CN114907606B - A method for surface modification of polymer brushes for active immobilized proteins - Google Patents

Abstract

The application discloses a polymer brush surface modification method for active immobilized protein. The polymer brush consists of a short chain of poly (glycidyl methacrylate) PGMA, wherein one end of the poly (glycidyl methacrylate) is fixed on the surface of a solid material, and the other end of the poly (glycidyl methacrylate) is used for supporting active proteins. The polymer brush for immobilizing and maintaining the activity of the protein of the application fixes the protein on the surface of a solid material through chemical bonds. Compared with the traditional physical adsorption protein, the polymer brush for immobilizing and maintaining the protein activity can not only stably immobilize the protein for a long time, but also effectively maintain the high activity of the protein, is suitable for various solid surfaces, has wide universality and has wide application prospect in the aspect of biomedical materials.

Description

A method for surface modification of polymer brushes for active immobilized proteins

Technical field

The invention relates to the field of biomedical materials, and in particular to a polymer brush surface modification method.

Background technique

Biological processes critical to life include chemical and biochemical reactions. Among them, protein molecules play an important role in these reactions, such as catalysis, signal transduction, etc. Apart from this, proteins are equally important in industrial production as they are widely used in environmental, food, medical and pharmaceutical industries.

The normal function of a protein depends on the integrity of its secondary and tertiary structure. Changes in environmental conditions may disrupt structural integrity, leading to protein denaturation/unfolding. Electrostatics, van der Waals, hydrogen bonding, hydrophobic/hydrophilic are the main non-covalent interactions behind changes in protein secondary and tertiary structure. Due to these interactions, loss of integrity of these secondary and tertiary structures may lead to misfolding/denaturation of enzymes, leading to protein-mediated diseases and abnormalities. Since proteins play an integral role in life, controlling protein stability and activity is crucial.

Proteins are unstable in non-physiological environments. Therefore, the approach of immobilizing proteins on different carriers is the most effective way to enhance protein stability and activity. The surface-modified polymer brush just becomes the medium for immobilizing proteins on the surface of different materials.

Polymer brush is a monomolecular layer formed by physical adsorption or chemical grafting of polymer on the surface of the material. One end of the polymer brush is bound to the surface of the solid material, and the other end extends outward, forming a brush-like configuration on the surface of the material. When end-group-modified macromolecules are grafted onto the surface through chemical grafting, the steric hindrance between the macromolecular chains forces the chains to vertically form a brush-like structure, thereby achieving control of the material surface. At the same time, by further adjusting the density, chain length, polymer type, etc. of the polymer brush grafted on the solid surface, adjusting the composition of the surface grafted polymer brush can control the stability of immobilized and adsorbed proteins.

Therefore, in order to enhance protein stability and activity, it is necessary to study methods of surface modification of polymer brushes and active immobilization of proteins.

Contents of the invention

A first object of the present invention is to provide a surface-modified polymer brush of solid material.

The second object of the present invention is to provide a method for surface modification of solid materials and active immobilization of proteins.

The third object of the present invention is to provide a surface modification of a polymer brush and the application of active immobilized protein.

A first aspect of the present invention provides a surface modification material, comprising:

solid matrix;

Polymer brush, the polymer brush is composed of polyglycidyl methacrylate (PGMA), wherein the polyglycidyl methacrylate is fixed on the surface of the solid substrate.

In the present invention, the surface of the solid substrate after being treated with oxygen ions contains abundant hydroxyl groups, and the hydroxyl groups react with the silicon-oxygen bonds on the silane coupling agent 3-(trimethoxysilyl)propyl methacrylate, causing the silane coupling agent to The coupling agent is anchored to the surface of the solid matrix, and then the double bonds on the glycidyl methacrylate monomer and the double bonds on the siloxane anchored on the solid matrix undergo free radical polymerization to make glycidyl methacrylate. The monomers form polymer brushes on the surface of the solid substrate.

In another preferred example, the solid matrix is an inorganic, organic or organic/inorganic composite material; preferably selected from metal (gold), glass, silicon (sheet), polymethyl methacrylate, polylactic acid-glycolic acid copolymer One or more of polyglycerol sebacate, polyglycerol sebacate/tricalcium phosphate composite scaffold, polycaprolactone, polylactic acid, and inorganic calcium phosphate.

In another preferred embodiment, the solid matrix is a solid matrix modified with a silane coupling agent, and the polymer brush is coupled with the silane coupling agent to obtain the surface modified material.

In another preferred example, the silane coupling agent is 3-(trimethoxysilyl)propyl methacrylate.

