CN107376036B - A method for constructing an enzyme-responsive multifunctional nanocoating - Google Patents

Abstract

The invention discloses a method for constructing an enzyme-responsive nano-coating. First, avidin molecules are immobilized on the stainless steel surface coated with dopamine. Biotinylated heparin/PEI nanoparticles were immobilized on the surface of the material, and further high concentrations of avidin molecules were used to bind to the residual biotin on the surface of the nanoparticle coating, thereby introducing new biotin binding sites; Biotinylated enzyme-responsive peptides are assembled on the surface of nanoparticles through the interaction between avidin and avidin, and finally, SDF-1α is covalently immobilized on the amino terminus of the peptide through EDC/NHS/MES coupling agent, thereby constructing Multifunctional nanocoatings with matrix metalloproteinase 9-responsive properties. The invention constructs a multifunctional layer with the ability of anticoagulation and inducing endothelial regeneration on the titanium surface, and significantly improves the blood compatibility of the material and the repairing ability of damaged endothelium.

Description

A method for constructing an enzyme-responsive multifunctional nanocoating

Technical field

The invention relates to the technical field of inorganic material surface modification technology, in particular to a construction method of an enzyme-responsive multifunctional nano-coating.

Background technique

Cardiovascular diseases such as coronary heart disease are a serious threat to human life and health. At present, the clinical application of metal vascular stents is mainly used to improve the blood supply of patients with coronary heart disease. However, due to insufficient biocompatibility, traditional bare metal stents and drug-eluting stents often lead to late-stage thrombosis and vascular intimal hyperplasia after implantation, resulting in implantation failure and even endangering the life of patients.

By biochemically modifying the surface of the material, it is a commonly used method to improve the biocompatibility of the material to endow the material with good anticoagulant ability and the ability to induce endothelial regeneration. However, in the complex environment in vivo, many biocoatings are difficult to maintain long-term functions, but rapidly lose their biological activities in the dynamic environment in vivo. In order to realize the long-term and effective function of the biological modification layer, it needs to be specially designed for the biological environment in which it is located.

Heparin is a widely used anticoagulant drug in clinical practice, and it is also a commonly used substance for surface modification of cardiovascular materials. However, since heparin molecules lack a site for direct reaction with the surface of the material, the present invention first introduces avidin molecules on the surface of the material, and then performs biotinylation modification on the heparin molecules to utilize the specific binding effect between biotin and avidin. , introducing heparin molecules to the surface of the material. In order to increase the loading of surface heparin, the present invention also introduces an amino-rich polycation electrolyte polyethyleneimine, which utilizes the characteristic that biotinylated heparin can interact electrostatically with polyethyleneimine to form nanoparticles. The nanoparticles are anchored to the surface of the material.

Mesenchymal cell-derived factor-1α (SDF-1α) is a chemokine that has a strong chemotactic effect on bone marrow CXCR4+ stem cells and endothelial progenitor cells (EPCs), and also stimulates endothelial cell growth and induces endothelial progenitor cells to enter the endothelium. function of cell differentiation. Studies have shown that SDF-1α can accelerate vascular injury repair and endothelial regeneration by inducing endothelial progenitor cells to aggregate and differentiate at the site of vascular injury.

PRQITAG polypeptide is a polypeptide with special response to matrix metalloproteinase 9 (MMP-9), which can be decomposed in the presence of MMP-9. MMP-9 is an important functional protein in the process of vascular injury repair. It can rapidly degrade the vascular basement membrane and extracellular matrix components, release a variety of proteins and growth factors, and stimulate the migration and proliferation of vascular cells. Therefore, MMP-9 can also be regarded as a signaling protein for vascular injury repair. Taking advantage of this feature, the present invention uses the PRQITAG polypeptide with MMP-9 response properties as a linking molecule, one end of which is fixed on the surface of the nanoparticle coating, and the other end is linked to the chemokine SDF-1α. The purpose is to induce the release of SDF-1α molecules on the surface of the material when the vascular tissue is damaged and secrete a large amount of MMP-9 to accelerate the repair of vascular damage.

