CN113735964B

CN113735964B - Pegylated collagen and preparation method and application thereof

Info

Publication number: CN113735964B
Application number: CN202111071337.8A
Authority: CN
Inventors: 张加慧; 王崇宇; 刘沐荣; 张军; 何超先
Original assignee: Xiwei Suzhou Biomedical Technology Co ltd
Current assignee: Xiwei Suzhou Biomedical Technology Co ltd
Priority date: 2021-09-13
Filing date: 2021-09-13
Publication date: 2023-05-05
Anticipated expiration: 2041-09-13
Also published as: WO2023035719A1; CN113735964A

Abstract

The invention relates to polyethylene glycol collagen and a preparation method and application thereof, belonging to the technical field of biology. The invention provides a polyethylene glycol collagen-like protein, which comprises collagen-like protein, and PEG-40k and PEG-20k modified on the collagen-like protein; the modification of PEG-40k and PEG-20k can obviously improve the crosslinking performance of the collagen-like protein, so that the strength of the cornea made of the polyethylene glycol collagen-like protein can be improved by about 20 times compared with that of the cornea made of the collagen-like protein, and the strength of the cornea made of the polyethylene glycol collagen-like protein is improved by about 12 times compared with that of the cornea made of other polyethylene glycol collagen-like proteins, and therefore, the polyethylene glycol collagen-like protein has extremely high application prospect in the preparation of bionic or regenerated biological materials such as bionic cornea and the like.

Description

Pegylated collagen and preparation method and application thereof

Technical Field

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

Background

The Cornea (Cornea) is the anterior-most convex highly transparent substance of the eye, is in the shape of a transverse oval, covers the iris, pupil and anterior chamber, and provides most of the refractive power to the eye. And the light can be accurately focused on the retina to form an image by adding the refractive power of the crystal. The cornea has very sensitive nerve endings, and if a foreign object contacts the cornea of the eye, the eyelid may not be closed autonomously to protect the eye. In order to remain transparent, the cornea is not vascularized and nutrients and oxygen are available through the tears and aqueous humor.

Cornea is fragile, and cornea lesions can be caused by ocular trauma, inflammation, anaphylaxis, physical injury, chemical burn, severe exercise, excessive use of eyes and the like. Once the cornea is diseased, it can cause significant ocular symptoms such as pain, photophobia, tearing, vision loss, etc., and severe blindness.

Cornea transplantation is a treatment method for replacing the existing diseased cornea of a patient with normal cornea to make the diseased eye recover or control the corneal disease, thereby improving vision or treating some cornea diseases. Some corneal diseases that cause severe vision impairment and even blindness in patients can be treated entirely by methods of performing corneal transplants, helping these unfortunate patients get away from suffering. Because the cornea itself contains no blood vessels, is in an 'immune privilege' position, so that the success rate of cornea transplantation is at the first of other allogeneic organ transplantation.

However, cornea resources are limited and far from meeting patient needs. To solve this problem, a keratoprosthesis has been proposed by researchers. Keratoplasty is a surgical method for restoring vision by surgically implanting a special optical device made of a transparent medical polymer material into corneal tissue to replace a portion of the corneal scar tissue. The problems of rejection reaction of cornea tissue to artificial synthetic materials and the like are not solved, the long-term effect is poor, and aqueous humor leakage at the transplanted position and falling of a transplanted piece are often caused, so that the cornea tissue cannot be widely applied at present. The artificial cornea is only suitable for binocular blindness after various serious cornea diseases, especially for all leucoma caused by serious chemical burn and repeated cornea transplantation failure, and can not be used for other operators.

Based on artificial cornea, researchers have proposed 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, propose a pegylated collagen, which is chemically crosslinked to obtain a biomimetic cornea. The bionic cornea has excellent biocompatibility, and can effectively solve the problems of strong rejection reaction of cornea tissue to artificial synthetic materials, and the like.

