Injectable hydrogel for sustained release of eye medicine as well as preparation method and application of injectable hydrogel

Abstract

The invention discloses injectable hydrogel for eye drug sustained release and a preparation method and application thereof.The method comprises the steps that a NaIO4 solution is dropwise added into a hyaluronic acid solution, after the reaction is conducted in a dark place at the room temperature, ethylene glycol is added for continuous reaction, and formylated hyaluronic acid is prepared; the preparation method comprises the following steps: adding adipic dihydrazide into a carboxymethyl cellulose solution, uniformly stirring at room temperature, adding 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and stirring to react, so as to prepare aminated carboxymethyl cellulose; and mixing the PBS solution of aminated carboxymethyl cellulose with the PBS solution of formylated hyaluronic acid, uniformly stirring, adding a to-be-carried drug and a FeCl3 solution, and carrying out a reaction to prepare the injectable hydrogel for sustained release of the eye drug. The hydrogel is convenient to use and high in gelation rate, effectively realizes controllable release of drugs, and has a wide application prospect in the field of biological medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of drug sustained-release material development, and relates to an injectable hydrogel for sustained-release of eye drugs, and a preparation method and application thereof. Background Art

[0002] As an intricate and unique organ, the eye plays a key role in our daily acquisition of information, and more than 95% of information is conveyed to us through the eye. Ocular diseases such as cataracts, dry eye syndrome (DES), glaucoma, and uveitis continue to affect the health and quality of life of patients with eye diseases, which has become a problem that cannot be ignored. In current clinical treatment, topical ocular instillation of drugs is considered to be the most ideal way to treat ocular diseases due to its convenience, relatively low cost, and high patient compliance. However, as a complex and fragile organ, the eye is protected by biological barriers (such as the corneal epithelium and blood-retinal barrier) and tear flow, which limits the penetration and retention of eye drops. It is reported that the ocular surface retention time of eye drops does not exceed 15 seconds, and only less than 5% of the drug can penetrate the corneal barrier and reach the intraocular tissue. Therefore, the absorption of drugs administered through the eye drop route is often limited, and it becomes crucial to prolong the residence time of the drug in front of the cornea to improve the bioavailability of the drug. To overcome this challenge, researchers have developed a variety of biomaterials suitable for ocular drug delivery, including contact lenses, nanoparticles, and hydrogels. The introduction of these innovative materials has brought new possibilities to the field of ocular drug delivery.

[0003] Hydrogel is a hydrophilic polymer with a three-dimensional network structure. It has excellent water absorption capacity, stable structure in aqueous media, and provides structural and biological support for cell growth, migration, and tissue formation. Due to its high biocompatibility, excellent biodegradability, and biomimetic properties such as adjustability for different needs, it has attracted much attention in the field of drug sustained release. In situ formed hydrogels are potential carriers of ocular drugs. Under physical stimulation such as temperature, pH, and ions, it is in a flowing liquid state during application to the eye, and gels to form a viscoelastic hydrogel after contact. However, since these environmentally sensitive hydrogel drug delivery systems usually require harsh external conditions, there may be risks of irritation and damage to the eye and conjunctival cells. In addition, there may be uneven distribution of drugs in the gel, and the release rate is difficult to accurately control. As a result, the drug is released too quickly or too slowly, affecting efficacy and safety. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides an injectable hydrogel for sustained release of ocular drugs and a preparation method thereof, thereby solving the problems in the prior art that the environmentally sensitive hydrogel drug delivery system requires harsh external conditions that may bring the risk of eye damage, and the drug distribution is uneven and the release rate is difficult to control, affecting the efficacy and safety.

