In-situ drug-loaded hydrogels and their preparation methods and uses

Abstract

The present invention relates to an in-situ drug-loaded hydrogel and its preparation and use. The preparation method of the multi-arm polyethylene glycol derivative complex includes pre-freezing a multi-arm polyethylene glycol derivative solution, followed by three drying steps in sequence. The in-situ drug-loaded hydrogel includes a multi-arm polyethylene glycol derivative complex, a phosphate buffer, a buffer salt solution, and a drug. The in-situ drug-loaded hydrogel of the present invention can be used to seal, repair, fill, or treat tissue wound surfaces.

Description

cross reference

[0001] This application claims priority from Chinese Patent Application No. 202110351993.7, filed on March 31, 2021, entitled "In-situ drug-loaded hydrogel and its preparation method and use," the entire disclosure of which is incorporated herein by reference. [Technical field]

[0002] The present invention relates to an in-situ drug-loaded hydrogel and its preparation method and use. [Background technology]

[0003] In-situ degradable hydrogels, as a diversified developing material, have a large amount of water content, good biocompatibility, controllable safety and performance, and the injectable in-situ hydrogels can be easily manipulated during surgery, so they have broad research prospects in the treatment of diseases. The hydrogel modification technology with PEG can be applied to tissue engineering, especially tissue sealants, which can help prevent tissue fluid leakage and prepare sealants with different clinical degradation cycles for ophthalmology, neurosurgery, spinal surgery, etc. Hydrogel products developed based on PEG derivatives have the advantages of good biocompatibility, non-toxicity and non-irritation, no postoperative removal is required in clinical applications, the products are gradually degraded as the patient recovers, and are metabolized by the kidney. Currently, there are various commercial products of this type, such as ReSureSealant and OcuSeal Liquid Ocular Bandage, which are used for sealing corneal incisions and sealants for conjunctival and scleral surgical wounds. Summary of the Invention [Problem to be solved by the invention]

[0004] The inventors have discovered that when a multi-arm polyethylene glycol derivative is used as the base material for a hydrogel, its stability decreases over time, the gelation time of the hydrogel becomes longer, and the need for rapid hardening cannot be met. [Means for solving the problem]

[0005] To address this issue, the present inventors have conducted extensive experimental research and found that by pretreating a multi-arm polyethylene glycol derivative, its stability can be improved, the gelation time of the hydrogel can be prevented from being prolonged, and the need for rapid curing can be met.

[0006] Specifically, the present invention provides a method for preparing a multi-arm polyethylene glycol derivative conjugate, comprising the steps of: dissolving the multi-arm polyethylene glycol derivative in tert-butyl alcohol to obtain a multi-arm polyethylene glycol derivative solution; adding a compound having an amino group (-NH2) at the end to the multi-arm polyethylene glycol derivative solution, mixing uniformly to obtain a mixed raw material, and pre-freezing the mixture at -45°C to -35°C; Next, the first drying step is performed at -15°C to -5°C until the tert-butyl alcohol content is 10wt% to 15wt% and the water content is 0.5wt% to 1wt%; the second drying step is performed at 10°C to 15°C until the tert-butyl alcohol content is 2wt% to 5wt% and the water content is ≦0.2wt% to 0.5wt%; and the third drying step is performed at 25°C to 30°C until the tert-butyl alcohol content is ≦0.1wt% and the water content is ≦0.1wt%. The preparation method includes the steps of: Effect of the Invention

