A hyaluronic acid-multi-arm polyethylene glycol hydrogel and its preparation method and application

Abstract

The present invention discloses a hyaluronic acid-multi-arm polyethylene glycol hydrogel and a preparation method and application thereof, and belongs to the technical field of ophthalmic medical biomaterials. The present invention uses hyaluronate and different types of multi-arm polyethylene glycol as raw materials, first prepares different types of hyaluronate-multi-arm polyethylene glycol precursor solutions, then mixes different types of hyaluronate-multi-arm polyethylene glycol precursor solutions, and obtains the result after cross-linking reaction; the prepared hyaluronic acid-multi-arm polyethylene glycol hydrogel has a short gelation time, excellent mechanical stability, shear resistance and biocompatibility, and is applied to the preparation of intraocular vitreous substitutes, which have a good fixing and supporting effect on the retina.

Description

Technical Field

[0001] The invention belongs to the technical field of ophthalmic medical biomaterials, and specifically relates to a hyaluronic acid-multi-arm polyethylene glycol hydrogel and a preparation method and application thereof. Background Art

[0002] The natural vitreous in the human eye is a transparent, colorless, avascular, and nerve-free gel-like substance, mainly composed of more than 98% water, less than 1% collagen and hyaluronic acid, and other trace elements and vitamins. It has important functions in the eye, such as supporting the retina, regulating optical properties, promoting material exchange, and providing barrier protection. However, the vitreous itself cannot regenerate. Some blinding eye diseases, such as retinal detachment, severe ocular trauma, and proliferative diabetic retinopathy, often require the removal of the diseased vitreous and filling it with alternative materials to maintain retinal support. Currently, the commonly used alternative materials in clinical practice include silicone oil, gas, and balanced salt solution, which are only temporary fillers with problems of short effective action time and poor biocompatibility. The prior art discloses a treatment method of using silicone oil and heavy water for intraocular filling after vitrectomy. However, silicone oil will emulsify after long-term use in the eye, and the tiny oil droplets formed after emulsification may enter the anterior chamber or other intraocular structures, causing inflammation or other complications; silicone oil cannot be naturally degraded and must be removed through a secondary operation, which increases the surgical risk and economic burden of the patient; the removal process may cause retinal detachment again or other postoperative complications; residual silicone oil may also cause retinal toxicity or irritation; the density of silicone oil is lower than that of water. If the retinal hole is in the posterior pole (such as the macular area), a specific body position (such as face down) must be maintained during treatment, which causes inconvenience to the patient's daily life, especially the elderly or patients with physical limitations. The prior art also discloses the use of foldable vitreous for intraocular filling. The foldable vitreous includes a balloon, a drainage tube and a drainage valve. When implanting the vitreous, a larger scleral incision needs to be made, and specific instruments are required to unfold and fill it into the vitreous cavity. This requires high operating skills of the doctor. The folding and unfolding process may add additional operating time and prolong the operation time. Due to the limitations of the material properties of the balloon, the mechanical properties of the vitreous may still not be able to fully simulate the elasticity and supporting capacity of the natural vitreous, especially when used for a long time, it may show fatigue or deformation. In addition, its optical properties cannot match those of the natural vitreous. After surgery, decreased vision quality, glare or other optical abnormalities may occur. Therefore, it is urgent to find an ideal artificial vitreous filler that can be used for long-term intraocular filling. Summary of the invention

[0003] In view of the above-mentioned prior art, the present invention discloses a hyaluronic acid-multi-arm polyethylene glycol hydrogel and a preparation method and application thereof, so as to solve the technical problems in the prior art that the in situ cross-linked gel-type vitreous substitute has a long gelling time after filling, has a weak supporting effect, and the gel precursor solution easily enters under the retinal hole to form a gel, resulting in the inability to heal the retinal hole or retinal detachment.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing a hyaluronic acid-multi-arm polyethylene glycol hydrogel, which comprises the following steps:

[0005] S1: dissolving hyaluronate in the first solution to obtain a hyaluronate solution with a concentration of 0.1-200 mg / mL; the molecular weight of the hyaluronate is 2000-4000000 Da; the first solution is an electrolyte solution or water;

[0006] S2: Dissolving different types of multi-arm polyethylene glycol in the second solution respectively to obtain multi-arm polyethylene glycol solutions with concentrations ranging from 0.1 to 200 mg / mL; different types of multi-arm polyethylene glycol include multi-arm polyethylene glycol containing double bonds, multi-arm polyethylene glycol containing triple bonds, multi-arm polyethylene glycol containing thiol groups, and multi-arm polyethylene glycol containing azide, and their molecular weights are all 1000 to 200000 Da; the second solution is an electrolyte solution or water;

[0007] S3: mixing the hyaluronate solution with different types of multi-arm polyethylene glycol solutions respectively to obtain a hyaluronate-multi-arm polyethylene glycol precursor solution; the hyaluronate-multi-arm polyethylene glycol precursor solution includes a hyaluronate-double bond multi-arm polyethylene glycol precursor solution, a hyaluronate-thiol multi-arm polyethylene glycol precursor solution, a hyaluronate-triple bond multi-arm polyethylene glycol precursor solution and a hyaluronate-azide multi-arm polyethylene glycol precursor solution;

[0008] S4: The hyaluronate-double-bond multi-arm polyethylene glycol precursor solution is mixed with the hyaluronate-thiol multi-arm polyethylene glycol precursor solution and reacted to obtain the product; or the hyaluronate-triple-bond multi-arm polyethylene glycol precursor solution is mixed with the hyaluronate-azide multi-arm polyethylene glycol precursor solution and reacted to obtain the product.