[0005] The present invention is achieved through the following technical solutions: A method for preparing an injectable hydrogel for sustained release of ocular drugs comprises the following steps: NaIO 4 The solution is dropped into the hyaluronic acid solution, and after the reaction is carried out at room temperature in the dark, ethylene glycol is added to continue the reaction, and after dialysis and freeze-drying, the aldehyde-modified hyaluronic acid is obtained; Adipic acid dihydrazide is added to the carboxymethyl cellulose solution, stirred evenly at room temperature, and 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are added, the pH value of the system is adjusted to 6-7, stirred for reaction, dialyzed and freeze-dried to obtain amino carboxymethyl cellulose; The PBS solution of the amino carboxymethyl cellulose and the PBS solution of the aldehyded hyaluronic acid were mixed, stirred evenly, and then the drug to be carried and FeCl 3 The solution is reacted to prepare the injectable hydrogel for sustained release of ocular drugs.

[0006] Preferably, the NaIO 4 The ratio to hyaluronic acid is (0.3~0.7):(0.8~1.2).

[0007] Preferably, NaIO 4 The solution was dropped into the hyaluronic acid solution and allowed to react at room temperature in the dark for 4.5 to 5 hours.

[0008] Preferably, ethylene glycol is added and the reaction is continued for 0.5 to 1 h.

[0009] Preferably, the ratio of adipic acid dihydrazide to carboxymethyl cellulose is (2.5-3.5):(0.5-1.5).

[0010] Preferably, the molar ratio of the carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:(1.9-2.1):(0.9-1.1).

[0011] Preferably, the aldehyde-modified hyaluronic acid, amino-modified carboxymethyl cellulose and FeCl 3The ratio of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is (3-7): (3-7): (0.8-1.2); the concentration of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 0.5%-0.7% (w / v), the FeCl 3 The concentration is 0.0005~0.0015M.

[0012] Preferably, the mass percentage of the drug to be carried is 1% to 4%.

[0013] An injectable hydrogel for sustained release of ocular drugs is prepared by the above method; the gelation time of the hydrogel is less than 3 minutes.

[0014] Application of the above-mentioned injectable hydrogel for sustained release of ocular drugs in the preparation of ocular drugs.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects: The invention discloses a method for preparing an injectable hydrogel for sustained release of ocular drugs. The method comprises the following steps: firstly, hyaluronic acid is aldehyde-modified by sodium periodate to obtain aldehyde-modified hyaluronic acid (HA-CHO); and carboxymethyl cellulose is modified by adipic acid dihydrazide to obtain amino carboxymethyl cellulose (CMC-NH 2 ), and finally, HA-CHO, CMC-NH 2 and FeCl 3 The solution was fully mixed and HC hydrogel was quickly formed at room temperature. During the hydrogel formation process, the aldehyde groups of HA-CHO and CMC-NH 2 Schiff base reaction between the amino group and the carboxyl group with Fe 3+ The metal coordination bonds formed between them together constitute a hydrogel with a dual dynamic cross-linked network. The hydrogel has high viscosity, strong shear resistance, and fast gelation speed, which effectively improves the retention of drugs on the ocular surface and effectively improves the duration of drug action. The hydrogel can quickly gel within 3 minutes without external stimulation. This time not only meets the reasonable dosing operation time, but also ensures the stable retention of hydrogel eye drops on the ocular surface. It not only has good injectability, but also shows a modulus close to that of ocular tissue, realizing the controlled release of carried drugs, which makes it have broad application prospects in the field of biomedicine.

[0016] Furthermore, the NaIO 4 The ratio of hyaluronic acid to ketone or aldehyde is (0.3~0.7): (0.8~1.2), which can effectively generate corresponding ketone or aldehyde from hyaluronic acid.

[0017] Furthermore, NaIO 4The solution is dropped into the hyaluronic acid solution and allowed to react at room temperature in the dark for 4.5 to 5 hours. This can effectively prevent sodium periodate from being decomposed by light, thereby reducing its oxidizing ability.

[0018] Furthermore, ethylene glycol is added to continue the reaction for 0.5 to 1 hour to react away excess sodium periodate.

[0019] Furthermore, the ratio of adipic acid dihydrazide to carboxymethyl cellulose is (2.5-3.5):(0.5-1.5), which can provide sufficient amino source in the amination reaction.

[0020] Furthermore, the molar ratio of the carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:(1.9-2.1):(0.9-1.1), which can effectively improve the efficiency and selectivity of the reaction.