[0007] Research has found that by subjecting the multi-arm polyethylene glycol derivatives to the above-mentioned pretreatment, their stability can be significantly improved, the gelation time of the hydrogel can be prevented from being prolonged, and the need for rapid curing can be met. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In some embodiments, the multi-arm polyethylene glycol derivative is selected from a multi-arm polyethylene glycol derivative comprising a succinimidyl carbonate group, a multi-arm polyethylene glycol derivative comprising a succinimidyl acetate group, a multi-arm polyethylene glycol derivative comprising a succinimidyl propionate group, a multi-arm polyethylene glycol derivative comprising a succinimidyl succinate group, a multi-arm polyethylene glycol derivative comprising a succinimidyl glutarate group, and a multi-arm polyethylene glycol derivative comprising a succinimidyl sebacate group. In some embodiments, the multi-arm includes 4-arm, 6-arm, or 8-arm. In some embodiments, the multi-arm polyethylene glycol derivative has a number average molecular weight of 10K to 40K (ie, 10,000 to 40,000). As used herein, a number average molecular weight of 10K means 10,000. In some embodiments, the multi-arm polyethylene glycol derivative is selected from 4-arm polyethylene glycol succinimidyl succinate (10k), 4-arm polyethylene glycol succinimidyl succinate (20k), 4-arm polyethylene glycol succinimidyl glutarate (20k), 8-arm polyethylene glycol succinimidyl glutarate (20k), and 8-arm polyethylene glycol succinimidyl glutarate (40k).

[0009] In some embodiments, the multi-arm polyethylene glycol derivative is dissolved in tert-butyl alcohol at 30 to 40° C. (eg, 35° C.) to obtain a multi-arm polyethylene glycol derivative solution. In some embodiments, the weight to volume ratio of the multi-arm polyethylene glycol derivative to tert-butyl alcohol in the multi-arm polyethylene glycol derivative solution, in g / ml, is 1:(2-4). Research has found that this concentration range can ensure not only effective stability, but also a fast dissolution rate.

[0010] In some embodiments, the weight ratio of the multi-arm polyethylene glycol derivative to the compound having an amino group at its terminal in the mixed raw material is 1:(0.01 to 0.06) in terms of g / ml. In some embodiments, the compound having an amino group (-NH2) at the end is at least one or a combination of at least one selected from polylysine or a polylysine salt, carboxymethylchitosan, and chitosan, and is preferably trilysine acetate. In some embodiments, the compound having an amino group (-NH2) at the end is dissolved or ultrasonically dispersed in the multi-arm polyethylene glycol derivative solution under conditions where the multi-arm polyethylene glycol derivative solution is at 30°C to 40°C (e.g., 35°C). In some embodiments, the pre-freezing temperature is -45°C, -40°C or -35°C. In some embodiments, the freeze dryer is pre-cooled to a range of -45°C to -35°C prior to the pre-freezing. In some embodiments, the pre-freezing time is 100 to 150 minutes, for example 120 minutes. In some embodiments, the mixed ingredients may be dried in a lyophilizer bottle. Research has found that pre-freezing treatment can produce small ice crystals with dense structure and smooth surface, which is beneficial for improving the stability of the multi-arm polyethylene glycol derivative.

[0011] In some embodiments, the temperature of the first drying is -15°C, -10°C, or -5°C. In some embodiments, the time for the first drying may be 1200 to 1800 minutes, for example 1500 minutes, and the specific time can be determined based on the content of tert-butyl alcohol and the water content required for the drying. In some embodiments, the temperature of the second drying is 10°C or 15°C. In some embodiments, the second drying time may be 240 to 720 minutes, for example, 360 minutes, and the specific time may be determined based on the content of tert-butyl alcohol and the water content required for the drying. In some embodiments, the temperature of the third drying is 25°C or 30°C. In some embodiments, the time for the third drying may be 60 to 600 minutes, for example 120 minutes, and the specific time may be determined based on the content of tert-butyl alcohol and the water content required for the drying. Research has found that the above three-stage drying process can produce a freeze-dried powder with a smooth and dense surface and low water content, which is beneficial for improving the stability of the multi-arm polyethylene glycol derivative.

[0012] In some embodiments, the temperature of the pre-freezing is −45° C. to −40° C., and the temperature of the first drying is −15° C. to −10° C. Experiments have found that this condition is more favorable for improving the stability of the polyethylene glycol derivative, and there is no significant decrease in the hardening time of the polyethylene glycol derivative after the treatment even after it is stored for 720 d.

