Preparation method of an agarose gel for injection

Abstract

The present invention relates to the technical field of medical aesthetics, and particularly to a preparation method of an injectable agarose gel. The preparation of the injectable agarose gel of the present invention is obtained by using raw materials including agarose, a sodium hyaluronate derivative gel solution, and a buffer solution. The preparation of the sodium hyaluronate derivative gel solution of the present invention includes crosslinking sodium hyaluronate using glycerol triglycidyl ether and neopentyl glycol diglycidyl ether. The gel obtained by the present invention has excellent enzyme resistance, a long in vivo retention time, low toxicity, low residue, and a small extrusion force.

Description

Technical Field

[0001] The present invention relates to the technical field of medical beauty, and particularly to a preparation method of an injectable agarose gel containing a sodium hyaluronate derivative. Background Art

[0002] Injectable beauty is a type of non-surgical plastic surgery. By injecting a biological material or a synthetic biocompatible material into the dermis or subcutaneous tissue, different mechanisms are used to reduce skin wrinkles or reshape the body, which is a type of plastic surgery method.

[0003] Hyaluronic acid is a linear high-molecular-weight viscous polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. Hyaluronic acid is an endogenous substance in the human body, with good biocompatibility; it has high viscoelasticity and non-Newtonian rheological properties; it is non-toxic, non-immunogenic, non-irritating, has high safety, and can be degraded and eliminated by injecting hyaluronidase. Therefore, it is widely used as a soft tissue filler in beauty. That is, injecting hyaluronic acid into the skin to increase the volume of soft tissue can achieve the purpose of removing wrinkles or reshaping the body.

[0004] However, since hyaluronic acid itself exists in a liquid form, it is very easy to be decomposed under the action of hyaluronidase and free radicals in the body. The hyaluronic acid-based gel is basically degraded and absorbed in the body in about 6 months, and consumers have to inject again, which increases the burden on consumers. Therefore, it is difficult to achieve the plastic effect and the filling effect has a short duration.

[0005] Therefore, it is an urgent problem to provide an injectable agarose gel with excellent enzyme resistance, long retention time in the body, low toxicity, low residue, and small extrusion force. Summary of the Invention

[0006] Based on the solution of the above technical problems, the present invention proposes the technical solution of the present invention. The technical solution of the present invention includes a preparation method of an injectable agarose gel containing a sodium hyaluronate derivative, the product obtained by the method, and the application of the product.

[0007] The preparation of the injectable agarose gel containing a sodium hyaluronate derivative of the present invention is prepared using raw materials including agarose, a sodium hyaluronate derivative gel solution, and a buffer solution. The preparation of the sodium hyaluronate derivative gel solution of the present invention includes crosslinking sodium hyaluronate using glycerol triglycidyl ether and neopentyl glycol diglycidyl ether. The gel obtained by the present invention has excellent enzyme resistance, long retention time in the body, low toxicity, low residue, and small extrusion force.

[0008] On the one hand, the present invention provides a preparation method of an injectable agarose gel containing a sodium hyaluronate derivative, including the following steps:

[0009] Step 1: Weigh raw materials: agarose, sodium hyaluronate derivative gel solution, and buffer solution.

[0010] Step 2: Place the buffer solution in a mixing container, then add agarose and sodium hyaluronate derivative gel solution, heat and mix evenly, cool, and sterilize to obtain the agarose gel for injection.

[0011] Furthermore, calculated by mass parts, the raw materials contain 4 - 8 parts of agarose, 20 - 40 parts of sodium hyaluronate derivative gel solution, and 180 - 300 parts of buffer solution.

[0012] Furthermore, the dosage of the agarose is any value or the range between any two values among 4 parts by mass, 4.5 parts by mass, 5 parts by mass, 5.5 parts by mass, 6 parts by mass, 6.5 parts by mass, 7 parts by mass, 7.5 parts by mass, and 8 parts by mass.

[0013] Furthermore, the dosage of the sodium hyaluronate derivative gel solution is any value or the range between any two values among 20 parts by mass, 25 parts by mass, 25 parts by mass, 30 parts by mass, 33 parts by mass, 35 parts by mass, 38 parts by mass, and 40 parts by mass.