According to the specific embodiment of the present invention, only when the mass ratio of the mass of the base material and the glycidyl methacrylate monomer is in the range of 1: (0.1-1), can the polyglycidyl methacrylate be guaranteed to be in the solid material The surface has a high grafting rate. If it is too large or too small, it will affect the grafting rate, which will affect the immobilization of proteins and the maintenance of their activity. Based on the quality of the base material, the preferred mass ratio of the base material to glycidyl methacrylate monomer is 1:0.3-0.8, 1:0.4-0.6, preferably 1:0.5.

In the present invention, polyglycidyl methacrylate is grafted onto the surface of a solid material through chemical grafting. The polymer brush produced has good biocompatibility and is suitable for various material surfaces. It can be used to modify the surface of biomedical materials. It has broad application prospects in sex.

A second aspect of the present invention provides a protein-immobilizing material, comprising:

The surface modification material described in the first aspect;

Protein, which is immobilized on the surface modified material.

In another preferred embodiment, the protein is immobilized on the surface modification material through covalent bonds.

The invention provides a polymer brush for surface modification of solid materials. The polymer brush is formed from a polyglycidyl methacrylate molecular chain, wherein one end of the polyglycidyl methacrylate molecular chain is fixed on the solid surface; the other end A covalent bond can be formed by a ring-opening reaction between the epoxy bond itself and the amino group on the protein, so that the protein can be stably immobilized on the surface of the solid material. Furthermore, the density and chain length of the polymer brush grafted on the solid surface can be further adjusted to control the immobilization. and the stability of adsorbed proteins.

In another preferred example, the protein is selected from: growth factors, enzymes, macromolecular proteins/polypeptides, preferably selected from: bovine serum albumin (BSA), bone morphogenetic protein-2 (BMP-2), peroxidase One of hydrogenase (CAT), glucose oxidase, horseradish peroxidase, and ovalbumin.

A third aspect of the present invention provides a method for preparing the surface modification material described in the first aspect, which includes the following steps:

(i) Put the solid substrate into oxygen plasma for surface modification treatment to introduce hydroxyl groups on the surface of the solid substrate;

(ii) immersing the solid matrix into which hydroxyl groups are introduced in a solution containing a silane coupling agent, and performing a grafting reaction to obtain a solid matrix modified by the silane coupling agent;

(iii) Immerse the solid matrix modified with the silane coupling agent in a reaction solution containing glycidyl methacrylate, and perform a coupling reaction to obtain a solid matrix grafted with polyglycidyl methacrylate, that is, the surface modification sexual material.

It should be noted that in step (i), the specifications of the solid substrate are not specifically limited and can be in any shape such as slices, blocks, tubes, balls, etc., but for the subsequent grafting reaction, the solid surface should be kept clean and dry. For example, before pretreatment, the solid can be sonicated in deionized water and ethanol for 5-60 minutes, and then dried. Then, solid materials of different materials are pretreated and surface treated in oxygen plasma for 5-60 minutes. By pretreating the solid substrate surface, different solid surfaces with exposed hydroxyl groups can be obtained, and the preparation method of the present invention has wide applicability.

According to the specific embodiment of the present invention, only when the mass ratio of the mass of the base material and the glycidyl methacrylate monomer is in the range of 1: (0.1-1), can the polyglycidyl methacrylate be guaranteed to be in the solid material The surface has a high grafting rate. If it is too large or too small, it will affect the grafting rate, which will affect the immobilization of proteins and the maintenance of their activity. Based on the quality of the base material, the preferred mass ratio of the base material to glycidyl methacrylate monomer is 1:0.3-0.8, 1:0.4-0.6, preferably 1:0.5.

In another preferred example, the silane coupling agent is 3-(trimethoxysilyl)propyl methacrylate.

In another preferred embodiment, the volume fraction of the silane coupling agent in the solution containing the silane coupling agent is 4%-10%.

In the present invention, the silane coupling agent is dissolved in a mixed solvent of absolute ethanol, deionized water and glacial acetic acid to prepare a silane coupling agent solution, in which the volume ratio of absolute ethanol, deionized water and glacial acetic acid is 95:4 :1. For example, add 19 ml of absolute ethanol, 0.2 ml of glacial acetic acid and 0.8 ml of deionized water into a beaker to prepare the required solvent. Then add 4%-10% volume fraction of 3-(trimethoxysilyl)propyl methacrylate according to the total amount of the aforementioned solvent to prepare the required silane coupling agent solution. The amount of the prepared solution needs to be prepared according to the actual situation.

In another preferred example, the silane coupling agent is dissolved in a mixed solvent of absolute ethanol, deionized water and glacial acetic acid to prepare a solution of the silane coupling agent, absolute ethanol, 3-(trimethoxysilyl)methyl The volume ratio of propyl acrylate, deionized water and glacial acetic acid is 95: (4～10): 4:1.

In another preferred example, the conditions for the grafting reaction are: the temperature is 15-25°C and the time is 2-4 hours.