[0013] In some embodiments, the preparation method further includes packaging the raw material obtained by the third drying step, and preferably packaging the raw material in an environment with a moisture content of ≦25 ppm (e.g., 20 ppm) and an oxygen (i.e., oxygen gas) content of ≦25 ppm (e.g., 20 ppm), which can further improve the stability of the multi-arm polyethylene glycol derivative and reduce the oxidation and hydrolysis of the multi-arm polyethylene glycol derivative.

[0014] In some embodiments, the method comprises the steps of: dissolving the polyethylene glycol derivative in tert-butyl alcohol at 35°C to obtain a multi-arm polyethylene glycol derivative solution in which the weight / volume ratio of the multi-arm polyethylene glycol derivative to tert-butyl alcohol is 1:(2-4) in terms of g / ml; the compound having an amino group at its terminal is dissolved or ultrasonically dispersed in the multi-arm polyethylene glycol derivative solution at 35°C to obtain a mixed raw material in which the weight ratio of the multi-arm polyethylene glycol derivative to the compound having an amino group at its terminal is 1:(0.01 to 0.06); Pre-freezing the mixed raw material at -45°C to -35°C; Next, the first drying is performed at −15° C. to −5° C. until the tert-butyl alcohol content is 10 wt% to 15 wt% and the water content is 0.5 wt% to 1 wt%; the second drying is performed at 10° C. to 15° C. until the tert-butyl alcohol content is 2 wt% to 5 wt% and the water content is ≦0.2 wt% to 0.5 wt%; and the third drying is performed at 25° C. to 30° C. until the tert-butyl alcohol content is ≦0.1 wt% and the water content is ≦0.1 wt%. Packaging must be done in an environment with moisture content ≦25ppm and oxygen content ≦25ppm It includes. The present inventors have discovered that the above-mentioned pre-freezing and freeze-drying treatments can relatively satisfactorily improve the stability of the polyethylene glycol derivative, and that even after the treated polyethylene glycol derivative is stored for 720 days, there is no significant decrease in its hardening time.

[0015] The present invention also includes a multi-arm polyethylene glycol derivative conjugate prepared by the above method, which has relatively good stability. The present invention also includes the use of the multi-arm polyethylene glycol derivative conjugate prepared by the above method in the preparation of a hydrogel.

[0016] The present invention further provides an in-situ hydrogel, the in-situ hydrogel comprising: a first component which is a multi-arm polyethylene glycol derivative conjugate prepared by the above method; a second component which is a phosphate buffer; and a third component, which is a buffered salt solution selected from a borax-phosphate buffer or a sodium carbonate-phosphate buffer; It includes. In some embodiments, the pH of the phosphate buffer is 6.5 to 7.5, which allows the acidity of the entire hydrogel to be maintained in a neutral or near-neutral range. In some embodiments, the phosphate buffer solution can be prepared with reference to the phosphate buffer solution with pH=7.3 described in Section 8004 of Part 4 of the Chinese Pharmacopoeia, 2015 Edition. In some embodiments, due to the toxicity of borax, it is preferred that the third component is a phosphate buffer that does not contain borax. In some embodiments, the pH of the buffer salt solution, which is the third component, is 9.5 to 10.0, which can ensure the setting time of the gel. In some examples, the borax-phosphate buffer or sodium carbonate-phosphate buffer can be prepared with phosphate buffer (see above) and borax or sodium carbonate, respectively, and adjusted to the desired pH. In some examples, a sodium carbonate-phosphate buffer is prepared with 0.1 M sodium carbonate solution and a phosphate buffer (see above, eg, pH=7.3).

[0017] The in-situ drug-loaded hydrogel of the present invention can be prepared immediately before use; specifically, the first component is dissolved in the second component, and then mixed with the third component to obtain the in-situ hydrogel.