[0009] Based on the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, in step S1, the operation of dissolving hyaluronate in the first solution is as follows: half of the hyaluronate is added to the first solution at a temperature of 20-30° C., and the solution is stirred at this temperature for 2-4 hours, then heated to 45-55° C., the other half of the hyaluronate is added and stirred at this temperature for 0.5-2 hours, then heated to 75-85° C., and stirred at this temperature for 25-35 minutes; the stirring speed is 50-300 rpm.

[0011] Further, in step S2, the operation of dissolving the multi-arm polyethylene glycol in the second solution is: adding the multi-arm polyethylene glycol to the second solution at a temperature of 20-30° C., and stirring for 25-35 minutes; the stirring speed is 50-300 rpm; the multi-arm polyethylene glycol containing double bonds is two-arm polyethylene glycol maleimide, four-arm polyethylene glycol maleimide, six-arm polyethylene glycol maleimide, eight-arm polyethylene glycol maleimide or sixteen-arm polyethylene glycol maleimide; the multi-arm polyethylene glycol containing triple bonds is two-arm polyethylene glycol diphenylcyclooctyne , four-arm polyethylene glycol diphenyl cyclooctyne, six-arm polyethylene glycol diphenyl cyclooctyne, eight-arm polyethylene glycol diphenyl cyclooctyne or sixteen-arm polyethylene glycol diphenyl cyclooctyne; the multi-arm polyethylene glycol containing thiol is two-arm polyethylene glycol thiol, four-arm polyethylene glycol thiol, six-arm polyethylene glycol thiol, eight-arm polyethylene glycol thiol or sixteen-arm polyethylene glycol thiol; the multi-arm polyethylene glycol containing azide is two-arm polyethylene glycol azide, four-arm polyethylene glycol azide, six-arm polyethylene glycol azide, eight-arm polyethylene glycol azide or sixteen-arm polyethylene glycol azide.

[0012] Furthermore, the electrolyte solution is phosphate buffer, balanced salt solution or Ringer's solution.

[0013] Furthermore, in step S3, the volume ratio of the hyaluronate solution to the multi-arm polyethylene glycol solution in the hyaluronate-multi-arm polyethylene glycol precursor solution is 1-100000:1000, and the temperature of the hyaluronate solution and the multi-arm polyethylene glycol solution during mixing is 20-40°C.

[0014] Furthermore, during the mixing reaction in step S4, the volume ratio of the hyaluronate-double-bond multi-arm polyethylene glycol precursor solution to the hyaluronate-thiol multi-arm polyethylene glycol precursor solution is 0.5~2:1; the volume ratio of the hyaluronate-triple-bond multi-arm polyethylene glycol precursor solution to the hyaluronate-azide multi-arm polyethylene glycol precursor solution is 0.5~2:1.

[0015] Furthermore, the hyaluronate is sodium hyaluronate or potassium hyaluronate.

[0016] Furthermore, in step S4, the reaction temperature is 20-37° C., and the reaction time is 2.5-3.5 min.

[0017] The invention also discloses that the hyaluronic acid-multi-arm polyethylene glycol hydrogel prepared by the preparation method is used for preparing an intraocular vitreous substitute.

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

[0019] 1. The present invention uses hyaluronate and multi-arm polyethylene glycol as raw materials to form a double-network hydrogel system, and utilizes the high molecular chain length and strong intermolecular interaction of hyaluronate to significantly increase the viscosity of the gel precursor solution, while shortening the gelation time, and preventing the gel precursor solution or gel from passing through retinal holes into the retinal neuroepithelial layer; the double-network structure also enhances the mechanical stability and shear resistance of the hydrogel, and improves the fixation and support effect on the retina.

[0020] 2. The hydrogel prepared by the present invention has optical properties similar to those of natural vitreous, with a refractive index of 1.34 and a transmittance in the visible light range of >90%. It has elastic modulus and storage modulus similar to those of natural vitreous, with an elastic modulus of 1~10Pa and a storage modulus of 0~2Pa.

[0021] 3. The patient only needs to undergo a traditional vitrectomy surgery, and then inject the gel precursor into the vitreous cavity through an intraocular double-chamber syringe. After cross-linking, the vitreous can be replaced to achieve a therapeutic effect. No complicated surgical operation is required and the operation time is short. The density of the hydrogel prepared in the present invention is close to that of water. Regardless of the location of the retinal tear or retinal detachment, there is no special position requirement for the patient after surgery.

[0022] 4. In the present invention, the gel precursor solution is cross-linked to form a hydrogel. After the hydrogel is formed, no droplets will be produced, there is no risk of entering the front or other structures in the eye, and no inflammatory response will be caused; the prepared hydrogel has good biocompatibility and will not cause toxicity or irritation to the retina or ciliary body. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the light transmittance measurement results of the hydrogel prepared in Example 1;

[0024] Figure 2 The figure is a measurement result diagram of the gelation time, elastic modulus (G') and storage modulus (G'') of the hydrogel prepared in Example 1 after gelation;

[0025] Figure 3 The figure is a graph showing the modulus measurement results of the hydrogel in Example 1 and the hydrogel in Comparative Example 2;

[0026] Figure 4 Graph showing the viscosity measurement results of the hydrogel in Example 1, the potassium hyaluronate solution in Comparative Example 1, and the hydrogel in Comparative Example 2;

