Pegylated collagen and preparation method and application thereof

文档序号：335743 发布日期：2021-12-03 浏览：28次中文

阅读说明：本技术 一种聚乙二醇化类胶原蛋白及其制备方法和应用 (Pegylated collagen and preparation method and application thereof ) 是由张加慧王崇宇刘沐荣张军何超先于 2021-09-13 设计创作，主要内容包括：本发明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。本发明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,使得使用本发明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。(The invention relates to a polyethylene glycol collagen and a preparation method and application thereof, belonging to the technical field of biology. The invention provides a PEGylated collagen, which comprises collagen, PEG-40k and PEG-20k modified on the collagen; the modification of PEG-40k and PEG-20k can obviously improve the crosslinking performance of collagen-like protein, so that the strength of the cornea prepared by using the polyethylene glycol collagen-like protein can be improved by nearly 20 times compared with the strength of the cornea prepared by using collagen-like protein, and is improved by nearly 12 times compared with the strength of the cornea prepared by using other existing polyethylene glycol collagen-like proteins, therefore, the polyethylene glycol collagen-like protein has extremely high application prospect in the preparation of bionic or regeneration biological materials such as bionic corneas and the like.)

1. The polyethylene glycol collagen is characterized by comprising collagen-like protein and polyethylene glycol derivatives modified on the collagen-like protein; the polyethylene glycol derivative comprises PEG-40k and PEG-20 k.

2. The pegylated collagen according to claim 1, wherein said polyethylene glycol derivative consists of PEG-40k and PEG-20 k.

3. The pegylated collagen according to claim 1, wherein said collagen has the amino acid sequence as shown in SEQ ID No. 1.

4. The pegylated collagen according to any one of claims 1 to 3, wherein the molar ratio of PEG-40k to PEG-20k is from 0.5 to 6: 1.

5. The pegylated collagen according to any one of claims 1 to 3, wherein the molar ratio of PEG-40k to PEG-20k is from 2 to 3: 1.

6. The PEGylated collagen according to any one of claims 1 to 3, wherein the number of the activating groups of PEG-40k is one or more than one of 8-arm, 4-arm, 2-arm and 1-arm; the number of the activated groups of the PEG-20k is one or more than one of 8-arm, 4-arm, 2-arm and 1-arm.

7. The pegylated collagen according to claim 6, wherein said PEG-40k has an activating group number of 4-arm or 8-arm; the number of the activating groups of the PEG-20k is 4-arm or 8-arm.

8. The pegylated collagen according to any one of claims 1 to 3, wherein the activating group of PEG-40k is one or more of-MAL, -NHS, -SG, -SPA, -SS or-EDC; the activating group of the PEG-20k is one or more than one of-MAL, -NHS, -SG, -SPA, -SS or-EDC.

9. The pegylated collagen according to claim 8, wherein said PEG-40k activating group is-MAL; the activating group of the PEG-20k is-MAL.

10. The pegylated collagen according to any one of claims 1 to 3, wherein a linker is attached to one end of said collagen; the polyethylene glycol derivative is modified on a linker through an activating group.

11. The pegylated collagen-like protein according to claim 10, wherein said linker is attached to the N-terminus of the collagen-like protein.

12. The pegylated collagen according to claim 10, wherein said linker has an amino acid sequence as set forth in SEQ ID No.2 or SEQ ID No. 3.

13. The pegylated collagen according to claim 12, wherein the modification site of the polyethylene glycol derivative on the linker is one or more of thiol, amino, carboxyl or imidazolyl.

14. The pegylated collagen according to claim 13, wherein when the amino acid sequence of the linker is as shown in SEQ ID No.2, the modification site of said polyethylene glycol derivative on the linker is thiol.

15. The pegylated collagen according to claim 13 or 14, wherein when the amino acid sequence of the linker is as shown in SEQ ID No.3, the modification site of said polyethylene glycol derivative on the linker is an amino group.

16. The pegylated collagen according to any one of claims 1 to 3, wherein the pegylated collagen has a mother nucleus conformation of one or more of HG and TP.