[0018] The present invention further provides an in-situ drug-loaded hydrogel, the in-situ drug-loaded hydrogel comprising: a first component which is a multi-arm polyethylene glycol derivative conjugate prepared by the above method; a second component which is a phosphate buffer; a third component, a buffered salt solution selected from a borax-phosphate buffer or a sodium carbonate-phosphate buffer; and The fourth ingredient is a drug It includes. In some embodiments, the second component, a phosphate buffer, and the third component, a buffered salt solution, are as described above. In some embodiments, the drug may be selected from commonly used medications, including, but not limited to, anti-inflammatory drugs (cyclosporine, erythromycin, moxifloxacin, etc.), debulking drugs (dexamethasone, cyclodextrin), moisturizers (hyaluronic acid, HPMC), glaucoma (carteolol hydrochloride, netarsudil, travoprost, bimatoprost, tafluprost), non-steroidal anti-inflammatory drugs, and the like.

[0019] The in-situ drug-loaded hydrogel of the present invention can be prepared immediately before use. Specifically, the first component is dissolved in the second component, the fourth component is dissolved in the third component, and the resulting raw material liquid is mixed to obtain the in-situ drug-loaded hydrogel.

[0020] Surprisingly, the present invention also overcomes the drawback that conventional hydrogels cannot carry drugs, and the multi-arm polyethylene glycol derivative conjugate prepared by the above method can carry drugs, especially ophthalmic drugs, and can not only realize sealing, repair and filling, but also achieve therapeutic effects.

[0021] The present invention also allows the control of different curing times by adjusting the ratio of the two kinds of buffer salt solutions (ie, the second component and the third component). In some embodiments, the first component can be dissolved in the second component, a phosphate buffer solution, to provide a raw material solution with a concentration of 50 to 300 mg / ml. In some embodiments, the fourth component can be dissolved in the third component, a buffer salt solution, to provide a raw material solution with a concentration of 6 to 200 mg / ml. In some embodiments, the content of the first component in the in-situ drug-loaded hydrogel is 2.5 wt% to 20 wt%. In some embodiments, the content of the drug in the in-situ drug-loaded hydrogel is 0.02 wt% to 10 wt%. In some embodiments, the in-situ drug-loaded hydrogel further comprises a fifth component, a color former, which may be selected from non-azo colorants. When a color former is included, the color former can be dissolved in the second or third component and then mixed with other raw material liquids. In some embodiments, the content of the color former in the in-situ drug-loaded hydrogel is 0.01 wt% to 0.1 wt%.

[0022] The present invention further provides a method for preparing the in-situ drug-loaded hydrogel, the method comprising: dissolving the first component with the second component (i.e., phosphate buffer solution) to obtain a first raw material solution; dissolving the fourth component in the third component (i.e., a buffer salt solution) to obtain a second raw material solution; Next, the first raw material liquid and the second raw material liquid are mixed to form an in-situ drug-loaded hydrogel. It includes.

[0023] The present invention uses freeze-drying of a multi-arm polyethylene glycol derivative and an amino group-containing compound together to improve their stability, meet the need for rapid hardening, and also improve their dissolution rate, shorten the operation time, realize time-controllable hardening and different drug loading at different sites of tissue, and realize sealing, repair, filling and treatment of multiple indications. The in-situ hydrogel and in-situ drug-loaded hydrogel of the present invention can be used for sealing, repairing, filling or treating tissue wound surfaces. The drug-loaded hydrogel is prepared just before use and applied to the corresponding site of the tissue before forming a gel (for example, via an accessory), thereby achieving tissue sealing, repair and filling while achieving therapeutic effects such as anti-inflammation, anti-infection, swelling relief, moisturizing, etc. This hydrogel can be molded in-situ, and the dissolution time of the first component can be controlled to <20s, and to form a gel within 0-10s depending on different tissue application sites, and the gel can be decomposed by hydrolysis. [Brief description of the drawings]

[0024] [Figure 1] 1 is a photograph showing the rabbit eye sealing effect in an experimental example of the present invention. [Diagram 2] 1 shows a sustained release curve of a drug in an experimental example of the present invention. EXAMPLES

[0025] The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, unless specific techniques or conditions are specified, the techniques and conditions are those described in the literature in the field or in accordance with the product instruction manual. For reagents and equipment used, unless the manufacturer is specified, they are all ordinary products that can be purchased through official sales channels.