However, the strength of the bionic cornea is very low, and only 0.022MPa, and if the bionic cornea is used for cornea transplantation, the problems of great transplantation difficulty, easy cornea cone generation and the like still 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-like protein, which comprises collagen-like protein and polyethylene glycol derivatives modified on the collagen-like protein; the polyethylene glycol derivative comprises PEG-40k and PEG-20k.

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

In one embodiment of the invention, the amino acid sequence of the collagen-like protein is shown as 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 present invention, the number of activating groups of PEG-40k is one or more of 8-arm, 4-arm, 2-arm or 1-arm; the number of the activating groups of the PEG-20k is one or more than one of 8-arm, 4-arm, 2-arm or 1-arm.

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

In one embodiment of the invention, the activating group of 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 than one of mercapto, amino, carboxyl or imidazolyl.

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 sulfhydryl; 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 amino.

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

In one embodiment of the present invention, the mother conformation 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-like protein is 15000 to 75000Da.

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

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

In one embodiment of the present invention, the lyophilized preparation is obtained by mixing the above-mentioned pegylated collagen-like protein with a lyoprotectant and 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-like protein, which comprises the following steps:

the reaction steps are as follows: reacting collagen and polyethylene glycol derivatives for 1-48 h 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.

In one embodiment of the invention, the reaction steps are: reacting collagen and polyethylene glycol derivative at pH 6.0-8.0 and temperature 2-8 deg.c for 5-8 hr.

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

In one embodiment of the present 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 reaction 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 present invention, the concentration of the dilute hydrochloric acid solution is 1 to 10mmol/L; the pH of the dilute hydrochloric acid solution is regulated to 6.0-8.0 by dilute alkali solution.

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

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

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

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

the reaction steps are as follows: reacting collagen and polyethylene glycol derivatives for 1-48 h 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;

and (3) freeze-drying: and freeze-drying the reaction product to obtain a freeze-dried preparation.

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

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

stage one: lyophilizing at-45deg.C under vacuum (vacuum) of 500 mTorr for 6 hr;

stage two: lyophilizing at-30deg.C under vacuum (vacuum) of 100 mTorr for 17 hr;

stage three: the mixture was lyophilized at 25℃under vacuum (vacuum) of 100 mTorr for 7 hours.

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

The invention also provides application of the polyethylene glycol collagen-like protein or the freeze-dried preparation or the polyethylene glycol collagen-like protein prepared by the method or the freeze-dried preparation prepared by the method in preparation of bionic or regenerated biological materials.

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 polyethylene glycol collagen-like protein, which comprises collagen-like protein, and PEG-40k and PEG-20k modified on the collagen-like protein; the modification of PEG-40k and PEG-20k can obviously improve the crosslinking performance of the collagen-like protein, so that the strength of the cornea made of the polyethylene glycol collagen-like protein can be improved by about 20 times compared with that of the cornea made of the collagen-like protein, and is improved by about 12 times compared with that of the cornea made of other polyethylene glycol collagen-like proteins (the cornea strength made of the polyethylene glycol collagen-like protein in the document Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants is only 0.022 MPa), and therefore, the polyethylene glycol collagen-like protein has extremely high application prospect in the preparation of bionic or regenerated 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 PEG modification in the molar ratio has better effect of improving the crosslinking performance of the collagen-like protein.

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

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

3. The invention provides a preparation method of the polyethylene glycol collagen, which takes dilute hydrochloric acid solution with the concentration of 1-10 mmol/L, pH of 6.0-8.0 as a reaction solvent, and reacts the collagen with PEG for 5-8 h at the temperature of 2-8 ℃ to prepare the polyethylene glycol collagen; the polyethylene glycol collagen-like protein prepared under the reaction condition has better crosslinking performance, and compared with the existing other polyethylene glycol collagen-like protein preparation methods, the preparation method has shorter reaction time, and the polyethylene glycol collagen-like protein capable of preparing hydrogel-formed cornea can be obtained only by reacting for 5-8 hours (in the document Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants, the preparation of the polyethylene glycol collagen-like protein capable of preparing hydrogel-formed cornea requires 4 weeks).