17. The pegylated collagen according to any one of claims 1 to 3, wherein the peg derivative has a core conformation of TP.

18. The pegylated collagen according to any one of claims 1 to 3, wherein the amino acid configuration of the pegylated collagen is in one or more of D-form or L-form.

19. The pegylated collagen according to any one of claims 1 to 3, wherein said pegylated collagen has a molecular weight of 15000 to 75000 Da.

20. The pegylated collagen according to claim 19, wherein said pegylated collagen has a molecular weight of from 30000Da to 75000 Da.

21. A lyophilized preparation obtained by lyophilizing the pegylated collagen according to any one of claims 1 to 20.

22. The lyophilized formulation of claim 21, wherein the lyophilized formulation is prepared by mixing the pegylated collagen with a lyoprotectant and then lyophilizing.

23. The lyophilized formulation of claim 22, wherein the lyoprotectant is one or more of mannitol, sucrose, or alanine.

24. A method for preparing the pegylated collagen-like protein of any one of claims 1 to 20, comprising the steps of:

the reaction steps are as follows: reacting the collagen-like protein and the polyethylene glycol derivative for 1-48 hours under the conditions that the pH is 4.0-10.0 and the temperature is 2-40 ℃ to obtain a reaction product; the reaction product contains the polyethylene glycol collagen of any one of claims 1 to 20.

25. The method of claim 24, wherein the reacting step is: and reacting the collagen-like protein and the polyethylene glycol derivative for 5-8 hours under the conditions that the pH is 6.0-8.0 and the temperature is 2-8 ℃.

26. The method of claim 24, wherein in the reacting step, the collagen-like protein and the polyethylene glycol derivative are fed in a molar ratio of 1-16: 1.

27. The method of claim 26, wherein in the reacting step, the collagen-like protein and the polyethylene glycol derivative are fed in a molar ratio of 8-12: 1.

28. The method according to any one of claims 24 to 26, wherein in the reacting step, the reaction solvent for the collagen-like protein and the polyethylene glycol derivative is water or a dilute hydrochloric acid solution.

29. The method of claim 28, wherein the dilute hydrochloric acid solution has a concentration of 1 to 10 mmol/L; and the pH of the dilute hydrochloric acid solution is adjusted to 6.0-8.0 by using a dilute alkali solution.

30. The method of claim 28, wherein the dilute alkaline solution is a sodium hydroxide solution or ammonia solution having a pH of 9.0 to 11.0.

31. The method according to any one of claims 24 to 26, wherein in the reacting step, the concentration of the collagen-like protein in the reaction solvent is 1 to 15 mg/mL.

32. The method of claim 31, wherein in the reacting step, the collagen-like protein is fed to the reaction solvent at a concentration of 8-10 mg/mL.

33. The method of any one of claims 24 to 26, wherein after the reacting step, the method further comprises a purifying step; the purification steps are as follows: and filtering and intercepting substances with the molecular weight more than or equal to 30000Da in the reaction product to obtain the pegylated collagen.

34. The method of claim 33, wherein the filtration is dialysis or ultrafiltration.

35. A method of preparing a lyophilized formulation according to any one of claims 21 to 23, comprising the steps of:

a freeze-drying step: freeze-drying the pegylated collagen prepared by the method of any one of claims 24 to 34 to obtain a freeze-dried formulation.

36. Use of a pegylated collagen according to any one of claims 1 to 20 or a lyophilized formulation according to any one of claims 21 to 23 or a pegylated collagen prepared according to the method of any one of claims 24 to 34 or a lyophilized formulation prepared according to the method of claim 35 for the preparation of a biomimetic or regenerative biomaterial.

37. The use of claim 36, wherein the biomimetic or regenerative biological material is a cornea.

Technical Field

The invention relates to a polyethylene glycol collagen and a preparation method and application thereof, belonging to the technical field of biology.