[0014] Furthermore, the dosage of the buffer solution is any value or the range between any two values among 180 parts by mass, 190 parts by mass, 200 parts by mass, 210 parts by mass, 220 parts by mass, 230 parts by mass, 240 parts by mass, 250 parts by mass, 260 parts by mass, 270 parts by mass, 280 parts by mass, 290 parts by mass, and 300 parts. The pH of the buffer solution A is 6.2 - 6.8.

[0015] Furthermore, the heating temperature in Step 2 is 35 - 60°C, or any value or the range between any two values among 35°C, 37°C, 39°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C.

[0016] Furthermore, the uniform mixing in Step 2 is in the way of stirring and mixing. The rotation speed of the stirring is 30 - 5000 revolutions per minute, 50 - 1500 revolutions per minute, 60 - 1000 revolutions per minute, 80 - 800 revolutions per minute, 100 - 700 revolutions per minute, or 100 - 500 revolutions per minute. The stirring time is 0.2 - 10 hours, 0.2 - 9 hours, 0.2 - 8 hours, 0.3 - 7 hours, 0.4 - 6 hours, 0.5 - 5 hours, or 0.6 - 4 hours.

[0017] The cooling temperature in step 3 is 15 - 30 °C, or any value among 15 °C, 17 °C, 19 °C, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C or the range between any two values.

[0018] Furthermore, the preparation of the sodium hyaluronate derivative gel solution includes crosslinking sodium hyaluronate using glycerol triglycidyl ether and neopentyl glycol diglycidyl ether.

[0019] Furthermore, the molecular weight of the sodium hyaluronate is 200,000 - 2,500,000 Daltons, and more preferably any value among 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,100,000, 1,200,000, 1,300,000, 1,400,000, 1,500,000, 1,600,000, 1,700,000, 1,800,000, 1,900,000, 2,000,000, 2,100,000, 2,200,000, 2,300,000, 2,400,000, 2,500,000 Daltons or the range between any two values of Daltons molecular weight.

[0020] Furthermore, the mass ratio of glycerol triglycidyl ether to neopentyl glycol diglycidyl ether is 1:(3 - 5), 1:(3.2 - 5), 1:(3.2 - 4.7), 1:(3.5 - 4.7), 1:(3.5 - 4.5) or 1:(3.7 - 4.2).

[0021] Furthermore, the mass ratio of glycerol triglycidyl ether to sodium hyaluronate is 1:(50 - 500), 1:(50 - 400), 1:(50 - 300), 1:(60 - 200), 1:(70 - 150) or 1:(80 - 120).

[0022] Furthermore, the preparation of the sodium hyaluronate derivative gel solution includes the following steps:

[0023] Step A: Mix sodium hyaluronate evenly with an alkaline solution;

[0024] Step B: Add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether, heat, and carry out a crosslinking reaction to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate;

[0025] Step C: Crush and screen the intermediate material, and then perform multiple dialysis using buffer B to remove the liquid to obtain a gel;

[0026] Step D: Add buffer C to the gel obtained in step C, and then screen it to obtain the sodium hyaluronate derivative gel solution.

[0027] Further, the mass ratio of sodium hyaluronate to the alkali solution in step A is 1:(5 - 20), 1:(5 - 18), 1:(6 - 15), 1:(6 - 12), or 1:(6 - 10).

[0028] Further, the alkali solution in step A is an aqueous solution of an alkali, and the alkali is at least one of sodium hydroxide and potassium hydroxide. The mass concentration of the alkali solution is 0.5 - 2.5%, 0.6 - 2.4%, 0.7 - 2.3%, 0.8 - 2.2%, 0.9 - 2.1%, or 1 - 2%.

[0029] Further, the mixing in step A is carried out by stirring. The rotation speed of the stirring is 30 - 5000 revolutions per minute, 50 - 1500 revolutions per minute, 60 - 1000 revolutions per minute, 80 - 800 revolutions per minute, 100 - 700 revolutions per minute, or 100 - 500 revolutions per minute. The stirring time is 0.2 - 10 hours, 0.2 - 9 hours, 0.2 - 8 hours, 0.3 - 7 hours, 0.4 - 6 hours, 0.5 - 5 hours, or 0.6 - 4 hours.

