WO2023035720A1 - 一种制备聚乙二醇化类胶原蛋白的方法及其应用 - Google Patents

一种制备聚乙二醇化类胶原蛋白的方法及其应用 Download PDF

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WO2023035720A1
WO2023035720A1 PCT/CN2022/099755 CN2022099755W WO2023035720A1 WO 2023035720 A1 WO2023035720 A1 WO 2023035720A1 CN 2022099755 W CN2022099755 W CN 2022099755W WO 2023035720 A1 WO2023035720 A1 WO 2023035720A1
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protein
collagen
cornea
freeze
pegylated
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PCT/CN2022/099755
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English (en)
French (fr)
Inventor
王崇宇
张加慧
张军
刘沐荣
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 WO2023035720A1 publication Critical patent/WO2023035720A1/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
    • A61L27/52—Hydrogels or hydrocolloids
    • 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 method for preparing pegylated collagen-like protein and its 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 method for preparing pegylated collagen-like protein, the method comprising the following steps:
  • Reaction steps react collagen-like protein and polyethylene glycol derivatives at a pH of 6.0-8.0 and a temperature of 2-8°C to obtain a reaction product; the reaction product contains the above-mentioned pegylated collagen-like protein.
  • 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 reaction time is 1-48 hours.
  • the reaction time is 5-8 hours.
  • 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 polyethylene glycol derivative comprises one or more of PEG-40k, PEG-20k, PEG-10k or PEG-5k.
  • the number of activated groups of the polyethylene glycol derivative is one or more of 8-arm, 4-arm, 2-arm or 1-arm.
  • the number of activated groups of the polyethylene glycol derivative is 4-arm or 8-arm.
  • the activating group of the polyethylene glycol derivative is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC.
  • the activating group of the polyethylene glycol derivative 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.
  • amino acid sequence of the linker is shown in SEQ ID NO.2.
  • the modification site of the polyethylene glycol derivative on the linker is one or more of thiol, amino, carboxyl or imidazole.
  • the modified site of the polyethylene glycol derivative on the linker is a sulfhydryl 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 PEGylated collagen-like protein, which is prepared by the above-mentioned method.
  • the present application also provides a method for preparing a freeze-dried preparation of PEGylated collagen, the method comprising the steps of:
  • Freeze-drying step freeze-dry the pegylated collagen-like protein prepared by the above method to obtain a freeze-dried preparation.
  • the freeze-drying step is: mixing the pegylated collagen-like protein and a freeze-drying protective 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 present application also provides a freeze-dried preparation of pegylated collagen-like protein, which is prepared by the above-mentioned method.
  • the present application also provides the application of the PEGylated collagen-like protein prepared by the above method or the freeze-dried preparation prepared by the above method in the preparation of biomimetic or regenerative biomaterials.
  • the biomimetic or regenerative biomaterial is cornea.
  • the application also provides a method for preparing a bionic cornea, said method comprising the steps of:
  • Cross-linking step dissolving the freeze-dried preparation prepared by the above method in the buffer solution to obtain a solution; mixing the solution with the MPC mother solution to obtain a mixed solution 1; mixing the mixed solution 1 and the DMTMM mother solution to obtain a mixed solution 2 ;
  • Solidification step pour the mixed solution 2 into the cornea mold and let it stand still to obtain a crude cornea.
  • the method further includes a soaking step; the soaking step is: adding the rough cornea together with the mold to the buffer for the first soaking, and the first soaking is over Finally, the mold is opened for the second soaking, and after the second soaking is finished, the mold is demoulded to obtain the finished cornea.
  • the pH of the buffer is 5.5-8.0.
  • the pH of the buffer in the cross-linking step, is 6.5-7.5.
  • the concentration of the buffer in the cross-linking step, is 0.5-0.7 mol/L.
  • the buffer in the cross-linking step, is MOPS Buffer, MES Buffer or PBS Buffer.
  • the concentration of pegylated collagen-like protein in the buffer solution is 5-40 g/mL.
  • the concentration of pegylated collagen-like protein in the buffer solution is 12-18 g/mL.
  • the concentration of the pegylated collagen-like protein in the buffer is 15 g/mL.
  • the mixing mass ratio of the dissolving solution and the MPC mother liquor is 2:1 ⁇ 4:1.
  • the mixing mass ratio of the mixed liquid 1 to the DMTMM mother liquid is 5:1 ⁇ 7:1.
  • the cross-linking step is completed at 25-60°C.
  • the cross-linking step is completed at 45-55°C.
  • the standing temperature is 4-35°C.
  • the standing temperature is 4-25°C.
  • the standing time is 8-20 hours.
  • the pH of the buffer is 5.5-8.0.
  • the pH of the buffer in the soaking step, is 6.5-7.5.
  • the buffer solution in the soaking step, has a concentration of 0.05 ⁇ 1 mol/L.
  • the buffer in the soaking step, is MOPS Buffer, MES Buffer or PBS Buffer.
  • the temperature of the first soaking is 4-35° C. and the time is 5-24 hours.
  • the temperature of the second soaking is 4-35° C. and the time is 3-10 hours.
  • the components of the MPC mother liquor include MPC (2-methacryloyloxyethyl phosphorylcholine), PEGDA (poly(ethylene glycol) diacrylate), TEMED (N, N,N',N'-tetramethylethylenediamine) and solvent.
  • the concentration of MPC in the MPC mother liquor, is 15-40 g/mL; in terms of volume percentage, in the MPC mother liquor, the concentration of PEGDA is 0.6-15%, and the concentration of TEMED is 0.05-2%.