This biofunctional layer with enzyme-responsive properties can not only improve the blood compatibility of the material surface, but also realize the on-demand release of biomolecules according to the changes in the intravascular environment, effectively improving the utilization rate and action time of biomolecules. Promotes vascular damage repair. However, there is no relevant report on the surface modification of this enzyme-responsive nano-coating cardiovascular material.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for constructing an enzyme-responsive multifunctional nano-coating, by which the biochemical modification of the surface of cardiovascular materials can realize the on-demand release of biological factors and effectively improve the biocompatibility of the materials and promote the ability to repair damage; creatively select the construction method of the multifunctional layer, and use the specific interaction between various biomolecules under a certain biomolecule concentration ratio to realize the orderly order of functional biomolecules on the surface of the material Assemble, and then achieve the characteristics that in a specific biological environment, the surface biomolecules are released on demand, and the surface biological functions are continuously and stably exerted; the construction process and fixing method of the biological microenvironment are simple and easy to operate, without expensive and complicated equipment, and the process cost is relatively high. Low, strong controllability and remarkable effect; the fixation of various biomolecules on the stainless steel surface is carried out by immersion, which can ensure that all parts of the material can be evenly fixed with biomolecules, which is conducive to the realization of various cardiovascular implantable devices with complex structures The functional modification of the surface is suitable for a wide range of applications.

The technical solution adopted by the present invention to achieve the above object is, a method for constructing an enzyme-responsive multifunctional nanocoating, the steps of which include:

A) After the surface of 316L stainless steel was polished and cleaned, it was immersed in a dopamine solution with a concentration of 2 mg/ml for 12 hours. After ultrasonic cleaning with double distilled water, the above steps were repeated twice to obtain three layers of polydopamine coating. surface, drying at 37°C;

B) Soak the samples with the polydopamine coating deposited in step A) in 0.1~0.5 mg/ml avidin solution, let them stand for 8~24 hours at 37 °C, and then wash the samples with double distilled water. save for use;

C) Blend the biotinylated heparin sodium solution with a concentration of 10-20 mg/ml and a polyethyleneimine solution with a concentration of 1-2 mg/ml in equal volumes, and ultrasonically treat it for 5 minutes at room temperature to obtain a nanoparticle suspension . Immerse the sample obtained in step B in the nanoparticle suspension, shake and react at 37°C for 8 to 24 hours, then rinse the sample with double distilled water, and store it for later use;

D) Immerse the sample obtained in step C) into a 1-3 mg/ml avidin solution, and react at 37°C for 1-3 hours; synthesize a biotinylated enzyme-responsive polypeptide whose structure is Biotin- PRQITAG- _NH2 . Soak the above samples in 10-50 μg/ml polypeptide solution, react at 37°C for 1-3 hours, rinse the samples with double distilled water, and store them for later use;

E) Add the EDC/NHS/MES crosslinker solution with a molar ratio of 2:1:1 to the SDF-1α solution with a concentration of 100~500 ng/ml dropwise, and immediately mix the sample obtained in D) step Soak in it, react at 37°C for 1 to 3 hours, and wash the sample with double-distilled water.

A further improvement scheme of the present invention is that the biomolecule solutions in the steps B), C), D), and E) are all phosphate buffers as the solvent.

A further improvement scheme of the present invention is that, in the steps B), C), D), and E), the samples are all refrigerated and stored at a temperature of 4°C under the condition of surface wetness.

A further improvement scheme of the present invention is that, in the step C), the molecular weight of polyethyleneimine is in the range of 60-75kDa.

A further improvement scheme of the present invention is that, in the step E), MES is 0.05mol.

A further improvement scheme of the present invention is, in the step E), in the EDC/NHS/MES crosslinking agent solution,

V _SDF-1 /V _crosslinker = 10/1.