[0030] Further, the heating temperature in step B is 35 - 60 °C, or any value or the range between any two values among 35 °C, 37 °C, 39 °C, 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C.

[0031] Further, the cross - linking reaction in step B is carried out while maintaining the stirring rotation speed of step A, and the reaction time is 0.5 - 20 hours, 3 - 18 hours, 5 - 16 hours, 6 - 15 hours, 7 - 14 hours, 8 - 14 hours, or 9 - 13 hours.

[0032] Further, the sieving in step C is through a sieve of 40 mesh, 50 mesh, 60 mesh, 70 mesh, 80 mesh, 90 mesh, or 100 mesh.

[0033] Further, buffer B in step C has a pH of 7.2 - 7.6. The total mass of buffer B is 50 - 500 times, 60 - 450 times, 65 - 400 times, 70 - 300 times, 80 - 250 times, or 90 - 200 times the mass of the material to be dialyzed. The number of dialysis times is 2 times, 3 times, 4 times, or more times.

[0034] Further, buffer C in step D has a pH of 6.7 - 7.2. The addition of buffer C makes the content of cross - linked sodium hyaluronate 20 - 50 mg / mL.

[0035] Further, the sieving in step D is through a sieve of 30 mesh, 40 mesh, 50 mesh, 60 mesh, 70 mesh, 80 mesh, or 90 mesh.

[0036] Furthermore, the preparation of the sodium hyaluronate derivative gel solution includes the following steps:

[0037] Step A: Sodium hyaluronate with a molecular weight of 200,000 - 2,500,000 daltons and an aqueous solution of sodium hydroxide and / or potassium hydroxide with a mass concentration of 0.5 - 2.5% are stirred at a rotation speed of 30 - 5000 revolutions per minute for 0.2 - 10 hours in a mass ratio of 1:(5 - 20) until evenly mixed;

[0038] Step B: While maintaining stirring, glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:(3 - 5) are added, heated to 35 - 60°C, and subjected to a cross-linking reaction for 0.5 - 20 hours to obtain an intermediate material of a gel containing cross-linked sodium hyaluronate; wherein, the mass ratio of glycerol triglycidyl ether to sodium hyaluronate is 1:(50 - 500);

[0039] Step C: The intermediate material is crushed and sieved, and then dialyzed multiple times using a buffer solution with a pH of 7.2 - 7.6 that is 50 - 500 times the mass of the sieved material to remove the liquid and obtain a gel;

[0040] Step D: A buffer solution with a pH of 6.7 - 7.2 is added to the gel obtained in Step C to adjust the content of cross-linked sodium hyaluronate to 20 - 50 mg / mL, and then sieved to obtain the sodium hyaluronate derivative gel solution.

[0041] On the other hand, the present invention provides an injectable agarose gel agent containing sodium hyaluronate derivatives prepared by the above preparation method.

[0042] On the other hand, the present invention provides an application, including the non-therapeutic use of the injectable agarose gel agent containing sodium hyaluronate derivatives in medical aesthetics.

[0043] Beneficial effects:

[0044] The preparation of the injectable agarose gel agent containing sodium hyaluronate derivatives of the present invention is prepared using raw materials including agarose, sodium hyaluronate derivative gel solution, and buffer solution. The preparation of the sodium hyaluronate derivative gel solution of the present invention includes cross-linking sodium hyaluronate using glycerol triglycidyl ether and neopentyl glycol diglycidyl ether. The gel agent obtained by the present invention has excellent enzyme resistance, a long in vivo retention time, low toxicity, low residue, and a small extrusion force.

[0045] The biocompatibility effect of the sodium hyaluronate derivative gel solution prepared by crosslinking sodium hyaluronate with glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in the present invention is good and has low toxicity; the sodium hyaluronate derivative gel solution prepared from glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:4 shows the optimal biocompatibility effect.

