WO2022206882A1 - 原位载药水凝胶及其制备方法与应用 - Google Patents

原位载药水凝胶及其制备方法与应用 Download PDF

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WO2022206882A1
WO2022206882A1 PCT/CN2022/084316 CN2022084316W WO2022206882A1 WO 2022206882 A1 WO2022206882 A1 WO 2022206882A1 CN 2022084316 W CN2022084316 W CN 2022084316W WO 2022206882 A1 WO2022206882 A1 WO 2022206882A1
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polyethylene glycol
arm polyethylene
glycol derivative
solution
component
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PCT/CN2022/084316
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English (en)
French (fr)
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冷鸿飞
徐小雨
陶秀梅
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北京诺康达医药科技股份有限公司
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Priority to JP2023552256A priority Critical patent/JP2024508482A/ja
Publication of WO2022206882A1 publication Critical patent/WO2022206882A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2371/00—Characterised by the use of polyethers obtained by reactions forming an ether link in the main chain; Derivatives of such polymers
    • C08J2371/02—Polyalkylene oxides

Definitions

  • the invention relates to an in-situ drug-carrying hydrogel and a preparation method and application thereof.
  • in situ degradable hydrogel is rich in water, has good biocompatibility, and its safety and performance are controllable, and the injectable in situ hydrogel is easy to operate during surgery, so There are broad research prospects in the treatment of diseases.
  • PEG-modified hydrogel technology can be applied in tissue engineering, especially in tissue sealants, which can help prevent tissue exudation, and can prepare sealants for different clinical degradation cycles such as ophthalmology, neurosurgery, and spine surgery.
  • the hydrogel products developed based on PEG derivatives have the advantages of good biocompatibility, non-toxic and non-irritating, no need to be removed after surgery in clinical applications, and the products will gradually degrade as the patients recover and metabolize through the kidneys.
  • ReSureSealant and OcuSeal Liquid Ocular Bandage which are used for corneal incision sealing, conjunctival and scleral surgical wound sealants.
  • the inventors found that when the multi-arm polyethylene glycol derivative is used as a hydrogel base material, its stability decreases with time, thereby prolonging the gel formation time of the hydrogel, which cannot meet the needs of rapid curing.
  • the inventors have found through a large number of experimental studies that pretreatment of multi-arm polyethylene glycol derivatives can improve their stability, avoid prolonging the gelation time of hydrogels, and meet the needs of rapid curing.
  • the present invention provides a preparation method of a multi-arm polyethylene glycol derivative complex, comprising:
  • the first drying at -15°C to -5°C in sequence, and dry to the tertiary butanol content of 10wt%-15wt% and the water content of 0.5wt%-1wt%; the second drying is performed at 10°C-15°C until the t The butanol content is 2wt%-5wt%, and the water content is ⁇ 0.2wt%-0.5wt%; the third drying is carried out at 25°C-30°C until the tert-butanol content is less than or equal to 0.1wt% and the water content is less than or equal to 0.1wt%.
  • the multi-arm polyethylene glycol derivative is selected from the group consisting of succinimidyl carbonate, succinimidyl acetate, succinimidyl propionate, succinimidyl succinimide Multi-arm polyethylene glycol derivatives of ester group, succinimide glutarate group, and succinimide sebacate group.
  • the multiple arms include four arms, six arms, and eight arms.
  • the multi-arm polyethylene glycol derivative has a number average molecular weight of 10K-40K (ie, 10000-40000).
  • the number-average molecular weight of 10K means 10,000.
  • the multi-arm polyethylene glycol derivative is selected from four-arm polyethylene glycol succinimidyl succinate (10k), four-arm polyethylene glycol succinimidyl succinate (20k) ), four-arm polyethylene glycol succinimide glutarate (20k), eight-arm polyethylene glycol succinimide glutarate (20k), eight-arm polyethylene glycol succinimide glutarate Acetate (40k).
  • the multi-arm polyethylene glycol derivative solution is prepared by dissolving the multi-arm polyethylene glycol derivative in tert-butanol at 30-40° C. (eg, 35° C.).
  • the weight-to-volume ratio of the multi-arm polyethylene glycol derivative to tert-butanol is 1:(2-4). The study found that within this concentration range, both effective stability and faster dissolution rate can be ensured.
  • the weight ratio of the multi-arm polyethylene glycol derivative to the compound with an amino group at the end is 1:(0.01-0.06).
  • the compound with an amino group (-NH 2 ) at the end is selected from at least one or more of polylysine or polylysine salt, carboxymethyl chitosan and chitosan A combination of species; preferably trilysine acetate.
  • the compound with an amino group (-NH 2 ) at the terminal is dissolved in or Sonication is dispersed in the multi-arm polyethylene glycol derivative solution.
  • the pre-freezing temperature is -45°C, -40°C or -35°C.
  • the lyophilizer is pre-cooled to a range of -45°C to -35°C before the pre-freezing.
  • the pre-freezing time is 100-150 min, such as 120 min.
  • the mixture can be dried in a lyophilized vial.
  • the temperature of the first drying is -15°C, -10°C or -5°C.
  • the first drying time may be 1200-1800 min, for example, 1500 min, and the specific time may be determined according to the tert-butanol content and water content required for the above drying.
  • the temperature of the second drying is 10°C or 15°C.
  • the time for the second drying may be 240-720 min, for example, 360 min, and the specific time may be determined according to the tert-butanol content and water content required for the above drying.
  • the temperature of the third drying is 25°C or 30°C.
  • the time of the third drying may be 60-600 min, for example, 120 min, and the specific time may be determined according to the tert-butanol content and water content required for the above drying.
  • the temperature of the pre-freezing is -45°C to -40°C
  • the temperature of the first drying is -15°C to -10°C.
  • the above-mentioned preparation method further includes the step of packaging the material obtained by the third drying, preferably in an environment with a moisture content ⁇ 25ppm (eg, 20ppm) and an oxygen (ie, oxygen) content ⁇ 25ppm (eg, 20ppm) .
