosslinked hyaluronic acid gel according to claim 6, characterized in that: The preparation method satisfies at least one of the following conditions (A) to (D): (A) In step (1), the activation time is 20 to 30 minutes; (B) In step (1), the activation temperature is room temperature; (C) In step (2), the mixing comprises the following steps: mixing the lysine and the buffer to obtain a lysine solution, and adding the lysine solution dropwise to the material A; preferably, the mass ratio of the lysine to the buffer is (0.05-0.15):1; (D) In ​​step (2), the mixing time is 10 to 30 minutes.

8. A lysine-crosslinked hyaluronic acid gel, characterized in that: The lysine-crosslinked hyaluronic acid gel is prepared by the preparation method of any one of claims 1 to 7.

9. Use of the lysine cross-linked hyaluronic acid gel as claimed in claim 8 as a raw material in the preparation of an injectable filling material; Preferably, the injectable filling material is selected from a nose bridge filling material, a nasolabial fold filling material, a neck wrinkle filling material, a forehead wrinkle filling material or a joint cavity filling material.

10. An injectable filling material, characterized in that: Comprising the lysine cross-linked hyaluronic acid gel as claimed in claim 8; Preferably, the injectable filling material also includes an anesthetic; More preferably, the anesthetic is selected from at least one of lidocaine hydrochloride, lidocaine carbonate, tetracaine, prilocaine, procaine, mepivacaine and bupivacaine.

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