[0046] The sodium hyaluronate derivative gel solution prepared by crosslinking sodium hyaluronate with glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in the present invention has a good effect of inhibiting hyaluronidase degradation and has a long retention time in vivo.

[0047] In the present invention, glycerol triglycidyl ether and neopentyl glycol diglycidyl ether are used to crosslink sodium hyaluronate in the mass ratio range of 1:(3 - 5), and the obtained sodium hyaluronate derivative gel solution has better in vitro enzyme stability.

[0048] The injectable agarose gel agent of the present invention is convenient to use, has a small pushing force, is safe to use, has a low incidence rate of adverse reactions, has no carcinogenicity, no genotoxicity, and the cytotoxicity and skin sensitization are not greater than grade I, meeting the regulations of the national industry standards for biomedical materials.

[0049] The preparation method of the product of the present invention is simple, green and environmentally friendly, and has excellent effects, having a good application prospect. Detailed implementation manners

[0050] The present invention will be described below in combination with the detailed implementation manners, and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners are used to illustrate the present invention rather than limit the present invention.

[0051] I. Preparation of sodium hyaluronate derivative gel solution

[0052] 1. Sodium hyaluronate derivative gel solution 1:

[0053] The preparation steps are as follows:

[0054] Step A: Sodium hyaluronate with a molecular weight of about 800,000 daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.2% are stirred at a rotation speed of 600 revolutions per minute for 2 hours in a mass ratio of 1:12 to be mixed evenly;

[0055] Step B: While maintaining stirring, glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:4 (both as crosslinking agents) are added, heated to 45 °C, and subjected to a crosslinking reaction for 9.5 hours to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; among them, the mass ratio of glycerol triglycidyl ether to sodium hyaluronate is 1:100, that is, the mass ratio of the crosslinking agent to sodium hyaluronate is 1:20;

[0056] Step C: Crush the intermediate material, sieve it through a 70-mesh sieve, and then perform dialysis 3 times with an average dosage using a phosphate buffer solution with a pH of 7.4 and a volume 100 times that of the sieved material to remove the liquid, obtaining a gel.

[0057] Step D: Add a phosphate buffer solution with a pH of 6.8 to the gel obtained in Step C to adjust the content of cross-linked sodium hyaluronate to 30 mg / mL, and then sieve it through a 60-mesh sieve to obtain the sodium hyaluronate derivative gel solution 1.

[0058] 2. Sodium hyaluronate derivative gel solution 2:

[0059] The preparation steps are as follows:

[0060] Step A: Mix sodium hyaluronate with a molecular weight of about 1 million Daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.5% at a mass ratio of 1:15 and stir at a rotation speed of 800 revolutions per minute for 2.2 hours to mix evenly.

[0061] Step B: Keep stirring, add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether at a mass ratio of 1:3.2, heat to 40 °C, and carry out a cross-linking reaction for 9 hours to obtain an intermediate material containing cross-linked sodium hyaluronate gel; among them, the mass ratio of glycerol triglycidyl ether to sodium hyaluronate is 1:95.

[0062] Step C: Crush the intermediate material, sieve it through a 60-mesh sieve, and then perform dialysis 4 times with an average dosage using a phosphate buffer solution with a pH of 7.5 and a volume 200 times that of the sieved material to remove the liquid, obtaining a gel.

[0063] Step D: Add a phosphate buffer solution with a pH of 7.2 to the gel obtained in Step C to adjust the content of cross-linked sodium hyaluronate to 30 mg / mL, and then sieve it through a 50-mesh sieve to obtain the sodium hyaluronate derivative gel solution 2.

[0064] 3. Sodium hyaluronate derivative gel solution 3:

[0065] The preparation steps are as follows:

[0066] Step A: Mix sodium hyaluronate with a molecular weight of about 800,000 Daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.2% at a mass ratio of 1:12 and stir at a rotation speed of 600 revolutions per minute for 2 hours to mix evenly.