  • a moisture content ⁇ 25ppm eg, 20ppm
  • an oxygen (ie, oxygen) content ⁇ 25ppm eg, 20ppm
  • the above-mentioned preparation method comprises:
  • the polyethylene glycol derivative was dissolved in tert-butanol at 35°C to prepare a multi-arm polyethylene glycol derivative solution; in g/ml, in the multi-arm polyethylene glycol derivative solution, the multi-arm polyethylene glycol derivative solution was The weight volume ratio of polyethylene glycol derivative and tert-butanol is 1:(2-4);
  • the first drying at -15°C to -5°C in sequence, and dry to the tertiary butanol content of 10wt%-15wt% and the water content of 0.5wt%-1wt%;
  • the second drying is performed at 10°C-15°C until the t
  • the butanol content is 2wt%-5wt%, the water content is ⁇ 0.2wt%-0.5wt%;
  • the third drying is carried out at 25°C-30°C until the tert-butanol content is less than or equal to 0.1wt% and the water content is less than or equal to 0.1wt%;
  • the present invention also includes the multi-arm polyethylene glycol derivative complex prepared by the above method, which has better stability.
  • the present invention also includes the application of the multi-arm polyethylene glycol derivative complex prepared by the above method in the preparation of hydrogel.
  • the present invention also provides an in-situ hydrogel, comprising:
  • the first component is the multi-arm polyethylene glycol derivative complex prepared by the above method
  • the second component is phosphate buffered solution
  • the third component is buffered saline solution; selected from borax-phosphate buffer solution or sodium carbonate-phosphate buffer solution.
  • the pH value of the phosphate buffer solution is 6.5-7.5, which can keep the pH value of the hydrogel as a whole in a neutral or near-neutral range.
  • the third component is preferably a phosphate buffered solution without borax in view of the possible toxicity of borax.
  • the pH value of the third component, the buffered salt solution is 9.5-10.0, which can ensure the curing time of the gel.
  • the borax-phosphate buffered solution or sodium carbonate-phosphate buffered solution can be formulated with phosphate buffered solution (see above) and borax or sodium carbonate, respectively, adjusted to the desired pH.
  • the in-situ drug-loaded hydrogel of the present invention can be configured before use, specifically, the first component can be dissolved with the second component, and then mixed with the third component to obtain the in-situ hydrogel.
  • the present invention also provides an in-situ drug-loaded hydrogel, comprising:
  • the first component is the multi-arm polyethylene glycol derivative complex prepared by the above method
  • the second component is phosphate buffered solution
  • the third component is buffered saline solution; selected from borax-phosphate buffer solution or sodium carbonate-phosphate buffer solution;
  • the fourth component is a drug.
  • the second component phosphate buffered solution and the third component is a buffered saline solution are the same as above.
  • the drugs can be selected from commonly used ophthalmic drugs, including but not limited to: anti-inflammatory drugs (cyclosporine, erythromycin, moxifloxacin, etc.), deedema drugs (dexamethasone, cyclodextrin, etc.) ), moisturizing (hyaluronic acid, HPMC), glaucoma (carteolol hydrochloride, nesudil, travoprost, bimeprost, tafluprost), non-steroidal anti-inflammatory drugs, etc.
  • anti-inflammatory drugs cyclosporine, erythromycin, moxifloxacin, etc.
  • deedema drugs diexamethasone, cyclodextrin, etc.
  • moisturizing hyaluronic acid, HPMC
  • glaucoma carteolol hydrochloride, nesudil, travoprost, bimeprost, tafluprost
  • the in-situ drug-loaded hydrogel of the present invention can be configured immediately before use. Specifically, the first component can be dissolved with the second component, the fourth component can be dissolved with the third component, and then the obtained materials and liquids can be mixed, that is, In situ drug-loaded hydrogels were obtained.
  • the present invention also overcomes the defect that traditional hydrogels cannot carry medicines, and the multi-arm polyethylene glycol derivative complex prepared by the above method can carry medicines, especially ophthalmic medicines. In addition to filling, it can also achieve therapeutic effects.
  • the present invention can also achieve different curing time control by adjusting the ratio of the two buffered salt solutions (ie, the second component and the third component).
  • the first component can be dissolved in a phosphate buffered solution of the second component to prepare a feed solution with a concentration of 50-300 mg/ml.
  • the fourth component can be dissolved in a buffered saline solution of the third component to prepare a feed solution with a concentration of 6-200 mg/ml.
  • the content of the first component in the in situ drug-loaded hydrogel is 2.5wt%-20wt%.
  • the content of the drug in the in situ drug-loaded hydrogel is 0.02wt%-10wt%.
  • the components of the in-situ drug-loaded hydrogel further include a fifth component, namely a color developer, which can be selected from non-azo colorants.
  • a color developer When a color developer is contained, the color developer can be dissolved with the second component or the third component, and then mixed with other feed liquids.
  • the content of the color developer in the in-situ drug-loaded hydrogel is 0.01wt%-0.1wt%.
  • the present invention also provides a preparation method of the above-mentioned in-situ drug-loaded hydrogel, comprising:
  • the fourth component is dissolved with the third component (that is, buffered saline solution) to make the second feed solution;
  • the first feed solution and the second feed solution are mixed to prepare an in-situ drug-loaded hydrogel.
  • the present invention can not only improve its stability, meet the needs of rapid solidification, but also improve its dissolution speed, shorten the operation time, and realize different tissue types.
  • the controllable time curing of the site and the loading of different drugs enable the closure, 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 wounds. It is configured before use, and the drug-loaded hydrogel is applied to the corresponding parts of the tissue before the gel is formed (for example, through accessories), which can achieve anti-inflammatory, anti-infection, and de-edema while satisfying tissue sealing, repairing, and filling. , moisturizing and other therapeutic effects.