[0026] Example 1 This example provides a method for preparing a multi-arm polyethylene glycol derivative conjugate, the method comprising: Dissolve 4-arm polyethylene glycol succinimidyl succinate (10k) in tert-butyl alcohol at 35°C to obtain a 4-arm polyethylene glycol succinimidyl succinate solution in which the ratio of 4-arm polyethylene glycol succinimidyl succinate to tert-butyl alcohol is 1g:2ml; ultrasonically dispersing trilysine acetate in a 4-arm polyethylene glycol succinimidyl succinate solution at 35°C and mixing uniformly to obtain a mixed raw material in which the weight ratio of 4-arm polyethylene glycol succinimidyl succinate to trilysine acetate is 1:0.03; The mixed raw material is charged into a freeze-drying bottle, and the freeze-drying machine is pre-cooled to -45°C to -35°C. The freeze-drying bottle is then placed on the partition plate of the freeze-drying machine and pre-frozen for 120 minutes. Then, a first drying step is performed at -15°C to -5°C, with a drying time of 1500 minutes, a second drying step is performed at 15°C, with a drying time of 360 minutes, and a third drying step is performed at 30°C, with a drying time of 120 minutes. The freeze-dried product must be packaged in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm. It includes. By controlling the pre-cooling temperature, the pre-freezing temperature, and the temperature of the first drying, Samples 1 to 3 were prepared, respectively, as shown in the following table.

[0027] [Table 1]

[0028] Example 2 This example provides a method for preparing a multi-arm polyethylene glycol derivative conjugate, the method comprising: Dissolving the multi-arm polyethylene glycol derivative in tert-butyl alcohol at 35°C to obtain a multi-arm polyethylene glycol derivative solution in which the ratio of the multi-arm polyethylene glycol derivative to tert-butyl alcohol is 1g:4ml; A compound having an amino group at its end is ultrasonically dispersed in a multi-arm polyethylene glycol derivative solution at 35°C, and the resulting mixture is mixed uniformly to obtain a mixed raw material. The mixed raw material is charged into a freeze-drying bottle, and the freeze-drying machine is pre-cooled to -45°C. The freeze-drying bottle is placed on the partition plate of the freeze-drying machine and pre-frozen for 120 minutes. Then, the first drying is performed at -15°C for 1500 minutes, and then the second drying is performed at 15°C for 360 minutes and the third drying is performed at 30°C for 120 minutes. The freeze-dried product must be packaged in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm. It includes. Different multi-arm polyethylene glycol derivatives and compounds having an amino group at the end were selected, and the weight ratio of the multi-arm polyethylene glycol derivative and the compound having an amino group at the end was controlled to prepare samples 4 to 9. The details are shown in the table below.

[0029] [Table 2]

[0030] Example 3 This example provides a method for preparing a multi-arm polyethylene glycol derivative conjugate, the method comprising: Dissolve 4-arm polyethylene glycol succinimidyl glutarate (10k) in tert-butyl alcohol at 35°C to obtain a 4-arm polyethylene glycol succinimidyl glutarate solution in which the ratio of 4-arm polyethylene glycol succinimidyl glutarate to tert-butyl alcohol is 1g:2ml; ultrasonically dispersing a compound having an amino group at its terminal into a 4-arm polyethylene glycol succinimidyl glutarate solution at 35°C and mixing uniformly to obtain a mixed raw material having a weight ratio of 4-arm polyethylene glycol succinimidyl glutarate to trilysine acetate of 1:0.03; The mixed raw material is charged into a freeze-drying bottle, and the freeze-drying machine is pre-cooled to -45°C. The freeze-drying bottle is placed on the partition plate of the freeze-drying machine and pre-frozen for 120 minutes. Then, the first drying is performed at -15°C for 1500 minutes, and then the second drying is performed at 15°C for 360 minutes and the third drying is performed at 30°C for 120 minutes. The freeze-dried product was packaged in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm, and used as sample 10. It includes.