Background

The Cornea (Cornea) is the most anterior convex highly transparent substance of the eye, is in the shape of a transverse ellipse, covers the iris, pupil and anterior chamber, and provides the eye with most of its refractive power. With the refractive power of the crystal, the light can be focused on the retina accurately to form an image. The cornea has very sensitive nerve endings, and if a foreign object contacts the cornea of the eye, the eyelids can be involuntarily closed to protect the eye. To maintain transparency, the cornea has no blood vessels and nutrients and oxygen are obtained through the tears and aqueous humor.

The cornea is very fragile, and the cornea is affected by trauma, inflammation, anaphylaxis, physical injury, chemical burn, strenuous exercise, overuse of the eye and the like. Once the cornea is diseased, it can cause significant ocular symptoms such as pain, photophobia, lacrimation, vision loss, and the like, and can also lead to blindness.

The cornea transplantation is a treatment method for replacing the existing pathological cornea of a patient with a normal cornea to restore the vision of an affected eye or control the pathological change of the cornea so as to achieve the purpose of improving the vision or treating certain corneal diseases. Some corneal diseases, which cause severe visual impairment and even blindness in patients, can be completely treated by corneal transplantation, helping these unfortunate patients to go away from suffering. Because the cornea itself is vascular-free, at the "immune privilege" level, the success rate of corneal transplantation is at the forefront of transplantation of other allogeneic organs.

However, corneal resources are limited and far from meeting patient needs. In order to solve this problem, artificial keratoplasty has been proposed by researchers. Artificial cornea transplantation is a surgical method for restoring vision by implanting it into corneal tissue to replace part of corneal scar tissue by using a special optical device made of transparent medical high-molecular material. Since the problems of rejection of corneal tissues to synthetic materials and the like are not solved yet, the long-term effect is not good, and the leakage of aqueous humor at the transplanted site and the falling of the transplanted piece are often caused, the wide application is not possible at present. The artificial cornea at the present stage is only suitable for binocular blindness after suffering from various serious corneal diseases, particularly for all corneal leukoplakias and multiple times of corneal transplantation failures caused by serious chemical burns, and other operators can not be done any more.

On the basis of artificial cornea, researchers have proposed a bionic cornea. For example, in the document "Short peptide analogues as alternative to collagen in pro-regenerative cornea implants", jangmardy, janamohan r. The bionic cornea has excellent biocompatibility, and can effectively solve the problems of strong rejection reaction of corneal tissues to artificially synthesized materials and the like.

However, the strength of the bionic cornea is low, only 0.022MPa, and if the bionic cornea is used for corneal transplantation, the problems of great difficulty in transplantation, easy generation of corneal conicity and the like exist.

Disclosure of Invention

In order to solve the problem of low strength of the existing bionic cornea, the invention provides a polyethylene glycol collagen, which comprises collagen-like protein and polyethylene glycol derivatives modified on the collagen-like protein; the polyethylene glycol derivative comprises PEG-40k and PEG-20 k.

In one embodiment of the present invention, the polyethylene glycol derivative consists of PEG-40k and PEG-20 k.

In one embodiment of the invention, the amino acid sequence of the collagen-like protein is shown in SEQ ID NO. 1. In SEQ ID NO.1, X is 4Hyp (4-hydroxyproline), namely:

the amino acid sequence of SEQ ID NO.1 is:

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

in one embodiment of the invention, the molar ratio of PEG-40k to PEG-20k is 0.5-6: 1.

In one embodiment of the invention, the molar ratio of PEG-40k to PEG-20k is 2-3: 1.

In one embodiment of the invention, the number of the activating groups of the PEG-40k is one or more than one of 8-arm, 4-arm, 2-arm and 1-arm; the number of the activated groups of the PEG-20k is one or more than one of 8-arm, 4-arm, 2-arm and 1-arm.

In one embodiment of the invention, the number of activating groups of the PEG-40k is 4-arm or 8-arm; the number of the activating groups of the PEG-20k is 4-arm or 8-arm.