  • the hydrogel can be formed in situ, the dissolution time of the first component is less than 20s, and the gel can be formed within 0-10s according to different tissue application sites, and the gel can be degraded by hydrolysis.
  • FIG. 1 is a photo of the rabbit eye sealing effect in the experimental example of the present invention.
  • Fig. 2 is the drug sustained-release curve in the experimental example of the present invention.
  • the present embodiment provides a method for preparing a multi-arm polyethylene glycol derivative complex, comprising:
  • the trilysine acetate is ultrasonically dispersed in the four-arm polyethylene glycol succinimidyl succinate solution at 35° C., and mixed uniformly to prepare a mixed material; wherein, in the mixed material, the four-arm polyethylene glycol
  • the weight ratio of diol succinimidyl succinate to trilysine acetate is 1:0.03;
  • the mixed raw materials were placed in a freeze-dried bottle; the freeze-drier was pre-cooled to -45°C to -35°C, and then the freeze-dried bottle was placed on the freeze-drier partition, pre-freeze for 120 minutes and then at -15°C
  • the first drying was performed at -5°C for 1500min, then the second drying was performed at 15°C for 360min, and the third drying was performed at 30°C for 120min;
  • the lyophilized product was packaged in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm.
  • samples 1-3 were prepared respectively, as shown in the following table:
  • the present embodiment provides a method for preparing a multi-arm polyethylene glycol derivative complex, comprising:
  • the multi-arm polyethylene glycol derivative is dissolved in tert-butanol at 35°C to make a multi-arm polyethylene glycol derivative solution; wherein, the ratio of the multi-arm polyethylene glycol derivative to the tert-butanol is 1 g: 4ml;
  • the compound with an amino group at the end is ultrasonically dispersed in the multi-arm polyethylene glycol derivative solution at 35°C, and mixed evenly to prepare a mixed material;
  • the mixed raw materials were placed in a freeze-dried bottle; the freeze-drier was pre-cooled to -45°C, and then the freeze-dried bottle was placed on the freeze-drier partition, pre-freeze for 120min, and then first dried at -15°C for 1500min , and then the second drying at 15°C for 360min and the third drying at 30°C for 120min;
  • the lyophilized product was packaged in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm.
  • samples 4-9 were prepared respectively, as shown in the following table. :
  • This embodiment provides a method for preparing a multi-arm polyethylene glycol derivative complex, including:
  • the compound with an amino group at the end was ultrasonically dispersed in the four-arm polyethylene glycol succinimide glutarate solution at 35°C, and mixed uniformly to prepare a mixed material; wherein, in the mixed material, four-arm polyethylene glycol
  • the weight ratio of alcohol succinimide glutarate to trilysine acetate is 1:0.03;
  • the mixed raw materials were placed in a freeze-dried bottle; the freeze-drier was pre-cooled to -45°C, then the freeze-dried bottle was placed on the freeze-drier partition, pre-freeze for 120min, and then first dried at -15°C for 1500min , and then the second drying at 15°C for 360min and the third drying at 30°C for 120min;
  • the freeze-dried product was packaged in an environment with a moisture content of 20 ppm and an oxygen content of 20 ppm to prepare Sample 10.
  • Example 1 The only difference from Example 1 is that the freeze-dried product was packaged in a normal environment with a relative humidity of 50% to prepare Sample 11.
  • samples 12-15 were prepared respectively by controlling the pre-cooling temperature, the pre-freezing temperature, the cooling rate and the first drying temperature, as shown in the following table:
  • the cooling rate refers to the rate at which the freeze dryer drops from the pre-cooling temperature to the pre-freezing temperature.
  • the trilysine acetate is ultrasonically dispersed in the four-arm polyethylene glycol succinimidyl succinate solution at 35° C., and mixed uniformly to prepare a mixed material; wherein, in the mixed material, the four-arm polyethylene glycol
  • the weight ratio of diol succinimidyl succinate to trilysine acetate is 1:0.03;
  • the mixed raw materials were placed in a freeze-dried bottle; the freeze-drier was pre-cooled to -45°C, then the freeze-dried bottle was placed on the freeze-drier separator, and the freeze-dried bottle was pre-frozen for 120 minutes at -15°C to -5°C.
  • Carry out the first drying the drying time is 1500min, and then the second drying is carried out at 15°C, the drying time is 360min, and the third drying time is 30°C, and the drying time is 120min;
  • the lyophilized 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, as shown in the following table:
  • freeze-drying machine was pre-cooled to -45°C, then the above freeze-dried bottles A and B were placed on the separator of the freeze-drying machine. Dry for 360min and 30°C for 120min.
  • Sample 1 was prepared as in Example 1. Add sample 1 to phosphate buffer solution with pH 7.5, shake until all dissolved, and prepare solution N with a concentration of 50-300 mg/ml. In addition, take an appropriate volume of sodium carbonate-phosphate buffer solution with a pH value of 10.0, and mix solution N and sodium carbonate-phosphate buffer solution to form a gel, numbered A-E, and record its curing time.
  • Sample 6 was prepared as in Example 2.
  • Sample 1 was prepared as in Example 1.
  • Sample 6 was prepared as in Example 2.
  • dexamethasone Take another dexamethasone, dissolve it with a sodium carbonate-phosphate buffer solution with a pH value of 10.0 to obtain solution Y, and mix solution X and solution Y to form a gel.
  • the content of dexamethasone in the gel was 5 wt%.
  • Detection method of drug sustained release take the solidified hydrogel loaded with the drug, according to the ratio of the volume of the hydrogel to the release medium is 1g: 50ml, put the sample in a water bath shaker at 37°C ⁇ 1°C, and operate according to the law, respectively for 1h , 2h, 6h, 1d, 3d, 6d, 9d, 12d, 15d, 18d, 21d, 24d, 27d, 30d, sample 1.0ml respectively, and add the same volume of fresh solution, filter the sampling solution, and take the filtrate as the supply.