[0027] Figure 5 The SEM images of the hydrogel prepared in Example 1 after immersion for 0 days and 30 days;

[0028] Figure 6 This is a micrograph of ARPE-19 cells. DETAILED DESCRIPTION

[0029] The intraocular double-chamber syringe used in the embodiment has been disclosed in the invention patent with publication number CN111529196B; the multi-arm polyethylene glycol used in the embodiment was purchased from Shanghai Tuoyang Biotechnology Co., Ltd.; the components of the balanced salt solution in the embodiment include: 0.64% sodium chloride, 0.075% potassium chloride, 0.048% calcium chloride dihydrate, 0.03% magnesium chloride hexahydrate, 0.39% sodium acetate trihydrate, 0.17% sodium citrate dihydrate, and the rest is sterile water; during preparation, the pH value is adjusted to 7.5 with sodium chloride or hydrochloric acid, and its osmotic pressure is 300mOsm / kg.

[0030] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0031] Example 1

[0032] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0033] S1: Add 5 mg of potassium hyaluronate powder with a molecular weight of 2500000Da to 10 mL of a balanced salt solution at 25°C, keep warm and stir for 4 hours, then heat to 50°C, add 5 mg of potassium hyaluronate powder with a molecular weight of 2500000Da and keep warm and stir for 1 hour, then heat to 80°C, keep warm and stir for 30 minutes to obtain a potassium hyaluronate solution with a concentration of 1 mg / mL, wherein the stirring speed is 80 rpm;

[0034] S2: 20 mg of eight-arm polyethylene glycol maleimide with a molecular weight of 20,000 Da and 20 mg of eight-arm polyethylene glycol thiol with a molecular weight of 20,000 Da were stirred and dissolved in 1 mL of a balanced salt solution at a temperature of 30° C. The stirring speed was 80 rpm. After keeping warm and stirring for 25 min, an eight-arm polyethylene glycol maleimide solution with a concentration of 20 mg / mL and an eight-arm polyethylene glycol thiol solution with a concentration of 20 mg / mL were obtained.

[0035] S3: The potassium hyaluronate solution is mixed with the eight-arm polyethylene glycol maleimide solution and the eight-arm polyethylene glycol thiol solution respectively. The solution temperature is 40°C during mixing. The volume ratio of the potassium hyaluronate solution to the multi-arm polyethylene glycol solution is 1:1. After mixing, a potassium hyaluronate-eight-arm polyethylene glycol maleimide precursor solution and a potassium hyaluronate-eight-arm polyethylene glycol thiol precursor solution are obtained.

[0036] S4: Load 1 mL of potassium hyaluronate-oct-arm polyethylene glycol maleimide precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of potassium hyaluronate-oct-arm polyethylene glycol thiol precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 37°C for 3 minutes to obtain the solution.

[0037] Example 2

[0038] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0039] S1: Heat 10 mL of phosphate buffer to 25°C, then add 1 g of sodium hyaluronate powder with a molecular weight of 2000 Da to the phosphate buffer, keep warm and stir for 2 hours, then heat to 50°C, add 1 g of sodium hyaluronate powder with a molecular weight of 2000 Da and keep warm and stir for 2 hours, then heat to 80°C, keep warm and stir for 30 minutes to obtain a sodium hyaluronate solution with a concentration of 200 mg / mL, wherein the stirring speed is 300 rpm;

[0040] S2: 0.8 g of two-arm polyethylene glycol maleimide with a molecular weight of 200,000 Da and 0.8 g of two-arm polyethylene glycol thiol with a molecular weight of 200,000 Da were stirred and dissolved in 10 mL of phosphate buffer at a temperature of 25° C. The stirring speed was 300 rpm. After keeping warm and stirring for 30 minutes, a two-arm polyethylene glycol maleimide solution with a concentration of 80 mg / mL and a two-arm polyethylene glycol thiol solution with a concentration of 80 mg / mL were obtained;

[0041] S3: The sodium hyaluronate solution is mixed with the two-arm polyethylene glycol maleimide solution and the two-arm polyethylene glycol thiol solution respectively. The solution temperature is 25°C during mixing. The volume ratio of the sodium hyaluronate solution to the multi-arm polyethylene glycol solution is 10:1. After mixing, a sodium hyaluronate-two-arm polyethylene glycol maleimide precursor solution and a sodium hyaluronate-two-arm polyethylene glycol thiol precursor solution are obtained.

[0042] S4: Load 1 mL of sodium hyaluronate-two-arm polyethylene glycol maleimide precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of sodium hyaluronate-two-arm polyethylene glycol thiol precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 20°C for 3 minutes to obtain the solution.

[0043] Example 3

[0044] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0045] S1: 0.4 g of sodium hyaluronate powder with a molecular weight of 2000 Da was added to 10 mL of pure water at 25°C, and the mixture was stirred for 3 h. The mixture was then heated to 50°C, and 0.4 g of sodium hyaluronate powder with a molecular weight of 2000 Da was added and stirred for 2 h. The mixture was then heated to 80°C and stirred for 30 min to obtain a sodium hyaluronate solution with a concentration of 80 mg / mL. The stirring speed was 200 rpm.

[0046] S2: 0.1 mg of 50,000 Da six-arm polyethylene glycol maleimide and 0.1 mg of 50,000 Da six-arm polyethylene glycol thiol were stirred and dissolved in 1 mL of pure water at 25°C, respectively, at a stirring speed of 300 rpm. After keeping warm and stirring for 30 min, a 0.1 mg / mL six-arm polyethylene glycol maleimide solution and a 0.1 mg / mL six-arm polyethylene glycol thiol solution were obtained.