  • the concentration of PEGDA in the MPC mother liquor is 8-12%.
  • the concentration of PEGDA in the MPC mother liquor is 10%.
  • the solvent of the MPC mother liquor is a buffer; the pH of the buffer is 6.0-8.0.
  • the buffer solution in the MPC mother liquor, has a pH of 6.5-7.5.
  • the buffer solution in the MPC mother liquor, has a concentration of 0.5-0.7 mol/L.
  • the buffer in the MPC mother liquor, is MOPS Buffer, MES Buffer or PBS Buffer.
  • the components of the DMTMM mother liquor include DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride), APS (ammonium persulfate) and solvent.
  • the concentration of DMTMM is 5-20 g/mL, and the concentration of APS is 0.5-5 g/mL.
  • the solvent of the DMTMM mother solution is a buffer; the pH of the buffer is 6.0-8.0.
  • the pH of the buffer in the DMTMM mother liquor, is 6.5-7.5.
  • the buffer solution in the DMTMM mother liquor, has a concentration of 0.5-0.7 mol/L.
  • the buffer in the DMTMM mother liquor, is MOPS Buffer, MES Buffer or PBS Buffer.
  • the present application also provides a bionic cornea, which is prepared by using the above method.
  • the present application provides a method for preparing PEGylated collagen-like protein, which involves carrying out collagen-like protein and polyethylene glycol derivatives at a pH of 6.0-8.0 and a temperature of 2-8°C. reaction to obtain PEGylated collagen-like protein; compared with other existing methods for preparing PEGylated collagen-like protein, the PEGylated collagen-like protein obtained by the method of the present application has better cross-linking properties, After the PEGylated collagen-like protein prepared by the method of the present application is made into a hydrogel to form a cornea, the strength can be improved by more than 10 times compared with the PEGylated collagen-like protein prepared by the existing method (using The strength of the cornea made from PEGylated collagen in the document "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants" is only 0.022MPa).
  • the reaction time for preparing PEGylated collagen-like proteins using the method of the present application is shorter, and only 5-8 hours of reaction is required to obtain Hydrogel-forming cornea-like pegylated collagens (in the literature "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants", the preparation of pegylated collagen-like proteins that can be made into hydrogels to form corneas requires response for 4 weeks).
  • the molar ratio of the collagen-like protein to the polyethylene glycol derivative is 8-12:1; the PEGylated collagen-like protein cross-linking performance obtained under this molar ratio is 8-12:1. better.
  • the feeding concentration of collagen-like protein in the reaction solvent is 8-10 mg/mL; the PEGylated collagen-like protein prepared under this feeding concentration has better cross-linking performance.
  • the method of the present application and the PEGylated collagen-like protein prepared by the method of the present application have a very high application prospect in the preparation of bionic or regenerative biomaterials such as bionic cornea.
  • the application provides a method for preparing a freeze-dried preparation of pegylated collagen-like protein.
  • the method is to mix the pegylated collagen-like protein prepared by the above-mentioned method with a freeze-drying protective agent and then carry out freeze-drying. Dry to obtain lyophilized preparations of pegylated collagen-like proteins; lyoprotectants can protect the pegylated collagen-like proteins prepared using the above method, so that the pegylated collagen-like proteins prepared using the above-mentioned method
  • the structure of the collagen will not be destroyed during the freeze-drying process, so that the strength of the freeze-dried preparation prepared by the method of the present application is further improved after the cornea is formed into a hydrogel.
  • the method of the present application and the freeze-dried preparation prepared by using the method of the present application have extremely high application prospects in the preparation of bionic or regenerative biomaterials such as bionic corneas.
  • the application provides a method for preparing a bionic cornea.
  • the method is to dissolve the freeze-dried preparation prepared by the above method in MES Buffer to obtain a solution, and then mix the solution with the MPC mother solution to obtain a mixed Solution 1, and then mix the mixed solution 1 and the DMTMM mother solution to obtain the mixed solution 2, and finally pour the mixed solution 2 into the corneal mold for standing to obtain the crude cornea; compared with other existing methods for preparing bionic corneas, using this method
  • the strength of the cornea prepared by the applied method is high, which can be increased by more than 10 times compared with the cornea prepared by other existing bionic cornea methods (the cornea prepared in the document "Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants" Strength is only 0.022MPa).
  • the lyophilized preparation is cross-linked under the conditions of pH 6.5-7.5 and temperature 45-55° C.; the bionic cornea prepared under this cross-linking condition has higher strength.
  • the method controls the concentration of the pegylated collagen to 12-18 g/mL; the bionic cornea prepared at this concentration of the pegylated collagen has higher strength.
  • the method controls the concentration of PEGDA in the MPC mother liquor at 8-12%; the bionic cornea prepared under this concentration of PEGDA has higher strength.
  • Example 1-1 A method for preparing pegylated collagen-like protein
  • the present embodiment provides a method for preparing pegylated collagen-like proteins, the method comprising the steps of:
  • Collagen-like protein (the amino acid sequence of the collagen-like protein is shown in SEQ ID NO.1, and the N-terminal of the collagen-like protein is connected with a linker with an amino acid sequence shown in SEQ ID NO.2) and polyethylene glycol Derivative (8-arm-PEG-40k-MAL) is dissolved in the dilute hydrochloric acid solution that concentration is 5mmol/L (the pH of dilute hydrochloric acid solution is adjusted to 6.5 with the sodium hydroxide solution of pH11.0), makes 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 ultrafiltered 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 method for preparing a freeze-dried preparation of pegylated collagen-like protein
  • This embodiment provides a method for preparing a freeze-dried preparation of pegylated collagen-like protein, the method comprising the following steps:
  • the PEGylated collagen-like protein 1 and mannitol prepared in Example 1 were mixed according to the mass ratio of 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.
