WO2023035719A1 - 一种聚乙二醇化类胶原蛋白及其制备方法和应用 - Google Patents

一种聚乙二醇化类胶原蛋白及其制备方法和应用 Download PDF

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WO2023035719A1
WO2023035719A1 PCT/CN2022/099673 CN2022099673W WO2023035719A1 WO 2023035719 A1 WO2023035719 A1 WO 2023035719A1 CN 2022099673 W CN2022099673 W CN 2022099673W WO 2023035719 A1 WO2023035719 A1 WO 2023035719A1
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collagen
protein
pegylated
peg
freeze
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PCT/CN2022/099673
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English (en)
French (fr)
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张加慧
王崇宇
刘沐荣
张军
何超先
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熹微(苏州)生物医药科技有限公司
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Publication of WO2023035719A1 publication Critical patent/WO2023035719A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea

Definitions

  • the application relates to a pegylated collagen-like protein and its preparation method and application, belonging to the field of biotechnology.
  • the cornea is the convex, highly transparent substance at the front of the eye. It is in the shape of a transverse ellipse, covering the iris, pupil and anterior chamber, and provides most of the refractive power for the eye. Coupled with the refractive power of the lens, the light can be accurately focused on the retina to form an image.
  • the cornea has very sensitive nerve endings. If something foreign comes into contact with the cornea, the eyelids will involuntarily close to protect the eyes. In order to maintain transparency, the cornea does not have blood vessels, and obtains nutrients and oxygen through tears and aqueous humor.
  • the cornea is very fragile, eye trauma, inflammation, allergic reaction, physical damage, chemical burns, strenuous exercise, overuse of the eyes, etc. can lead to corneal lesions. Once the cornea is damaged, it will lead to obvious ocular symptoms, such as eye pain, photophobia, tearing, vision loss, etc., and even blindness in severe cases.
  • Corneal transplantation is to replace the patient's existing diseased cornea with a normal cornea, so as to restore the eyesight of the affected eye or control the corneal disease, so as to improve vision or treat certain corneal diseases.
  • Some corneal diseases that cause severe visual impairment or even blindness in patients can be completely cured through corneal transplantation, helping these unfortunate patients stay away from pain.
  • the cornea itself does not contain blood vessels, it is in the status of "immune amnesty", which makes the success rate of corneal transplantation rank first among other allogeneic organ transplants.
  • Artificial keratoplasty is a special optical device made of transparent medical polymer material, which is implanted into corneal tissue through surgery to replace part of corneal scar tissue and restore vision. Since the rejection of corneal tissue to artificial synthetic materials has not been resolved, the long-term effect is not good, often causing leakage of aqueous humor at the transplant site and shedding of the graft, so it is not yet widely used.
  • artificial corneas are only suitable for blindness after suffering from various severe corneal diseases, especially those who have all corneal leukoplakia caused by severe chemical burns and failed multiple keratoplasty operations, and cannot perform other operations.
  • bionic cornea On the basis of the artificial cornea, some researchers have proposed a bionic cornea.
  • a bionic cornea For example, in the document "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants", Jangamreddy, Jaganmohan R. et al proposed a PEGylated collagen-like protein, which was chemically By cross-linking, a bionic cornea can be obtained.
  • the bionic cornea has excellent biocompatibility and can effectively solve the problems of strong rejection of corneal tissue to artificial synthetic materials.
  • this bionic cornea is very low, only 0.022 MPa. If this bionic cornea is used for corneal transplantation, there are still problems such as difficult transplantation and easy corneal cones.
  • the application provides a PEGylated collagen-like protein
  • the PEGylated collagen-like protein includes collagen-like protein and polyethylene glycol modified on the collagen-like protein Derivatives; the polyethylene glycol derivatives include PEG-40k and PEG-20k.
  • the polyethylene glycol derivative consists of PEG-40k and PEG-20k.
  • the amino acid sequence of the collagen-like protein is shown in SEQ ID NO.1.
  • X is 4Hyp (4-hydroxyproline), namely:
  • amino acid sequence of SEQ ID NO.1 is:
  • the molar ratio of PEG-40k to PEG-20k is 0.5-6:1.
  • the molar ratio of PEG-40k to PEG-20k is 2-3:1.
  • the number of activating groups of the PEG-40k is one or more of 8-arm, 4-arm, 2-arm or 1-arm; the PEG-20k The number of activated groups is one or more of 8-arm, 4-arm, 2-arm or 1-arm.
  • the number of activating groups of the PEG-40k is 4-arm or 8-arm; the number of activating groups of the PEG-20k is 4-arm or 8-arm.
  • the activating group of the PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC; the PEG- The activating group of 20k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC.
  • the activating group of the PEG-40k is -MAL; the activating group of the PEG-20k is -MAL.
  • a linker is connected to one end of the collagen-like protein; the polyethylene glycol derivative is modified on the linker by an activation group.
  • the linker is connected to the N-terminus of the collagen-like protein.
  • amino acid sequence of the linker is shown in SEQ ID NO.2 or SEQ ID NO.3.
  • the modification site of the polyethylene glycol derivative on the linker is one or more of thiol, amino, carboxyl or imidazole.
  • the modification site of the polyethylene glycol derivative on the linker is a sulfhydryl group
  • the amino acid sequence of the linker is as shown in SEQ ID NO.2
  • the modified site of the polyethylene glycol derivative on the linker is an amino group
  • the core conformation of the PEGylated collagen-like protein is one or more of HG or TP.
  • the core conformation of the polyethylene glycol derivative in the PEGylated collagen-like protein is TP.
  • the amino acid configuration of the collagen-like protein in the PEGylated collagen-like protein is one or more of D-type or L-type.
  • the molecular weight of the PEGylated collagen-like protein is 15000-75000 Da.
  • the molecular weight of the PEGylated collagen-like protein is 30000-75000 Da.
  • the present application also provides a freeze-dried preparation, which is obtained by freeze-drying the above-mentioned pegylated collagen-like protein.
  • the freeze-dried preparation is obtained by mixing the above-mentioned pegylated collagen-like protein with a freeze-drying protective agent and then freeze-drying.
  • the lyoprotectant is one or more of mannitol, sucrose or alanine.
  • the present application also provides a method for preparing the above-mentioned PEGylated collagen, said method comprising the steps of:
  • Reaction steps react collagen-like protein and polyethylene glycol derivatives at a pH of 4.0-10.0 and a temperature of 2-40°C for 1-48 hours to obtain a reaction product; the reaction product contains the above-mentioned PEGylated Collagen-like.
  • the reaction step is: reacting the collagen-like protein and the polyethylene glycol derivative at a pH of 6.0-8.0 and a temperature of 2-8° C. for 5-8 hours.
  • the molar ratio of the collagen-like protein and the polyethylene glycol derivative is 1-16:1.