[0031] Example 4 The only difference from Example 1 was that the freeze-dried product was packaged in a normal environment with a relative humidity of 50%, and designated as Sample 11.

[0032] Comparative Example 1 The only difference from Example 1 is that Samples 12 to 15 were prepared by controlling the pre-cooling temperature, pre-freezing temperature, temperature drop rate, and first drying temperature, as shown in the table below.

[0033] [Table 3]

[0034] Comparative Example 2 4-arm polyethylene glycol succinimidyl succinate (10k) was dissolved in tert-butyl alcohol at 35°C to prepare a 4-arm polyethylene glycol succinimidyl succinate solution. Trilysine acetate was ultrasonically dispersed in a 4-arm polyethylene glycol succinimidyl succinate solution at 35° C. and mixed uniformly to obtain a mixed raw material in which the weight ratio of 4-arm polyethylene glycol succinimidyl succinate to trilysine acetate was 1:0.03. The mixed raw material was loaded into a freeze-drying bottle, and the freeze-drying machine was pre-cooled to -45°C. The freeze-drying bottle was then placed on the partition plate of the freeze-drying machine and pre-frozen for 120 min. After that, a first drying step was carried out at -15°C to -5°C with a drying time of 1500 min. Thereafter, a second drying step was carried out at 15°C with a drying time of 360 min., and a third drying step was carried out at 30°C with a drying time of 120 min. The freeze-dried product was packaged in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm. Samples 16-17 were prepared by controlling the ratio of 4-arm polyethylene glycol succinimidyl succinate to tert-butyl alcohol, as shown in the table below.

[0035] [Table 4]

[0036] Comparative Example 3 4-arm polyethylene glycol succinimidyl succinate (10k) was dissolved in tert-butyl alcohol at 35°C to give a solution with a ratio of 4-arm polyethylene glycol succinimidyl succinate to tert-butyl alcohol of 1 g:2 ml, and the prepared solution was placed in freeze-drying bottle A. The trilysine salt was dispersed in tert-butyl alcohol at 35° C. to prepare a mixed raw material having a weight ratio of trilysine salt to tert-butyl alcohol of 0.03 g:2 ml. The prepared mixed raw material was placed in a freeze-drying bottle B. After pre-cooling the freeze-dryer to -45°C, the freeze-drying bottles A and B were placed on the partition plate of the freeze-dryer and pre-frozen at -45°C for 120 min, then dried at -15°C for 1,500 min, and then dried at 15°C for 360 min and 30°C for 120 min, respectively. Next, 4-arm polyethylene glycol succinimidyl succinate and trilysine acetate were mixed and packaged in a weight ratio of 1:0.03 in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm to obtain Sample 18.

[0037] Comparative Example 4 4-arm polyethylene glycol succinimidyl succinate (10k) and 2,6-di-tert-butyl-4-methylphenol (BHT) as an antioxidant were mixed and dried until the moisture content was <0.1%. Then, trilysine acetate was mixed under an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm to obtain sample 19. The weight ratio of 4-arm polyethylene glycol succinimidyl succinate to trilysine acetate was 1:0.03, and the content of the antioxidant BHT in sample 19 was 0.05 wt%.

[0038] Experimental Example 1: Stability study of multi-arm polyethylene glycol derivatives The samples of the above examples and comparative examples were packaged and stored in a stability test box at 2 to 8° C., and after 720 days, their dissolution and hardening times were measured. Measurement method: Phosphate buffer solution of pH 7.5 was added to each of the above samples and shaken until completely dissolved to prepare a 200 mg / ml solution M. In addition, a sodium carbonate-phosphate buffer solution of pH 10.0 with the same volume as the phosphate buffer solution used was separately taken, and the solution M and the sodium carbonate-phosphate buffer solution were mixed to form a gel, and the hardening time was recorded. The experimental results are shown in the table below.