In another preferred example, in step (iii), the reaction conditions are: the temperature is 80°C and the time is 0.5-2 hours.

In another preferred embodiment, the reaction solution containing glycidyl methacrylate consists of methanol, glycidyl methacrylate, potassium persulfate, and deionized water.

In another preferred example, potassium persulfate (K ₂ S ₂ O ₈ ) is used as the catalyst for the coupling reaction, and methanol is used as the solvent of glycidyl methacrylate (GMA).

A fourth aspect of the present invention provides a method for preparing a protein-immobilizing material or a protein-immobilizing method described in the second aspect, including the following steps:

(i) Put the solid substrate into oxygen plasma for surface modification treatment to introduce hydroxyl groups on the surface of the solid substrate;

(ii) immersing the solid matrix into which hydroxyl groups are introduced in a solution containing a silane coupling agent, and performing a grafting reaction to obtain a solid matrix modified by the silane coupling agent;

(iii) Immerse the solid matrix modified with the silane coupling agent in a reaction solution containing glycidyl methacrylate, and perform a coupling reaction to obtain a solid matrix grafted with polyglycidyl methacrylate, that is, the surface modification sexual material;

(iv) Put the solid matrix grafted with polyglycidyl methacrylate into a solution containing protein (phosphate buffer), gently immobilize the protein at room temperature, and then wash away the unimmobilized protein to obtain the solid matrix. Protein-laden materials.

In another preferred example, the silane coupling agent is 3-(trimethoxysilyl)propyl methacrylate.

In another preferred example, the conditions for the grafting reaction are: the temperature is 15-25°C and the time is 2-4 hours.

In another preferred example, in step (iii), the reaction conditions are: the temperature is 80°C and the time is 0.5-2 hours.

In another preferred embodiment, the reaction solution containing glycidyl methacrylate consists of methanol, glycidyl methacrylate, potassium persulfate, and deionized water. In another preferred example, potassium persulfate (K ₂ S ₂ O ₈ ) is used as the catalyst for the coupling reaction, and methanol is used as the solvent of glycidyl methacrylate (GMA).

In another preferred example, in step (iv), the conditions for the immobilized protein reaction (ring-opening reaction of epoxy bond and amino group) are: temperature is 15-25°C; time is 3-24 hours.

In another preferred embodiment, the protein-containing solution is a phosphate buffer solution containing protein.

In another preferred embodiment, the concentration of protein in the protein-containing solution is 0.01 mg/mL-10 mg/mL.

The present invention combines the surface modification of solid materials with the immobilization and activity maintenance of proteins through a simple three-step synthesis method. It can not only modify the surface of various solid materials, but also immobilize and maintain the high activity of various proteins. .

A fifth aspect of the present invention provides the use of the surface-modified material described in the first aspect or the protein-immobilizing material described in the second aspect for preparing biomedical materials.

By pre-treating different solid surfaces, the polymer brush can be further grafted on different solid surfaces to endow the different solids with the function of immobilizing and maintaining protein activity. For example, it has certain applications in 3D printing of bone repair scaffolds. potential.

Beneficial effects of the invention

The technology of surface modification of the polymer brush and active immobilization of protein in the present invention not only increases the content of immobilized protein on the surface of solid materials, but also has the effect of maintaining protein activity.

The polymer brush of the present invention has a better immobilizing effect on proteins such as bovine serum albumin (BSA), bone morphogenetic protein-2 (BMP-2), catalase (CAT), etc., and is different from traditional physical adsorption protein methods. In comparison, this method can stabilize the protein on the surface of the material for a long time and maintain better activity.

The invention adopts a simple three-step synthesis method, has mild synthesis conditions, easy-to-obtain raw materials, is suitable for industrial production, and has certain economic benefits.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of the drawings

Figure 1 shows a schematic diagram of the technical process of surface modification of polymer brushes and active immobilization of proteins in Example 1.

Figure 2 shows a Fourier transform infrared spectrum.

Figure 3 shows a Raman spectrum.

Figure 4 shows digital photos of PLGA tablets (a), 3D printed PLGA scaffold (b) and 3D printed PGS/β-TCP scaffold (c).

Figure 5 shows SEM photos of the surface morphology of the PLGA tablet (a) and its surface-grafted polyglycidyl methacrylate polymer brush (b) in Example 1.

Figure 6 shows SEM photos of the surface morphology of the 3D printed PLGA scaffold (a) and its surface-grafted polyglycidyl methacrylate polymer brush (b) in Example 2.

Figure 7 shows the SEM images of the surface morphology of the 3D printed PGS/β-TCP scaffold (a) and its surface-grafted polyglycidyl methacrylate polymer brush (b).

Figure 8 shows a comparison of PLGA tablets treated with PGMA polymer brushes and untreated PLGA tablets loaded with different proteins.