[0021] Furthermore, the aldehyde-modified hyaluronic acid, amino-modified carboxymethyl cellulose and FeCl 3 The ratio of is (3-7):(3-7):(0.8-1.2), which can effectively form a dual dynamic network; the concentration of the amino carboxymethyl cellulose and aldehyde hyaluronic acid solution is 0.5%-0.7% (w / v), the FeCl 3 The concentration is 0.0005~0.0015M, which can make the hydrogel have an appropriate modulus.

[0022] Furthermore, the amount of the drug to be carried is 1% to 4% (w / w), which can ensure that the hydrogel has good sustained-release properties without affecting its mechanical properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The synthetic route of the injectable hydrogel for sustained release of ocular drugs in the present invention is as follows; Figure 2 The infrared spectra (FT-IR) of hyaluronic acid, aldehyde-modified hyaluronic acid, carboxymethyl cellulose, amino-modified carboxymethyl cellulose and the hydrogels prepared in Examples 1 to 3 of the present invention are shown; Figure 3A This is a test chart of the gel time of the injectable hydrogel for sustained release of ocular drugs prepared in Example 1 of the present invention; Figure 3B This is a test chart of the gel time of the injectable hydrogel for sustained release of ocular drugs prepared in Example 2 of the present invention; Figure 3C This is a test chart of the gel time of the injectable hydrogel for sustained release of ocular drugs prepared in Example 3 of the present invention; Figure 4 This is an OCT test image of the injectable hydrogel for sustained release of ocular drugs in the present invention; Figure 5 This is a test chart of the swelling rate of the injectable hydrogel for sustained release of ocular drugs prepared in Examples 1 to 3 of the present invention; Figure 6 This is a test graph of in vitro drug release rate of the injectable hydrogel for sustained release of ocular drugs prepared in Examples 1 to 3 of the present invention; Figure 7 This is a live-dead fluorescent staining test diagram of the injectable hydrogel for sustained release of ocular drugs prepared by the present invention. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0026] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0027] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0028] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0029] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0030] like Figure 1 As shown, the present invention provides a method for preparing an injectable hydrogel for sustained release of ocular drugs, comprising the following steps: Disperse HA in deionized water and stir on a magnetic stirrer for 2-3 hours at room temperature. 4 Dissolve in deionized water and drop into HA solution, react at room temperature in the dark for 4.5-5 hours, then add blocking agent EG and react for another 0.5-1 hour. Dialyze for 3 days and freeze-dry to obtain the final aldehyde-modified hyaluronic acid (HA-CHO); Wherein, the NaIO 4 The ratio of NaIO to hyaluronic acid is (0.3-0.7): (0.8-1.2), preferably NaIO 4 The ratio with hyaluronic acid is 0.5:1.

[0031] CMC was dissolved in deionized water and ADH was added. Stirring was performed at room temperature to form a well-dispersed mixture. HOBt and EDC were added to the mixed solution. The pH value was maintained at 6.8 with sodium hydroxide and stirred overnight. Finally, the mixture was dialyzed with a 3500 MWCO membrane for 3 days and the final amino carboxymethyl cellulose (CMC-NH 2 ); Wherein, the ratio of adipic acid dihydrazide to carboxymethyl cellulose is (2.5-3.5):(0.5-1.5), and the preferred ratio of adipic acid dihydrazide to carboxymethyl cellulose is 3:1; The molar ratio of the carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:(1.9-2.1):(0.9-1.1), preferably, the molar ratio of the carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:2.0:1.

[0032] HOBt and EDC were used as condensation reagents.

[0033] Mix the PBS solution of amino carboxymethyl cellulose and the PBS solution of aldehyded hyaluronic acid, stir evenly, and then add the drug to be carried and FeCl 3The solution is subjected to Schiff base reaction and metal coordination to obtain the injectable hydrogel for sustained release of ocular drugs.

[0034] Wherein, the aldehyde-modified hyaluronic acid, amino-modified carboxymethyl cellulose and FeCl 3 The ratio is (3~7):(3~7):(0.8~1.2); the concentration of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 0.5%~0.7% (w / v), preferably, the concentration of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 0.5% (w / v).