  • the molar ratio of the collagen-like protein and the polyethylene glycol derivative is 8-12:1.
  • the reaction solvent of the collagen-like protein and the polyethylene glycol derivative is water or dilute hydrochloric acid solution.
  • the concentration of the dilute hydrochloric acid solution is 1-10 mmol/L; the pH of the dilute hydrochloric acid solution is adjusted to 6.0-8.0 with a dilute alkaline solution.
  • the dilute alkali solution is sodium hydroxide solution or ammonia water with a pH of 9.0-11.0.
  • the feeding concentration of the collagen-like protein in the reaction solvent is 1-15 mg/mL.
  • the feeding concentration of the collagen-like protein in the reaction solvent is 8-10 mg/mL.
  • the method further includes a purification step; the purification step is: to obtain pegylated collagen-like proteins by filtering and intercepting substances with a molecular weight greater than or equal to 30,000 Da in the reaction product.
  • the filtration is dialysis or ultrafiltration.
  • the present application also provides a method for preparing the above-mentioned lyophilized preparation, the method comprising the following steps:
  • Reaction steps react collagen-like protein and polyethylene glycol derivatives at a pH of 4.0-10.0 and a temperature of 2-40°C for 1-48 hours to obtain a reaction product; the reaction product contains the above-mentioned PEGylated Collagen-like protein;
  • Freeze-drying step freeze-dry the reaction product to obtain a freeze-dried preparation.
  • the reaction step is: reacting the collagen-like protein and the polyethylene glycol derivative at a pH of 6.0-8.0 and a temperature of 2-8° C. for 5-8 hours.
  • the molar ratio of the collagen-like protein and the polyethylene glycol derivative is 1-16:1.
  • the molar ratio of the collagen-like protein and the polyethylene glycol derivative is 8-12:1.
  • the reaction solvent of the collagen-like protein and the polyethylene glycol derivative is water or dilute hydrochloric acid solution.
  • the concentration of the dilute hydrochloric acid solution is 1-10 mmol/L; the pH of the dilute hydrochloric acid solution is adjusted to 6.0-8.0 with a dilute alkaline solution.
  • the dilute alkali solution is sodium hydroxide solution or ammonia water with a pH of 9.0-11.0.
  • the feeding concentration of the collagen-like protein in the reaction solvent is 1-15 mg/mL.
  • the feeding concentration of the collagen-like protein in the reaction solvent is 8-10 mg/mL.
  • the freeze-drying step is: mixing the reaction product and the freeze-drying protection agent and then freeze-drying to obtain a freeze-dried preparation.
  • the lyoprotectant is one or more of mannitol, sucrose or alanine.
  • the lyophilization includes the following stages:
  • Stage 1 Freeze-drying for 6 hours at a temperature of -45°C and a vacuum of 500m Torr;
  • Stage 2 Freeze-drying for 17 hours at a temperature of -30°C and a vacuum of 100 m Torr;
  • Stage 3 Freeze-drying for 7 hours at a temperature of 25° C. and a vacuum of 100 m Torr.
  • the method further includes a purification step; the purification step is: by filtering and intercepting substances with a molecular weight greater than or equal to 30000Da in the reaction product to obtain polyethylene glycol Alcoholated Collagen.
  • the filtration is dialysis or ultrafiltration.
  • the application also provides the application of the above-mentioned PEGylated collagen-like protein or the above-mentioned lyophilized preparation or the PEGylated collagen-like protein prepared by the above-mentioned method or the lyophilized preparation prepared by the above-mentioned method in the preparation of bionic or regenerative biomaterials .
  • the biomimetic or regenerative biomaterial is a biomimetic cornea.
  • the application provides a PEGylated collagen-like protein
  • the PEGylated collagen-like protein comprises a collagen-like protein and PEG-40k and PEG-20k modified on the collagen-like protein; PEG-40k and PEG
  • the modification of -20k can significantly improve the cross-linking performance of the collagen-like protein, so that the strength of the cornea made of the PEGylated collagen-like protein of the present application can be increased by nearly 20 times compared with the strength of the cornea made of the collagen-like protein, which is higher than that of using
  • the strength of corneas made of other PEGylated collagens is nearly 12 times higher (corneas made with PEGylated collagens in the literature "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants" Strength is only 0.022MPa), therefore, the pegylated collagen-like protein has a very high application prospect in the preparation of bionic or regenerative biomaterials such as bionic cornea.
  • the molar ratio of PEG-40k to PEG-20k is 2-3:1; PEG modification at this molar ratio has a better effect of improving the cross-linking performance of collagen-like proteins.
  • the PEG-40k is 8-arm-PEG-40k-MAL
  • the PEG-20k is 4-arm-PEG-20k-MAL; the PEG modification under this formula improves the effect of collagen-like cross-linking properties better.
  • the application provides a freeze-dried preparation, which is obtained by mixing the above-mentioned pegylated collagen and a freeze-drying protection agent; the freeze-drying protection agent can protect the above-mentioned poly
  • the freeze-drying protection agent can protect the above-mentioned poly
  • the pegylated collagen-like protein is protected so that the structure of the above-mentioned pegylated collagen-like protein will not be destroyed during the freeze-drying process, so that the strength of the cornea made by using the freeze-dried preparation is further improved.
  • the application provides a method for preparing the above-mentioned PEGylated collagen-like protein.
  • the method uses dilute hydrochloric acid solution with a concentration of 1-10 mmol/L and a pH of 6.0-8.0 as the reaction solvent, and the collagen-like protein and PEG in React at 2-8°C for 5-8 hours to prepare PEGylated collagen; the PEGylated collagen produced under this reaction condition has better cross-linking performance, and is different from other existing PEGylated collagens.
  • the reaction time of the preparation method of the present application is shorter, and only need to react for 5-8 hours to obtain the PEGylated collagen-like protein that can be made into hydrogel to form cornea (document "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants”, the preparation of PEGylated collagen that can be made into a hydrogel to form a cornea requires 4 weeks of reaction).
  • the present application provides a preparation method for the above-mentioned lyophilized preparation, the method is to lyophilize the above-mentioned PEGylated collagen under the protection of a lyoprotectant; the lyoprotectant can protect the above-mentioned PEGylated collagen Collagen is protected so that the structure of the above-mentioned PEGylated collagen-like protein will not be destroyed during the freeze-drying process, thereby making the cross-linking performance of the above-mentioned freeze-dried preparation better.
  • Example 1-1 A PEGylated collagen-like protein and its preparation
  • This embodiment provides a PEGylated collagen-like protein
  • the PEGylated collagen-like protein consists of a collagen-like protein with an amino acid sequence as shown in SEQ ID NO.1, and the amino acid sequence connected to the N-terminal of the collagen-like protein is as follows
  • the linker shown in SEQ ID NO.2 and the composition of 8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL modified on the thiol of the linker; wherein, 8-arm-PEG-40k-MAL
  • the molar ratio of PEG and 4-arm-PEG-20k-MAL is 2:1
  • the core conformation of the polyethylene glycol derivative in the PEGylated collagen is TP
  • the molecular weight of the PEGylated collagen is 30000 ⁇ 75000 Da.