Figure 9 is a graph showing alkaline phosphatase activity (ALP) detection results.

Figure 10 shows the difference in surface morphology of PLGA tablets modified with different masses of glycidyl methacrylate monomers.

Detailed ways

After extensive and in-depth research, the inventor of the present application developed a method of surface-modifying a polymer brush of solid materials and an active method of immobilizing proteins. The polymer brush is composed of polyglycidyl methacrylate (PGMA), in which One end of polyglycidyl methacrylate is fixed on the surface of the solid material; the other end can form a covalent bond through a ring-opening reaction between the epoxy bond it carries and the amino group on the protein, so that the protein can be stably fixed on the surface of the solid material and further adjusted The density and chain length of polymer brushes grafted on solid surfaces can control the stability of immobilized and adsorbed proteins. The inventor combines the surface modification of solid materials with the immobilization and activity maintenance of proteins through a simple three-step method. It can not only modify the surface of a variety of solid materials, but also immobilize and maintain the high activity of a variety of proteins. On this basis, the present invention was completed.

It should also be noted that, unless otherwise specified, any range described in the present invention includes the end value and any value between the end value and any sub-range constituted by the end value or any value between the end value. The preparation methods in the present invention are conventional methods unless otherwise specified. The raw materials used can be obtained from open commercial channels or prepared according to existing technologies unless otherwise specified. The percentages mentioned are mass percentages unless otherwise specified. The solutions are all aqueous solutions unless otherwise specified.

The present invention is described in detail below through examples. It is necessary to point out that this example is only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the field can make reference to the above invention. Some non-essential improvements and adjustments have been made to the content. The embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

Example 1

Preparation of PLGA tablets that immobilize and maintain high activity of BMP-2

The preparation process is shown in Figure 1. After the surface of polylactic acid-glycolic acid copolymer (PLGA) is plasma treated, hydroxyl groups are introduced on the surface. After MPTS modification, polyglycidyl methacrylate (PGMA) polymer brush is grafted, and then Fixed BMP-2.

(1) Surface treatment of solid: Press polylactic acid-glycolic acid copolymer (PLGA) into tablets (1g), ultrasonicate in deionized water and ethanol for 5 minutes each, blow dry in _N2 atmosphere, and then in oxygen plasma Perform surface treatment for 5 minutes to introduce hydroxyl groups on the solid surface and set aside (the photo of the base material is shown in a in Figure 4, and the SEM photo is shown in a in Figure 5);

(2) Grafted silane coupling agent: Immerse the PLGA tablet with introduced hydroxyl groups in ethanol/water/glacial acetic acid with a volume fraction of 4% 3-(trimethoxysilyl)propyl methacrylate (MPTS) In the mixed solution, stir for 2 hours at 15-25°C, wash twice with ethanol and deionized water in sequence, and blow dry in an _N2 atmosphere to obtain MPTS-modified PLGA tablets, which are recorded as PLGA tablets/MPTS (infrared spectrum is as follows: As shown in Figure 2, the Raman spectrum is shown in Figure 3);

(3) Grafted polyglycidyl methacrylate (PGMA) polymer brush: Dissolve 0.5mL (0.5g) glycidyl methacrylate (GMA) in 10ml methanol, and then weigh 5mg K ₂ S ₂ O ₈ was dissolved in 5 ml of ultrapure water, and the two were evenly mixed. The MPTS-modified PLGA tablets were immersed in the mixed solution at 80°C, condensed and refluxed, and stirred for 1 hour. After the reaction was completed, the materials were taken out and placed in methanol and deionized water respectively. Ultrasonic for 5 minutes, rinse with a large amount of absolute ethanol, and then blow dry in an _N2 atmosphere to obtain a PLGA tablet grafted with PGMA, which is recorded as PLGA tablet/PGMA (the infrared spectrum is shown in Figure 2, and the SEM photo is shown in Figure 5 (shown in b).

As shown in the infrared spectrum of Figure 2, comparing PLGA tablets and PLGA tablets/MPTS, PLGA tablets/PGMA has a characteristic peak of the epoxy group at 910 cm ^-1 , indicating that PGMA has been successfully modified on the surface of PLGA tablets.

The characteristic peaks appearing at 1637cm ^-1 and 1723cm ^-1 in the Raman spectrum in Figure 3 also confirm the successful modification of PGMA on the surface of PLGA tablets.

As shown in the SEM image in Figure 5, the surface of unmodified PLGA tablets is smooth, while the surface of PLGA tablets/PGMA has an obvious layered structure, which can also confirm the successful modification of PGMA on the surface of PLGA tablets.