[0047] S3: mixing the sodium hyaluronate solution with the six-arm polyethylene glycol maleimide solution and the six-arm polyethylene glycol thiol solution respectively, the solution temperature during mixing is 25°C, the volume ratio of the sodium hyaluronate solution to the multi-arm polyethylene glycol solution during mixing is 1:1, and after mixing, a sodium hyaluronate-six-arm polyethylene glycol maleimide precursor solution and a sodium hyaluronate-six-arm polyethylene glycol thiol precursor solution are obtained;

[0048] S4: Load 0.5 mL of sodium hyaluronate-six-arm polyethylene glycol maleimide precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of sodium hyaluronate-six-arm polyethylene glycol thiol precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 37°C for 2.5 minutes to obtain the solution.

[0049] Example 4

[0050] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0051] S1: 40 mg of sodium hyaluronate powder with a molecular weight of 1000000 Da was added to 2 mL of a balanced salt solution at 25°C, and the mixture was stirred for 4 h. The mixture was then heated to 50°C, and 40 mg of sodium hyaluronate powder with a molecular weight of 1000000 Da was added and stirred for 1 h. The mixture was then heated to 80°C and stirred for 30 min to obtain a sodium hyaluronate solution with a concentration of 40 mg / mL. The stirring speed was 150 rpm.

[0052] S2: 200 mg of two-arm polyethylene glycol diphenylcyclooctyne with a molecular weight of 10000 Da and 200 mg of two-arm polyethylene glycol azide with a molecular weight of 10000 Da were stirred and dissolved in 1 mL of a balanced salt solution at a temperature of 25° C. The stirring speed was 150 rpm. After keeping warm and stirring for 30 min, a two-arm polyethylene glycol diphenylcyclooctyne solution with a concentration of 200 mg / mL and a two-arm polyethylene glycol azide solution with a concentration of 200 mg / mL were obtained;

[0053] S3: The sodium hyaluronate solution is mixed with the two-arm polyethylene glycol diphenylcyclooctyne solution and the two-arm polyethylene glycol azide solution respectively. The solution temperature is 25°C during mixing. The volume ratio of the sodium hyaluronate solution to the multi-arm polyethylene glycol solution is 1:100. After mixing, a sodium hyaluronate-two-arm polyethylene glycol diphenylcyclooctyne precursor solution and a sodium hyaluronate-two-arm polyethylene glycol azide precursor solution are obtained.

[0054] S4: Load 2 mL of sodium hyaluronate-two-arm polyethylene glycol diphenylcyclooctyne precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of sodium hyaluronate-two-arm polyethylene glycol azide precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 40°C for 3 minutes to obtain the result.

[0055] Example 5

[0056] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0057] S1: 0.5 mg of sodium hyaluronate powder with a molecular weight of 4,000,000 Da was added to 10 mL of Ringer's solution at 25°C, stirred at this temperature for 2 h, then heated to 50°C, and then 0.5 mg of sodium hyaluronate powder with a molecular weight of 4,000,000 Da was added and stirred at this temperature for 0.5 h, then heated to 80°C, and stirred at this temperature for 30 min to obtain a sodium hyaluronate solution with a concentration of 0.1 mg / mL, wherein the stirring speed was 50 rpm;

[0058] S2: 40 mg of 1000Da six-arm polyethylene glycol diphenylcyclooctyne and 40 mg of 1000Da six-arm polyethylene glycol azide were stirred and dissolved in 1 mL of Ringer's solution at 25°C, respectively, with a stirring speed of 50 rpm. After keeping warm and stirring for 30 minutes, a 40 mg / mL six-arm polyethylene glycol diphenylcyclooctyne solution and a 40 mg / mL six-arm polyethylene glycol azide solution were obtained.

[0059] S3: The sodium hyaluronate solution is mixed with the six-arm polyethylene glycol diphenyl cyclooctyne solution and the six-arm polyethylene glycol azide solution respectively. The solution temperature is 25°C during mixing. The volume ratio of the sodium hyaluronate solution to the multi-arm polyethylene glycol solution is 1:1000. After mixing, a sodium hyaluronate-six-arm polyethylene glycol diphenyl cyclooctyne precursor solution and a sodium hyaluronate-six-arm polyethylene glycol azide precursor solution are obtained.

[0060] S4: Load 1 mL of sodium hyaluronate-six-arm polyethylene glycol diphenylcyclooctyne precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of sodium hyaluronate-six-arm polyethylene glycol azide precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 25°C for 3 minutes to obtain the result.

[0061] Example 6

[0062] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0063] S1: Add 40 mg of potassium hyaluronate powder with a molecular weight of 2000 Da to 1 mL of phosphate buffer at 25°C, keep warm and stir for 3 h, then heat to 50°C, add 40 mg of potassium hyaluronate powder with a molecular weight of 2000 Da and keep warm and stir for 0.5 h, then heat to 80°C, keep warm and stir for 30 min to obtain a potassium hyaluronate solution with a concentration of 80 mg / mL, wherein the stirring speed is 300 rpm;

[0064] S2: 1 mg of 1000Da eight-arm polyethylene glycol maleimide and 1 mg of 1000Da eight-arm polyethylene glycol thiol were stirred and dissolved in 10 mL of 25°C phosphate buffer at a stirring speed of 300 rpm. After stirring for 30 min, an eight-arm polyethylene glycol maleimide solution with a concentration of 0.1 mg / mL and an eight-arm polyethylene glycol thiol solution with a concentration of 0.1 mg / mL were obtained.