In one embodiment of the invention, the activating group of PEG-40k is one or more of-MAL, -NHS, -SG, -SPA, -SS or-EDC; the activating group of the PEG-20k is one or more than one of-MAL, -NHS, -SG, -SPA, -SS or-EDC.

In one embodiment of the invention, the activating group of the PEG-40k is-MAL; the activating group of the PEG-20k is-MAL.

In one embodiment of the invention, one end of the collagen-like protein is connected with a linker; the polyethylene glycol derivative is modified on a linker through an activating group.

In one embodiment of the invention, the linker is attached to the N-terminus of the collagen-like protein.

In one embodiment of the invention, the amino acid sequence of the linker is shown as SEQ ID NO.2 or SEQ ID NO. 3.

In one embodiment of the invention, the modification site of the polyethylene glycol derivative on the linker is one or more of a sulfhydryl group, an amino group, a carboxyl group or an imidazolyl group.

In one embodiment of the invention, when the amino acid sequence of the linker is shown as SEQ ID NO.2, the modification site of the polyethylene glycol derivative on the linker is a sulfydryl group; when the amino acid sequence of the linker is shown as SEQ ID NO.3, the modification site of the polyethylene glycol derivative on the linker is an amino group.

In one embodiment of the present invention, the mother nucleus conformation of the pegylated collagen is one or more of HG or TP.

In one embodiment of the present invention, the conformation of the mother nucleus of the polyethylene glycol derivative in the pegylated collagen is TP.

In one embodiment of the present invention, 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.

In one embodiment of the invention, the molecular weight of the pegylated collagen is 15000-75000 Da.

In one embodiment of the invention, the molecular weight of the pegylated collagen is 30000-75000 Da.

The invention also provides a freeze-dried preparation which is obtained by freeze-drying the polyethylene glycol collagen.

In an embodiment of the present invention, the lyophilized preparation is obtained by mixing the pegylated collagen with a lyoprotectant and then lyophilizing.

In one embodiment of the present invention, the lyoprotectant is one or more of mannitol, sucrose, or alanine.

The invention also provides a method for preparing the polyethylene glycol collagen, which comprises the following steps:

In one embodiment of the present invention, the reaction step is: and reacting the collagen-like protein and the polyethylene glycol derivative for 5-8 hours under the conditions that the pH is 6.0-8.0 and the temperature is 2-8 ℃.

In one embodiment of the invention, in the reaction step, the feeding molar ratio of the collagen-like protein to the polyethylene glycol derivative is 1-16: 1.

In one embodiment of the invention, in the reaction step, the feeding molar ratio of the collagen-like protein to the polyethylene glycol derivative is 8-12: 1.

In one embodiment of the present invention, in the reacting step, the reaction solvent of the collagen-like protein and the polyethylene glycol derivative is water or a dilute hydrochloric acid solution.

In one embodiment of the invention, the concentration of the dilute hydrochloric acid solution is 1-10 mmol/L; and the pH of the dilute hydrochloric acid solution is adjusted to 6.0-8.0 by using a dilute alkali solution.

In one embodiment of the present invention, the dilute alkali solution is a sodium hydroxide solution or ammonia water with a pH of 9.0 to 11.0.

In one embodiment of the present invention, in the reaction step, the feeding concentration of the collagen-like protein in the reaction solvent is 1-15 mg/mL.

In one embodiment of the present invention, in the reaction step, the feeding concentration of the collagen-like protein in the reaction solvent is 8-10 mg/mL.

In one embodiment of the invention, after the reacting step, the method further comprises a purifying step; the purification steps are as follows: and filtering and intercepting substances with the molecular weight more than or equal to 30000Da in the reaction product to obtain the pegylated collagen.

In one embodiment of the invention, the filtration is dialysis or ultrafiltration.

The invention also provides a method for preparing the freeze-dried preparation, which comprises the following steps:

a freeze-drying step: and (4) freeze-drying the reaction product to obtain a freeze-dried preparation.

In one embodiment of the invention, in the reaction step, the feeding molar ratio of the collagen-like protein to the polyethylene glycol derivative is 1-16: 1.