(4) Take 5 mL of prepared 0.01 mg/ml phosphate buffer containing bone morphogenetic protein-2 (BMP-2) and place it in a beaker. Press the PGMA-grafted PLGA into tablets in the phosphate buffer containing BMP-2. Immerse in the liquid and incubate at 15-25°C for 12 hours. After incubation, change the medium and wash away the unimmobilized protein to obtain a PLGA tablet loaded with BMP-2, which is recorded as PLGA tablet/PGMA/BMP-2.

Example 2

Preparation of 3D printed PLGA scaffold that immobilizes and maintains high activity of BMP-2

(1) Surface treatment of solid: The 3D printed PLGA scaffold (1g) was sonicated in deionized water and ethanol for 5 minutes each, dried in _N2 atmosphere, and then surface treated in oxygen plasma for 5 minutes. Introduce hydroxyl groups on the solid surface and wait for use (the photo of the base material is shown in b in Figure 4, and the SEM photo is shown in a in Figure 6);

(2) Grafted silane coupling agent: Immerse the PLGA scaffold with hydroxyl groups introduced in absolute ethanol/water/ice with a volume fraction of 4% 3-(trimethoxysilyl)propyl methacrylate (MPTS) In the acetic acid mixed solution, stir for 2 hours at 15-25°C, wash twice with ethanol and deionized water in sequence, and blow dry in an _N2 atmosphere to obtain an MPTS-modified PLGA scaffold, which is recorded as PLGA scaffold/MPTS;

(3) Grafted polyglycidyl methacrylate (PGMA) polymer brush: Dissolve 0.5mL (0.5g) glycidyl methacrylate (GMA) in 10ml methanol, and then weigh 5mg K ₂ S ₂ O ₈ was dissolved in 5 ml of ultrapure water, and the two were evenly mixed. The MPTS-modified PLGA scaffold was immersed in the mixed solution at 80°C, condensed and refluxed, and stirred for 1 hour. After the reaction was completed, the material was taken out and ultrasonicated in methanol and deionized water respectively. 5 min, then rinse with a large amount of absolute ethanol, and then blow dry in an _N2 atmosphere to obtain a PLGA scaffold grafted with PGMA polymer brush, which is recorded as PLGA scaffold/PGMA (SEM photo is shown in b in Figure 6);

As shown in the SEM image in Figure 6, the surface of the unmodified PLGA scaffold is smooth, while a large number of point-like protruding structures appear on the surface of the PLGA scaffold/PGMA, which confirms the successful modification of PGMA on the surface of the PLGA scaffold.

(4) Take 5 ml of prepared 0.01 mg/ml phosphate buffer containing bone morphogenetic protein-2 (BMP-2) and place it in a beaker. Place the PGMA-grafted PLGA scaffold in the phosphate buffer containing BMP-2. Immerse in medium and incubate at 15-25°C for 12 hours. After incubation, change the medium and wash away the unimmobilized protein to obtain a PLGA scaffold loaded with BMP-2, which is recorded as PLGA scaffold/PGMA/BMP-2.

Example 3

Preparation of polyglyceryl sebacate/β-tricalcium phosphate composite scaffold that immobilizes and maintains high activity of BMP-2

(1) Solid surface treatment: 3D printed polyglyceryl sebacate/β-tricalcium phosphate (PGS/β-TCP) composite scaffold (1g) was sonicated in deionized water and ethanol for 5 minutes each, N _2. Blow dry in the atmosphere, and then perform surface treatment in oxygen plasma for 5 minutes to introduce hydroxyl groups on the solid surface, ready for use (the photo of the base material is shown as c in Figure 4, and the SEM photo is shown as a in Figure 7);

(2) Grafted silane coupling agent: The (PGS/β-TCP) composite scaffold with introduced hydroxyl groups is immersed in 3-(trimethoxysilyl)propyl methacrylate (MPTS) with a volume fraction of 4%. In ethanol/water/glacial acetic acid mixed solution, stir for 2 hours at 15-25°C, wash twice with ethanol and deionized water in sequence, and blow dry in _N2 atmosphere to obtain MPTS-modified (PGS/β-TCP) composite scaffold. , recorded as (PGS/β-TCP)/MPTS;

(3) Grafted polyglycidyl methacrylate (PGMA): Dissolve 0.5mL (0.5g) glycidyl methacrylate (GMA) in 10ml methanol, then weigh 5mg K ₂ S ₂ O ₈ and dissolve in 5ml of ultrapure water, mix the two evenly, immerse the MPTS-modified (PGS/β-TCP) composite scaffold in the mixed solution, 80°C, condense and reflux, stir for 1 hour, after the reaction is completed, take out the material, respectively in methanol, Ultrasonicate in deionized water for 5 minutes, rinse with a large amount of absolute ethanol, and then blow dry in an _N2 atmosphere to obtain a GMA-grafted (PGS/β-TCP) composite scaffold, which is recorded as (PGS/β-TCP)/PGMA ( The SEM photo is shown in b in Figure 7);

The SEM picture in Figure 7 shows that the surface of the unmodified (PGS/β-TCP) composite scaffold is in a state of tightly arranged particles, while a large number of hemispherical protrusions appear on the surface of (PGS/β-TCP)/PGMA, which confirms that PGMA The modification on the surface of (PGS/β-TCP) composite scaffold was successful.