[0067] Step B: Keep stirring, add glycerol triglycidyl ether (as a crosslinking agent), heat to 45 °C, and carry out a crosslinking reaction for 9.5 hours to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; wherein, the mass ratio of the crosslinking agent (i.e., glycerol triglycidyl ether) to sodium hyaluronate is 1:20;

[0068] Step C: Crush the intermediate material, pass it through a 70-mesh sieve, and then perform dialysis 3 times with an average dosage using a phosphate buffer solution with a pH of 7.4 that is 100 times the mass of the sieved material to remove the liquid and obtain a gel;

[0069] Step D: Add a phosphate buffer solution with a pH of 6.8 to the gel obtained in Step C to adjust the content of crosslinked sodium hyaluronate to 30 mg / mL, and then pass it through a 60-mesh sieve to obtain the sodium hyaluronate derivative gel solution 3.

[0070] 4. Sodium hyaluronate derivative gel solution 4:

[0071] The preparation steps are as follows:

[0072] Step A: Mix sodium hyaluronate with a molecular weight of about 800,000 Daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.2% at a mass ratio of 1:12, and stir at a rotation speed of 600 revolutions per minute for 2 hours to mix evenly;

[0073] Step B: Keep stirring, add neopentyl glycol diglycidyl ether (as a crosslinking agent), heat to 45 °C, and carry out a crosslinking reaction for 9.5 hours to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; wherein, the mass ratio of the crosslinking agent to sodium hyaluronate is 1:20;

[0074] Step C: Crush the intermediate material, pass it through a 70-mesh sieve, and then perform dialysis 3 times with an average dosage using a phosphate buffer solution with a pH of 7.4 that is 100 times the mass of the sieved material to remove the liquid and obtain a gel;

[0075] Step D: Add a phosphate buffer solution with a pH of 6.8 to the gel obtained in Step C to adjust the content of crosslinked sodium hyaluronate to 30 mg / mL, and then pass it through a 60-mesh sieve to obtain the sodium hyaluronate derivative gel solution 4.

[0076] 5. Sodium hyaluronate derivative gel solution 5:

[0077] The preparation steps are as follows:

[0078] Step A: Mix sodium hyaluronate with a molecular weight of about 800,000 Daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.2% at a mass ratio of 1:12, and stir at a rotation speed of 600 revolutions per minute for 2 hours to mix evenly;

[0079] Step B: Keep stirring, add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:1 (both as crosslinking agents), heat to 45 °C, and carry out a crosslinking reaction for 9.5 hours to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; wherein, the mass ratio of the crosslinking agent to sodium hyaluronate is 1:20;

[0080] Step C: Crush the intermediate material, pass it through a 70-mesh sieve, and then perform dialysis 3 times with an average dosage using a phosphate buffer solution with a pH of 7.4 that is 100 times the mass of the sieved material to remove the liquid and obtain a gel;

[0081] Step D: Add a phosphate buffer solution with a pH of 6.8 to the gel obtained in Step C to adjust the content of crosslinked sodium hyaluronate to 30 mg / mL, and then pass it through a 60-mesh sieve to obtain the sodium hyaluronate derivative gel solution 5.

[0082] 6. Sodium hyaluronate derivative gel solution 6:

[0083] The preparation steps are as follows:

[0084] Step A: Mix sodium hyaluronate with a molecular weight of about 800,000 daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.2% at a mass ratio of 1:12, and stir at a rotation speed of 600 revolutions per minute for 2 hours to mix evenly;

[0085] Step B: Keep stirring, add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:2 (both as crosslinking agents), heat to 45 °C, and carry out a crosslinking reaction for 9.5 hours to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; wherein, the mass ratio of the crosslinking agent to sodium hyaluronate is 1:20;

[0086] Step C: Crush the intermediate material, pass it through a 70-mesh sieve, and then perform dialysis 3 times with an average dosage using a phosphate buffer solution with a pH of 7.4 that is 100 times the mass of the sieved material to remove the liquid and obtain a gel;

[0087] Step D: Add a phosphate buffer solution with a pH of 6.8 to the gel obtained in Step C to adjust the content of crosslinked sodium hyaluronate to 30 mg / mL, and then pass it through a 60-mesh sieve to obtain the sodium hyaluronate derivative gel solution 6.