In one embodiment of the invention, in the reaction step, the feeding molar ratio of the collagen-like protein to the polyethylene glycol derivative is 8-12: 1.

In one embodiment of the present invention, the dilute alkali solution is a sodium hydroxide solution or ammonia water with a pH of 9.0 to 11.0.

In one embodiment of the present invention, in the reaction step, the feeding concentration of the collagen-like protein in the reaction solvent is 1-15 mg/mL.

In one embodiment of the present invention, in the reaction step, the feeding concentration of the collagen-like protein in the reaction solvent is 8-10 mg/mL.

In one embodiment of the present invention, the lyophilization step is: and mixing the reaction product with a freeze-drying protective agent, and freeze-drying to obtain a freeze-dried preparation.

In one embodiment of the present invention, the lyoprotectant is one or more of mannitol, sucrose, or alanine.

In one embodiment of the invention, the lyophilization comprises the following stages:

stage one: lyophilizing at-45 deg.C and vacuum degree (vacuum) of 500m Torr for 6 hr;

and a second stage: freeze-drying at-30 deg.C and vacuum degree (vacuum) of 100m Torr for 17 hr;

and a third stage: lyophilizing at 25 deg.C and vacuum degree (vacuum) of 100m Torr for 7 hr.

In one embodiment of the present invention, the method further comprises a purification step after the reaction step and before the lyophilization step; the purification steps are as follows: and filtering and intercepting substances with the molecular weight more than or equal to 30000Da in the reaction product to obtain the pegylated collagen.

In one embodiment of the invention, the filtration is dialysis or ultrafiltration.

The invention also provides the application of the pegylated collagen or the freeze-dried preparation or the pegylated collagen prepared by the method or the freeze-dried preparation prepared by the method in preparing bionic or regenerative biomaterials.

In one embodiment of the invention, the biomimetic or regenerative biological material is a biomimetic cornea.

The technical scheme of the invention has the following advantages:

1. the invention provides a PEGylated collagen, which comprises collagen, PEG-40k and PEG-20k modified on the collagen; modification of PEG-40k and PEG-20k can obviously improve the crosslinking performance of collagen-like protein, so that the strength of the cornea prepared by using the pegylated collagen protein can be improved by nearly 20 times compared with the strength of the cornea prepared by using collagen-like protein, and the strength of the cornea prepared by using other existing pegylated collagen proteins is improved by nearly 12 times (the strength of the cornea prepared by using the pegylated collagen protein in the literature' Short peptide analogues to collagen in pro-regenerative cornea is only 0.022MPa), therefore, the pegylated collagen protein has extremely high application prospect in the preparation of bionic or regenerative biological materials such as bionic cornea and the like.

Further, the molar ratio of the PEG-40k to the PEG-20k is 2-3: 1; the effect of improving the cross-linking performance of the collagen-like protein by PEG modification under the molar ratio is better.

Further, the PEG-40k is 8-arm-PEG-40k-MAL, and the PEG-20k is 4-arm-PEG-20 k-MAL; the effect of improving the cross-linking performance of the collagen-like protein by PEG modification under the formula is better.

2. The invention provides a freeze-dried preparation, which is prepared by mixing the polyethylene glycol collagen and a freeze-drying protective agent and then freeze-drying the mixture; the freeze-drying protective agent can protect the pegylated collagen, so that the structure of the pegylated collagen cannot be damaged in the freeze-drying process, and the strength of the cornea prepared by using the freeze-drying preparation is further improved.

3. The invention provides a preparation method of the polyethylene glycol collagen, which takes a dilute hydrochloric acid solution with the concentration of 1-10 mmol/L, pH of 6.0-8.0 as a reaction solvent, and the polyethylene glycol collagen is prepared by reacting the collagen with PEG for 5-8 hours at the temperature of 2-8 ℃; the crosslinking performance of the polyethylene glycol collagen prepared under the reaction condition is better, and compared with other existing preparation methods of polyethylene glycol collagen, the preparation method provided by the invention has shorter reaction time, and can obtain the polyethylene glycol collagen capable of preparing hydrogel-forming cornea only by reacting for 5-8 hours (in the literature, "Short peptide collagen as an analog to collagen in pro-regenerative cornea", the polyethylene glycol collagen capable of preparing hydrogel-forming cornea needs to react for 4 weeks).