  • the release amount was detected according to the method specified in the content detection of dexamethasone under the second part of the Chinese Pharmacopoeia 2015 edition.
  • the sustained-release curve of the drug is shown in Figure 2, and the results show that the hydrogel has a longer sustained-release effect on dexamethasone.
  • the invention provides an in-situ drug-carrying hydrogel and a preparation method and application thereof.
  • the preparation method of the multi-arm polyethylene glycol derivative complex comprises the steps of pre-freezing the multi-arm polyethylene glycol derivative solution, and then performing three-stage drying in sequence.
  • In situ drug-loaded hydrogels include multi-arm polyethylene glycol derivative complexes, phosphate buffered solutions, buffered saline solutions and drugs.
  • the in-situ drug-carrying hydrogel of the invention can be used for sealing, repairing, filling or treating tissue wounds, and has good economic value and application prospect.

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Abstract

本发明涉及一种原位载药水凝胶及其制备方法与应用。多臂聚乙二醇衍生物复合物的制备方法，包括将多臂聚乙二醇衍生物溶液预冻，然后依次进行三阶段的干燥。原位载药水凝胶包括多臂聚乙二醇衍生物复合物，磷酸盐缓冲溶液，缓冲盐溶液和药物。本发明原位载药水凝胶可用于组织创面封闭、修复、填充或治疗。

Description

原位载药水凝胶及其制备方法与应用
交叉引用
本申请要求2021年3月31日提交的专利名称为“原位载药水凝胶及其制备方法与应用”的第202110351993.7号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及一种原位载药水凝胶及其制备方法与应用。
背景技术
原位可降解水凝胶作为一种多尺度发展的材料，富含大量水分，生物相容性良好，其安全和性能可控，且可注射原位水凝胶在手术过程中易操作，因此在疾病的治疗中有广阔的研究前景。
PEG多样化修饰的水凝胶技术可应用在组织工程方面，特别是组织密封剂方面，有助于预防组织渗液、可制备针对眼科、神经外科、脊柱外科等不同临床降解周期需求的密封剂。基于PEG衍生物开发的水凝胶产品具有生物相容性好，无毒、无刺激，临床应用中术后无需再移除，产品会随着患者的康复逐渐降解并通过肾脏代谢等优点。目前该类产品中已有多种上市品，如ReSureSealant和OcuSeal Liquid Ocular Bandage等用于角膜切口封闭、结膜和巩膜手术伤口的封闭剂。
发明内容
发明人发现，多臂聚乙二醇衍生物作为水凝胶基材时其稳定性随着时间的延长而下降，从而延长了水凝胶的成胶时间，不能满足快速固化的需要。针对该技术问题，发明人通过大量实验研究发现，对多臂聚乙二醇衍生物进行预处理，可以提高其稳定性，能够避免延长水凝胶的成胶时间，满足快速固化的需要。
具体而言，本发明提供一种多臂聚乙二醇衍生物复合物的制备方法，包括：
将多臂聚乙二醇衍生物溶解于叔丁醇中，制成多臂聚乙二醇衍生物溶液；
向所述多臂聚乙二醇衍生物溶液中加入末端带氨基(-NH 2)的化合物，混合均匀，制成混合物料；于-45℃至-35℃预冻；
然后依次于-15℃至-5℃进行第一干燥，干燥至叔丁醇含量10wt％-15wt％，含水量0.5wt％-1wt％；于10℃-15℃进行第二干燥，干燥至叔丁醇含量2wt％-5wt％，含水量≤0.2wt％-0.5wt％；于25℃-30℃进行第三干燥，干燥至叔丁醇含量≤0.1wt％，含水量≤0.1wt％。
研究发现，对多臂聚乙二醇衍生物进行如上预处理，可以显著提高其稳定性，能够避免水凝胶成胶时间的延长，满足快速固化的需要。
在一些实施例中，所述多臂聚乙二醇衍生物选自含有琥珀酰亚胺碳酸酯基、琥珀酰亚胺乙酸酯基、琥珀酰亚胺丙酸酯基、琥珀酰亚胺琥珀酸酯基、琥珀酰亚胺戊二酸酯基、琥珀酰亚胺葵二酸酯基的多臂聚乙二醇衍生物。