Referring to Figure 1 of the description, the reaction process of the present invention includes the following steps. The first step is to graft avidin on the stainless steel surface. First, by depositing a polydopamine coating on the surface of 316L stainless steel, the quinone group on the surface of dopamine can react with the amino group in the avidin molecule to undergo Schiff base reaction, and the avidin is immobilized on the surface of the material; the second step, the use of molecular Biotinylated heparin/polyethyleneimine nanoparticles are synthesized by electrostatic interaction between the two; the third step is to fix the nanoparticles to The surface of the material; the fourth step, using a high concentration of avidin molecules to bind the remaining biotin molecules on the nanocoating surface, while introducing avidin binding sites; the fifth step, synthesizing biotinylated matrix metalloproteinase 9 in response to It uses the specific recognition function of the biotin molecule and the avidin binding site on the surface of the material to immobilize the peptide on the surface of the material; the sixth step is to activate the carboxyl group in the SDF-1α molecule with EDC/NHS/MES cross-linking agent , and a dehydration condensation reaction occurs with the amino group at the end of the polypeptide molecule on the surface of the material, thereby immobilizing SDF-1α on the surface of the material.

Compared with the prior art, the beneficial effects of the present invention are:

First, the construction method of the enzyme-responsive multifunctional nanocoating layer creatively selects the construction method of the multifunctional layer, and uses the specific interaction between various biomolecules to realize the function under a certain biomolecule concentration ratio. The orderly assembly of biomolecules on the surface of the material, and then achieve the characteristics that in a specific biological environment, the biomolecules on the surface of the material are released on demand, and the surface biological functions are continuously and stably exerted.

Second, the construction method of the enzyme-responsive multifunctional nanocoating, the construction process of the biological microenvironment and the fixation method are all simple and easy to operate, without the need for expensive and complex equipment, the process cost is low, the controllability is strong, and the effect is remarkable.

Third, the construction method of the enzyme-responsive multifunctional nano-coating, the immobilization of various biomolecules on the stainless steel surface is carried out by immersion, which can ensure that all parts of the material can be uniformly immobilized on biomolecules, which is conducive to the realization of various complex structures. The functional modification of the surface of cardiovascular implanted devices has a wide range of applications.

Description of drawings

The method of the present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

Figure 1 is a schematic diagram of each step in the construction of a matrix metalloproteinase 9-responsive nanocoating in the method of the present invention. (1) Deposition of dopamine coating on 316L stainless steel surface and assembly of avidin; (2) Preparation of biotinylated heparin/polyethyleneimine nanoparticles; (3) Immobilization of nanoparticles on the material surface; (4) Nanoparticles Assembly of avidin molecules on the coating surface; (5) assembly of biotinylase-responsive polypeptides on the surface of the material; (6) immobilization of SDF-1α on the surface of the material.

Figure 2 shows the effect of MMP9 on the release kinetics of SDF-1 under simulated in vivo flow field conditions.

Figure 3 shows the fluorescent staining results of platelet adhesion on the surface of different samples for 2 hours.

Figure 4 shows the results of the homing of endothelial progenitor cells induced by enzyme-responsive nanocoatings in the presence of MMP9 in the chemotactic chamber model. Dopamine-coated samples were blank controls.

Detailed ways

Example 1

Referring to Fig. 1, a first specific embodiment of the present invention is a method for constructing an enzyme-responsive nanocoating, the steps of which are:

A) After the surface of 316L stainless steel was polished and cleaned, it was immersed in a dopamine solution with a concentration of 2 mg/ml for 12 hours. After ultrasonic cleaning with double distilled water, the above steps were repeated twice to obtain three layers of polydopamine coating. surface, drying at 37°C;

B) Soak the sample deposited with polydopamine coating in step A) in 0.1 mg/ml avidin solution, let it stand for 8 hours at 37°C, and then wash the sample with double distilled water and save it for use;

C) The biotinylated heparin sodium solution with a concentration of 10 mg/ml and the polyethyleneimine solution with a concentration of 1 mg/ml were blended in equal volumes, and ultrasonically treated for 5 minutes at room temperature to obtain a nanoparticle suspension. Immerse the sample obtained in step B in the nanoparticle suspension, shake and react at 37°C for 8 hours, then rinse the sample with double distilled water, and save it for later use;