[0088] 7. Sodium hyaluronate derivative gel solution 7:

[0089] The preparation steps are as follows:

[0090] Step A: Mix sodium hyaluronate with a molecular weight of about 800,000 daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.2% at a mass ratio of 1:12, and stir at a rotation speed of 600 revolutions per minute for 2 hours to mix evenly;

[0091] Step B: Keep stirring, add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:6 (both as crosslinking agents), heat to 45°C, and carry out a crosslinking reaction for 9.5 hours to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; wherein, the mass ratio of the crosslinking agent to sodium hyaluronate is 1:20;

[0092] Step C: Crush the intermediate material, pass it through a 70-mesh sieve, and then perform dialysis 3 times with an average dosage using a phosphate buffer solution with a pH of 7.4 that is 100 times the mass of the sieved material to remove the liquid and obtain a gel;

[0093] Step D: Add a phosphate buffer solution with a pH of 6.8 to the gel obtained in Step C to adjust the content of crosslinked sodium hyaluronate to 30 mg / mL, and then pass it through a 60-mesh sieve to obtain the sodium hyaluronate derivative gel solution 7.

[0094] 8. Sodium hyaluronate derivative gel solution 8:

[0095] The preparation steps are as follows:

[0096] Step A: Mix sodium hyaluronate with a molecular weight of about 800,000 daltons and an aqueous sodium hydroxide solution with a mass concentration of 1.2% at a mass ratio of 1:12, and stir at a speed of 600 revolutions per minute for 2 hours to mix evenly;

[0097] Step B: Keep stirring, add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:4 (both as crosslinking agents), heat to 45°C, and carry out a crosslinking reaction for 9.5 hours to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; wherein, the mass ratio of the crosslinking agent to sodium hyaluronate is 1:20;

[0098] Step C: Crush the intermediate material, pass it through a 70-mesh sieve, and then perform dialysis 3 times with an average dosage using a phosphate buffer solution with a pH of 7.4 that is 100 times the mass of the sieved material to remove the liquid and obtain a gel;

[0099] Step D: Add a phosphate buffer solution with a pH of 6.8 to the gel obtained in Step C to adjust the content of crosslinked sodium hyaluronate to 30 mg / mL, and then pass it through a 60-mesh sieve to obtain the sodium hyaluronate derivative gel solution 8.

[0100] 9. Perform a cytotoxicity test on the above sodium hyaluronate derivative gel solutions 1-8.

[0101] Irradiate the above sodium hyaluronate derivative gel solutions 1-8 with γ-rays for sterilization (dose 25 kGy), and then perform a cytotoxicity test.

[0102] The cytotoxicity test was carried out by referring to the standard of "Biological Evaluation of Medical Devices - In Vitro Cytotoxicity Test" to detect the cell proliferation rate. In this experiment, the extraction method was adopted, and L929 cells were used for the cytotoxicity test. The absorbance value was measured by an enzyme-linked immunosorbent assay tester to calculate the relative cell proliferation rate (RCR, %). The higher the RCR, the better the biocompatibility and the lower the toxicity. The cytotoxicity results of the above sodium hyaluronate derivative gel solution are shown in Table 1.

[0103] Table 1: Cytotoxicity Test of Sodium Hyaluronate Derivative Gel Solution

[0104]

[0105] From the test results in Table 1, it can be seen that the sodium hyaluronate derivative gel solution prepared by cross-linking sodium hyaluronate with glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in the present invention has good biocompatibility and low toxicity; the sodium hyaluronate derivative gel solution prepared from glycerol triglycidyl ether and neopentyl glycol diglycidyl ether with a mass ratio of 1:4 shows the best biocompatibility effect.

[0106] 10. The above sodium hyaluronate derivative gel solutions 1-8 were subjected to an in vitro enzyme stability test.

[0107] The above sodium hyaluronate derivative gel solutions 1-8 were sterilized by γ-ray irradiation (dose 25 kGy), and then an in vitro enzyme stability test was carried out.