在一些实施例中，所述多臂包括四臂、六臂、八臂。
在一些实施例中，所述多臂聚乙二醇衍生物的数均分子量为10K-40K(即10000-40000)。
本文中，数均分子量10K意思是10000。
在一些实施例中，所述多臂聚乙二醇衍生物选自四臂聚乙二醇琥珀酰亚胺琥珀酸酯(10k)、四臂聚乙二醇琥珀酰亚胺琥珀酸酯(20k)、四臂聚乙二醇琥珀酰亚胺戊二酸酯(20k)、八臂聚乙二醇琥珀酰亚胺戊二酸酯(20k)、八臂聚乙二醇琥珀酰亚胺戊二酸酯(40k)。
在一些实施例中，将多臂聚乙二醇衍生物溶解于30-40℃(例如35℃)叔丁醇中，制成多臂聚乙二醇衍生物溶液。
在一些实施例中，以g/ml计，所述多臂聚乙二醇衍生物溶液中，多臂聚乙二醇衍生物与叔丁醇的重量体积比为1:(2-4)。研究发现，在该浓度范围内既可以保证有效的稳定性，又保证较快的溶解速度。
在一些实施例中，以g/ml计，所述混合物料中，所述多臂聚乙二醇衍生物与所述末端带氨基的化合物的重量比为1:(0.01-0.06)。
在一些实施例中，所述末端带氨基(-NH 2)的化合物选自多聚赖氨酸或多聚赖氨酸盐、羧甲基壳聚糖、壳聚糖中的至少一种或几种的组合；优选三赖氨酸醋酸盐。
在一些实施例中，是在所述多臂聚乙二醇衍生物溶液在30℃-40℃(例如35℃)的条件下，将所述末端带氨基(-NH 2)的化合物溶解于或超声分散于所述多臂聚乙二醇衍生物溶液中。
在一些实施例中，所述预冻的温度为-45℃、-40℃或-35℃。
在一些实施例中，在所述预冻前，事先将冻干机预冷至-45℃至-35℃范围内。
在一些实施例中，所述预冻的时间为100-150min，例如120min。
在一些实施例中，可将所述混合物料置于冻干瓶中进行干燥。
研究发现，通过预冻处理可以获得结构致密、表面光滑的小冰晶，有利于提高所述多臂聚乙二醇衍生物的稳定性。
在一些实施例中，所述第一干燥的温度为-15℃、-10℃或-5℃。
在一些实施例中，所述第一干燥的时间可为1200-1800min，例如1500min，具体时间可根据上述干燥所需的叔丁醇含量和含水量而确定。
在一些实施例中，所述第二干燥的温度为10℃或15℃。
在一些实施例中，所述第二干燥的时间可为240-720min，例如360min，具体时间可根据上述干燥所需的叔丁醇含量和含水量而确定。
在一些实施例中，所述第三干燥的温度为25℃或30℃。
在一些实施例中，所述第三干燥的时间可为60-600min，例如120min，具体时间可根据上述干燥所需的叔丁醇含量和含水量而确定。
研究发现，通过以上三阶段的干燥可以获得表面光滑致密、含水量低的冻干粉末，并有利于提高所述多臂聚乙二醇衍生物的稳定性。
在一些实施例中，在一些实施例中，所述预冻的温度为-45℃至-40℃， 所述第一干燥的温度为-15℃至-10℃。实验发现，在该条件下，更有利于提高聚乙二醇衍生物的稳定性，处理后的聚乙二醇衍生物在存放720d后其固化时间无明显下降。
在一些实施例中，上述制备方法还包括将经第三干燥所得物料进行包装的步骤，优选在水分含量≤25ppm(例如20ppm)、氧(即氧气)含量≤25ppm(例如20ppm)的环境中包装。这样可以进一步提高多臂聚乙二醇衍生物的稳定性，降低多臂聚乙二醇衍生物的氧化和水解。
在一些实施例中，上述制备方法包括：
将聚乙二醇衍生物于35℃溶解于叔丁醇中，制成多臂聚乙二醇衍生物溶液；以g/ml计，所述多臂聚乙二醇衍生物溶液中，多臂聚乙二醇衍生物与叔丁醇的重量体积比为1:(2-4)；
将所述末端带氨基的化合物于35℃溶解于或超声分散于所述多臂聚乙二醇衍生物溶液中，制成混合物料；所述混合物料中，所述多臂聚乙二醇衍生物与所述末端带氨基的化合物的重量比为1:(0.01-0.06)；
将所述混合物料于-45℃至-35℃预冻；
然后依次于-15℃至-5℃进行第一干燥，干燥至叔丁醇含量10wt％-15wt％，含水量0.5wt％-1wt％；于10℃-15℃进行第二干燥，干燥至叔丁醇含量2wt％-5wt％，含水量≤0.2wt％-0.5wt％；于25℃-30℃进行第三干燥，干燥至叔丁醇含量≤0.1wt％，含水量≤0.1wt％；
在水分含量≤25ppm、氧含量≤25ppm的环境中包装。
发明人发现，通过以上预冻及冻干处理可以较好的提高聚乙二醇衍生物的稳定性，处理后的聚乙二醇衍生物在存放720d后其固化时间无明显下降。
本发明还包括上述方法制备的多臂聚乙二醇衍生物复合物，其具有较好的稳定性。
本发明还包括上述方法制备的多臂聚乙二醇衍生物复合物在制备水凝胶中的应用。
本发明还提供一种原位水凝胶，其包括：
第一组分，为上述方法制备的多臂聚乙二醇衍生物复合物；
第二组分，为磷酸盐缓冲溶液；
第三组分，为缓冲盐溶液；选自硼砂-磷酸盐缓冲溶液或碳酸钠-磷酸盐缓冲溶液。
在一些实施例中，所述磷酸盐缓冲溶液的pH值为6.5-7.5，这样可以保持水凝胶整体的酸碱性为中性或接近中性范围内。
在一些实施例中，所述磷酸盐缓冲溶液可参照中国药典2015版四部8004项下pH＝7.3磷酸盐缓冲液进行配置。
在一些实施例中，考虑到硼砂可能存在毒性，所述第三组分优选不含硼砂的磷酸盐缓冲溶液。
在一些实施例中，所述第三组分即缓冲盐溶液pH值为9.5-10.0，这样可以保证凝胶的固化时间。
在一些实施例中，所述硼砂-磷酸盐缓冲溶液或碳酸钠-磷酸盐缓冲溶液可分别用磷酸盐缓冲溶液(参见上文)和硼砂或碳酸钠进行配制，调节至所需的pH。