[0035] The FeCl 3 The concentration of FeCl is 0.0005~0.0015M, preferably, FeCl 3 The concentration is 0.001M.

[0036] The mass percentage of the drug to be carried is 1% to 4%. The mass percentage is 1% to 4% of the total mass of the system.

[0037] The above English abbreviations are: HA: Hyaluronic Acid.

[0038] NaIO 4 : Sodium Periodate.

[0039] EG: Ethylene Glycol.

[0040] CMC: Carboxymethyl Cellulose.

[0041] ADH: Adipic Dihydrazide.

[0042] HOBt: 1-Hydroxybenzotriazole.

[0043] EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride.

[0044] The invention also discloses an injectable hydrogel for sustained release of ocular drugs prepared by the method, wherein the gelation time of the hydrogel is less than 3 minutes.

[0045] In addition, the present invention also discloses the use of the above-mentioned injectable hydrogel for sustained release of ocular drugs in the preparation of ocular drugs.

[0046] The invention discloses a method for preparing an injectable hydrogel for sustained release of ocular drugs, and belongs to the field of drug sustained release material preparation methodology. The preparation method specifically comprises the following steps: hyaluronic acid aldehyde modification: hyaluronic acid is oxidized with sodium periodate to finally obtain aldehyded hyaluronic acid. Carboxymethyl cellulose amination modification: carboxymethyl cellulose is modified by adipic acid dihydrazide to obtain amino carboxymethyl cellulose. Schiff base reaction: hydrogel is quickly formed by the Schiff base reaction between the amino group of the amino carboxymethyl cellulose and the aldehyde group of the aldehyded hyaluronic acid. The preparation process has the advantages of being simple and easy to operate, safe and environmentally friendly, low cost, strong operation repeatability, and easy large-scale preparation.

[0047] The hydrogel prepared by the method of the present invention can quickly gel within 3 minutes without external stimulation (3 minutes not only meets the reasonable drug administration operation time, but also ensures the stable retention of the hydrogel eye drops on the ocular surface), not only has good injectability, but also shows a modulus close to that of eye tissue. In addition, the hydrogel also has high transparency, good biocompatibility and antibacterial properties. It has broad application prospects in the field of drug sustained release.

[0048] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0049] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.

[0050] Example 1 A method for preparing an injectable hydrogel for sustained release of ocular drugs comprises the following steps: Control HA-CHO:CMC-NH 2 =3:7 Step 1: Disperse HA in deionized water and stir on a magnetic stirrer for 2.5 h at room temperature. 4Dissolve in deionized water and drop it into the HA solution, react in the dark for 5 hours, and finally add the blocking agent EG to react for another 1 hour. Dialyze for 3 days and freeze-dry to obtain the final aldehyde-modified hyaluronic acid; Step 2, dissolve CMC in deionized water and add ADH. Stir at room temperature to form a well-dispersed mixture. Add HOBt and EDC to the mixed solution. Use sodium hydroxide to maintain the pH value at 6.8 and stir the reaction overnight. Dialyze with a 3500MWCO membrane for 3 days and obtain the final amino carboxymethyl cellulose by freeze drying; Step 3, at room temperature, evenly dispersing 1 g of amino carboxymethyl cellulose in 100 mL of deionized water to obtain an amino carboxymethyl cellulose solution; at room temperature, evenly dispersing 1 g of aldehyded hyaluronic acid in 100 mL of deionized water to obtain an aldehyded hyaluronic acid solution; Step 4: At room temperature, add 7 mL of amino carboxymethyl cellulose solution and 3 mL of aldehyded hyaluronic acid solution into the same beaker, mix them thoroughly, and add 1 mL of FeCl 3 The solution (0.001 M) was subjected to a Schiff base reaction to rapidly form an injectable hydrogel for sustained release of ocular drugs. The sample was labeled as hydrogel H3C7.