[0108] Take 0.5 g of the above sodium hyaluronate derivative gel solution, add 2 mL of hyaluronidase solution with an activity of 300 U / mL, and then enzymatically hydrolyze at 37 °C for 40 hours; after enzymatic hydrolysis, add PBS buffer to 5 mL; then take 1 mL, add 4 mL of absolute ethanol and mix well, and then centrifuge at 10000 r / min for 12 min; take 2 mL of the supernatant after centrifugation and make up the volume to 5 mL with PBS buffer to obtain solution K1.

[0109] Take 0.5 g of the above sodium hyaluronate derivative gel solution, add 10 mL of sulfuric acid solution with a concentration of 0.5 mol / L, hydrolyze in a boiling water bath for 15 min, and then dilute with water to 100 mL to obtain solution K2.

[0110] Take 1 mL each of solution K1 and solution K2, and measure the glucuronic acid content in solution K1 and solution K2 by the modified carbazole colorimetric method.

[0111] Calculate the in vitro anti-enzyme degradation coefficient R of the gel solution. The calculation formula is: R = 100 * (1 - 0.625T1 / T2), where T1 is the glucuronic acid content of solution K1 and T2 is the glucuronic acid content of solution K2.

[0112] Among them, the higher the value of the in vitro anti-enzyme degradation coefficient R, the better the in vitro anti-enzyme degradation performance and the more stable the gel. The results of the in vitro enzyme stability test of the above sodium hyaluronate derivative gel solutions 1-8 are shown in Table 2.

[0113] Table 2: In vitro enzyme stability test of sodium hyaluronate derivative gel solution

[0114]

[0115] It can be seen from the test results in Table 2 that the sodium hyaluronate derivative gel solution prepared by crosslinking sodium hyaluronate with glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in the present invention has good inhibitory effect on the degradation of hyaluronidase and has a long retention time in vivo. From the comparison results of gel solutions 1, 5-8, it can be obtained that when glycerol triglycidyl ether and neopentyl glycol diglycidyl ether are used to crosslink sodium hyaluronate in the mass ratio range of 1:(3-5), the in vitro enzyme stability of the obtained sodium hyaluronate derivative gel solution is better. It is speculated that this may be because the crosslinking degree of the molecular chains, the strength and chain length of the crosslinking chains brought by different crosslinking agents affect the physical and chemical properties of the crosslinked products, and the properties of the crosslinked products obtained by doping different crosslinking agents for crosslinking are more stable.

[0116] II. Preparation of injectable agarose gel containing sodium hyaluronate derivative

[0117] The preparation of the injectable agarose gel is as follows:

[0118] Step 1: Weigh the raw materials in parts by mass: 5 parts of agarose, 35 parts of sodium hyaluronate derivative gel solution, and 220 parts of acetate buffer solution with a pH of 6.7;

[0119] Step 2: Place the buffer solution in a mixing container, then add agarose and sodium hyaluronate derivative gel solution, heat to 50°C, stir at 500 revolutions per minute for 1 hour to mix evenly, cool to 25°C, and sterilize by γ-ray irradiation (dose 28 kGy) to obtain the injectable agarose gel;

[0120] Among them, the injectable agarose gels prepared using the above sodium hyaluronate derivative gel solutions 1-8 are sequentially named injectable agarose gels 1-8. Then, performance tests of the products are carried out, and the test results are shown in Table 3.

[0121] Table 3: Performance test of gel

[0122]

[0123] As can be seen from the test content in Table 3, the agarose gel for injection of the present invention is convenient to use, has a small pushing force, is safe to use, has a low incidence of adverse reactions, has no carcinogenicity, no genotoxicity, and the cytotoxicity and skin sensitization are not more than Grade I, meeting the requirements of the national industry standard for biomedical materials.