  • This PEGylated collagen-like protein was named PEGylated collagen-like protein 1.
  • the preparation method of the above-mentioned PEGylated collagen-like protein 1 comprises the following steps:
  • the collagenoid and polyethylene glycol derivatives are dissolved in a dilute hydrochloric acid solution with a concentration of 5mmol/L (the pH of the dilute hydrochloric acid solution is adjusted to 6.5 with a sodium hydroxide solution of pH 11.0), so that in the dilute hydrochloric acid solution,
  • the molar concentrations of collagen-like protein and polyethylene glycol derivatives were 2.4mmol/L and 0.3mmol/L respectively, and the reaction system was obtained; the reaction system was reacted for 12h under the conditions of pH 6.5 and temperature 5°C, and the reaction product was obtained ;
  • the reaction product is subjected to ultrafiltration at 5° C. with an ultrafiltration membrane with a pore size of 30,000 Da, and substances with a molecular weight greater than or equal to 30,000 Da in the reaction product are intercepted to obtain PEGylated collagen-like protein 1.
  • Example 2-1 A freeze-dried preparation and its preparation
  • This embodiment provides a freeze-dried preparation, which is composed of the pegylated collagen-like protein 1 prepared in Example 1-1 and mannitol.
  • This lyophilized formulation was designated as lyophilized formulation 1.
  • the preparation method of the above-mentioned freeze-dried preparation 1 comprises the following steps:
  • the PEGylated collagen-like protein and mannitol prepared in Example 1 were mixed according to the ratio of mass ratio 1:5 to obtain a lyophilized system; the lyophilized system was lyophilized to obtain a lyophilized preparation 1;
  • lyophilization comprises the following stages:
  • Stage 1 Freeze-drying for 6 hours at a temperature of -45°C and a vacuum of 500m Torr;
  • Stage 2 Freeze-drying for 17 hours at a temperature of -30°C and a vacuum of 100 m Torr;
  • Stage 3 Freeze-drying for 7 hours at a temperature of 25° C. and a vacuum of 100 m Torr.
  • Example 1-2 A PEGylated collagen-like protein and its preparation
  • This embodiment provides a PEGylated collagen-like protein.
  • the PEG formula (mol 8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL with a ratio of 2:1) are replaced by:
  • the PEGylated collagen-like proteins were named as PEGylated collagen-like proteins 2-9 in sequence.
  • the preparation method of the above PEGylated collagen-like proteins 2-9 is the same as that of PEGylated collagen-like protein 1.
  • Embodiment 2-2 A kind of lyophilized preparation and its preparation
  • This example provides a freeze-dried preparation, which is prepared on the basis of the freeze-dried preparation 1 in Example 2-1, by substituting pegylated collagen-like protein 1 with Example 1-2 respectively. PEGylated collagen-like proteins 2-9.
  • freeze-dried preparations were named as freeze-dried preparations 2-9 in sequence.
  • the preparation method of the above-mentioned lyophilized preparations 2-9 is the same as that of the lyophilized preparation 1.
  • Embodiment 1-3 A kind of PEGylated collagen-like protein and its preparation
  • This embodiment provides a PEGylated collagen-like protein
  • the PEGylated collagen-like protein consists of a collagen-like protein with an amino acid sequence as shown in SEQ ID NO.1, and the amino acid sequence connected to the N-terminal of the collagen-like protein is as follows
  • the linker shown in SEQ ID NO.3 and the composition of 8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL modified on the linker amino group; wherein, 8-arm-PEG-40k-MAL
  • the molar ratio of PEG and 4-arm-PEG-20k-MAL is 2:1
  • the core conformation of the polyethylene glycol derivative in the PEGylated collagen is TP
  • the molecular weight of the PEGylated collagen is 30000 ⁇ 75000 Da.
  • This PEGylated collagen-like protein was named PEGylated collagen-like protein 10.
  • the preparation method of the PEGylated collagen-like protein 10 is the same as that of the PEGylated collagen-like protein 1.
  • Embodiment 2-3 A kind of lyophilized preparation and its preparation
  • This example provides a freeze-dried preparation, which is based on the freeze-dried preparation 1 of Example 2-1, and replaces the pegylated collagen-like protein 1 with the one prepared in Example 1-3. PEGylated Collagen-like 10.
  • lyophilized preparations were named as lyophilized preparation 10 in turn.
  • the preparation method of the above-mentioned lyophilized preparation 10 is the same as that of the lyophilized preparation 1.
  • Embodiment 1-4 A kind of PEGylated collagen-like protein and its preparation
  • This embodiment provides a PEGylated collagen-like protein, on the basis of the PEGylated collagen-like protein 1 in Example 1-1, the temperature in the preparation method ( 5°C) were replaced by: 2°C, 8°C, 25°C, 37°C, respectively.
  • the PEGylated collagen-like proteins were named as PEGylated collagen-like proteins 11-14 in sequence.
  • Embodiment 2-4 A kind of lyophilized preparation and its preparation
  • This example provides a freeze-dried preparation, which is prepared on the basis of the freeze-dried preparation 1 in Example 2-1, by substituting pegylated collagen-like protein 1 with Examples 1-4, respectively. PEGylated collagen-like proteins 11-14.
  • freeze-dried preparations were named as freeze-dried preparations 11-14 in sequence.
  • the preparation method of the above-mentioned lyophilized preparations 11-14 is the same as that of the lyophilized preparation 1.
  • Embodiment 1-5 A kind of PEGylated collagen-like protein and its preparation
  • This embodiment provides a PEGylated collagen-like protein.
  • the pH ( 6.5) are replaced by: pH 2.5, pH 4.5, pH 6.0, pH 6.5, pH 7.0, pH 8.0, pH 8.5, pH 10.5.
  • PEGylated collagen-like proteins were sequentially named as PEGylated collagen-like proteins 15-22.
  • Embodiment 2-5 A kind of lyophilized preparation and its preparation
  • This example provides a freeze-dried preparation, which is prepared on the basis of the freeze-dried preparation 1 in Example 2-1, by substituting pegylated collagen-like protein 1 with Examples 1-5, respectively. PEGylated collagen 15-22.
  • freeze-dried preparations were named as freeze-dried preparations 15-22 in sequence.
  • the preparation method of the above-mentioned lyophilized preparations 15-22 is the same as that of the lyophilized preparation 1.
  • Embodiment 2-6 A kind of lyophilized preparation and its preparation
  • This example provides a lyophilized preparation, which is based on the lyophilized preparation 1 in Example 2-1, and the lyoprotectant (mannitol) in the preparation method is replaced by: sucrose, alanine.