[0065] S3: The potassium hyaluronate solution is mixed with the eight-arm polyethylene glycol maleimide solution and the eight-arm polyethylene glycol thiol solution respectively. The solution temperature is 25°C during mixing. The volume ratio of the potassium hyaluronate solution to the multi-arm polyethylene glycol solution is 1:100. After mixing, a potassium hyaluronate-eight-arm polyethylene glycol maleimide precursor solution and a potassium hyaluronate-eight-arm polyethylene glycol thiol precursor solution are obtained.

[0066] S4: Load 1 mL of potassium hyaluronate-oct-arm polyethylene glycol maleimide precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of potassium hyaluronate-oct-arm polyethylene glycol thiol precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 37°C for 2.5 minutes to obtain the solution.

[0067] Example 7

[0068] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0069] S1: 100 mg of potassium hyaluronate powder with a molecular weight of 20,000 Da was added to 1 mL of pure water at 25°C, and the mixture was stirred for 4 h. The mixture was then heated to 55°C, and 100 mg of potassium hyaluronate powder with a molecular weight of 20,000 Da was added and stirred for 1 h. The mixture was then heated to 85°C and stirred for 30 min to obtain a potassium hyaluronate solution with a concentration of 200 mg / mL. The stirring speed was 200 rpm.

[0070] S2: 40 mg of 16-arm polyethylene glycol maleimide with a molecular weight of 10000 Da and 40 mg of 16-arm polyethylene glycol thiol with a molecular weight of 10000 Da were stirred and dissolved in 1 mL of pure water at a temperature of 25° C. The stirring speed was 200 rpm. After keeping warm and stirring for 30 minutes, a 40 mg / mL 16-arm polyethylene glycol maleimide solution and a 40 mg / mL 16-arm polyethylene glycol thiol solution were obtained.

[0071] S3: The potassium hyaluronate solution is mixed with the hexadecane-polyethylene glycol maleimide solution and the hexadecane-polyethylene glycol thiol solution respectively. The solution temperature is 25°C during mixing. The volume ratio of the potassium hyaluronate solution to the multi-arm polyethylene glycol solution is 1:10. After mixing, a potassium hyaluronate-hexadecane-polyethylene glycol maleimide precursor solution and a potassium hyaluronate-hexadecane-polyethylene glycol thiol precursor solution are obtained.

[0072] S4: Load 1 mL of potassium hyaluronate-hexadecyl-polyethylene glycol maleimide precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of potassium hyaluronate-hexadecyl-polyethylene glycol thiol precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 30°C for 3 minutes to obtain the solution.

[0073] Example 8

[0074] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0075] S1: 40 mg of sodium hyaluronate powder with a molecular weight of 2000000Da was added to 2 mL of a balanced salt solution at 20°C, and the mixture was stirred for 4 hours, then heated to 45°C, and then 40 mg of sodium hyaluronate powder with a molecular weight of 1000000Da was added and stirred for 1 hour, and then heated to 75°C and stirred for 25 minutes to obtain a sodium hyaluronate solution with a concentration of 40 mg / mL, wherein the stirring speed was 150 rpm;

[0076] S2: 80 mg of eight-arm polyethylene glycol diphenylcyclooctyne with a molecular weight of 10000 Da and 80 mg of eight-arm polyethylene glycol azide with a molecular weight of 10000 Da were stirred and dissolved in 1 mL of a balanced salt solution at a temperature of 25° C. The stirring speed was 150 rpm. After keeping warm and stirring for 30 min, an eight-arm polyethylene glycol diphenylcyclooctyne solution with a concentration of 80 mg / mL and an eight-arm polyethylene glycol azide solution with a concentration of 80 mg / mL were obtained;

[0077] S3: The sodium hyaluronate solution is mixed with the eight-arm polyethylene glycol diphenyl cyclooctyne solution and the eight-arm polyethylene glycol azide solution respectively. The solution temperature is 20°C during mixing. The volume ratio of the sodium hyaluronate solution to the multi-arm polyethylene glycol solution is 10:1. After mixing, a sodium hyaluronate-eight-arm polyethylene glycol diphenyl cyclooctyne precursor solution and a sodium hyaluronate-eight-arm polyethylene glycol azide precursor solution are obtained.

[0078] S4: Load 1 mL of sodium hyaluronate-octahedral polyethylene glycol diphenylcyclooctyne precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of sodium hyaluronate-octahedral polyethylene glycol azide precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 25°C for 3 minutes to obtain the result.