4. The invention provides a preparation method of the freeze-dried preparation, which is used for freeze-drying the pegylated collagen under the protection of a freeze-drying protective agent; the freeze-drying protective agent can protect the pegylated collagen, so that the structure of the pegylated collagen cannot be damaged in the freeze-drying process, and the cross-linking performance of the freeze-drying preparation is better.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially. The synthesis of collagen-like protein and the linkage between collagen-like protein and linker in the following examples were performed by Shanghanbo Biotechnology, Inc. (AmbioPharm. PEG was purchased from Xiamen Nippon Biotech, Inc. in the following examples.

Example 1-1: pegylated collagen and preparation thereof

The embodiment provides a PEGylated collagen, which consists of a collagen-like protein with an amino acid sequence shown as SEQ ID No.1, a linker with an amino acid sequence shown as SEQ ID No.2 and connected to the N end of the collagen-like protein, and 8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL which are modified on the thiol group of the linker; wherein the molar ratio of the 8-arm-PEG-40k-MAL to the 4-arm-PEG-20k-MAL is 2:1, the parent nuclear conformation of the polyethylene glycol derivative in the polyethylene glycol collagen is TP, and the molecular weight of the polyethylene glycol collagen is 30000-75000 Da. The pegylated collagen was named pegylated collagen 1.

The preparation method of the pegylated collagen 1 comprises the following steps:

the reaction steps are as follows: dissolving collagen-like protein and polyethylene glycol derivatives in a dilute hydrochloric acid solution with the concentration of 5mmol/L (the pH of the dilute hydrochloric acid solution is adjusted to 6.5 by using a sodium hydroxide solution with the pH of 11.0), so that the molar concentrations of the collagen-like protein and the polyethylene glycol derivatives in the dilute hydrochloric acid solution are respectively 2.4mmol/L and 0.3mmol/L, and obtaining a reaction system; reacting the reaction system for 12 hours under the conditions that the pH is 6.5 and the temperature is 5 ℃ to obtain a reaction product;

a purification step: and (3) carrying out ultrafiltration on the reaction product at 5 ℃ by using an ultrafiltration membrane with the aperture of 30000Da, and intercepting substances with the molecular weight of more than or equal to 30000Da in the reaction product to obtain the pegylated collagen 1.

Example 2-1: freeze-dried preparation and preparation thereof

This example provides a lyophilized formulation consisting 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 freeze-dried preparation 1 comprises the following steps:

mixing the polyethylene glycol collagen prepared in the example 1 and mannitol according to the mass ratio of 1:5 to obtain a freeze-drying system; freeze-drying the freeze-drying system to obtain a freeze-dried preparation 1;

wherein, the freeze-drying comprises the following stages:

stage one: lyophilizing at-45 deg.C and vacuum degree (vacuum) of 500m Torr for 6 hr;

and a second stage: freeze-drying at-30 deg.C and vacuum degree (vacuum) of 100m Torr for 17 hr;

and a third stage: lyophilizing at 25 deg.C and vacuum degree (vacuum) of 100m Torr for 7 hr.

Examples 1 to 2: pegylated collagen and preparation thereof

This example provides a pegylated collagen, which is obtained by replacing the PEG formulations (8-arm-PEG-40 k-MAL and 4-arm-PEG-20k-MAL in a molar ratio of 2: 1) used with the following PEG formulations on the basis of the pegylated collagen 1 of example 1-1:

8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL in a molar ratio of 1: 0;

8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL in a molar ratio of 0: 1;

8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL in a molar ratio of 6: 1;

8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL in a molar ratio of 4: 1;

8-arm-PEG-40k-MAL and 4-arm-PEG-40k-MAL in a molar ratio of 2: 1;

8-arm-PEG-40k-MAL, 4-arm-PEG-20k-MAL and 4-arm-PEG-40k-MAL in a molar ratio of 4:2: 1;

8-arm-PEG-40k-MAL, 4-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL in a molar ratio of 2:2: 1;

8-arm-PEG-40k-MAL, 4-arm-PEG-40k-MAL and 8-arm-PEG-20k-MAL in a molar ratio of 2:2: 1.