  • Embodiment 1-2 A kind of method for preparing pegylated collagen-like protein
  • This example provides a method for preparing PEGylated collagen-like proteins.
  • the reaction temperature (5°C) is replaced by: 2°C, 8°C, At 25°C and 37°C, PEGylated collagen-like proteins 2-5 were prepared.
  • Example 2-2 A method for preparing a freeze-dried preparation of pegylated collagen-like protein
  • This example provides a method for preparing a freeze-dried preparation of PEGylated collagen-like protein, which is based on the method in Example 2-1, replacing PEGylated collagen-like protein 1 with The PEGylated collagen-like proteins 2-5 obtained in Example 1-2 were used to prepare freeze-dried preparations 2-5.
  • Embodiment 1-3 A kind of method for preparing pegylated collagen-like protein
  • This embodiment provides a method for preparing PEGylated collagen-like proteins.
  • the reaction pH (6.5) is replaced by: pH 2.5, pH 4.5, pH 5.5, pH 6.0, pH 7.0, pH 8.0, pH 8.5, pH 10.5, to obtain PEGylated collagen 6-13.
  • Example 2-3 A method for preparing a freeze-dried preparation of pegylated collagen-like protein
  • This example provides a method for preparing a freeze-dried preparation of PEGylated collagen-like protein, which is based on the method in Example 2-1, replacing PEGylated collagen-like protein 1 with The PEGylated collagen-like proteins 6-13 prepared in Example 1-3 were used to prepare freeze-dried preparations 6-13.
  • Embodiment 1-4 A kind of method for preparing pegylated collagen-like protein
  • This embodiment provides a method for preparing PEGylated collagen-like protein.
  • the preparation of collagen-like protein and polyethylene glycol derivatives in dilute hydrochloric acid solution The molar concentrations are replaced by:
  • the molar concentrations of collagen-like protein and polyethylene glycol derivatives are 3.0mmol/L and 0.375mmol/L respectively;
  • the molar concentrations of collagenoid and polyethylene glycol derivatives are 1.8mmol/L and 0.225mmol/L respectively;
  • the molar concentrations of collagenoid and polyethylene glycol derivatives are 1.2mmol/L and 0.15mmol/L respectively,
  • PEGylated collagen-like proteins 14-16 were prepared.
  • Example 2-4 A method for preparing a freeze-dried preparation of pegylated collagen-like protein
  • This example provides a method for preparing a freeze-dried preparation of PEGylated collagen-like protein, which is based on the method in Example 2-1, replacing PEGylated collagen-like protein 1 with The PEGylated collagen-like proteins 14-16 obtained in Examples 1-4 were used to prepare freeze-dried preparations 14-16.
  • Embodiment 1-5 A kind of method for preparing pegylated collagen-like protein
  • This example provides a method for preparing PEGylated collagen-like proteins.
  • the reaction time (12h) is replaced by: 4h, 8h, 16h, 24h , to prepare PEGylated collagen 17-20.
  • Example 2-5 A method for preparing a freeze-dried preparation of pegylated collagen-like protein
  • This example provides a method for preparing a freeze-dried preparation of PEGylated collagen-like protein, which is based on the method in Example 2-1, replacing PEGylated collagen-like protein 1 with The PEGylated collagen-like proteins 17-20 prepared in Examples 1-5 were used to prepare freeze-dried preparations 17-20.
  • Example 2-6 A method for preparing a freeze-dried preparation of pegylated collagen-like protein
  • This embodiment provides a method for preparing a lyophilized preparation of PEGylated collagen-like protein.
  • the lyoprotectant mannitol
  • Sucrose and alanine were used to prepare freeze-dried preparations 21-22.
  • This experimental example provides an experiment on the influence of the preparation process on the cross-linking properties of pegylated collagen and its freeze-dried preparations.
  • 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 1-22 were made into corneas respectively to obtain corneas 1-22.
  • Use a universal tensile machine to test the strength of corneas 1-22, 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 the mold) to PBS Buffer with a concentration of 0.1mol/L and pH 6.5, soak at 4°C for 24 hours, open the mold, continue soaking at 4°C for 4 hours, and remove the mold to obtain the cornea finished product;
  • the formula of MPC mother liquor is: 30g/mL MPC, 10% PEGDA (v/v), 1% TEMED (v/v), the solvent is 0.5mol/L, MOPS Buffer with pH 5.5;
  • the formula of DMTMM mother liquor is: 10g/mL DMTMM, 15g/mL APS, the solvent is 0.5mol/L, MOPS Buffer with pH 5.5.
  • the preparation process will affect the cross-linking performance of PEGylated collagen-like protein and its freeze-dried preparation, and then affect the formation of cornea after making hydrogel, and the freeze-dried auxiliary material is composed of polyethylene glycol Alcoholated collagen provides skeleton support and protection, which affects the spatial conformation and biological activity of the polymer, resulting in differences in the hardness of the film after formation.
  • the strength of the corneas 1-22 made by using the freeze-dried preparations 1-22 prepared in Examples 2-1-2-6 is better, and the biocompatibility of the product is improved at the same time. It has a very high application prospect in the preparation of .