  • freeze-dried preparations were named as freeze-dried preparations 23-24 in sequence.
  • This experimental example provides an experiment on the influence of PEG formula and preparation process on the crosslinking performance of PEGylated collagen and its freeze-dried preparation.
  • the experimental process is as follows:
  • the collagen-like protein whose amino acid sequence is shown in SEQ ID NO.
  • the obtained freeze-dried preparations 1-24 were prepared into corneas respectively to obtain corneas 1-24.
  • Use a universal tensile machine to test the strength of corneas 1-24, and the test results are shown in Table 1;
  • Cross-linking step dissolve the lyophilized preparation in MES Buffer with a concentration of 0.5mol/L and pH 5.5, so that the concentration of PEGylated collagen in the MES Buffer is 12.5g/mL to obtain a solution; according to the mass Mix the solution and the MPC mother liquor at a ratio of 5:1 to obtain a mixture 1; mix the mixture 1 and the DMTMM mother liquor at a mass ratio of 7:1 to obtain a mixture 2; the entire crosslinking process is completed at 45°C;
  • Curing step pour the mixed solution 2 into a cornea mold, and place it at 25°C for 12 hours to obtain a crude cornea;
  • Soaking steps Add the crude cornea (with mold) to PBS Buffer with a concentration of 0.1mol/L and pH 5.5, soak at 4°C for 24 hours, open the mold, continue soaking at 4°C for 4 hours, and remove the mold to obtain cornea finished product;
  • MPC mother liquor is: 30g/mL MPC (2-methacryloyloxyethyl phosphorylcholine), 10% PEGDA (poly(ethylene glycol) diacrylate, v/v), 1% TEMED ( N,N,N′,N′-Tetramethylethylenediamine, v/v), the solvent is 0.5mol/L, MOPS Buffer with pH 5.5;
  • DMTMM mother liquor 10g/mL DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride), 15g/mLAPS (ammonium persulfate),
  • the solvent is MOPS Buffer with 0.5mol/L and pH 5.5.
  • both the combination of PEG-modified formula and the preparation process will affect the cross-linking properties of PEGylated collagen and its freeze-dried preparations, and then affect the formation of corneas by making hydrogels; among them, PEG-modified
  • the combination of formulations will significantly affect the strength and elasticity of the cornea; the preparation process, such as reaction temperature, pH, etc., is directly related to the conjugation rate between collagen-like protein and PEG, which can become a modification by affecting the formation of the polymer network structure.
  • Whether the product can be made into a hydrogel is one of the key factors to form a cornea; in addition, the composition of lyophilized auxiliary materials provides skeletal support and protection for PEGylated collagen, thereby affecting the spatial conformation and biological activity of the polymer, resulting in There are differences in hardness.
  • corneas 1-13, 17-21, and 23-24 have better strength and good biocompatibility, and have great application prospects in the field of corneal transplantation.

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Abstract

提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白包含类胶原蛋白以及修饰在类胶原蛋白上的PEG-40k和PEG-20k；PEG-40k和PEG-20k的修饰可显著提高类胶原蛋白的交联性能，使得使用本申请聚乙二醇化类胶原蛋白制成角膜的强度可较使用类胶原蛋白制成的角膜的强度提高将近20倍，较使用现有其他聚乙二醇化类胶原蛋白制成角膜的强度提高将近12倍，因此，所述聚乙二醇化类胶原蛋白在仿生角膜等仿生或再生生物材料的制备中具有极高的应用前景。

Description

一种聚乙二醇化类胶原蛋白及其制备方法和应用
相关申请的交叉引用