[0079] Example 9

[0080] A hyaluronic acid-multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0081] S1: Add 5 mg of sodium hyaluronate powder with a molecular weight of 4,000,000 Da to 1 mL of Ringer's solution at 30°C, keep warm and stir for 3 hours, then heat to 50°C, add 5 mg of sodium hyaluronate powder with a molecular weight of 4,000,000 Da and keep warm and stir for 0.5 hours, then heat to 75°C, keep warm and stir for 35 minutes to obtain a sodium hyaluronate solution with a concentration of 10 mg / mL, wherein the stirring speed is 50 rpm;

[0082] S2: 200 mg of 16-arm polyethylene glycol diphenylcyclooctyne with a molecular weight of 200,000 Da and 200 mg of 16-arm polyethylene glycol azide with a molecular weight of 200,000 Da were stirred and dissolved in 1 mL of Ringer's solution at a temperature of 20° C. The stirring speed was 50 rpm. After keeping warm and stirring for 35 minutes, a 16-arm polyethylene glycol diphenylcyclooctyne solution with a concentration of 200 mg / mL and a 16-arm polyethylene glycol azide solution with a concentration of 200 mg / mL were obtained;

[0083] S3: The sodium hyaluronate solution is mixed with the hexadecyl polyethylene glycol diphenylcyclooctyne solution and the hexadecyl polyethylene glycol azide solution respectively. The solution temperature is 40°C during mixing. The volume ratio of the sodium hyaluronate solution to the multi-arm polyethylene glycol solution is 100:1. After mixing, a sodium hyaluronate-hexadecyl polyethylene glycol diphenylcyclooctyne precursor solution and a sodium hyaluronate-hexadecyl polyethylene glycol azide precursor solution are obtained.

[0084] S4: Load 1 mL of sodium hyaluronate-hexadecyl-polyethylene glycol diphenylcyclooctyne precursor solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of sodium hyaluronate-hexadecyl-polyethylene glycol azide precursor solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 20°C for 3.5 minutes to obtain the solution.

[0085] Comparative Example 1

[0086] S1: Add 5 mg of potassium hyaluronate powder with a molecular weight of 2500000Da to 10 mL of a balanced salt solution at 25°C, keep warm and stir for 4 hours, then heat to 50°C, add 5 mg of potassium hyaluronate powder with a molecular weight of 2500000Da and keep warm and stir for 1 hour, then heat to 80°C, keep warm and stir for 30 minutes to obtain a potassium hyaluronate solution with a concentration of 1 mg / mL, wherein the stirring speed is 80 rpm;

[0087] S2: Load 1 mL of potassium hyaluronate solution into the injection chamber 1 of the intraocular double-chamber syringe. After the solution is injected through the syringe, it becomes a viscous solution at 37°C.

[0088] Comparative Example 2

[0089] A multi-arm polyethylene glycol hydrogel, the preparation method of which comprises the following steps:

[0090] S1: 20 mg of eight-arm polyethylene glycol maleimide with a molecular weight of 20,000 Da and 20 mg of eight-arm polyethylene glycol thiol with a molecular weight of 20,000 Da were stirred and dissolved in 1 mL of a balanced salt solution at a temperature of 30° C. The stirring speed was 80 rpm. After keeping warm and stirring for 25 min, an eight-arm polyethylene glycol maleimide solution with a concentration of 20 mg / mL and an eight-arm polyethylene glycol thiol solution with a concentration of 20 mg / mL were obtained.

[0091] S2: Load 1 mL of eight-arm polyethylene glycol maleimide solution into the injection chamber 1 of the intraocular double-chamber syringe, and then load 1 mL of eight-arm polyethylene glycol thiol solution into the injection chamber 2 of the intraocular double-chamber syringe. After the solution is injected through the syringe, cross-linking reaction is carried out at 37°C for 3 minutes to obtain the result.

[0092] Experimental example

[0093] The hyaluronic acid-multi-arm polyethylene glycol hydrogel prepared in the embodiments of the present invention has similar performance. Taking Embodiment 1 as an example, the performance of the related products is described.

[0094] ① The water content, refractive index and transmittance of the hydrogel prepared in Example 1 were measured using an Abbe refractometer and an ultraviolet spectrophotometer. The refractive index of the hydrogel in Example 1 was 1.41, the water content was 98%, and Figure 1 As shown, its transmittance in the visible light range of 300~700nm is greater than 90%.

[0095] ② The gelation time, elastic modulus (G'), storage modulus (G'') and viscosity of the hydrogel after gelation in Example 1 were measured using a rotational rheometer. Figure 2 It can be seen that after uniform mixing, G' is less than G'', and G' is equal to G'' at 3 minutes, indicating that gel is formed at this time, so the hydrogel gelation time is about 3 minutes; then the modulus of the hydrogel after gelation is stable is measured; from Figure 3 It can be seen that the G' of the hydrogel in Example 1 is about 15Pa, and G'' is about 1.3Pa, while the G' of the hydrogel in Comparative Example 2 is about 2Pa, and G'' is about 0.2Pa. The low-concentration potassium hyaluronate solution in Comparative Example 1 cannot form a gel, indicating that potassium hyaluronate provides strength support for the gel; Figure 4 It can be seen that when the shear rate is 0~1000s -1 When the viscosity of the hydrogel in Example 1 is 15-400 mPa·s, the viscosity of the potassium hyaluronate solution in Comparative Example 1 is 5-20 mPa·s, and the viscosity of the hydrogel prepared in Comparative Example 2 is 1-5 mPa·s.

[0096] ③ The hydrogel in Example 1 was immersed in a balanced salt solution at 37°C for 0 days and 30 days, and then the hydrogel was scanned by electron microscopy. The electron microscopy images are as follows: Figure 5 As shown, Figure 5 A is the electron microscopy image of the hydrogel immersed for 0 days. Figure 5 B is an electron micrograph of the hydrogel after immersion for 30 days. It can be seen from the figure that the gel presents a dense porous structure on day 0, and the gel network structure is still maintained after immersion for 30 days, but it is looser than on day 0.