  • Cornea 4 0.025 Cornea 16 0.052 Cornea 5 / (no film formation) Cornea 17 0.016 Cornea 6 / (no film formation) Cornea 18 0.037 Cornea 7 0.019 Cornea 19 0.084 Cornea 8 0.036 Cornea 20 0.082 Cornea 9 0.074 Cornea 21 0.061 Cornea 10 0.072 Cornea 22 0.047 Cornea 11 0.062 Cornea (blank control) 0.014 Cornea 12 0.035 the the
  • Example 3-1 A method of preparing a bionic cornea
  • the present embodiment provides a kind of preparation bionic cornea method, described method comprises the steps:
  • Cross-linking step dissolve the lyophilized preparation prepared in Example 2-1 in MES Buffer with a concentration of 0.5 mol/L and pH 6.5, so that the concentration of pegylated collagen-like protein in the MES Buffer is 15 g/mL , to obtain the solution; mix the solution with the MPC mother liquor according to the mass ratio of 4:1 to obtain the mixed solution 1; mix the mixed solution 1 and the DMTMM mother solution according to the mass ratio of 7:1 to obtain the mixed solution 2; the whole crosslinking process is in Finished 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 the mold) to PBS Buffer with a concentration of 0.1mol/L and pH 6.5, soak at 4°C for 24 hours, open the mold, continue soaking at 4°C for 4 hours, and remove the mold to obtain the bionic cornea 23;
  • the formula of MPC mother liquor is: 30% MPC (w/v, g/mL), 10% PEGDA (v/v), 1% TEMED (v/v), the solvent is MES of 0.5mol/L, pH 6.5 Buffer;
  • the formula of DMTMM mother liquor is: 10% DMTMM (w/v, g/mL), 15% APS (w/v, g/mL), the solvent is 0.5mol/L, MES Buffer with pH 6.5.
  • Example 3-2 A method of preparing a bionic cornea
  • This example provides a method for preparing a bionic cornea.
  • the crosslinking temperature (45°C) is replaced by: 20°C, 25°C, 40°C, and 50°C , 55°C, 60°C, 65°C, 24-30 bionic corneas were prepared.
  • Embodiment 3-3 A kind of method for preparing bionic cornea
  • This example provides a method for preparing a bionic cornea.
  • the pH (6.5) of Buffer is replaced by: 5, 5.5, 6, 7, 7.5, 8, 8.5 and 9, bionic corneas 31-38 were prepared.
  • Embodiment 3-4 A kind of method for preparing bionic cornea
  • This embodiment provides a method for preparing a bionic cornea.
  • the method is based on the method in Example 3-1, and the concentration of PEGylated collagen in MES Buffer (15g/mL) is replaced by: 1g /mL, 5g/mL, 10g/mL, 20g/mL, 25g/mL, 30g/mL, 35g/mL, 40g/mL, 45g/mL, bionic corneas 39-47 were prepared.
  • Embodiment 3-5 A kind of method for preparing bionic cornea
  • This embodiment provides a method for preparing a bionic cornea.
  • the concentration of PEGDA in the MPC mother liquor (10%, v/v) is replaced by: 0.5%, 0.6 %, 1%, 5%, 8%, 12%, 15% (v/v), 48-54 bionic corneas were prepared.
  • This experimental example provides an experiment on the influence of the preparation process on the strength of the cornea.
  • the experimental process is as follows:
  • the strength of corneas 23-54 was tested with a universal tensile machine, and the test results are shown in Table 2.
  • Cornea 29 0.154 Cornea 45 0.163 Cornea 30 / (no film formation) Cornea 46 / (no film formation) Cornea 31 0.046 Cornea 47 / (no film formation) Cornea 32 0.082 Cornea 48 0.024 Cornea 33 0.169 Cornea 49 0.037 Cornea 34 0.213 Cornea 50 0.109 Cornea 35 0.201 Cornea 51 0.143 Cornea 36 0.133 Cornea 52 0.206 Cornea 37 / (no film formation) Cornea 53 0.211 Cornea 38 / (no film formation) Cornea 54 0.213