在一些实施例中，用0.1M碳酸钠溶液和磷酸盐缓冲液(参见上文，例如pH＝7.3)配制碳酸钠-磷酸盐缓冲溶液。
本发明原位载药水凝胶可在临用前进行配置，具体可将第一组分用第二组分溶解，然后再与第三组分混合即得原位水凝胶。
本发明还提供一种原位载药水凝胶，其包括：
第一组分，为上述方法制备的多臂聚乙二醇衍生物复合物；
第二组分，为磷酸盐缓冲溶液；
第三组分，为缓冲盐溶液；选自硼砂-磷酸盐缓冲溶液或碳酸钠-磷酸盐缓冲溶液；
第四组分，为药物。
在一些实施例中，所述第二组分磷酸盐缓冲溶液及第三组分为缓冲盐 溶液与上文相同。
在一些实施例中，所述药物可选自眼科常用药，包括但不限于：抗炎药物(环孢素、红霉素、莫西沙星等)、去水肿药物(地塞米松、环糊精)、保湿(透明质酸、HPMC)、青光眼(盐酸卡替洛尔、奈舒地尔、曲伏前列素、贝美前列素、他氟前列素)、非甾体抗炎药等。
本发明原位载药水凝胶可在临用前进行配置，具体可将第一组分用第二组分溶解，将第四组分用第三组分溶解，然后将所得料液混合，即得原位载药水凝胶。
令人惊喜的是，本发明还克服了传统水凝胶不能载药的缺陷，采用上述方法制备的多臂聚乙二醇衍生物复合物能够负载药物尤其是眼科用药，除实现封闭、修复、填充外，还能达到治疗效果。
此外，本发明还可以通过调整两种缓冲盐溶液(即第二组分和第三组分)的比例实现不同的固化时间控制。
在一些实施例中，可将第一组分用第二组分磷酸盐缓冲溶液溶解，制成浓度为50-300mg/ml的料液。
在一些实施例中，可将第四组分用第三组分缓冲盐溶液溶解，制成浓度为6-200mg/ml的料液。
在一些实施例中，第一组分在所述原位载药水凝胶中的含量为2.5wt％-20wt％。
在一些实施例中，所述药物在所述原位载药水凝胶中的含量为0.02wt％-10wt％。
在一些实施例中，所述原位载药水凝胶的组分还包括包括第五组分即显色剂，可选自非偶氮类着色剂。当含有显色剂时，可将显色剂用第二组分或第三组分溶解，然后再与其他料液混合。在一些实施例中，显色剂在所述原位载药水凝胶中的含量为0.01wt％-0.1wt％。
本发明还提供上述原位载药水凝胶的制备方法，包括：
将第一组分用第二组分(即磷酸盐缓冲溶液)溶解，制成第一料液；
将第四组分用第三组分(即缓冲盐溶液)溶解，制成第二料液；
然后将第一料液和第二料液混合，制成原位载药水凝胶。
本发明通过对多臂聚乙二醇衍生物和含有氨基的化合物进行共同冻干处理，不仅可以提高其稳定性，满足快速固化的需要，而且可以提高其溶解速度，缩短操作时间，实现组织不同部位的可控时间固化和不同药物的负载，实现多种适应症的封闭、修复、填充和治疗。
本发明原位水凝胶、原位载药水凝胶可用于组织创面封闭、修复、填充或治疗。在临用前进行配置，在形成凝胶之前(例如可通过配件)将载药水凝胶用于组织相应部位，在满足组织封闭、修复、填充的同时，起到抗炎、抗感染、去水肿、保湿等治疗效果。该水凝胶可原位成型，第一组分溶解时间＜20s，根据不同组织应用部位可控在0-10s内形成凝胶，凝胶可通过水解降解。
附图说明
图1为本发明实验例中兔眼封闭效果的照片。
图2为本发明实验例中药物缓释曲线。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
实施例1
本实施例提供一种多臂聚乙二醇衍生物复合物的制备方法，包括：
将四臂聚乙二醇琥珀酰亚胺琥珀酸酯(10k)于35℃溶解于叔丁醇中，制成四臂聚乙二醇琥珀酰亚胺琥珀酸酯溶液；其中，四臂聚乙二醇琥珀酰亚胺琥珀酸酯与叔丁醇的比例为1g:2ml；
将三赖氨酸醋酸盐于35℃超声分散于四臂聚乙二醇琥珀酰亚胺琥珀酸酯溶液中，混合均匀，制成混合物料；其中，所述混合物料中，四臂聚 乙二醇琥珀酰亚胺琥珀酸酯与三赖氨酸醋酸盐的重量比例为1:0.03；
将所述混合原料置于冻干瓶中；将冻干机预冷至-45℃至-35℃，然后将上述冻干瓶置于冻干机隔板上，预冻120min后于-15℃至-5℃进行第一干燥，干燥时间为1500min，然后分别在15℃进行第二干燥，干燥时间为360min、在30℃进行第三干燥，干燥时间为120min；
将冻干后的产品在水分含量20ppm、氧含量20ppm的环境中包装。
通过控制预冷温度、预冻温度以及第一干燥温度，分别制备样品1-3，具体见下表：
样品 预冷温度，℃ 预冻温度，℃ 预冻时间，min 第一干燥温度
1 -45 -45 120 -15
2 -35 -35 120 -15
3 -35 -35 120 -5
实施例2
本实施例提供一种多臂聚乙二醇衍生物复合物的制备方法，包括：
将多臂聚乙二醇衍生物于35℃溶解于叔丁醇中，制成多臂聚乙二醇衍生物溶液；其中，多臂聚乙二醇衍生物与叔丁醇的比例为1g:4ml；
将末端带氨基的化合物于35℃超声分散于多臂聚乙二醇衍生物溶液中，混合均匀，制成混合物料；
将所述混合原料置于冻干瓶中；将冻干机预冷至-45℃，然后将上述冻干瓶置于冻干机隔板上，预冻120min后于-15℃第一干燥1500min，然后分别在15℃第二干燥360min、30℃第三干燥120min；
将冻干后的产品在水分含量20ppm、氧含量20ppm的环境中包装。
通过选择不同的多臂聚乙二醇衍生物、末端带氨基的化合物，以及控制多臂聚乙二醇衍生物与末端带氨基的化合物的重量比，分别制备样品4-9，具体见下表：
Figure PCTCN2022084316-appb-000001