The polyethylene glycol collagen is named as polyethylene glycol collagen 2-9 in sequence.

The preparation method of the pegylated collagen 2-9 is the same as that of the pegylated collagen 1.

Example 2-2: freeze-dried preparation and preparation thereof

The embodiment provides a freeze-dried preparation, and on the basis of the freeze-dried preparation 1 in the embodiment 2-1, the pegylated collagen 1 is replaced by the pegylated collagens 2-9 prepared in the embodiment 1-2.

The freeze-dried preparations are named freeze-dried preparations 2-9 in sequence.

The preparation method of the freeze-dried preparation 2-9 is the same as that of the freeze-dried preparation 1.

Examples 1 to 3: pegylated collagen and preparation thereof

The embodiment provides a PEGylated collagen, which consists of a collagen-like protein with an amino acid sequence shown as SEQ ID No.1, a linker with an amino acid sequence shown as SEQ ID No.3 and connected to the N end of the collagen-like protein, and 8-arm-PEG-40k-MAL and 4-arm-PEG-20k-MAL modified on the amino group of the linker; wherein the molar ratio of the 8-arm-PEG-40k-MAL to the 4-arm-PEG-20k-MAL is 2:1, the parent nuclear conformation of the polyethylene glycol derivative in the polyethylene glycol collagen is TP, and the molecular weight of the polyethylene glycol collagen is 30000-75000 Da. This pegylated collagen was named pegylated collagen 10.

The preparation method of the polyethylene glycol collagen 10 is the same as that of the polyethylene glycol collagen 1.

Examples 2 to 3: freeze-dried preparation and preparation thereof

This example provides a lyophilized preparation in which the pegylated collagen-like protein 1 was replaced with the pegylated collagen-like protein 10 prepared in examples 1-3, based on the lyophilized preparation 1 of example 2-1.

The above lyophilized preparation was sequentially named lyophilized preparation 10.

The preparation method of the lyophilized preparation 10 is the same as that of the lyophilized preparation 1.

Examples 1 to 4: pegylated collagen and preparation thereof

This example provides a pegylated collagen, which is prepared by replacing the temperature (5 ℃) in the preparation method with the following steps on the basis of the pegylated collagen 1 of example 1-1: 2 ℃, 8 ℃, 25 ℃ and 37 ℃.

The polyethylene glycol collagen is sequentially named as polyethylene glycol collagen 11-14.

Examples 2 to 4: freeze-dried preparation and preparation thereof

The embodiment provides a freeze-dried preparation, and on the basis of the freeze-dried preparation 1 in the embodiment 2-1, the pegylated collagen 1 is respectively replaced by the pegylated collagens 11 to 14 prepared in the embodiment 1-4.

The freeze-dried preparations are named as freeze-dried preparations 11-14 in sequence.

The preparation method of the freeze-dried preparations 11-14 is the same as that of the freeze-dried preparation 1.

Examples 1 to 5: pegylated collagen and preparation thereof

This example provides a pegylated collagen, which is prepared by replacing the pH (6.5) in the preparation method with the following pH (6.5) based on the pegylated collagen 1 of example 1-1: pH 2.5, pH 4.5, pH 6.0, pH 6.5, pH 7.0, pH 8.0, pH 8.5, pH 10.5.

The polyethylene glycol collagen is named as 15-22 polyethylene glycol collagen in sequence.

Examples 2 to 5: freeze-dried preparation and preparation thereof

The freeze-dried preparations are named as freeze-dried preparations 15-22 in sequence.