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Abstract

本申请涉及一种制备聚乙二醇化类胶原蛋白的方法及其应用，属于生物技术领域。本申请提供了一种制备聚乙二醇化类胶原蛋白的方法，所述方法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℃的条件下反应以制得聚乙二醇化类胶原蛋白；与现有其他制备聚乙二醇化类胶原蛋白的方法相比，使用本申请的方法制得的聚乙二醇化类胶原蛋白交联性能更佳，将使用本申请的方法制得的聚乙二醇化类胶原蛋白制成水凝胶形成角膜后，在强度上可较使用现有方法制得的聚乙二醇化类胶原蛋白提高10倍以上，并且，使用本申请的方法制备聚乙二醇化类胶原蛋白的反应时间更短，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。

Description

一种制备聚乙二醇化类胶原蛋白的方法及其应用
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、申请号为202111071336.3、发明名称为“一种制备聚乙二醇化类胶原蛋白的方法及其应用”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
本申请涉及一种制备聚乙二醇化类胶原蛋白的方法及其应用，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，本申请提供了一种制备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6.0～8.0、温度为2～8℃的条件下进行反应，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。
在本申请的一种实施方式中，所述反应的时间为1～48h。
在本申请的一种实施方式中，所述反应的时间为5～8h。
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，所述聚乙二醇衍生物包含PEG-40k、PEG-20k、PEG-10k或PEG-5k中的一种或一种以上。
在本申请的一种实施方式中，所述聚乙二醇衍生物的活化基团数为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，所述聚乙二醇衍生物的活化基团数为4-arm或8-arm。
在本申请的一种实施方式中，所述聚乙二醇衍生物的活化基团为-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，所述聚乙二醇衍生物的活化基团为-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linker的氨基酸序列如SEQ ID NO.2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，所述聚乙二醇衍生物在linker上的修饰位点为巯基。
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聚乙二醇化类胶原蛋白，所述聚乙二醇化类胶原蛋白是使用上述方法制备得到的。
本申请还提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法包括如下步骤：
冻干步骤：将使用上述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。
在本申请的一种实施方式中，所述冻干步骤为：将聚乙二醇化类胶原蛋白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。
本申请还提供了一种聚乙二醇化类胶原蛋白的冻干制剂，所述冻干制剂是是使用上述方法制备得到的。
本申请还提供了上述方法制备得到的聚乙二醇化类胶原蛋白或上述方法制备得到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角膜。
本申请还提供了一种制备仿生角膜的方法，所述方法包括如下步骤：
交联步骤：将使用上述方法制备得到的冻干制剂溶于缓冲液中，得到溶解液；将溶解液和MPC母液混合，得到混合液1；将混合液1和DMTMM母液混合，得到混合液2；
固化步骤：将混合液2浇注在角膜模具中进行静置，得到角膜粗品。
在本申请的一种实施方式中，固化步骤后，所述方法还包括浸泡步骤；所述浸泡步骤为：将角膜粗品连带模具一起添加至缓冲液中进行第一次浸泡，第一次浸泡结束后，打开模具，进行第二次浸泡，第二次浸泡结束后，脱模，得到角膜成品。
在本申请的一种实施方式中，交联步骤中，所述缓冲液的pH为5.5～8.0。
在本申请的一种实施方式中，交联步骤中，所述缓冲液的pH为6.5～7.5。
在本申请的一种实施方式中，交联步骤中，所述缓冲液的浓度为0.5～0.7mol/L。
在本申请的一种实施方式中，交联步骤中，所述缓冲液为MOPS Buffer、MES Buffer或PBS Buffer。
在本申请的一种实施方式中，交联步骤中，所述缓冲液中，聚乙二醇化类胶原蛋白的浓度为5～40g/mL。
在本申请的一种实施方式中，交联步骤中，所述缓冲液中，聚乙二醇化类胶原蛋白的浓度为12～18g/mL。
在本申请的一种实施方式中，交联步骤中，所述缓冲液中，聚乙二醇化类胶原蛋白的浓度为15g/mL。
在本申请的一种实施方式中，交联步骤中，所述溶解液和MPC母液的混合质量比为2:1～4:1。
在本申请的一种实施方式中，交联步骤中，所述混合液1和DMTMM母液的混合质量比为5:1～7:1。
在本申请的一种实施方式中，所述交联步骤在25～60℃下完成。
在本申请的一种实施方式中，所述交联步骤在45～55℃下完成。
在本申请的一种实施方式中，固化步骤中，所述静置的温度为4～35℃。
在本申请的一种实施方式中，固化步骤中，所述静置的温度为4～25℃。
在本申请的一种实施方式中，固化步骤中，所述静置的时间为8～20h。
在本申请的一种实施方式中，浸泡步骤中，所述缓冲液的pH为5.5～8.0。
在本申请的一种实施方式中，浸泡步骤中，所述缓冲液的pH为6.5～7.5。
在本申请的一种实施方式中，浸泡步骤中，所述缓冲液的浓度为0.05～1mol/L。
在本申请的一种实施方式中，浸泡步骤中，所述缓冲液为MOPS Buffer、MES Buffer或PBS Buffer。
在本申请的一种实施方式中，所述第一次浸泡的温度为4～35℃、时间为5～24h。
在本申请的一种实施方式中，所述第二次浸泡的温度为4～35℃、时间为3～10h。
在本申请的一种实施方式中，所述MPC母液的成分包含MPC(2-甲基丙烯酰氧乙基磷酸胆碱)、PEGDA(聚(乙二醇)二丙烯酸酯)、TEMED(N,N,N′,N′-四甲基乙二胺)以及溶剂。
在本申请的一种实施方式中，所述MPC母液中，MPC的浓度为15～40g/mL；以体积百分比计，所述MPC母液中，PEGDA的浓度为0.6～15％，TEMED的浓度为0.05～2％。