本申请要求在2021年9月13日提交中国专利局、申请号202111071337.8、发明名称为“一种聚乙二醇化类胶原蛋白及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用的方式并入本文中。
技术领域
本申请涉及一种聚乙二醇化类胶原蛋白及其制备方法和应用，属于生物技术领域。
背景技术
角膜(Cornea)是眼睛最前面的凸形高度透明物质，呈横椭圆形，覆盖虹膜、瞳孔及前房，并为眼睛提供大部分屈光力。加上晶体的屈光力，光线便可准确地聚焦在视网膜上构成影像。角膜有十分敏感的神经末梢，如有外物接触眼角膜，眼睑便会不由自主地合上以保护眼睛。为了保持透明，角膜并没有血管，透过泪液及房水获取养份及氧气。
角膜十分脆弱，眼部外伤、炎症、过敏反应、物理损伤、化学灼伤、剧烈运动、用眼过度等均会导致角膜病变。角膜一旦病变，会导致明显的眼部症状，比如眼睛的疼痛、畏光、流泪、视力下降等，严重的还会导致失明。
角膜移植就是用正常的角膜替换患者现有的病变角膜，使患眼复明或控制角膜病变，达到增进视力或治疗某些角膜疾患的治疗方法。一些引起患者严重视力受损甚至是失明的角膜疾病，通过进行角膜移植的方法，完全可以治疗，帮助这些不幸的患者远离痛苦。因为角膜本身不含血管，处于“免疫赦免”地位，使角膜移植的成功率位于其他同种异体器官移植之首。
然而，角膜资源有限，远不能满足患者需求。为解决这一问题，有研究者提出了人工角膜移植术。人工角膜移植术是用透明的医用高分子材料制成的特殊光学装置，通过手术将它植入角膜组织中，以取代部分角膜瘢痕组织，而重新恢复视力的一种手术方法。由于角膜组织对人工合成材料的排异反应等问题尚未解决，远期效果不佳， 常造成移植处的房水渗漏及移植片的脱落，故目前尚不可能广泛应用。现阶段人工角膜仅适用于患各种严重角膜疾患后的双目失明，特别是严重的化学烧伤引起的全部角膜白斑和多次角膜移植术失败，无法再做其他手术者。
在人工角膜的基础上，又有研究者提出了仿生角膜。例如，文献“Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants”中，Jangamreddy,Jaganmohan R.等人提出了一种聚乙二醇化类胶原蛋白，将此聚乙二醇化类胶原蛋白经化学交联，即可获得仿生角膜。此仿生角膜生物相容性极好，可有效解决角膜组织对人工合成材料的排异反应强烈等问题。
但是，此仿生角膜的强度很低，仅有0.022MPa，若将此仿生角膜用于角膜移植，尚存在移植难度大、容易引起角膜圆锥等问题。
发明内容
为解决现有仿生角膜强度低的问题，本申请提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白包含类胶原蛋白以及修饰在类胶原蛋白上的聚乙二醇衍生物；所述聚乙二醇衍生物包含PEG-40k和PEG-20k。
在本申请的一种实施方式中，所述聚乙二醇衍生物由PEG-40k和PEG-20k组成。
在本申请的一种实施方式中，所述类胶原蛋白的氨基酸序列如SEQ ID NO.1所示。SEQ ID NO.1中，X为4Hyp(4-羟脯氨酸)，即：
SEQ ID NO.1的氨基酸序列为：
H-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-OH
在本申请的一种实施方式中，所述PEG-40k和PEG-20k的摩尔比为0.5～6:1。
在本申请的一种实施方式中，所述PEG-40k和PEG-20k的摩尔比为2～3:1。
在本申请的一种实施方式中，所述PEG-40k的活化基团数为8-arm、4-arm、2-arm或1-arm中的一种或一种以上；所述PEG-20k的活化基团数为8-arm、4-arm、2-arm或1-arm中的一种或一种以上。
在本申请的一种实施方式中，所述PEG-40k的活化基团数为4-arm或8-arm；所述PEG-20k的活化基团数4-arm或8-arm。
在本申请的一种实施方式中，所述PEG-40k的活化基团为-MAL、-NHS、-SG、-SPA、-SS或-EDC中的一种或一种以上；所述PEG-20k的活化基团为-MAL、-NHS、- SG、-SPA、-SS或-EDC中的一种或一种以上。
在本申请的一种实施方式中，所述PEG-40k的活化基团为-MAL；所述PEG-20k的活化基团为-MAL。
在本申请的一种实施方式中，所述类胶原蛋白的其中一端连接有linker；所述聚乙二醇衍生物通过活化基团修饰在linker上。
在本申请的一种实施方式中，所述linker连接在类胶原蛋白的N端。
在本申请的一种实施方式中，所述linker的氨基酸序列如SEQ ID NO.2或SEQ ID NO.3所示。
在本申请的一种实施方式中，所述聚乙二醇衍生物在linker上的修饰位点为巯基、氨基、羧基或咪唑基中的一种或一种以上。
在本申请的一种实施方式中，当linker的氨基酸序列如SEQ ID NO.2所示时，所述聚乙二醇衍生物在linker上的修饰位点为巯基；当linker的氨基酸序列如SEQ ID NO.3所示时，所述聚乙二醇衍生物在linker上的修饰位点为氨基。
在本申请的一种实施方式中，所述聚乙二醇化类胶原蛋白的母核构象为HG或TP中的一种或一种以上。
在本申请的一种实施方式中，所述聚乙二醇化类胶原蛋白中聚乙二醇衍生物的母核构象为TP。
在本申请的一种实施方式中，所述聚乙二醇化类胶原蛋白中类胶原蛋白的氨基酸构型为D型或L型中的一种或一种以上。
在本申请的一种实施方式中，所述聚乙二醇化类胶原蛋白的分子量为15000～75000Da。
在本申请的一种实施方式中，所述聚乙二醇化类胶原蛋白的分子量为30000～75000Da。
本申请还提供了一种冻干制剂，所述冻干制剂是通过将上述聚乙二醇化类胶原蛋白进行冻干而得的。
在本申请的一种实施方式中，所述冻干制剂是通过将上述聚乙二醇化类胶原蛋白与冻干保护剂混合后进行冻干而得的。
在本申请的一种实施方式中，所述冻干保护剂为甘露醇、蔗糖或丙氨酸中的一种或一种以上。
本申请还提供了一种制备上述聚乙二醇化类胶原蛋白的方法，所述方法包括如下 步骤：
反应步骤：将类胶原蛋白和聚乙二醇衍生物于pH为4.0～10.0、温度为2～40℃的条件下反应1～48h，得到反应产物；所述反应产物中含有上述聚乙二醇化类胶原蛋白。
在本申请的一种实施方式中，所述反应步骤为：将类胶原蛋白和聚乙二醇衍生物于pH为6.0～8.0、温度为2～8℃的条件下反应5～8h。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的投料摩尔数比为1～16:1。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的投料摩尔数比为8～12:1。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的反应溶剂为水或稀盐酸溶液。
在本申请的一种实施方式中，所述稀盐酸溶液的浓度为1～10mmol/L；所述稀盐酸溶液的pH用稀碱溶液调节至6.0～8.0。
在本申请的一种实施方式中，所述稀碱溶液为pH 9.0～11.0的氢氧化钠溶液或氨水。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白在反应溶剂中的投料浓度为1～15mg/mL。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白在反应溶剂中的投料浓度为8～10mg/mL。
在本申请的一种实施方式中，反应步骤后，所述方法还包含纯化步骤；所述纯化步骤为：通过过滤截留反应产物中分子量大于等于30000Da的物质，得到聚乙二醇化类胶原蛋白。
在本申请的一种实施方式中，所述过滤为透析或超滤。
本申请还提供了一种制备上述冻干制剂的方法，所述方法包括如下步骤：
反应步骤：将类胶原蛋白和聚乙二醇衍生物于pH为4.0～10.0、温度为2～40℃的条件下反应1～48h，得到反应产物；所述反应产物中含有上述聚乙二醇化类胶原蛋白；