[0097] ④Cell biocompatibility test

[0098] In this experiment, rhesus monkey choroidal retinal vascular endothelial cells (RF / 6A cells) and human retinal pigment epithelial cells (ARPE-19 cells) were used as the test system, and the hydrogel prepared in Example 1 was added for toxicity evaluation. The groups were set as: negative control group, different concentration gradient hydrogel extraction groups (5 mg / mL, 7.5 mg / mL, 10 mg / mL, 12.5 mg / mL, 15 mg / mL), and the specific process included the following steps in sequence:

[0099] (1) RF / 6A cells and ARPE-19 cells were routinely cultured using DMEM / F12 medium with fetal bovine serum (final concentration of 10%), and cells from passage 3 to 6 were used for the experiment;

[0100] (2) Digest the cells with 0.25% trypsin, mix well, and prepare a cell suspension. After digestion, press 5×10 5 / mL was inoculated in a sterile 6-well culture plate, 1×10 5 / mL was inoculated into a sterile 96-well culture plate and cultured in a constant temperature incubator at 37°C and 5% CO2 until the cells were fused to 50% of the bottom area of ​​the well, and then cultured with serum-free culture medium for 12 h;

[0101] (3) Discard the culture medium, add fresh serum-free culture medium and different concentration gradient hydrogel extracts to each well, and continue culturing in a constant temperature incubator at 37°C and 5% CO2;

[0102] (4) (Cell morphology evaluation) After culturing for 24 h and 48 h in the 6-well plate, the cells were washed twice with PBS, fixed with 4% paraformaldehyde, stained with crystal violet solution, washed twice with PBS again, and finally bright-field imaging was performed using an inverted phase contrast microscope.

[0103] (5) (CCK8 cell proliferation evaluation) After the 96-well plate is cultured for 24h and 48h, 10μL CCK-8 reagent is added to each well and the 96-well plate is gently shaken to ensure that the reagent is evenly distributed. The 96-well plate is placed in a 37°C, 5% CO2 incubator for 2h and the absorbance is measured by a microplate reader.

[0104] (6) (Cell apoptosis evaluation) After culturing the 6-well plate for 48 hours, the cells were digested and collected, washed with pre-cooled PBS, and centrifuged at 1000 rpm for 5 minutes. 1×10 6pc / mL cell suspension. First add 100μL BindingBuffer to each tube, then add nucleic acid dye and Annexin V in proportion. Resuspend the cells, shake and mix, and react at room temperature (20-25℃) in the dark for 15min. Add 400μL 1xBindingBuffer. Flow cytometry was used to detect and analyze the apoptosis rate of ARPE-19 cells in each group.

[0105] In the cell morphology evaluation, no obvious morphological changes were observed in the cells of each group, such as Figure 6 As shown, the cell number growth was good; in the cell proliferation evaluation, the cell survival rate of each group was greater than 70%; in the cell apoptosis evaluation, there was no significant difference in the apoptosis rate of each group; the above results indicate that the hydrogel prepared by the present invention has no obvious cytotoxic reaction.

[0106] ⑤ Safety test of single intravitreal hydrogel implantation in blue rabbits

[0107] Select blue rabbits for animal experiments, set up BSS (balanced salt solution) group and experimental group, the specific process includes the following steps:

[0108] (1) Anesthetize the rabbit;

[0109] (2) Cut open half of the conjunctiva along the corneal margin and create an infusion hole and a vitrectomy hole with a 19G scleral puncture knife;

[0110] (3) After inserting the perfusion tip, start the perfusion. The perfusion solution formula is as follows: add 4 mL of 50% glucose, 1.6 mL of dexamethasone (5 mg / mL), 0.5 mL of epinephrine hydrochloride (1 mg / mL), and 0.2 mL of tobramycin (80,000 U / mL) to 500 mL of lactated Ringer's solution;

[0111] (4) The vitrectomy head is placed into the vitreous cavity to perform vitrectomy;

[0112] (5) injecting the precursor solution prepared in Example 1 into the vitreous cavity according to step S4, and also injecting a control BSS into the vitreous cavity;

[0113] (6) Suturing the scleral opening and bulbar conjunctiva;

[0114] (7) Apply Telibitol eye ointment to the conjunctival sac.

[0115] Six months after the operation, slit lamp and fundus examination showed that the retina and choroid structures were normal, without pathological changes such as hemorrhage or exudation. The results of full-field flash electrophysiology (F-ERG) test showed that there was no obvious abnormality in the electrophysiological activity of cones and rods in the experimental eyes of the experimental group under light and dark conditions. Retinal optical coherence tomography (OCT) showed that there were no significant abnormalities in the retina. When the rabbit eyes were dissected, transparent or amorphous material characteristics were visible in the vitreous cavity, and there was no exudative membrane or hemorrhage covering the surface. Hematoxylin-eosin (HE) staining showed that the structures of all levels of the retina were normal, the cells were tightly arranged, and there were no abnormalities such as edema or hemorrhage.

[0116] In summary, cell experiments show that the hyaluronic acid and multi-arm polyethylene glycol hydrogel provided by the present invention have good biocompatibility; animal experiments show that during the observation period of up to half a year, the hydrogel maintains a gel state and transparency in the rabbit eye after vitrectomy, and the retinal and choroidal tissue structures are intact, without pathological changes such as hemorrhage and edema. More importantly, the retinal function remains basically normal. Animal in vivo experiments further prove that the material has high safety as an intraocular filler.