在本申请的一种实施方式中，所述MPC母液中，PEGDA的浓度为8～12％。
在本申请的一种实施方式中，所述MPC母液中，PEGDA的浓度为10％。
在本申请的一种实施方式中，所述MPC母液的溶剂为缓冲液；所述缓冲液的pH为6.0～8.0。
在本申请的一种实施方式中，MPC母液中，所述缓冲液的pH为6.5～7.5。
在本申请的一种实施方式中，MPC母液中，所述缓冲液的浓度为0.5～0.7mol/L。
在本申请的一种实施方式中，MPC母液中，所述缓冲液为MOPS Buffer、MES Buffer或PBS Buffer。
在本申请的一种实施方式中，所述DMTMM母液的成分包含DMTMM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)、APS(过硫酸铵)以及溶剂。
在本申请的一种实施方式中，所述DMTMM母液中，DMTMM的浓度为5～20g/mL，APS的浓度为0.5～5g/mL。
在本申请的一种实施方式中，所述DMTMM母液的溶剂为缓冲液；所述缓冲液的pH为6.0～8.0。
在本申请的一种实施方式中，DMTMM母液中，所述缓冲液的pH为6.5～7.5。
在本申请的一种实施方式中，DMTMM母液中，所述缓冲液的浓度为0.5～0.7mol/L。
在本申请的一种实施方式中，DMTMM母液中，所述缓冲液为MOPS Buffer、MES Buffer或PBS Buffer。
本申请还提供了一种仿生角膜，所述仿生角膜是是使用上述方法制备得到的。
本申请技术方案，具有如下优点：
1、本申请提供了一种制备聚乙二醇化类胶原蛋白的方法，所述方法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℃的条件下进行反应以制得聚乙二醇化类胶原蛋白；与现有其他制备聚乙二醇化类胶原蛋白的方法相比，使用本申请的方法制得的聚乙二醇化类胶原蛋白交联性能更佳，将使用本申请的方法制得的聚乙二醇化类胶原蛋白制成水凝胶形成角膜后，在强度上可较使用现有方法制得的聚乙二醇化类胶原蛋白提高10倍以上(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)。
并且，与现有其他制备聚乙二醇化类胶原蛋白的方法相比，使用本申请的方法制备聚乙二醇化类胶原蛋白的反应时间更短，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)。
进一步地，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；此投料摩尔数比下制得的聚乙二醇化类胶原蛋白交联性能更佳。
进一步地，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；此投料浓度下制得的聚乙二醇化类胶原蛋白交联性能更佳。
综上，本申请的方法及使用本申请的方法制得的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。
2、本申请提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法为将使用上述方法制得的聚乙二醇化类胶原蛋白和冻干保护剂混合后进行冻干以制得聚乙二醇化类胶原蛋白的冻干制剂；冻干保护剂可对使用上述方法制得的聚乙二醇化类胶原蛋白进行保护，使得使用上述方法制得的聚乙二醇化类胶原蛋白的结构在冻干过程不会被破坏，进而使得使用本申请的方法制得的冻干制剂制成水凝胶形成角膜后，强度进一步提高。
综上，本申请的方法及使用本申请的方法制得的冻干制剂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。
3、本申请提供了一种制备仿生角膜的方法，所述方法为先将使用上述方法制备得到的冻干制剂溶于MES Buffer中，得到溶解液，然后将溶解液和MPC母液混合，得 到混合液1，再将混合液1和DMTMM母液混合，得到混合液2，最后将混合液2浇注在角膜模具中进行静置，得到角膜粗品；与现有其他制备仿生角膜的方法相比，使用本申请的方法制得的角膜强度高，可较使用现有其他制备仿生角膜的方法制得的角膜提高10倍以上(文献“Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants”制得的角膜强度仅有0.022MPa)。
进一步地，所述方法将冻干制剂于pH为6.5～7.5、温度为45～55℃的条件下进行交联；此交联条件下制得的仿生角膜强度更高。
进一步地，所述方法将聚乙二醇化类胶原蛋白的浓度控制在12～18g/mL；此聚乙二醇化类胶原蛋白浓度下制得的仿生角膜强度更高。
进一步地，所述方法将MPC母液中PEGDA的浓度控制在8～12％；此PEGDA浓度下制得的仿生角膜强度更高。
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。下述实施例中的聚乙二醇衍生物均购自厦门赛诺邦格生物科技股份有限公司。
实施例1-1：一种制备聚乙二醇化类胶原蛋白的方法
本实施例提供了一种制备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(类胶原蛋白的氨基酸序列如SEQ ID NO.1所示，且类胶原蛋白的N端连接有氨基酸序列如SEQ ID NO.2所示的linker)和聚乙二醇衍生物(8-arm-PEG-40k-MAL)溶于浓度为5mmol/L的稀盐酸溶液(稀盐酸溶液的pH用pH11.0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2-1：一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法包括如下步骤：
将实施例1制得的聚乙二醇化类胶原蛋白1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1-2：一种制备聚乙二醇化类胶原蛋白的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的方法，所述方法在实施例1-1的方法的基础上，将反应温度(5℃)分别替换为：2℃、8℃、25℃、37℃，制得聚乙二醇化类胶原蛋白2～5。
实施例2-2：一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法在实施例2-1的方法的基础上，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2～5，制得冻干制剂2～5。
实施例1-3：一种制备聚乙二醇化类胶原蛋白的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的方法，所述方法在实施例1-1的方法的基础上，将反应pH(6.5)分别替换为：pH 2.5、pH 4.5、pH 5.5、pH 6.0、pH 7.0、pH 8.0、pH 8.5、pH 10.5，制得聚乙二醇化类胶原蛋白6～13。
实施例2-3：一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法在实施例2-1的方法的基础上，将聚乙二醇化类胶原蛋白1分别替换为实施例1-3制得的 聚乙二醇化类胶原蛋白6～13，制得冻干制剂6～13。