[0051] Example 2 Control HA-CHO:CMC-NH 2 =5:5 Step 1: Disperse HA in deionized water and stir on a magnetic stirrer for 2.5 h at room temperature. 4 Dissolve in deionized water and drop it into the HA solution, react in the dark for 5 hours, and finally add the blocking agent EG to react for another 1 hour. Dialyze for 3 days and freeze-dry to obtain the final aldehyde-modified hyaluronic acid; Step 2, dissolve CMC in deionized water and add ADH. Stir at room temperature to form a well-dispersed mixture. Add HOBt and EDC to the mixed solution. Use sodium hydroxide to maintain the pH value at 6.8 and stir the reaction overnight. Dialyze with a 3500MWCO membrane for 3 days and obtain the final amino carboxymethyl cellulose by freeze drying; Step 3, at room temperature, evenly dispersing 1 g of amino carboxymethyl cellulose in 100 mL of deionized water to obtain an amino carboxymethyl cellulose solution; at room temperature, evenly dispersing 1 g of aldehyded hyaluronic acid in 100 mL of deionized water to obtain an aldehyded hyaluronic acid solution; Step 4: At room temperature, add 5 mL of amino carboxymethyl cellulose solution and 5 mL of aldehyded hyaluronic acid solution into the same beaker, mix them thoroughly, and add 1 mL of FeCl 3Solution (0.001M). It was subjected to Schiff base reaction to rapidly form an injectable hydrogel for sustained release of ocular drugs, and the sample was labeled as hydrogel H5C5.

[0052] Example 3 Control HA-CHO:CMC-NH 2 =7:3 Step 1: Disperse HA in deionized water and stir on a magnetic stirrer for 2.5 h at room temperature. 4 Dissolve in deionized water and drop it into the HA solution, react in the dark for 5 hours, and finally add the blocking agent EG to react for another 1 hour. Dialyze for 3 days and freeze-dry to obtain the final aldehyde-modified hyaluronic acid; Step 2, dissolve CMC in deionized water and add ADH. Stir at room temperature to form a well-dispersed mixture. Add HOBt and EDC to the mixed solution. Use sodium hydroxide to maintain the pH value at 6.8 and stir the reaction overnight. Dialyze with a 3500MWCO membrane for 3 days and obtain the final amino carboxymethyl cellulose by freeze drying; Step 3, at room temperature, evenly dispersing 1 g of amino carboxymethyl cellulose in 100 mL of deionized water to obtain an amino carboxymethyl cellulose solution; at room temperature, evenly dispersing 1 g of aldehyded hyaluronic acid in 100 mL of deionized water to obtain an aldehyded hyaluronic acid solution; Step 4: At room temperature, add 3 mL of amino carboxymethyl cellulose solution and 7 mL of aldehyded hyaluronic acid solution into the same beaker, mix them thoroughly, and add 1 mL of FeCl 3 Solution (0.001M). It was subjected to Schiff base reaction to rapidly form an injectable hydrogel for sustained release of ocular drugs, and the sample was labeled as hydrogel H7C3.

[0053] Example 4 A method for preparing an injectable hydrogel for sustained release of ocular drugs comprises the following steps: Disperse hyaluronic acid in deionized water and stir on a magnetic stirrer at room temperature for 2 h. 4 Dissolve in deionized water and drop into the hyaluronic acid solution, react at room temperature in the dark for 4.5 hours, and finally add the blocking agent EG to react for another 0.5 hour. Dialyze for 3 days and freeze-dry to obtain the final aldehyde-modified hyaluronic acid (HA-CHO); Wherein, the NaIO 4 The ratio with hyaluronic acid is 0.3:0.8.

[0054] Dissolve carboxymethyl cellulose in deionized water and add adipic acid dihydrazide. Stir at room temperature to form a well-dispersed mixture. Add 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the mixed solution. Keep the pH at 6.8 with sodium hydroxide and stir the reaction overnight. Finally, dialyze with a 3500 MWCO membrane for 3 days and obtain the final amino carboxymethyl cellulose (CMC-NH 2 ); Wherein, the ratio of adipic acid dihydrazide to carboxymethyl cellulose is 2.5:0.5; The molar ratio of the carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:1.9:0.9.