Figure PCTCN2022084316-appb-000002
实施例3
本实施例提供多臂聚乙二醇衍生物复合物的制备方法，包括：
将四臂聚乙二醇琥珀酰亚胺戊二酸酯(10k)于35℃溶解于叔丁醇中，制成四臂聚乙二醇琥珀酰亚胺戊二酸酯溶液；其中，四臂聚乙二醇琥珀酰亚胺戊二酸酯与叔丁醇的比例为1g:2ml；
将末端带氨基的化合物于35℃超声分散于四臂聚乙二醇琥珀酰亚胺戊二酸酯溶液中，混合均匀，制成混合物料；其中，所述混合物料中，四臂聚乙二醇琥珀酰亚胺戊二酸酯与三赖氨酸醋酸盐的重量比为1:0.03；
将所述混合原料置于冻干瓶中；将冻干机预冷至-45℃，然后将上述冻干瓶置于冻干机隔板上，预冻120min后于-15℃第一干燥1500min，然后分别在15℃第二干燥360min、30℃第三干燥120min；
将冻干后的产品在水分含量20ppm、氧含量20ppm的环境中包装，制成样品10。
实施例4
与实施例1的区别仅在于，将冻干后的产品在相对湿度50％普通环境包装，制成样品11。
对比例1
与实施例1的区别仅在于，通过控制预冷温度、预冻温度、降温速率以及第一干燥温度，分别制备样品12-15，具体见下表：
样品 预冷温度，℃ 预冻温度，℃ 降温速率，℃/min 预冻时间，min 第一干燥温度
12 -35 -35 / 120 0
13 -25 -45 1 120 -15
14 室温 -45 1 120 -15
15 室温 -45 0.5 120 -15
注：降温速率是指冻干机从预冷温度降至预冻温度的速率。
对比例2
将四臂聚乙二醇琥珀酰亚胺琥珀酸酯(10k)于35℃溶解于叔丁醇中，制成四臂聚乙二醇琥珀酰亚胺琥珀酸酯溶液；
将三赖氨酸醋酸盐于35℃超声分散于四臂聚乙二醇琥珀酰亚胺琥珀酸酯溶液中，混合均匀，制成混合物料；其中，所述混合物料中，四臂聚乙二醇琥珀酰亚胺琥珀酸酯与三赖氨酸醋酸盐的重量比为1:0.03；
将所述混合原料置于冻干瓶中；将冻干机预冷至-45℃，然后将上述冻干瓶置于冻干机隔板上，预冻120min后于-15℃至-5℃进行第一干燥，干燥时间为1500min，然后分别在15℃进行第二干燥，干燥时间为360min、在30℃进行第三干燥，干燥时间为120min；
将冻干后的产品在水分含量20ppm、氧含量20ppm的环境中包装。
通过控制四臂聚乙二醇琥珀酰亚胺琥珀酸酯与叔丁醇的比例，制成样品16-17，具体见下表：
Figure PCTCN2022084316-appb-000003
对比例3
将四臂聚乙二醇琥珀酰亚胺琥珀酸酯(10k)于35℃溶解于叔丁醇中，制成溶液；其中，四臂聚乙二醇琥珀酰亚胺琥珀酸酯与叔丁醇的比例为1g:2ml；将制成的溶液置于冻干瓶A中。
将三赖氨酸盐于35℃分散于叔丁醇中，制成混合物料；其中，三赖氨酸盐与叔丁醇的重量比为0.03g:2ml；将制成的混合物料置于冻干瓶B中。
将冻干机预冷至-45℃，然后将上述冻干瓶A和B分别置于冻干机隔板上，-45℃预冻120min后，于-15℃干燥1500min，然后分别在15℃干燥360min、30℃干燥120min。
然后按照四臂聚乙二醇琥珀酰亚胺琥珀酸酯与三赖氨酸醋酸盐重量比为1:0.03，在水分含量20ppm、氧含量20ppm的环境中混合、包装，制成样品18。
对比例4
将四臂聚乙二醇琥珀酰亚胺琥珀酸酯(10k)与抗氧剂2,6-二叔丁基-4-甲基苯酚(BHT)混合，干燥至含水量＜0.1％。然后与三赖氨酸醋酸盐在水分含量20ppm、氧含量20ppm的环境中混合，制成样品19。其中，四臂聚乙二醇琥珀酰亚胺琥珀酸酯与三赖氨酸醋酸盐重量比为1:0.03，样品19中抗氧剂BHT含量为0.05wt％。
实验例1 多臂聚乙二醇衍生物的稳定性研究
将上述实施例和对比例样品包装后存放于2-8℃的稳定性试验箱中，720d后测试其溶解和固化时间。
测试方法：
将以上样品分别加pH值7.5的磷酸盐缓冲溶液，振摇至全部溶解，配成浓度200mg/ml的溶液M；另取与所用磷酸盐缓冲溶液等量体积的pH值为10.0的碳酸钠-磷酸盐缓冲溶液，将溶液M和碳酸钠-磷酸盐缓冲溶液混合后形成凝胶，记录其固化时间。
实验结果见下表：
样品 溶解时间，s 固化时间，s 样品 溶解时间，s 固化时间，s
1 16 0-1 11 14 不成胶
2 15 4 12 19 7
3 16 5 13 20 8
4 15 2 14 21 15
5 13 6 15 23 18
6 15 5 16 21 4
7 14 7 17 12 11
8 12 8 18 15 52
9 15 5 19 35 5
10 16 2      
实验例2
按实施例1方法制备样品1。将样品1加pH值7.5的磷酸盐缓冲溶液，振摇至全部溶解，配成浓度50-300mg/ml的溶液N。另取与适量体积的pH值为10.0的碳酸钠-磷酸盐缓冲溶液，将溶液N和碳酸钠-磷酸盐缓冲溶液混合后形成凝胶，编号为A-E，记录其固化时间。
Figure PCTCN2022084316-appb-000004
实验例3 生物相容性结果
按实施例2方法制备样品6。
取四臂聚乙二醇琥珀酰亚胺戊二酸酯(20k，与样品6的原料相同)，未经冻干等处理，作为对照样品。
参照GB 16886系列标准对以上样品进行生物学评价，结果如下表。
材料生物相容性结果
Figure PCTCN2022084316-appb-000005
Figure PCTCN2022084316-appb-000006