实施例1-4：一种制备聚乙二醇化类胶原蛋白的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的方法，所述方法在实施例1-1的方法的基础上，将类胶原蛋白和聚乙二醇衍生物在稀盐酸溶液中的摩尔浓度分别替换为：
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3.0mmol/L和0.375mmol/L；
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1.8mmol/L和0.225mmol/L；
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1.2mmol/L和0.15mmol/L，
制得聚乙二醇化类胶原蛋白14～16。
实施例2-4：一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法在实施例2-1的方法的基础上，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14～16，制得冻干制剂14～16。
实施例1-5：一种制备聚乙二醇化类胶原蛋白的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的方法，所述方法在实施例1-1的方法的基础上，将反应时间(12h)分别替换为：4h、8h、16h、24h，制得聚乙二醇化类胶原蛋白17～20。
实施例2-5：一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法在实施例2-1的方法的基础上，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17～20，制得冻干制剂17～20。
实施例2-6：一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法
本实施例提供了一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，所述方法在实施例2-1的方法的基础上，将冻干保护剂(甘露醇)分别替换为：蔗糖、丙氨酸， 制得冻干制剂21～22。
实验例1：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1～22分别制成角膜，得到角膜1～22。使用万能拉力机检测角膜1～22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 6.5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、10％PEGDA(v/v)、1％TEMED(v/v)，溶剂为0.5mol/L、pH 5.5的MOPS Buffer；
DMTMM母液的配方为：10g/mL DMTMM、15g/mL APS，溶剂为0.5mol/L、pH 5.5的MOPS Buffer。
由表1可知，制备工艺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，并且，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。使用实施例2-1～2-6制得的冻干制剂1～22制成的角膜1～22强度都较佳，同时提高了产品的生物相容性，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。
表1角膜1～22的强度
组别 强度(MPa) 组别 强度(MPa)
角膜1 0.083 角膜13 /(不成膜)
角膜2 0.080 角膜14 0.076
角膜3 0.075 角膜15 0.067
角膜4 0.025 角膜16 0.052
角膜5 /(不成膜) 角膜17 0.016
角膜6 /(不成膜) 角膜18 0.037
角膜7 0.019 角膜19 0.084
角膜8 0.036 角膜20 0.082
角膜9 0.074 角膜21 0.061
角膜10 0.072 角膜22 0.047
角膜11 0.062 角膜(空白对照) 0.014
角膜12 0.035    
实施例3-1：一种制备仿生角膜的方法
本实施例提供了一种制备仿生角膜方法，所述方法包括如下步骤：
交联步骤：将实施例2-1制得的冻干制剂溶于浓度为0.5mol/L、pH 6.5的MES Buffer中，使得MES Buffer中，聚乙二醇化类胶原蛋白的浓度为15g/mL，得到溶解液；按照质量比4:1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 6.5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，得到仿生角膜23；
其中，MPC母液的配方为：30％MPC(w/v，g/mL)、10％PEGDA(v/v)、1％TEMED(v/v)，溶剂为0.5mol/L、pH 6.5的MES Buffer；
DMTMM母液的配方为：10％DMTMM(w/v，g/mL)、15％APS(w/v，g/mL)，溶剂为0.5mol/L、pH 6.5的MES Buffer。
实施例3-2：一种制备仿生角膜的方法
本实施例提供了一种制备仿生角膜方法，所述方法在实施例3-1的方法的基础上，将交联温度(45℃)分别替换为：20℃、25℃、40℃、50℃、55℃、60℃、65℃，制得仿生角膜24～30。
实施例3-3：一种制备仿生角膜的方法
本实施例提供了一种制备仿生角膜方法，所述方法在实施例3-1的方法的基础上，将Buffer的pH(6.5)分别替换为：5、5.5、6、7、7.5、8、8.5、9，制得仿生角膜31～38。
实施例3-4：一种制备仿生角膜的方法
本实施例提供了一种制备仿生角膜方法，所述方法在实施例3-1的方法的基础上，将MES Buffer中聚乙二醇化类胶原蛋白的浓度(15g/mL)分别替换为：1g/mL、5g/mL、10g/mL、20g/mL、25g/mL、30g/mL、35g/mL、40g/mL、45g/mL，制得仿生角膜39～47。
实施例3-5：一种制备仿生角膜的方法
本实施例提供了一种制备仿生角膜方法，所述方法在实施例3-1的方法的基础上，将MPC母液中PEGDA的浓度(10％，v/v)分别替换为：0.5％、0.6％、1％、5％、8％、12％、15％(v/v)，制得仿生角膜48～54。
实验例2：制备工艺对角膜强度性能的影响实验
本实验例提供了制备工艺对角膜强度性能的影响实验，实验过程如下：
使用万能拉力机检测角膜23～54的强度，检测结果见表2。
由表2可知，制备工艺会对角膜的强度性能造成影响，其中，聚乙二醇化类胶原蛋白的浓度太低会导致角膜强度变差，浓度太高导致角膜固化时间极短，无法完成后面的浇注步骤；由于聚乙二醇化类胶原蛋白的等电点为偏中性，当交联pH远离等电点时，参与反应的基团无法彼此靠近，会导致角膜强度变低或聚乙二醇化类胶原蛋白不发生凝胶的现象；聚乙二醇化类胶原蛋白制成的水凝胶为温敏型水凝胶，温度低时，其溶解度不好，会导致角膜固形物浓度偏低强度变小，温度高时，会导致其交联速度极快，进而导致无法浇注到模具中，提前成胶；MPC母液中PEGDA的浓度过低会导致角膜强度变差，浓度过高会导致聚乙二醇化类胶原蛋白过度交联，使聚乙二醇化类胶原蛋白制成的水凝胶脆性增加、韧性降低，进而使角膜强度变差。表1中，角膜23、25～29、32～36、40～45以及50～54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2角膜23～54的强度