冻干步骤：将反应产物进行冻干，得到冻干制剂。
在本申请的一种实施方式中，所述反应步骤为：将类胶原蛋白和聚乙二醇衍生物于pH为6.0～8.0、温度为2～8℃的条件下反应5～8h。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的 投料摩尔数比为1～16:1。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的投料摩尔数比为8～12:1。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的反应溶剂为水或稀盐酸溶液。
在本申请的一种实施方式中，所述稀盐酸溶液的浓度为1～10mmol/L；所述稀盐酸溶液的pH用稀碱溶液调节至6.0～8.0。
在本申请的一种实施方式中，所述稀碱溶液为pH 9.0～11.0的氢氧化钠溶液或氨水。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白在反应溶剂中的投料浓度为1～15mg/mL。
在本申请的一种实施方式中，所述反应步骤中，类胶原蛋白在反应溶剂中的投料浓度为8～10mg/mL。
在本申请的一种实施方式中，所述冻干步骤为：将反应产物和冻干保护剂混合后进行冻干，得到冻干制剂。
在本申请的一种实施方式中，所述冻干保护剂为甘露醇、蔗糖或丙氨酸中的一种或一种以上。
在本申请的一种实施方式中，所述冻干包括如下阶段：
阶段一：于温度为-45℃、真空度(vacuum)为500m Torr的条件下冻干6h；
阶段二：于温度为-30℃、真空度(vacuum)为100m Torr的条件下冻干17h；
阶段三：于温度为25℃、真空度(vacuum)为100m Torr的条件下冻干7h。
在本申请的一种实施方式中，反应步骤后、冻干步骤前，所述方法还包含纯化步骤；所述纯化步骤为：通过过滤截留反应产物中分子量大于等于30000Da的物质，得到聚乙二醇化类胶原蛋白。
在本申请的一种实施方式中，所述过滤为透析或超滤。
本申请还提供了上述聚乙二醇化类胶原蛋白或上述冻干制剂或上述方法制得的聚乙二醇化类胶原蛋白或上述方法制得的冻干制剂在制备仿生或再生生物材料中的应用。
在本申请的一种实施方式中，所述仿生或再生生物材料为仿生角膜。
本申请技术方案，具有如下优点：
1、本申请提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白包含类胶原蛋白以及修饰在类胶原蛋白上的PEG-40k和PEG-20k；PEG-40k和PEG-20k的修饰可显著提高类胶原蛋白的交联性能，使得使用本申请聚乙二醇化类胶原蛋白制成角膜的强度可较使用类胶原蛋白制成的角膜的强度提高将近20倍，较使用现有其他聚乙二醇化类胶原蛋白制成角膜的强度提高将近12倍(用文献“Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants”中的聚乙二醇化类胶原蛋白制得的角膜强度仅有0.022MPa)，因此，所述聚乙二醇化类胶原蛋白在仿生角膜等仿生或再生生物材料的制备中具有极高的应用前景。
进一步地，所述PEG-40k和PEG-20k的摩尔比为2～3:1；此摩尔比下的PEG修饰提高类胶原蛋白交联性能的效果更佳。
进一步地，所述PEG-40k为8-arm-PEG-40k-MAL，所述PEG-20k为4-arm-PEG-20k-MAL；此配方下的PEG修饰提高类胶原蛋白交联性能的效果更佳。
2、本申请提供了一种冻干制剂，所述冻干制剂是通过将上述聚乙二醇化类胶原蛋白和冻干保护剂混合后进行冻干而得的；冻干保护剂可对上述聚乙二醇化类胶原蛋白进行保护，使得上述聚乙二醇化类胶原蛋白的结构在冻干过程不会被破坏，进而使得使用冻干制剂制成的角膜的强度进一步提高。
3、本申请提供了上述聚乙二醇化类胶原蛋白的制备方法，所述方法以浓度为1～10mmol/L、pH为6.0～8.0的稀盐酸溶液作为反应溶剂，将类胶原蛋白和PEG于2～8℃下反应5～8h以制得聚乙二醇化类胶原蛋白；此反应条件下制得的聚乙二醇化类胶原蛋白交联性能更佳，并且，与现有其他聚乙二醇化类胶原蛋白的制备方法相比，本申请的制备方法反应时间更短，仅需反应5～8h即可获得能制成水凝胶形成角膜的聚乙二醇化类胶原蛋白(文献“Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants”中，制备能制成水凝胶形成角膜的聚乙二醇化类胶原蛋白需要反应4周)。
4、本申请提供了上述冻干制剂的制备方法，所述方法在冻干保护剂的保护下对上述聚乙二醇化类胶原蛋白进行冻干；冻干保护剂可对上述聚乙二醇化类胶原蛋白进行保护，使得上述聚乙二醇化类胶原蛋白的结构在冻干过程不会被破坏，进而使得上述冻干制剂的交联性能更佳。
具体实施方式
提供下述实施例是为了更好地进一步理解本申请，并不局限于所述最佳实施方式，不对本申请的内容和保护范围构成限制，任何人在本申请的启示下或是将本申请与其他现有技术的特征进行组合而得出的任何与本申请相同或相近似的产品，均落在本申请的保护范围之内。
下述实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。下述实施例中类胶原蛋白的合成以及类胶原蛋白与linker之间的连接均由上海昂博生物技术有限公司(AmbioPharm.InC.)完成。下述实施例中的PEG均购自厦门赛诺邦格生物科技股份有限公司。
实施例1-1：一种聚乙二醇化类胶原蛋白及其制备
本实施例提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白由氨基酸序列如SEQ ID NO.1所示的类胶原蛋白，连接在类胶原蛋白N端的氨基酸序列如SEQ ID NO.2所示的linker，以及，修饰在linker巯基上的8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL组成；其中，8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL的摩尔比为2:1，聚乙二醇化类胶原蛋白中聚乙二醇衍生物的母核构象为TP，且聚乙二醇化类胶原蛋白的分子量为30000～75000Da。将此聚乙二醇化类胶原蛋白命名为聚乙二醇化类胶原蛋白1。
上述聚乙二醇化类胶原蛋白1的制备方法包括如下步骤：
反应步骤：将类胶原蛋白和聚乙二醇衍生物溶于浓度为5mmol/L的稀盐酸溶液(稀盐酸溶液的pH用pH 11.0的氢氧化钠溶液调节至6.5)，使得稀盐酸溶液中，类胶原蛋白和聚乙二醇衍生物的摩尔浓度分别为2.4mmol/L和0.3mmol/L，得到反应体系；将反应体系于pH为6.5、温度为5℃的条件下反应12h，得到反应产物；
纯化步骤：将反应产物用孔径为30000Da的超滤膜，于5℃下进行超滤，截留反应产物中分子量大于等于30000Da的物质，得到聚乙二醇化类胶原蛋白1。
实施例2-1：一种冻干制剂及其制备
本实施例提供了一种冻干制剂，所述冻干制剂由实施例1-1制得的聚乙二醇化类胶原蛋白1以及甘露醇组成。将此冻干制剂命名为冻干制剂1。
上述冻干制剂1的制备方法包括如下步骤：
将实施例1制得的聚乙二醇化类胶原蛋白和甘露醇按照质量比1:5的比例混合，得 到冻干体系；将冻干体系进行冻干，得到冻干制剂1；
其中，冻干包括如下阶段：
阶段一：于温度为-45℃、真空度(vacuum)为500m Torr的条件下冻干6h；
阶段二：于温度为-30℃、真空度(vacuum)为100m Torr的条件下冻干17h；
阶段三：于温度为25℃、真空度(vacuum)为100m Torr的条件下冻干7h。
实施例1-2：一种聚乙二醇化类胶原蛋白及其制备
本实施例提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白在实施例1-1的聚乙二醇化类胶原蛋白1的基础上，将使用的PEG配方(摩尔比为2:1的8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL)分别替换为：