[0055] Mix the PBS solution of amino carboxymethyl cellulose and the PBS solution of aldehyded hyaluronic acid, stir evenly, and then add the drug to be carried and FeCl 3 The solution is subjected to Schiff base reaction and metal coordination to obtain the injectable hydrogel for sustained release of ocular drugs.

[0056] Wherein, the aldehyde-modified hyaluronic acid, amino-modified carboxymethyl cellulose and FeCl 3 The ratio of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 3:3:0.8; the concentration of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 0.5% (w / v), the FeCl 3 The concentration is 0.0005M, and the mass percentage of the drug to be carried is 1%.

[0057] Example 5 A method for preparing an injectable hydrogel for sustained release of ocular drugs comprises the following steps: Disperse hyaluronic acid in deionized water and stir on a magnetic stirrer at room temperature for 3 h. 4 Dissolve in deionized water and drop into the hyaluronic acid solution, react at room temperature in the dark for 5 hours, and finally add the blocking agent EG to react for another hour. After dialysis for 3 days, the final aldehyde-modified hyaluronic acid (HA-CHO) was obtained by freeze drying. Wherein, the NaIO 4 The ratio to hyaluronic acid is 0.7:1.2.

[0058] Dissolve carboxymethyl cellulose in deionized water and add adipic acid dihydrazide. Stir at room temperature to form a well-dispersed mixture. Add 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the mixed solution. Keep the pH at 6.8 with sodium hydroxide and stir the reaction overnight. Finally, dialyze with a 3500 MWCO membrane for 3 days and obtain the final amino carboxymethyl cellulose (CMC-NH 2 ); Wherein, the ratio of adipic acid dihydrazide to carboxymethyl cellulose is 3.5:1.5; The molar ratio of the carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:2.1:1.1.

[0059] Mix the PBS solution of amino carboxymethyl cellulose and the PBS solution of aldehyded hyaluronic acid, stir evenly, and then add the drug to be carried and FeCl 3 The solution is subjected to Schiff base reaction and metal coordination to obtain the injectable hydrogel for sustained release of ocular drugs.

[0060] Wherein, the aldehyde-modified hyaluronic acid, amino-modified carboxymethyl cellulose and FeCl 3 The ratio of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 7:7:1.2; the concentration of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 0.7% (w / v), the FeCl 3 The concentration is 0.0015M, and the mass percentage of the drug to be carried is 4%.

[0061] Example 6 A method for preparing an injectable hydrogel for sustained release of ocular drugs comprises the following steps: Disperse hyaluronic acid in deionized water and stir on a magnetic stirrer for 2.5 h at room temperature. 4 Dissolve in deionized water and drop into the hyaluronic acid solution, react at room temperature in the dark for 5 hours, and finally add the blocking agent EG to react for another 0.8 hours. After dialysis for 3 days, the final aldehyde-modified hyaluronic acid (HA-CHO) was obtained by freeze drying. Wherein, the NaIO 4 The ratio with hyaluronic acid is 0.5:1.

[0062] Dissolve carboxymethyl cellulose in deionized water and add adipic acid dihydrazide. Stir at room temperature to form a well-dispersed mixture. Add 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the mixed solution. Keep the pH at 6.8 with sodium hydroxide and stir the reaction overnight. Finally, dialyze with a 3500 MWCO membrane for 3 days and obtain the final amino carboxymethyl cellulose (CMC-NH 2 ); The ratio of adipic acid dihydrazide to carboxymethyl cellulose is 3:1; the molar ratio of carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:2.0:1.

[0063] Mix the PBS solution of amino carboxymethyl cellulose and the PBS solution of aldehyded hyaluronic acid, stir evenly, and then add the drug to be carried and FeCl 3 The solution is subjected to Schiff base reaction and metal coordination to obtain the injectable hydrogel for sustained release of ocular drugs.