组别 强度(MPa) 组别 强度(MPa)
角膜23 0.271 角膜39 /(不成膜)
角膜24 /(不成膜) 角膜40 0.097
角膜25 0.093 角膜41 0.125
角膜26 0.201 角膜42 0.232
角膜27 0.255 角膜43 0.213
角膜28 0.196 角膜44 0.179
角膜29 0.154 角膜45 0.163
角膜30 /(不成膜) 角膜46 /(不成膜)
角膜31 0.046 角膜47 /(不成膜)
角膜32 0.082 角膜48 0.024
角膜33 0.169 角膜49 0.037
角膜34 0.213 角膜50 0.109
角膜35 0.201 角膜51 0.143
角膜36 0.133 角膜52 0.206
角膜37 /(不成膜) 角膜53 0.211
角膜38 /(不成膜) 角膜54 0.213
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. 一种制备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6.0～8.0、温度为2～8℃的条件下进行反应，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。
  2. 如权利要求1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。
  3. 如权利要求2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。
  4. 如权利要求1～3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。
  5. 如权利要求4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。
  6. 如权利要求5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。
  7. 如权利要求1～3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。
  8. 如权利要求7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。
  9. 如权利要求1～3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。
  10. 如权利要求9所述的方法，其特征在于，所述过滤为透析或超滤。
  11. 如权利要求1～3任一项所述的方法，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。
  12. 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是使用权利要求1～11任一项所述的方法制备得到的。
  13. 一种制备聚乙二醇化类胶原蛋白的冻干制剂的方法，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1～11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。
  14. 如权利要求13所述的方法，其特征在于，所述冻干步骤为：将聚乙二醇化类胶原蛋白和冻干保护剂混合后进行冻干，得到冻干制剂。
  15. 如权利要求14所述的方法，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。
  16. 如权利要求13～15任一项所述的方法，其特征在于，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。
  17. 一种聚乙二醇化类胶原蛋白的冻干制剂，其特征在于，所述冻干制剂是是使用权利要求13～16任一项所述的方法制备得到的。
  18. 权利要求1～11任一项所述的方法制备得到的聚乙二醇化类胶原蛋白或权利要求12所述的聚乙二醇化类胶原蛋白或权利要求13～16任一项所述的方法制备得到的冻干制剂或权利要求17所述的冻干制剂在制备仿生或再生生物材料中的应用。
  19. 如权利要求18所述的应用，其特征在于，所述仿生或再生生物材料为角膜。
  20. 一种制备仿生角膜的方法，其特征在于，所述方法包括如下步骤：
    交联步骤：将使用权利要求13～16任一项所述的方法制备得到的冻干制剂溶于缓冲液中，得到溶解液；将溶解液和MPC母液混合，得到混合液1；将混合液1和DMTMM母液混合，得到混合液2；
    固化步骤：将混合液2浇注在角膜模具中进行静置，得到角膜粗品。
  21. 如权利要求20所述的方法，其特征在于，固化步骤后，所述方法还包括浸泡步骤；所述浸泡步骤为：将角膜粗品连带模具一起添加至缓冲液中进行第一次浸泡，第一次浸泡结束后，打开模具，进行第二次浸泡，第二次浸泡结束后，脱模，得到角膜成品。
  22. 如权利要求20或21所述的方法，其特征在于，交联步骤中，所述缓冲液的pH为5.5～8.0。
  23. 如权利要求20或21所述的方法，其特征在于，交联步骤中，所述缓冲液中，聚乙二醇化类胶原蛋白的浓度为5～40g/mL。
  24. 如权利要求20或21所述的方法，其特征在于，交联步骤中，所述溶解液和MPC母液的混合质量比为2:1～4:1。
  25. 如权利要求20或21所述的方法，其特征在于，交联步骤中，所述混合液1和 DMTMM母液的混合质量比为5:1～7:1。
  26. 如权利要求20或21所述的方法，其特征在于，所述交联步骤在25～60℃下完成。
  27. 如权利要求20或21所述的方法，其特征在于，固化步骤中，所述静置的温度为4～35℃。
  28. 如权利要求20或21所述的方法，其特征在于，浸泡步骤中，所述缓冲液的pH为5.5～8.0。
  29. 如权利要求21所述的方法，其特征在于，所述第一次浸泡的温度为4～35℃、时间为5～24h。
  30. 如权利要求21所述的方法，其特征在于，所述第二次浸泡的温度为4～35℃、时间为3～10h。
  31. 如权利要求20或21所述的方法，其特征在于，所述MPC母液的成分包含MPC、PEGDA、TEMED以及溶剂。
  32. 如权利要求31所述的方法，其特征在于，所述MPC母液中，MPC的浓度为15～40g/mL；以体积百分比计，所述MPC母液中，PEGDA的浓度为0.6～15％，TEMED的浓度为0.05～2％。
  33. 如权利要求31所述的方法，其特征在于，所述MPC母液的溶剂为缓冲液；所述缓冲液的pH为6～8。
  34. 如权利要求20或21所述的方法，其特征在于，所述DMTMM母液的成分包含DMTMM、APS以及溶剂。
  35. 如权利要求34所述的方法，其特征在于，所述DMTMM母液中，DMTMM的浓度为5～20g/mL，APS的浓度为0.5～5g/mL。
  36. 如权利要求34所述的方法，其特征在于，所述DMTMM母液的溶剂为缓冲液；所述缓冲液的pH为6.0～8.0。
  37. 一种仿生角膜，其特征在于，所述仿生角膜是是使用权利要求20～36任一项所述的方法制备得到的。
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