[0039] [Table 5]

[0040] Experimental Example 2 Sample 1 was prepared by the method of Example 1. A phosphate buffer solution of pH 7.5 was added to Sample 1 and shaken until it was completely dissolved to prepare a solution N of a concentration of 50 to 300 mg / ml. In addition, an appropriate volume of a sodium carbonate-phosphate buffer solution of pH 10.0 was separately collected, and the solution N and the sodium carbonate-phosphate buffer solution were mixed to form gels, which were numbered A to E and their hardening times were recorded.

[0041] [Table 6]

[0042] Experimental Example 3 Biocompatibility Results Sample 6 was prepared according to the method of Example 2. 4-arm polyethylene glycol succinimidyl glutarate (20k, the same as the raw material for sample 6) was taken and not subjected to any treatment such as freeze-drying, and used as a control sample. The biological evaluation of the above samples was carried out according to the GB 16886 series standards, and the results are shown in the table below.

[0043] [Table 7]

[0044] Experimental Example 4: In-situ drug-loaded hydrogel and its use Sample 1 was prepared according to the method of Example 1. 4 ml of pH 7.5 phosphate buffer was added to 600 mg of sample 1 and shaken until completely dissolved to prepare solution X. 10 mg of dexamethasone was separately collected and dissolved in 2 ml of pH 10.0 sodium carbonate-phosphate buffer to obtain solution Y. After mixing solution X and solution Y, a gel was formed. Animal experiment: A rabbit eye model was selected, and the gel was applied to the wound surface of the rabbit eye cornea with a length of about 10 mm (3 to 9 places) in a horizontal direction to seal the incision. The postoperative incision sealing effect and the occurrence of local inflammation were observed for 7 days after surgery, and the results showed that the incision damage tended to heal, and the inflammatory reaction gradually recovered, and finally recovered completely. The rabbit eye sealing effect is shown in Figure 1.

[0045] Experimental Example 5: In-situ drug-loaded hydrogel and measurement of its sustained release Sample 6 was prepared according to the method of Example 2. A phosphate buffer solution of pH 7.5 was added to sample 6 and shaken until completely dissolved to prepare solution X with a concentration of 300 mg / ml. In addition, dexamethasone was separately collected and dissolved in a sodium carbonate-phosphate buffer solution of pH 10.0 to obtain solution Y. Solution X and solution Y were mixed together to form a gel. The content of dexamethasone in this gel was 5 wt%. Drug sustained release detection method: The hydrogel loaded with the drug was collected, and the hydrogel and dissolution medium were mixed in a volume ratio of 1g:50ml, and the sample was placed in a water bath oscillator at 37℃±1℃, and operated according to the regulations. 1.0ml was sampled at 1h, 2h, 6h, 1d, 3d, 6d, 9d, 12d, 15d, 18d, 21d, 24d, 27d, and 30d, respectively, and the same volume of fresh solution was added, and the sampled solution was filtered, and the following filtrate was taken as the test solution, and the release amount was detected according to the method specified in the Chinese Pharmacopoeia 2015 Edition Part 2 Dexamethasone Content Detection. The drug release curves are shown in Figure 2, and the results reveal that the hydrogel has a relatively long sustained release effect on dexamethasone.

[0046] Although the present invention has been described in detail above through general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications and improvements can be made based on the present invention. Therefore, any modifications or improvements made within the scope of the present invention that do not deviate from the spirit of the present invention are within the scope of the present invention. [Industrial Applicability]

[0047] The present invention provides an in-situ drug-loaded hydrogel and its preparation method and use. The preparation method of the multi-arm polyethylene glycol derivative complex includes pre-freezing the multi-arm polyethylene glycol derivative solution, followed by three steps of drying in sequence. The in-situ drug-loaded hydrogel includes a multi-arm polyethylene glycol derivative complex, a phosphate buffer, a buffer salt solution, and a drug. The in-situ drug-loaded hydrogel of the present invention can be used to seal, repair, fill or treat tissue wound surfaces, and has relatively good economic value and use prospects.