摩尔比为1:0的8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL；
摩尔比为0:1的8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL；
摩尔比为6:1的8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL；
摩尔比为4:1的8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL；
摩尔比为2:1的8-arm-PEG-40k-MAL和4-arm-PEG-40k-MAL；
摩尔比为4:2:1的8-arm-PEG-40k-MAL、4-arm-PEG-20k-MAL和4-arm-PEG-40k-MAL；
摩尔比为2:2:1的8-arm-PEG-40k-MAL、4-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL；
摩尔比为2:2:1的8-arm-PEG-40k-MAL、4-arm-PEG-40k-MAL和8-arm-PEG-20k-MAL。
将上述聚乙二醇化类胶原蛋白依次命名为聚乙二醇化类胶原蛋白2～9。
上述聚乙二醇化类胶原蛋白2～9的制备方法同聚乙二醇化类胶原蛋白1。
实施例2-2：一种冻干制剂及其制备
本实施例提供了一种冻干制剂，所述冻干制剂在实施例2-1的冻干制剂1的基础上，将聚乙二醇化类胶原蛋白1分别替换为实施例1-2制得的聚乙二醇化类胶原蛋白2～9。
将上述冻干制剂依次命名为冻干制剂2～9。
上述冻干制剂2～9的制备方法同冻干制剂1。
实施例1-3：一种聚乙二醇化类胶原蛋白及其制备
本实施例提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白由氨基酸序列如SEQ ID NO.1所示的类胶原蛋白，连接在类胶原蛋白N端的氨基酸序列如SEQ ID NO.3所示的linker，以及，修饰在linker氨基上的8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL组成；其中，8-arm-PEG-40k-MAL和4-arm-PEG-20k-MAL的摩尔比为2:1，聚乙二醇化类胶原蛋白中聚乙二醇衍生物的母核构象为TP，且聚乙二醇化类胶原蛋白的分子量为30000～75000Da。将此聚乙二醇化类胶原蛋白命名为聚乙二醇化类胶原蛋白10。
上述聚乙二醇化类胶原蛋白10的制备方法同聚乙二醇化类胶原蛋白1。
实施例2-3：一种冻干制剂及其制备
本实施例提供了一种冻干制剂，所述冻干制剂在实施例2-1的冻干制剂1的基础上，将聚乙二醇化类胶原蛋白1替换为实施例1-3制得的聚乙二醇化类胶原蛋白10。
将上述冻干制剂依次命名为冻干制剂10。
上述冻干制剂10的制备方法同冻干制剂1。
实施例1-4：一种聚乙二醇化类胶原蛋白及其制备
本实施例提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白在实施例1-1的聚乙二醇化类胶原蛋白1的基础上，将制备方法中的温度(5℃)分别替换为：2℃、8℃、25℃、37℃。
将上述聚乙二醇化类胶原蛋白依次命名为聚乙二醇化类胶原蛋白11～14。
实施例2-4：一种冻干制剂及其制备
本实施例提供了一种冻干制剂，所述冻干制剂在实施例2-1的冻干制剂1的基础上，将聚乙二醇化类胶原蛋白1分别替换为实施例1-4制得的聚乙二醇化类胶原蛋白11～14。
将上述冻干制剂依次命名为冻干制剂11～14。
上述冻干制剂11～14的制备方法同冻干制剂1。
实施例1-5：一种聚乙二醇化类胶原蛋白及其制备
本实施例提供了一种聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白在实施例1-1的聚乙二醇化类胶原蛋白1的基础上，将制备方法中的pH(6.5)分别替换为： pH 2.5、pH 4.5、pH 6.0、pH 6.5、pH 7.0、pH 8.0、pH 8.5、pH 10.5。
将上述聚乙二醇化类胶原蛋白依次命名为聚乙二醇化类胶原蛋白15～22。
实施例2-5：一种冻干制剂及其制备
本实施例提供了一种冻干制剂，所述冻干制剂在实施例2-1的冻干制剂1的基础上，将聚乙二醇化类胶原蛋白1分别替换为实施例1-5制得的聚乙二醇化类胶原蛋白15～22。
将上述冻干制剂依次命名为冻干制剂15～22。
上述冻干制剂15～22的制备方法同冻干制剂1。
实施例2-6：一种冻干制剂及其制备
本实施例提供了一种冻干制剂，所述冻干制剂在实施例2-1的冻干制剂1的基础上，将制备方法中的冻干保护剂(甘露醇)分别替换为：蔗糖、丙氨酸。
将上述冻干制剂依次命名为冻干制剂23～24。
实验例1：PEG配方及制备工艺对聚乙二醇化类胶原蛋白及其冻干制剂交联性能的影响实验
本实验例提供了PEG配方及制备工艺对聚乙二醇化类胶原蛋白及其冻干制剂交联性能的影响实验，实验过程如下：
参照冻干制剂1的制备方法将氨基酸序列如SEQ ID NO.1所示的类胶原蛋白直接制备成冻干制剂，以此冻干制剂作为空白对照，将实施例2-1～2-6制得的冻干制剂1～24分别制成角膜，得到角膜1～24。使用万能拉力机检测角膜1～24的强度，检测结果见表1；
其中，角膜的制备工艺如下：
交联步骤：将冻干制剂溶于浓度为0.5mol/L、pH 5.5的MES Buffer中，使得MES Buffer中，聚乙二醇化类胶原蛋白的浓度为12.5g/mL，得到溶解液；按照质量比5:1将溶解液和MPC母液混合，得到混合液1；按照质量比7:1将混合液1和DMTMM母液混合，得到混合液2；整个交联过程均在45℃下完成；
固化步骤：将混合液2浇注在角膜模具中，于25℃下放置12h，得到角膜粗品；
浸泡步骤：将角膜粗品(连带模具)添加至浓度为0.1mol/L、pH 5.5的PBS Buffer中，于4℃下浸泡24h后，打开模具，于4℃下继续浸泡4h，脱模，得到角膜成品；
其中，MPC母液的配方为：30g/mL MPC(2-甲基丙烯酰氧乙基磷酸胆碱)、10％PEGDA(聚(乙二醇)二丙烯酸酯，v/v)、1％TEMED(N,N,N′,N′-四甲基乙二胺，v/v)，溶剂为0.5mol/L、pH 5.5的MOPS Buffer；
DMTMM母液的配方为：10g/mL DMTMM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)、15g/mLAPS(过硫酸铵)，溶剂为0.5mol/L、pH 5.5的MOPS Buffer。
由表1可知，PEG修饰配方组合及制备工艺均会对聚乙二醇化类胶原蛋白及其冻干制剂的交联性能造成影响，进而对制成水凝胶形成角膜造成影响；其中，PEG修饰配方组合会显著影响角膜的强度和弹性；制备工艺，例如反应温度、pH等，则与类胶原蛋白和PEG之间的缀合率有直接关联，能通过影响聚合物网络结构的形成，成为修饰产物是否能制成水凝胶形成角膜的关键因素之一；另外，冻干辅料组成为聚乙二醇化胶原蛋白提供骨架支撑和保护，从而影响聚合物的空间构象和生物活性，导致成膜后的硬度存在差异。表1中，角膜1～13、17～21以及23～24的强度都较佳，且生物相容性也很好，在角膜移植领域极具应用前景。
表1 角膜1～24的强度
组别 强度(MPa)
角膜1 0.255
角膜2 0.083
角膜3 0.074
角膜4 0.138
角膜5 0.201
角膜6 0.088
角膜7 0.173
角膜8 0.211
角膜9 0.197
角膜10 0.204
角膜11 0.253
角膜12 0.245
角膜13 0.123
角膜14 /(不成膜)
角膜15 /(不成膜)
角膜16 0.027
角膜17 0.241