4. The invention provides a preparation method of the freeze-dried preparation, which comprises the steps of freeze-drying the polyethylene glycol collagen-like protein under the protection of a freeze-drying protective agent; the freeze-drying protective agent can protect the polyethylene glycol collagen-like protein, so that the structure of the polyethylene glycol collagen-like protein can not be damaged in the freeze-drying process, and further, the cross-linking performance of the freeze-drying preparation is better.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge. The synthesis of collagen-like proteins and the connection between collagen-like proteins and linker in the following examples were all carried out by Shanghai OnBo Biotechnology Co., ltd (AmbioPharm. InC.). The PEG in the examples below were purchased from Xiaomenobang Biotech Co.

Example 1-1: pegylated collagen and preparation thereof

The embodiment provides a polyethylene glycol collagen-like protein, 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 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 thiol 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 mother nucleus conformation of the polyethylene glycol derivative in the polyethylene glycol collagen-like protein is TP, and the molecular weight of the polyethylene glycol collagen-like protein is 30000-75000 Da. This pegylated collagen was designated as pegylated collagen 1.

The preparation method of the polyethylene glycol collagen 1 comprises the following steps:

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

and (3) purification: ultrafiltering the reaction product with ultrafilter membrane with aperture of 30000Da at 5deg.C to intercept the substance with molecular weight greater than or equal to 30000Da in the reaction product to obtain polyethylene glycol collagen 1.

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

The present example provides a lyophilized formulation consisting of the pegylated collagen-like protein 1 prepared in example 1-1 and mannitol. This lyophilized preparation was designated as lyophilized preparation 1.

The preparation method of the freeze-dried preparation 1 comprises the following steps:

mixing the polyethylene glycol collagen-like protein prepared in the example 1 with mannitol according to a mass ratio of 1:5 to obtain a freeze-drying system; lyophilizing the lyophilized system to obtain lyophilized preparation 1;

wherein, the freeze-drying comprises the following stages:

stage one: lyophilizing at-45deg.C under vacuum (vacuum) of 500 mTorr for 6 hr;

Examples 1-2: pegylated collagen and preparation thereof

The present example provides a pegylated collagen, wherein the PEG formulation (molar ratio of 8-arm-PEG-40k-MAL and 4-arm-PEG-20 k-MAL) used in the pegylated collagen 1 of example 1-1 was replaced with:

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 polyethylene glycol collagen-like proteins 2 to 9 is the same as that of the polyethylene glycol collagen-like protein 1.

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

The present example provides a lyophilized preparation, wherein the PEGylated collagen-like protein 1 is replaced with the PEGylated collagen-like proteins 2 to 9 prepared in the examples 1 to 2, respectively, based on the lyophilized preparation 1 in the examples 2 to 1.

The above freeze-dried preparations were named freeze-dried preparations 2 to 9 in order.

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

Examples 1-3: pegylated collagen and preparation thereof

The embodiment provides a polyethylene glycol collagen-like protein, 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 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 mother nucleus conformation of the polyethylene glycol derivative in the polyethylene glycol collagen-like protein is TP, and the molecular weight of the polyethylene glycol collagen-like protein is 30000-75000 Da. This pegylated collagen was designated as pegylated collagen 10.

The preparation method of the polyethylene glycol collagen 10 is similar to that of the polyethylene glycol collagen 1.

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

The present example provides a lyophilized formulation wherein the pegylated collagen-like protein 1 was replaced with the pegylated collagen-like protein 10 prepared in examples 1-3 based on the lyophilized formulation 1 of example 2-1.

The above lyophilized preparation was designated as lyophilized preparation 10 in order.

The preparation method of the freeze-dried preparation 10 is the same as that of the freeze-dried preparation 1.

Examples 1 to 4: pegylated collagen and preparation thereof

The present example provides a pegylated collagen, wherein the temperature (5 ℃) in the preparation method is replaced with: 2 ℃,8 ℃, 25 ℃, 37 ℃.

The polyethylene glycol collagen-like proteins are named as polyethylene glycol collagen-like proteins 11 to 14 in sequence.