(4) Take 5 ml of prepared 0.01 mg/ml phosphate buffer containing bone morphogenetic protein-2 (BMP-2) and place it in a beaker. Place the PGMA-grafted (PGS/β-TCP) composite scaffold in the Immerse BMP-2 in phosphate buffer and incubate at 15-25°C for 12 hours. After incubation, change the medium and wash away the unimmobilized protein to obtain the (PGS/β-TCP) composite scaffold immobilized with BMP-2. Record It is (PGS/β-TCP) scaffold/PGMA/BMP-2.

Example 4

Preparation of PLGA sheets that immobilize and maintain high activity of catalase

(1) Surface treatment of solid: The PLGA tablets (1g) prepared by tableting were sonicated in deionized water and ethanol for 5 minutes respectively, dried in _N2 atmosphere, and then surface treated in oxygen plasma for 5 minutes. The treatment introduces hydroxyl groups on the solid surface and is ready for use;

(2) Grafted silane coupling agent: Immerse the PLGA sheet with introduced hydroxyl groups in a mixed solution of 3-(trimethoxysilyl)propyl methacrylate (MPTS)/water/glacial acetic acid with a volume fraction of 4% , stir for 2 hours at 15-25°C, wash twice with ethanol and deionized water and blow dry in _N2 atmosphere to obtain MPTS-modified PLGA tablets, recorded as PLGA tablets/MPTS;

(3) Grafted polyglycidyl methacrylate (PGMA) polymer brush: Dissolve 0.5mL (0.5g) glycidyl methacrylate (GMA) in 10ml methanol, and then weigh 5mgK ₂ S ₂ O ₈ Dissolve in 5 ml of ultrapure water, mix the two evenly, immerse the MPTS-modified PLGA scaffold in the mixed solution, 80°C, condense and reflux, stir for 1 hour, after the reaction is completed, take out the material and sonicate in methanol and deionized water for 5 minutes. , then rinse with a large amount of absolute ethanol, and then blow dry in an _N2 atmosphere to obtain PLGA grafted with PGMA polymer brush, which is recorded as PLGA tableting/PGMA;

(4) Take 5 ml of prepared 0.01 mg/ml phosphate buffer containing catalase (CAT) and place it in a beaker. Immerse the PGMA-grafted PLGA scaffold in the phosphate buffer containing CAT for 15-25 Incubate at ℃ for 12 hours. After incubation, change the medium and wash away the unimmobilized protein to obtain a CAT-immobilized PLGA tablet, which is recorded as PLGA tablet/PGMA/CAT.

Example 5

Same as Example 1, except that the solid material used is polymethylmethacrylate. The name of the obtained sample was recorded as PMMA/PGMA/BMP-2.

Example 6

Same as Embodiment 1, except that the solid material is a silicon wafer. The name of the obtained sample was recorded as Si-PGMA-BMP-2.

Example 7

Same as Example 1, except that the immobilized protein is bovine serum albumin (BSA). The name of the obtained sample is recorded as PLGA tableting/PGMA/BSA.

Comparative example 8

Same as Example 1, the difference is that in this example only the PLGA tablets are pressed and washed and then placed in the BMP-2 protein solution, so that the BMP-2 protein is simply adsorbed on the surface of the stent, recorded as PLGA tablets/BMP-2, as a control example .

The difference in protein loading is shown in Figure 8. The immobilized capacity of bovine serum albumin (BSA), bone morphogenetic protein-2 (BMP-2), and catalase (CAT) of PGMA-treated PLGA sheets is compared with The untreated PLGA sheet has a significant improvement, indicating that PGMA treatment can effectively increase the protein loading capacity.

Example 9

Activity detection of PGMA polymer brush-immobilized protein

The protein activity of bone morphogenetic protein-2 (BMP-2) can be quantitatively detected by the alkaline phosphatase activity (ALP) of C2C12 cells.

The BMP-2 immobilized in PLGA tablets/PGMA/BMP-2 in Example 1 was selected and compared with the culture medium prepared from the BMP-2 stock solution to detect its biological activity.

The BMP-2 content immobilized in PLGA tablets is calculated using the following method. The protein content of the initial BMP-2 solution before immobilization is subtracted from the remaining protein content in the solution after immobilization and the amount of unimmobilized protein in the cleaning solution to obtain each tablet. Content of BMP-2 immobilized in PLGA tablets. In this experiment, the mass of BMP-2 immobilized on PLGA tablets was 1.35 μg.