The preparation method of the freeze-dried preparation 15-22 is the same as that of the freeze-dried preparation 1.

Examples 2 to 6: freeze-dried preparation and preparation thereof

This example provides a lyophilized formulation, which is prepared by replacing the lyoprotectant (mannitol) in the preparation method with: sucrose and alanine.

The freeze-dried preparations are named as freeze-dried preparations 23-24 in sequence.

Experimental example 1: experiment for influence of PEG formula and preparation process on crosslinking performance of polyethylene glycol collagen and freeze-dried preparation thereof

The experimental example provides an experiment for the influence of the PEG formula and the preparation process on the crosslinking performance of the PEGylated collagen and the lyophilized preparation thereof, and the experimental process comprises the following steps:

with reference to the preparation method of the lyophilized preparation 1, the collagen-like protein with the amino acid sequence shown as SEQ ID No.1 is directly prepared into the lyophilized preparation, and the lyophilized preparation is used as a blank control, and the lyophilized preparations 1 to 24 prepared in examples 2-1 to 2-6 are respectively prepared into corneas to obtain the corneas 1 to 24. Detecting the strength of the cornea by 1-24 by using a universal tension machine, wherein the detection result is shown in a table 1;

wherein, the preparation process of the cornea comprises the following steps:

a crosslinking step: dissolving the freeze-dried preparation in MES Buffer with the concentration of 0.5mol/L, pH 5.5.5 to ensure that the concentration of the polyethylene glycol collagen in the MES Buffer is 12.5g/mL to obtain a dissolved solution; mixing the dissolved solution and the MPC mother solution according to the mass ratio of 5:1 to obtain a mixed solution 1; mixing the mixed solution 1 with the DMTMM mother solution according to the mass ratio of 7:1 to obtain a mixed solution 2; the whole cross-linking process is completed at 45 ℃;

and (3) curing: pouring the mixed solution 2 into a cornea mold, and standing at 25 ℃ for 12h to obtain a cornea crude product;

soaking: adding the cornea crude product (with a mould) into PBS Buffer with the concentration of 0.1mol/L, pH 5.5.5, soaking for 24h at 4 ℃, opening the mould, continuing to soak for 4h at 4 ℃, and demoulding to obtain a cornea finished product;

wherein, the formula of the MPC mother liquor is as follows: 30g/mL MPC (2-methacryloyloxyethyl phosphorylcholine), 10% PEGDA (poly (ethylene glycol) diacrylate, v/v), 1% TEMED (N, N, N ', N' -tetramethylethylenediamine, v/v), solvent 0.5mol/L, pH 5.5.5 MOPS Buffer;

the formula of the DMTMM mother solution is as follows: 10g/mL DMTMM (4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride), 15g/mL APS (ammonium persulfate) in a MOPS Buffer at 0.5mol/L, pH 5.5.5.

As can be seen from table 1, both the PEG-modified formulation combination and the preparation process affect the crosslinking performance of the pegylated collagen and the lyophilized preparation thereof, and further affect the formation of the hydrogel into the cornea; wherein the PEG modified formula combination can significantly affect the strength and elasticity of the cornea; the preparation process, such as reaction temperature, pH and the like, has direct correlation with the conjugation rate between the collagen-like protein and PEG, and can become one of the key factors for modifying whether a product can be made into hydrogel to form the cornea or not by influencing the formation of a polymer network structure; in addition, the freeze-drying auxiliary material provides framework support and protection for the pegylated collagen, so that the spatial conformation and the biological activity of the polymer are influenced, and the difference exists in the hardness after the film is formed. In Table 1, the cornea 1-13, 17-21 and 23-24 have better strength and good biocompatibility, and have great application prospect in the field of cornea transplantation.

TABLE 1 corneal Strength 1-24

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

<110> xi Zhu Mi (Suzhou) biomedical science and technology Co., Ltd

<120> polyethylene glycol collagen and preparation method and application thereof

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (14)..(14)