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

The present example provides a lyophilized preparation, wherein the PEGylated collagen-like protein 1 is replaced with the PEGylated collagen-like proteins 11 to 14 prepared in examples 1 to 4, respectively, based on the lyophilized preparation 1 of example 2 to 1.

The above lyophilized preparations were designated as lyophilized preparations 11 to 14 in this order.

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

Examples 1 to 5: pegylated collagen and preparation thereof

The present example provides a pegylated collagen, wherein based on the pegylated collagen 1 of example 1-1, the pH (6.5) in the preparation method is replaced with: 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 15-22 of polyethylene glycol collagen.

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

The present example provides a lyophilized preparation, wherein the PEGylated collagen-like protein 1 is replaced with the PEGylated collagen-like proteins 15 to 22 prepared in examples 1 to 5, respectively, based on the lyophilized preparation 1 of example 2 to 1.

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

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

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

The present example provides a lyophilized preparation, wherein the lyoprotectant (mannitol) in the preparation method is replaced by: sucrose, alanine.

The above lyophilized preparations were designated as lyophilized preparations 23 to 24 in sequence.

Experimental example 1: experiment of influence of PEG formula and preparation process on cross-linking performance of PEGylated collagen and freeze-dried preparation thereof

The experimental example provides an experiment for influencing the crosslinking performance of polyethylene glycol collagen and a freeze-dried preparation thereof by a PEG formula and a preparation process, and the experimental process is as follows:

referring to the preparation method of the freeze-dried preparation 1, collagen-like proteins with the amino acid sequences shown as SEQ ID NO.1 are directly prepared into freeze-dried preparations, the freeze-dried preparations 1 to 24 prepared in examples 2-1 to 2-6 are used as blank controls, and the cornea is prepared respectively to obtain the cornea 1 to 24. The strength of the corneas 1 to 24 was measured using a universal tensile machine, and the measurement results are shown in table 1;

the cornea preparation process comprises the following steps:

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

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

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

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

the formula of the DMTMM mother liquor is as follows: 10g/mL DMTMM (4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride), 15g/mL APS (ammonium persulfate) and a solvent of 0.5mol/L, pH 5.5.5.5 MOPS Buffer.

As shown in table 1, both the PEG-modified formulation and the preparation process affect the crosslinking performance of the pegylated collagen and the lyophilized preparation thereof, and further affect the formation of cornea from the hydrogel; wherein, the combination of PEG modified formulas 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 key factors for forming cornea by influencing the formation of a polymer network structure to form a modified product or not; in addition, the freeze-drying auxiliary material composition provides skeleton support and protection for the polyethylene glycol collagen, so that the spatial conformation and the biological activity of the polymer are affected, and the hardness after film formation is different. In Table 1, the cornea 1-13, 17-21 and 23-24 are all good in strength and good in biocompatibility, and have great application prospects in the field of cornea transplantation.

Table 1 intensity of cornea 1 to 24

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It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Sequence listing

<110> micro (Suzhou) biomedical technologies Co., ltd

<120> polyethylene glycol collagen-like protein, and preparation method and application thereof

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Claims

1. A pegylated collagen-like protein, wherein the pegylated collagen-like protein comprises collagen-like protein and polyethylene glycol derivatives modified on the collagen-like protein; the polyethylene glycol derivative comprises PEG-40k and PEG-20k; the molar ratio of the PEG-40k to the PEG-20k is 2-6:1.

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

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

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

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

6. The pegylated collagen according to claim 5, wherein the number of activating groups of PEG-40k is 4-arm or 8-arm; the number of the activating groups of the PEG-20k is 4-arm or 8-arm.

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

8. The pegylated collagen according to claim 7, wherein said PEG-40k activating group is-MAL; the activating group of the PEG-20k is-MAL.

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

10. The pegylated collagen-like protein according to claim 9, wherein said linker is attached to the N-terminus of the collagen-like protein.

11. The pegylated collagen according to claim 9, wherein the amino acid sequence of said linker is shown in SEQ ID No.2 or SEQ ID No. 3.