[0124] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. Application of the combination of glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in synergistically reducing the cytotoxicity of sodium hyaluronate derivative gel solution, characterized in that, The preparation of the sodium hyaluronate derivative gel solution comprises the following steps: Step A: Mix sodium hyaluronate and an alkali solution evenly; Step B: Add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether, heat, and carry out a crosslinking reaction to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; Step C: Crush and screen the intermediate material, and then perform multiple dialysis using buffer B to remove the liquid to obtain a gel; Step D: Add buffer C to the gel obtained in Step C, and then screen again to obtain the sodium hyaluronate derivative gel solution; The mass ratio of glycerol triglycidyl ether to neopentyl glycol diglycidyl ether is 1:(3 - 5); The mass ratio of glycerol triglycidyl ether to sodium hyaluronate is 1:(50 - 500); The molecular weight of the sodium hyaluronate is 200,000 - 2,500,000 Daltons.

2. Application of the combination of glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in synergistically enhancing the in vitro enzyme stability of sodium hyaluronate derivative gel solution, characterized in that, The preparation of the sodium hyaluronate derivative gel solution comprises the following steps: Step A: Mix sodium hyaluronate and an alkali solution evenly; Step B: Add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether, heat, and carry out a crosslinking reaction to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; Step C: Crush and screen the intermediate material, and then perform multiple dialysis using buffer B to remove the liquid to obtain a gel; Step D: Add buffer C to the gel obtained in Step C, and then screen again to obtain the sodium hyaluronate derivative gel solution; The mass ratio of glycerol triglycidyl ether to neopentyl glycol diglycidyl ether is 1:(3 - 5); The mass ratio of glycerol triglycidyl ether to sodium hyaluronate is 1:(50 - 500); The molecular weight of the sodium hyaluronate is 200,000 - 2,500,000 Daltons.

3. Application of glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in a mass ratio of 1:(3 - 5) in further reducing the cytotoxicity of sodium hyaluronate derivative gel solution, characterized in that, The preparation of the sodium hyaluronate derivative gel solution comprises the following steps: Step A: Mix sodium hyaluronate and an alkali solution evenly; Step B: Add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether, heat, and carry out a crosslinking reaction to obtain an intermediate material of a gel containing crosslinked sodium hyaluronate; Step C: Crush and screen the intermediate material, and then perform multiple dialysis using buffer B to remove the liquid to obtain a gel; Step D: Add buffer C to the gel obtained in Step C, and then screen again to obtain the sodium hyaluronate derivative gel solution; The mass ratio of glycerol triglycidyl ether to neopentyl glycol diglycidyl ether is 1:(3 - 5); The mass ratio of glycerol triglycidyl ether to sodium hyaluronate is 1:(50 - 500); The molecular weight of the sodium hyaluronate is 200,000 - 2,500,000 Daltons.

4. Application of glycerol triglycidyl ether and neopentyl glycol diglycidyl ether in further enhancing the in vitro enzyme stability of the sodium hyaluronate derivative gel liquid in a mass ratio of 1:(3 - 5), characterized in that The preparation of the sodium hyaluronate derivative gel liquid comprises the following steps: Step A: Mix the sodium hyaluronate evenly with an alkaline solution; Step B: Add glycerol triglycidyl ether and neopentyl glycol diglycidyl ether, heat, and carry out a cross-linking reaction to obtain an intermediate material of a gel containing cross-linked sodium hyaluronate; Step C: Crush and sieve the intermediate material, and then perform multiple dialyses using buffer B to remove the liquid to obtain a gel; Step D: Add buffer C to the gel obtained in Step C, and then sieve to obtain the sodium hyaluronate derivative gel liquid; The mass ratio of the glycerol triglycidyl ether to the neopentyl glycol diglycidyl ether is 1:(3 - 5); The mass ratio of the glycerol triglycidyl ether to the sodium hyaluronate is 1:(50 - 500); The molecular weight of the sodium hyaluronate is 200,000 - 2,500,000 Daltons.

5. The application according to any one of claims 1-4, characterized in that, In Step 1, the pH of the buffer A is 6.2 - 6.

8.

6. The application according to any one of claims 1-4, characterized in that: The heating temperature in Step 2 is 35 - 60 °C.

7. The application according to any one of claims 1-4, characterized in that: The cooling temperature in Step 3 is 15 - 30 °C.

8. The application according to any one of claims 1-4, characterized in that: The mass ratio of the glycerol triglycidyl ether to the sodium hyaluronate is 1:(60 - 200).

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