Claims

1. 1. A method for preparing a multi-arm polyethylene glycol derivative conjugate, comprising: dissolving the multi-arm polyethylene glycol derivative in tert-butyl alcohol to obtain a multi-arm polyethylene glycol derivative solution; adding a compound having an amino group at a terminal to the multi-arm polyethylene glycol derivative solution, uniformly mixing the resulting mixture to obtain a mixed raw material, and pre-freezing the mixture at −45° C. to −35° C.; Next, the following steps are carried out in order: a first drying step is performed at −15° C. to −5° C. until the tert-butyl alcohol content is 10 wt % to 15 wt % and the water content is 0.5 wt % to 1 wt %; a second drying step is performed at 10° C. to 15° C. until the tert-butyl alcohol content is 2 wt % to 5 wt % and the water content is ≦0.2 wt % to 0.5 wt %; and a third drying step is performed at 25° C. to 30° C. until the tert-butyl alcohol content is ≦0.1 wt % and the water content is ≦0.1 wt %. Then, packaging in an environment with a moisture content of ≦25 ppm and an oxygen content of ≦25 ppm; Including, the multi-arm polyethylene glycol derivative is selected from 4-arm polyethylene glycol succinimidyl succinate (10k), 4-arm polyethylene glycol succinimidyl succinate (20k), 4-arm polyethylene glycol succinimidyl glutarate (20k), 8-arm polyethylene glycol succinimidyl glutarate (20k), 8-arm polyethylene glycol succinimidyl glutarate (40k); the compound having an amino group at its terminal is at least one selected from the group consisting of polylysine or a polylysine salt, carboxymethylchitosan, and chitosan, or a combination of a plurality of such compounds; The preparation method.

2. g / ml, the weight / volume ratio of the multi-arm polyethylene glycol derivative to tert-butyl alcohol in the multi-arm polyethylene glycol derivative solution is 1:(2-4); and / or 2. The method for preparing a multi-arm polyethylene glycol derivative conjugate according to claim 1, wherein the weight ratio of the multi-arm polyethylene glycol derivative to the compound having an amino group at its terminal in the mixed raw material is 1:(0.01 to 0.06), in terms of g / ml.

3. The pre-freezing time is 100 to 150 min, and / or The first drying time is 1200 to 1800 min, and / or The second drying time is 240 to 720 min, and / or The method for preparing a multi-arm polyethylene glycol derivative conjugate according to claim 1 or 2, wherein the third drying time is 60 to 600 min.

4. A multi-arm polyethylene glycol derivative conjugate prepared by the method of any one of claims 1 to 3.

5. 5. Use of the multi-arm polyethylene glycol derivative conjugate according to claim 4 in the preparation of a hydrogel.

6. A first component which is a multi-arm polyethylene glycol derivative conjugate according to claim 4; a second component which is a phosphate buffer having a pH of 6.5 to 7.5; and a third component, a buffered salt solution selected from a borax-phosphate buffer or a sodium carbonate-phosphate buffer, wherein the pH of the buffered salt solution is between 9.5 and 10.

0.

7. A first component which is a multi-arm polyethylene glycol derivative conjugate according to claim 4; a second component which is a phosphate buffer having a pH of 6.5 to 7.5; a third component, a buffered salt solution selected from a borax-phosphate buffer or a sodium carbonate-phosphate buffer, the pH of the buffered salt solution being between 9.5 and 10.0; A fourth ingredient, which is a drug; An in-situ drug-loaded hydrogel comprising:

8. dissolving the first component in the second component to obtain a first raw material liquid; dissolving the fourth component in the third component to obtain a second raw material liquid; Next, the first raw material liquid and the second raw material liquid are mixed to form an in-situ drug-loaded hydrogel. A method for preparing an in-situ drug-loaded hydrogel according to claim 7, comprising:

Images