[0064] Wherein, the aldehyde-modified hyaluronic acid, amino-modified carboxymethyl cellulose and FeCl 3 The ratio of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 5:5:1; the concentration of the amino carboxymethyl cellulose and the aldehyde hyaluronic acid solution is 0.5% (w / v), the FeCl 3 The concentration is 0.001M, and the mass percentage of the drug to be carried is 2%.

[0065] Figure 2 The infrared spectra (FT-IR) of hyaluronic acid, aldehyde-modified hyaluronic acid, carboxymethyl cellulose, amino-modified carboxymethyl cellulose and the hydrogels prepared in Examples 1 to 3 of the present invention are shown in the figure. As can be seen from the figure, HA-CHO has a peak at 1735 cm −1 There is a new peak at , which corresponds to the characteristic peak of C=O in HA-CHO, indicating that the aldehyde-modified hyaluronic acid was successfully prepared; CMC-NH 2 At 3345 cm −1 There is a new peak at , corresponding to CMC-NH 2 NH 2 The characteristic peaks of

[0066] In order to characterize the performance of the hydrogel prepared by the present invention, the following tests were performed for verification: 1. Perform a gel time test on the prepared hydrogel sample, specifically: To study the sol-gel transition behavior of the hydrogel, a disc-shaped sample with a thickness of ≈2 mm and a diameter of 15 mm was placed on a plate with a diameter of 25 mm and coated with silicone oil to prevent water evaporation. A time scan was performed at 37°C. The strain was maintained at 10% and 100% for 60 s, respectively, and the frequency was set to 10.0 rad s -1 The gelation process was determined by measuring the storage modulus (G') and loss modulus (G") over time. Figure 3A~3C As shown, the intersection points (gel time) between G′ and G″ of the hydrogel prepared by the present invention are 130, 160 and 150 s, respectively, which has a fast gel time and is convenient for use.

[0067] 2. Perform OCT test on the hydrogel sample, specifically: Corneal clearance after blinking was determined by using optical coherence tomography. Sprague Dawley rats were anesthetized and fixed in front of the camera to obtain stable images. Eye drops and hydrogel solution (5 μL) were topically instilled and images were obtained immediately after manual blinking twice.

[0068] Figure 4 This is an OCT test image of the injectable hydrogel for sustained release of ocular drugs in the present invention. It can be seen from the figure that conventional eye drop preparations are more easily removed during blinking, while the rapid gelation of the prepared hydrogel drops leads to the formation of a thin regular coating on the ocular surface, which persists on the ocular surface after blinking.

[0069] 3. Swelling test of the prepared hydrogel samples The swelling rate of the hydrogel was determined by the gravimetric method. The freeze-dried xerogel sample was weighed and placed in a PBS solution. The sample was then incubated at 37°C for 24 hours, and the gel was taken out of the PBS solution, excess water was removed with a paper towel, and the gel was weighed.

[0070]

[0071] Where W 0 and W t represent the initial mass of the sample and the mass of the sample after swelling, respectively.

[0072] Figure 5 This is a swelling rate test graph of the injectable hydrogel for sustained release of ocular drugs prepared in Examples 1 to 3 of the present invention. It can be seen from the figure that the injectable hydrogel for sustained release of ocular drugs prepared in the present invention has a strong swelling ability, and its swelling rate is 520% ​​to 700%. The colloidal structure is loose, and the drug is easy to penetrate. At the same time, the strong swelling ability has a positive effect on the drug release rate, duration and efficiency.

[0073] 4. Conduct in vitro drug sustained release test on the prepared hydrogel samples In the preparation process of the hydrogels of Examples 1 to 3, 1 mL of doxorubicin hydrochloride solution (1 mg / mL) was added as the drug to be carried to prepare the hydrogel loaded with doxorubicin hydrochloride. The drug-loaded hydrogel was immersed in simulated tear fluid and incubated on a constant temperature shaker (37°C, 100RPM). At a specific time point, 2 mL of the supernatant solution was aspirated and analyzed by ultraviolet-visible (UV-vis) using an ELISA instrument. All in vitro release tests were performed three times.