The PLGA-PGMA sheets immobilized with BMP-2 were placed in a 24-well plate. The PLGA-PGMA sheets without BMP-2 were used as the control group. C2C12 cells were seeded in the 24-well plate at a density of 3.0×10 ⁴ /well. On the plate, incubate for 1 day in an incubator at 37°C and 5% _CO2 , and wash twice with PBS.

The BMP-2 solution was prepared with cell culture medium at concentrations of 2, 1.35, 1, 0.5, 0.25, 0.125, and 0 μg/mL, 1 mL per well, and added to the remaining 24-well plates. Place in a 37°C, 5% _CO2 incubator for 3 days.

After the culture, carefully aspirate the culture medium, rinse once with PBS, add 200 μl NP-40 (cell lysate) to each well, and place in a 37°C constant temperature shaking box for 1 hour. Pipette 2 μl of lysis solution from each well (10 μl in total) into a new 96-well plate, add 18 μl of ultrapure water, and 200 μl of BCA reagent, place in a 37°C incubator for 30 minutes, and use a microplate reader to read at 562 nm Measure the OD value and calculate the total protein amount.

At the same time, take 50 μL of lysis solution from the 24-well plate into a new 96-well plate. Take 3 times from each well of the 24-well plate. Add 50 μL of 2.5% PNPP-Na ALP substrate to the 96-well plate for color development. solution and place in a 37°C thermostat for 30 minutes away from light.

Finally, add 100 μl/well and 0.1 M NaOH to terminate the color reaction, and measure the OD value at a wavelength of 405 nm with a microplate reader.

Calculate alkaline phosphatase activity through the formula ALP=OD value/time/total protein content (mg), and then compare the BMP-2 protein activity before and after release.

In the ALP staining experiment, the cell culture method is similar. After the culture, each well plate is fixed with 300 μL/well of 2.5% glutaraldehyde (original 25% glutaraldehyde diluted 10 times) for 10 minutes, and rinsed twice with PBS. ; After the last wash, remove the washing solution and add an appropriate amount of BCIP/NBT staining working solution, about 200 μL/well, to ensure that the sample can be fully covered. Incubate at room temperature in the dark for 5-30 minutes until the color develops to the desired depth. Remove the BCIP/NBT staining working solution, wash with water 1-2 times to terminate the color reaction, and invert the microscope to take pictures.

As can be seen from Figure 9, compared to the BMP-2 in the solution (BMP-2 stock solution and culture medium, the highest activity state), the ALP activity of BMP-2 immobilized in PLGA tablets/PGMA is the same as that of the same content. Compared with BMP-2 solution, the activity is about 87% of the activity of solution BMP-2. This shows that BMP-2 after immobilization can still maintain its high biological activity. ALP staining also showed that PGMA-BMP-2 had deeper ALP staining, which also proved that BMP-2 still had high biological activity after immobilization.

Comparative example 1

In order to verify the influence of the monomer concentration of the PGMA polymer brush coating prepared in the present invention on its grafting rate and its immobilized protein, the following comparative experiments were performed:

The experimental steps are as in Example 1. In step 4, the amount of glycidyl methacrylate (GMA) monomer is changed, and 0.1 mL (0.1 g) and 1 mL (1.0 g) of GMA monomer are added respectively for grafting reaction to obtain PLGA tableting/0.1PGMA (a) and PLGA tableting/1.0PGMA (c).

As shown in Figure 10, compared with the PLGA tablet/0.5PGMA obtained in Example 1, it is found that the surface of the PLGA tablet/0.1PGMA SEM image has no obvious structure, which indicates that the grafting rate is low, while the PLGA tablet/1.0PGMA SEM image There is a thick layer of attachment on the surface, and there is peeling off.

After that, the obtained two sets of materials were incubated in a BSA protein solution with a concentration of 1 mg/mL for 4 hours, and the differences in protein immobilization of materials modified with different concentrations of monomers were compared. The results showed that the two monomer concentration modifications in Comparative Example 1 The protein immobilization capacity of the material is reduced compared with the protein immobilization capacity of the material obtained in Example 1.

That is, preferably, the best effect can be achieved when the mass ratio of the base material and the glycidyl methacrylate monomer is about 1:0.5.

Comparative example 2

Proteins are immobilized through chemical covalent binding, and proteins are easily inactivated. (Langmuir 2018, 34, 9298-9306) The rhBMP-6 protein is covalently bound to the surface of biological materials through siloxanes, esters, and imines. The immobilized rhBMP-6 has no biological activity at all and only has covalent bonds. After hydrolysis, its biological activity is partially restored. In the work of Theresa L.M. Pohl (Acta Biomaterialia, 2012, 8, 772-780.), BMP-2 was immobilized through a self-assembled monolayer structure. The activity of the immobilized BMP-2 in this method was approximately that of the solution. 60% of BMP-2 activity.