角膜18 0.245
角膜19 0.243
角膜20 0.234
角膜21 0.163
角膜22 /(不成膜)
角膜23 0.234
角膜24 0.180
角膜(空白对照) 0.014
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本申请创造的保护范围之中。

Claims (37)

  1. 一种聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇化类胶原蛋白包含类胶原蛋白以及修饰在类胶原蛋白上的聚乙二醇衍生物；所述聚乙二醇衍生物包含PEG-40k和PEG-20k。
  2. 如权利要求1所述的聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇衍生物由PEG-40k和PEG-20k组成。
  3. 如权利要求1所述的聚乙二醇化类胶原蛋白，其特征在于，所述类胶原蛋白的氨基酸序列如SEQ ID NO.1所示。
  4. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述PEG-40k和PEG-20k的摩尔比为0.5～6:1。
  5. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述PEG-40k和PEG-20k的摩尔比为2～3:1。
  6. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述PEG-40k的活化基团数为8-arm、4-arm、2-arm或1-arm中的一种或一种以上；所述PEG-20k的活化基团数为8-arm、4-arm、2-arm或1-arm中的一种或一种以上。
  7. 如权利要求6所述的聚乙二醇化类胶原蛋白，其特征在于，所述PEG-40k的活化基团数为4-arm或8-arm；所述PEG-20k的活化基团数4-arm或8-arm。
  8. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述PEG-40k的活化基团为-MAL、-NHS、-SG、-SPA、-SS或-EDC中的一种或一种以上；所述PEG-20k的活化基团为-MAL、-NHS、-SG、-SPA、-SS或-EDC中的一种或一种以上。
  9. 如权利要求8所述的聚乙二醇化类胶原蛋白，其特征在于，所述PEG-40k的活化基团为-MAL；所述PEG-20k的活化基团为-MAL。
  10. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述类胶原蛋白的其中一端连接有linker；所述聚乙二醇衍生物通过活化基团修饰在linker上。
  11. 如权利要求10所述的聚乙二醇化类胶原蛋白，其特征在于，所述linker连接在类胶原蛋白的N端。
  12. 如权利要求10所述的聚乙二醇化类胶原蛋白，其特征在于，所述linker的氨基酸序列如SEQ ID NO.2或SEQ ID NO.3所示。
  13. 如权利要求12所述的聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇衍生 物在linker上的修饰位点为巯基、氨基、羧基或咪唑基中的一种或一种以上。
  14. 如权利要求13所述的聚乙二醇化类胶原蛋白，其特征在于，当linker的氨基酸序列如SEQ ID NO.2所示时，所述聚乙二醇衍生物在linker上的修饰位点为巯基。
  15. 如权利要求13或14所述的聚乙二醇化类胶原蛋白，其特征在于，当linker的氨基酸序列如SEQ ID NO.3所示时，所述聚乙二醇衍生物在linker上的修饰位点为氨基。
  16. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇化类胶原蛋白的母核构象为HG或TP中的一种或一种以上。
  17. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇化类胶原蛋白中聚乙二醇衍生物的母核构象为TP。
  18. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇化类胶原蛋白中类胶原蛋白的氨基酸构型为D型或L型中的一种或一种以上。
  19. 如权利要求1～3任一项所述的聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇化类胶原蛋白的分子量为15000～75000Da。
  20. 如权利要求19所述的聚乙二醇化类胶原蛋白，其特征在于，所述聚乙二醇化类胶原蛋白的分子量为30000～75000Da。
  21. 一种冻干制剂，其特征在于，所述冻干制剂是通过将权利要求1～20任一项所述的聚乙二醇化类胶原蛋白进行冻干而得的。
  22. 如权利要求21所述的冻干制剂，其特征在于，所述冻干制剂是通过将上述聚乙二醇化类胶原蛋白与冻干保护剂混合后进行冻干而得的。
  23. 如权利要求22所述的冻干制剂，其特征在于，所述冻干保护剂为甘露醇、蔗糖或丙氨酸中的一种或一种以上。
  24. 一种制备权利要求1～20任一项所述的聚乙二醇化类胶原蛋白的方法，其特征在于，所述方法包括如下步骤：
    反应步骤：将类胶原蛋白和聚乙二醇衍生物于pH为4.0～10.0、温度为2～40℃的条件下反应1～48h，得到反应产物；所述反应产物中含有权利要求1～20任一项所述的聚乙二醇化类胶原蛋白。
  25. 如权利要求24所述的方法，其特征在于，所述反应步骤为：将类胶原蛋白和聚乙二醇衍生物于pH为6.0～8.0、温度为2～8℃的条件下反应5～8h。
  26. 如权利要求24所述的方法，其特征在于，所述反应步骤中，类胶原蛋白和聚乙 二醇衍生物的投料摩尔数比为1～16:1。
  27. 如权利要求26所述的方法，其特征在于，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的投料摩尔数比为8～12:1。
  28. 如权利要求24～26任一项所述的方法，其特征在于，所述反应步骤中，类胶原蛋白和聚乙二醇衍生物的反应溶剂为水或稀盐酸溶液。
  29. 如权利要求28所述的方法，其特征在于，所述稀盐酸溶液的浓度为1～10mmol/L；所述稀盐酸溶液的pH用稀碱溶液调节至6.0～8.0。
  30. 如权利要求28所述的方法，其特征在于，所述稀碱溶液为pH 9.0～11.0的氢氧化钠溶液或氨水。
  31. 如权利要求24～26任一项所述的方法，其特征在于，所述反应步骤中，类胶原蛋白在反应溶剂中的投料浓度为1～15mg/mL。
  32. 如权利要求31所述的方法，其特征在于，所述反应步骤中，类胶原蛋白在反应溶剂中的投料浓度为8～10mg/mL。
  33. 如权利要求24～26任一项所述的方法，其特征在于，反应步骤后，所述方法还包含纯化步骤；所述纯化步骤为：通过过滤截留反应产物中分子量大于等于30000Da的物质，得到聚乙二醇化类胶原蛋白。
  34. 如权利要求33所述的方法，其特征在于，所述过滤为透析或超滤。
  35. 一种制备权利要求21～23任一项所述的冻干制剂的方法，其特征在于，所述方法包括如下步骤：
    冻干步骤：将使用权利要求24～34任一项所述的方法制得的聚乙二醇化类胶原蛋白进行冻干，得到冻干制剂。
  36. 权利要求1～20任一项所述的聚乙二醇化类胶原蛋白或权利要求21～23任一项所述的冻干制剂或权利要求24～34任一项所述的方法制得的聚乙二醇化类胶原蛋白或权利要求35所述的方法制得的冻干制剂在制备仿生或再生生物材料中的应用。
  37. 如权利要求36所述的应用，其特征在于，所述仿生或再生生物材料为角膜。
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