实验例4 原位载药水凝胶及其应用实验
按实施例1方法制备样品1。
将600mg样品1加4ml的pH值7.5的磷酸盐缓冲溶液，振摇至全部溶解，配成溶液X。另取10mg的地塞米松，用2ml的pH值为10.0的碳酸钠-磷酸盐缓冲溶液溶解，得溶液Y，将溶液X和溶液Y混合后形成凝胶。
动物实验：选择兔眼模型，于兔眼角膜行长约10mm(3-9点位)的横向切口，采用涂抹器将凝胶涂抹至创面部位使切口封闭。术后7天观察术后切口封闭效果和局部炎症发生情况，结果表明：切口损伤可见愈合趋势，炎症反应逐渐恢复至最终完全恢复。兔眼封闭效果见图1。
实验例5 原位载药水凝胶及其缓释性能测试
按实施例2方法制备样品6。
将样品6加pH值7.5的磷酸盐缓冲溶液，振摇至全部溶解，配成浓度300mg/ml的溶液X；
另取地塞米松，用pH值为10.0的碳酸钠-磷酸盐缓冲溶液溶解，得溶液Y，将溶液X和溶液Y混合后形成凝胶。该凝胶中地塞米松的含量为5wt％。
药物缓释的检测方法：取载药固化的水凝胶，按照水凝胶与释放介质体积的比例为1g:50ml，将样品置37℃±1℃水浴振荡器中，依法操作，分别在1h、2h、6h、1d、3d、6d、9d、12d、15d、18d、21d、24d、27d、30d时分别取样1.0ml，并补充同等体积的新鲜溶液，取样溶液滤过，取 续滤液作为供试品溶液，按照中国药典2015版二部地塞米松项下含量检测规定的方法进行释放量检测。
药物缓释曲线见图2，结果表明水凝胶对地塞米松有较长的缓释效果。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种原位载药水凝胶及其制备方法与应用。多臂聚乙二醇衍生物复合物的制备方法，包括将多臂聚乙二醇衍生物溶液预冻，然后依次进行三阶段的干燥。原位载药水凝胶包括多臂聚乙二醇衍生物复合物，磷酸盐缓冲溶液，缓冲盐溶液和药物。本发明原位载药水凝胶可用于组织创面封闭、修复、填充或治疗，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种多臂聚乙二醇衍生物复合物的制备方法，其包括：
    将多臂聚乙二醇衍生物溶解于叔丁醇中，制成多臂聚乙二醇衍生物溶液；
    向所述多臂聚乙二醇衍生物溶液中加入末端带氨基的化合物，混合均匀，制成混合物料；于-45℃至-35℃预冻；
    然后依次于-15℃至-5℃进行第一干燥，干燥至叔丁醇含量10wt％-15wt％，含水量0.5wt％-1wt％；于10℃-15℃进行第二干燥，干燥至叔丁醇含量2wt％-5wt％，含水量≤0.2wt％-0.5wt％；于25℃-30℃进行第三干燥，干燥至叔丁醇含量≤0.1wt％，含水量≤0.1wt％。
  2. 根据权利要求1所述多臂聚乙二醇衍生物复合物的制备方法，其中，所述多臂聚乙二醇衍生物选自含有琥珀酰亚胺碳酸酯基、琥珀酰亚胺乙酸酯基、琥珀酰亚胺丙酸酯基、琥珀酰亚胺琥珀酸酯基、琥珀酰亚胺戊二酸酯基、琥珀酰亚胺葵二酸酯基的多臂聚乙二醇衍生物；和/或，
    所述末端带氨基的化合物选自多聚赖氨酸或多聚赖氨酸盐、羧甲基壳聚糖、壳聚糖中的至少一种或几种的组合；优选三赖氨酸醋酸盐。
  3. 根据权利要求1或2所述多臂聚乙二醇衍生物复合物的制备方法，其中，
    以g/ml计，所述多臂聚乙二醇衍生物溶液中，多臂聚乙二醇衍生物与叔丁醇的重量体积比为1:(2-4)；和/或，
    以g/ml计，所述混合物料中，所述多臂聚乙二醇衍生物与所述末端带氨基的化合物的重量比为1:(0.01-0.06)。
  4. 根据权利要求1-3任一项所述多臂聚乙二醇衍生物复合物的制备方法，其中，所述预冻的时间为100-150min；和/或，
    所述第一干燥的时间为1200-1800min；和/或，
    所述第二干燥的时间为240-720min；和/或，
    所述第三干燥的时间为60-600min。
  5. 根据权利要求1-4任一项所述多臂聚乙二醇衍生物复合物的制备方法，还包括将经第三干燥所得物料进行包装的步骤，优选在水分含量≤25ppm、氧含量≤25ppm的环境中包装。
  6. 权利要求1-5任一项所述方法制备的多臂聚乙二醇衍生物复合物。
  7. 权利要求6所述多臂聚乙二醇衍生物复合物在制备水凝胶中的应用。
  8. 一种原位水凝胶，其包括：
    第一组分，为权利要求6所述多臂聚乙二醇衍生物复合物；
    第二组分，为磷酸盐缓冲溶液；pH值优选为6.5-7.5；
    第三组分，为缓冲盐溶液；选自硼砂-磷酸盐缓冲溶液或碳酸钠-磷酸盐缓冲溶液；所述缓冲盐溶液pH值优选为9.5-10.0。
  9. 一种原位载药水凝胶，其包括：
    第一组分，为权利要求6所述多臂聚乙二醇衍生物复合物；
    第二组分，为磷酸盐缓冲溶液；pH值优选为6.5-7.5；
    第三组分，为缓冲盐溶液；选自硼砂-磷酸盐缓冲溶液或碳酸钠-磷酸盐缓冲溶液；所述缓冲盐溶液pH值优选为9.5-10.0；
    第四组分，为药物。
  10. 权利要求9所述原位载药水凝胶的制备方法，其包括：
    将所述第一组分用所述第二组分溶解，制成第一料液；
    将所述第四组分用所述第三组分溶解，制成第二料液；
    然后将所述第一料液和第二料液混合，制成原位载药水凝胶。
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