The method of this application can be confirmed by ALP activity detection that the activity of BMP-2 immobilized on PGMA polymer brushes can reach 87% of the activity of BMP-2 in the solution, which is better than the chemical covalent binding method disclosed in the prior art documents. The effect of immobilized proteins.

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (12)

1. A method for preparing surface modified materials, characterized in that: The surface modification materials include: A solid matrix, the solid matrix is selected from the group consisting of: polylactic acid-glycolic acid copolymer, polyglycerol sebacate/tricalcium phosphate composite scaffold, and polymethylmethacrylate; and A polymer brush, the polymer brush is composed of polyglycidyl methacrylate, wherein the polyglycidyl methacrylate is fixed on the surface of the solid substrate; And the preparation method includes the following steps: (i) Put the solid substrate into oxygen plasma for surface modification treatment to introduce hydroxyl groups on the surface of the solid substrate; (ii) Immerse the solid matrix into which hydroxyl groups are introduced in a solution containing a silane coupling agent, and perform a grafting reaction to obtain a solid matrix modified by the silane coupling agent; wherein the silane coupling agent is 3-(trimethoxy Silyl) propyl methacrylate, the hydroxyl group on the surface of the solid substrate reacts with the silicon-oxygen bond on the silane coupling agent 3-(trimethoxysilyl) propyl methacrylate to graft to obtain silane Coupling agent modified solid matrix; Wherein, the solvent of the solution containing the silane coupling agent is a mixed solvent of absolute ethanol, deionized water and glacial acetic acid; and the conditions of the grafting reaction are: the temperature is 15-25°C and the time is 2-4 Hour; (iii) Immerse the solid matrix modified with the silane coupling agent in a reaction solution containing glycidyl methacrylate, and perform a coupling reaction to obtain a solid matrix grafted with polyglycidyl methacrylate, that is, the surface modification sexual material; Wherein, the reaction solution containing glycidyl methacrylate is composed of methanol, glycidyl methacrylate, potassium persulfate and deionized water; And among them, the double bond on the glycidyl methacrylate monomer and the double bond on the 3-(trimethoxysilyl) methacrylate propyl ester on the solid matrix undergo a free radical polymerization reaction, causing the methacrylic acid to shrink. The glyceryl ester monomer forms a polymer brush polyglycidyl methacrylate on the surface of the solid matrix, thereby obtaining a solid matrix grafted with polyglycidyl methacrylate. 2. The preparation method according to claim 1, wherein the volume fraction of the silane coupling agent in the solution containing the silane coupling agent is 4%-10%. 3. The preparation method according to claim 1, wherein the volume ratio of absolute ethanol, deionized water and glacial acetic acid in the mixed solvent is 95:4:1. 4. The preparation method according to claim 1, characterized in that in step (iii), the reaction conditions are: temperature is 80°C and time is 0.5-2 hours. 5. The preparation method according to claim 1, characterized in that potassium persulfate is used as a catalyst for the coupling reaction, and methanol is used as a solvent for glycidyl methacrylate. 6. The preparation method as claimed in claim 1, characterized in that the silane coupling agent is dissolved in a mixed solvent of absolute ethanol, deionized water and glacial acetic acid to prepare a solution of the silane coupling agent, and anhydrous ethanol, 3 -The volume ratio of (trimethoxysilyl)propyl methacrylate, deionized water and glacial acetic acid is 95: (4～10): 4:1. 7. A surface modification material prepared by the method of claim 1. 8. The surface modification material according to claim 7, wherein the mass ratio of the solid matrix to the glycidyl methacrylate monomer is 1:0.1-1. 9. The surface modification material according to claim 7, characterized in that the mass ratio of the solid matrix to the glycidyl methacrylate monomer is 1:0.3-0.8. 10. The surface modification material according to claim 7, characterized in that the mass ratio of the solid matrix to the glycidyl methacrylate monomer is 1:0.4-0.6. 11. A protein-immobilizing material, characterized in that the protein-immobilizing material contains: The surface modification material as claimed in claim 7; Protein, the protein is immobilized on the surface modified material; Wherein, the polymer brush of the surface modification material is formed by a polyglycidyl methacrylate molecular chain, wherein one end of the polyglycidyl methacrylate molecular chain is fixed on the surface of the solid matrix, and the other end is looped by itself. The oxygen bond and the amino group on the protein undergo a ring-opening reaction to form a covalent bond, allowing the protein to be stably immobilized on the surface of the solid matrix; The protein is one of bovine serum albumin, bone morphogenetic protein-2, and catalase. 12. Application of the surface modified material according to claim 7 or the protein-immobilized material according to claim 11, characterized in that it is used to prepare biomedical materials.