[0117] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing a hyaluronic acid-multi-arm polyethylene glycol hydrogel, characterized in that: The following steps are involved: S1: dissolving hyaluronate in the first solution to obtain a hyaluronate solution with a concentration of 0.1-200 mg / mL; the molecular weight of the hyaluronate is 2000-4000000 Da; the first solution is an electrolyte solution or water; the hyaluronate is sodium hyaluronate or potassium hyaluronate; S2: Dissolving different types of multi-arm polyethylene glycol in the second solution respectively to obtain a plurality of multi-arm polyethylene glycol solutions with a concentration of 0.1-200 mg / mL; the different types of multi-arm polyethylene glycol include multi-arm polyethylene glycol containing double bonds, multi-arm polyethylene glycol containing triple bonds, multi-arm polyethylene glycol containing thiol groups and multi-arm polyethylene glycol containing azide, and their molecular weights are all 1000-200000 Da; the second solution is an electrolyte solution or water; S3: mixing the hyaluronate solution with different types of multi-arm polyethylene glycol solutions respectively to obtain a hyaluronate-multi-arm polyethylene glycol precursor solution; the hyaluronate-multi-arm polyethylene glycol precursor solution includes a hyaluronate-double bond multi-arm polyethylene glycol precursor solution, a hyaluronate-thiol multi-arm polyethylene glycol precursor solution, a hyaluronate-triple bond multi-arm polyethylene glycol precursor solution and a hyaluronate-azide multi-arm polyethylene glycol precursor solution; S4: The hyaluronate-double-bond multi-arm polyethylene glycol precursor solution is mixed with the hyaluronate-thiol multi-arm polyethylene glycol precursor solution and reacted to obtain the product; or the hyaluronate-triple-bond multi-arm polyethylene glycol precursor solution is mixed with the hyaluronate-azide multi-arm polyethylene glycol precursor solution and reacted to obtain the product.

2. The method for preparing the hyaluronic acid-multi-arm polyethylene glycol hydrogel according to claim 1, characterized in that: The operation of dissolving the hyaluronate in the first solution in step S1 is as follows: half of the hyaluronate is added to the first solution at a temperature of 20-30° C., and the solution is stirred at this temperature for 2-4 hours, then heated to 45-55° C., the other half of the hyaluronate is added and stirred at this temperature for 0.5-2 hours, then heated to 75-85° C., and stirred at this temperature for 25-35 minutes; the stirring speed is 50-300 rpm.

3. The method for preparing the hyaluronic acid-multi-arm polyethylene glycol hydrogel according to claim 1, characterized in that: The operation of dissolving the multi-arm polyethylene glycol in the second solution in step S2 is: adding the multi-arm polyethylene glycol to the second solution at a temperature of 20-30° C., and stirring for 25-35 minutes; the stirring speed is 50-300 rpm; the multi-arm polyethylene glycol containing double bonds is two-arm polyethylene glycol maleimide, four-arm polyethylene glycol maleimide, six-arm polyethylene glycol maleimide, eight-arm polyethylene glycol maleimide or sixteen-arm polyethylene glycol maleimide; the multi-arm polyethylene glycol containing triple bonds is two-arm polyethylene glycol diphenyl cyclooctyne, tetradecene The multi-arm polyethylene glycol containing thiol is a two-arm polyethylene glycol thiol, a four-arm polyethylene glycol thiol, a six-arm polyethylene glycol thiol, an eight-arm polyethylene glycol thiol or a sixteen-arm polyethylene glycol thiol; the multi-arm polyethylene glycol containing azide is a two-arm polyethylene glycol azide, a four-arm polyethylene glycol azide, a six-arm polyethylene glycol azide, an eight-arm polyethylene glycol azide or a sixteen-arm polyethylene glycol azide.

4. The method for preparing the hyaluronic acid-multi-arm polyethylene glycol hydrogel according to any one of claims 1 to 3, characterized in that: The electrolyte solution is phosphate buffer, balanced salt solution or Ringer's solution.

5. The method for preparing the hyaluronic acid-multi-arm polyethylene glycol hydrogel according to claim 4, characterized in that: In step S3, the volume ratio of the hyaluronate solution to the multi-arm polyethylene glycol solution in the hyaluronate-multi-arm polyethylene glycol precursor solution is 1-100000:1000, and the temperature of the hyaluronate solution and the multi-arm polyethylene glycol solution during mixing is 20-40°C.

6. The method for preparing the hyaluronic acid-multi-arm polyethylene glycol hydrogel according to claim 5, characterized in that: During the mixing reaction in step S4, the volume ratio of the hyaluronate-double bond multi-arm polyethylene glycol precursor solution to the hyaluronate-thiol multi-arm polyethylene glycol precursor solution is 0.5~2:1; the volume ratio of the hyaluronate-triple bond multi-arm polyethylene glycol precursor solution to the hyaluronate-azide multi-arm polyethylene glycol precursor solution is 0.5~2:

1.

7. The method for preparing the hyaluronic acid-multi-arm polyethylene glycol hydrogel according to claim 1, characterized in that: In step S4, the reaction temperature is 20-37° C., and the reaction time is 2.5-3.5 min.

8. A hyaluronic acid-multi-arm polyethylene glycol hydrogel, characterized in that: The hyaluronic acid-multi-arm polyethylene glycol hydrogel is prepared by the preparation method of any one of claims 1 to 7.

9. An application of a hyaluronic acid-multi-arm polyethylene glycol hydrogel, characterized in that: The hyaluronic acid-multi-arm polyethylene glycol hydrogel according to claim 8 is used to prepare an intraocular vitreous substitute.

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