12. The pegylated collagen according to claim 11, wherein the modification site of the polyethylene glycol derivative on linker is one or more of mercapto, amino, carboxyl or imidazolyl.

13. The pegylated collagen according to claim 12, wherein when the 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 a sulfhydryl group.

14. A pegylated collagen according to claim 12 or 13, wherein when the amino acid sequence of the linker is shown in SEQ ID No.3, the modification site of said polyethylene glycol derivative on the linker is an amino group.

15. A pegylated collagen according to any one of claims 1 to 3, wherein the mother conformation of said pegylated collagen is one or more of HG or TP.

16. A pegylated collagen according to any one of claims 1 to 3, wherein the parent conformation of the polyethylene glycol derivative in said pegylated collagen is TP.

17. A pegylated collagen according to any one of claims 1 to 3, wherein the amino acid configuration of said collagen is one or more of D-type and L-type.

18. A pegylated collagen according to any one of claims 1 to 3, wherein the molecular weight of said pegylated collagen is 15000 Da to 75000Da.

19. The pegylated collagen according to claim 18, wherein said pegylated collagen has a molecular weight of 30000Da to 75000Da.

20. A lyophilized formulation obtained by lyophilizing the pegylated collagen-like protein of any one of claims 1 to 19.

21. The lyophilized formulation according to claim 20, wherein the lyophilized formulation is obtained by mixing the pegylated collagen-like protein with a lyoprotectant and lyophilizing.

22. The lyophilized formulation according to claim 21, wherein the lyoprotectant is one or more of mannitol, sucrose or alanine.

23. A method of preparing the pegylated collagen-like protein of any one of claims 1 to 19, comprising the steps of:

the reaction steps are as follows: reacting collagen and polyethylene glycol derivatives for 1-48 h under the conditions that the pH is 6.0-8.5 and the temperature is 2-25 ℃ to obtain a reaction product; the reaction product comprising the pegylated collagen-like protein of any one of claims 1-19.

24. The method of claim 23, wherein the reacting step is: reacting collagen and polyethylene glycol derivative at pH 6.0-8.0 and temperature 2-8 deg.c for 5-8 hr.

25. The method of claim 23, wherein in the reacting step, the collagen-like protein and the polyethylene glycol derivative are fed in a molar ratio of 1 to 16:1.

26. The method of claim 25, wherein in the reacting step, the collagen-like protein and the polyethylene glycol derivative are fed in a molar ratio of 8-12:1.

27. The method according to any one of claims 23 to 26, wherein 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.

28. The method of claim 27, wherein the dilute hydrochloric acid solution has a concentration of 1 to 10mmol/L; the pH of the dilute hydrochloric acid solution is regulated to 6.0-8.0 by dilute alkali solution.

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

30. The method according to any one of claims 23 to 26, wherein in the reacting step, the collagen-like protein is added to the reaction solvent at a concentration of 1 to 15mg/mL.

31. The method of claim 30, wherein in the reacting step, the collagen-like protein is added to the reaction solvent at a concentration of 8 to 10mg/mL.

32. The method of any one of claims 23 to 26, wherein after the reacting step, the method further comprises a purifying step; the purification steps are as follows: filtering and intercepting substances with molecular weight of more than or equal to 30000Da in the reaction product to obtain the polyethylene glycol collagen.

33. The method of claim 32, wherein the filtration is dialysis or ultrafiltration.

34. A method of preparing a lyophilized formulation according to any one of claims 20 to 22, comprising the steps of:

and (3) freeze-drying: lyophilizing the pegylated collagen produced by the method of any one of claims 23-33 to obtain a lyophilized formulation.

35. Use of a pegylated collagen-like protein according to any one of claims 1 to 19 or a lyophilized formulation according to any one of claims 20 to 22 or a pegylated collagen-like protein obtainable by a method according to any one of claims 23 to 33 or a lyophilized formulation obtainable by a method according to claim 34 for the preparation of a biomimetic or regenerative biomaterial.

36. The use of claim 35, wherein the biomimetic or regenerative biological material is cornea.