[0074] Figure 6 This is a test diagram of the in vitro drug release rate of the injectable hydrogel for ocular drug sustained release prepared in Examples 1 to 3 of the present invention. It can be seen from the figure that the injectable hydrogel for ocular drug sustained release can still achieve stable drug sustained release ability on the 7th day, indicating that it has good sustained release performance.

[0075] 5. Cytotoxicity test of the prepared hydrogel samples The cytotoxicity of the hydrogel powder was tested by co-culturing it with HT-22, HUVEC, L929, and ARPE-19 cell lines. Different concentrations of hydrogel were placed in a sterilized 96-well plate. HT-22, HUVEC, L929, and ARPE-19 cells were seeded in 96-well plates. Cell viability was determined at 24, 48, and 72 hours by cell counting kit (CCK-8) and live-dead fluorescence staining.

[0076] Figure 7 The live-dead fluorescence staining test diagram of the injectable hydrogel for ocular drug sustained release prepared by the present invention shows that a large number of live cells (green fluorescence) can be observed in the injectable hydrogel for ocular drug sustained release in different cell lines after 1 day, 2 days and 3 days of culture, while dead cells are almost invisible. These results confirm that the hydrogel has no adverse effect on cell growth and proliferation, indicating that their cytotoxicity is very low.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an injectable hydrogel for sustained release of ocular drugs, characterized in that: The following steps are involved: The NaIO4 solution is dropped into the hyaluronic acid solution, and after the reaction is carried out at room temperature in the dark, ethylene glycol is added to continue the reaction, and after dialysis and freeze-drying, the aldehyde-modified hyaluronic acid is obtained; Adipic acid dihydrazide is added to the carboxymethyl cellulose solution, stirred evenly at room temperature, and 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are added, the pH value of the system is adjusted to 6-7, stirred for reaction, dialyzed and freeze-dried to obtain amino carboxymethyl cellulose; The PBS solution of the amino carboxymethyl cellulose and the PBS solution of the aldehyded hyaluronic acid are mixed, stirred evenly, and then the drug to be carried and the FeCl3 solution are added to react to obtain the injectable hydrogel for sustained release of ocular drugs.

2. The method for preparing an injectable hydrogel for sustained release of ocular drugs according to claim 1, characterized in that: The ratio of NaIO4 to hyaluronic acid is (0.3-0.7):(0.8-1.2).

3. The method for preparing an injectable hydrogel for sustained release of ocular drugs according to claim 1, characterized in that: Drop the NaIO4 solution into the hyaluronic acid solution and allow to react at room temperature in the dark for 4.5 to 5 hours.

4. The method for preparing an injectable hydrogel for sustained release of ocular drugs according to claim 1, characterized in that: Add ethylene glycol and continue the reaction for 0.5~1h.

5. The method for preparing an injectable hydrogel for sustained release of ocular drugs according to claim 1, characterized in that: The ratio of adipic acid dihydrazide to carboxymethyl cellulose is (2.5-3.5):(0.5-1.5).

6. The method for preparing an injectable hydrogel for sustained release of ocular drugs according to claim 1, characterized in that: The molar ratio of the carboxymethyl cellulose, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.01:(1.9-2.1):(0.9-1.1).

7. The method for preparing an injectable hydrogel for sustained release of ocular drugs according to claim 1, characterized in that: The ratio of the aldehyde-modified hyaluronic acid, amino-carboxymethyl cellulose and FeCl3 is (3-7):(3-7):(0.8-1.2); the concentration of the amino-carboxymethyl cellulose and aldehyde-modified hyaluronic acid solution is 0.5%-0.7% (w / v), and the concentration of the FeCl3 is 0.0005-0.0015M.

8. The method for preparing an injectable hydrogel for sustained release of ocular drugs according to claim 1, characterized in that: The mass percentage of the medicine to be carried is 1% to 4%.

9. An injectable hydrogel for sustained release of ocular drugs, characterized in that: The hydrogel is prepared by the method according to any one of claims 1 to 7; the gelation time of the hydrogel is less than 3 minutes.

10. Use of the injectable hydrogel for sustained release of ocular drugs as claimed in claim 9 in the preparation of ocular drugs.

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