D) Immerse the sample obtained in step C) into a 1 mg/ml avidin solution, and react at 37°C for 1 hour; synthesize biotinylated enzyme-responsive polypeptide, whose structure is Biotin-PRQITAG-NH ₂ . Soak the above samples in 10 μg/ml polypeptide solution, react at 37°C for 1 hour, rinse the samples with double-distilled water, and store them for later use;

E) Add dropwise the EDC/NHS/MES crosslinker solution with a molar ratio of 2:1:1 to the SDF-1α solution with a concentration of 100 ng/ml, and immediately soak the sample obtained in step D) in it after mixing evenly , After 1 hour of reaction at 37°C, the sample was washed with double distilled water.

A further improvement scheme of the present invention is that, in the step C), the molecular weight of polyethyleneimine is in the range of 60-75kDa.

A further improvement scheme of the present invention is that, in the step E), MES is 0.05mol.

A further improvement scheme of the present invention is, in the step E), in the EDC/NHS/MES crosslinking agent solution,

V _SDF-1 /V _crosslinker = 10/1.

Embodiment 2

A method for constructing an enzyme-responsive nano-coating, the steps of which are:

A) After the surface of 316L stainless steel was polished and cleaned, it was immersed in a dopamine solution with a concentration of 2 mg/ml for 12 hours. After ultrasonic cleaning with double distilled water, the above steps were repeated twice to obtain three layers of polydopamine coating. surface, drying at 37℃;

B) Soak the samples with polydopamine coating deposited in step A) in 0.5 mg/ml avidin solution, let stand at 37°C for 8-24 hours, then wash the samples with double distilled water and store them for later use;

C) The biotinylated heparin sodium solution with a concentration of 20 mg/ml and the polyethyleneimine solution with a concentration of 2 mg/ml were blended in equal volumes, and ultrasonically treated for 5 minutes at room temperature to obtain a nanoparticle suspension. Immerse the sample obtained in step B in the nanoparticle suspension, shake and react at 37°C for 24 hours, then rinse the sample with double distilled water, and save it for use;

D) Immerse the sample obtained in step C) into a 3 mg/ml avidin solution and react at 37°C for 3 hours; synthesize a biotinylated enzyme-responsive polypeptide whose structure is Biotin-PRQITAG-NH ₂ . Soak the above samples in 50 μg/ml polypeptide solution, react at 37°C for 3 hours, rinse the samples with double-distilled water, and store them for later use;

E) Add the EDC/NHS/MES crosslinker solution with a molar ratio of 2:1:1 to the SDF-1α solution with a concentration of 500 ng/ml dropwise, and immediately after mixing evenly, soak the sample obtained in step D) in it , after 3 hours of reaction at 37°C, the sample was washed with double distilled water.

A further improvement scheme of the present invention is that, in the step C), the molecular weight of polyethyleneimine is in the range of 60-75kDa.

A further improvement scheme of the present invention is that, in the step E), MES is 0.05mol.

A further improvement scheme of the present invention is, in the step E), in the EDC/NHS/MES crosslinking agent solution,

V _SDF-1 /V _crosslinker = 10/1.

Embodiment 3

A method for constructing an enzyme-responsive nano-coating, the steps of which are:

A) After the surface of 316L stainless steel was polished and cleaned, it was immersed in a dopamine solution with a concentration of 2 mg/ml for 12 hours. After ultrasonic cleaning with double distilled water, the above steps were repeated twice to obtain three layers of polydopamine coating. surface, drying at 37℃;

B) Soak the sample with polydopamine coating deposited in step A) in 0.3 mg/ml avidin solution, let it stand at 37°C for 12 hours, then wash the sample with double distilled water and save it for use;

C) An equal volume of biotinylated heparin sodium solution with a concentration of 15 mg/ml and a polyethyleneimine solution with a concentration of 1.5 mg/ml was mixed, and ultrasonically treated at room temperature for 5 minutes to obtain a nanoparticle suspension. Immerse the sample obtained in step B in the nanoparticle suspension, shake and react at 37°C for 12 hours, then rinse the sample with double distilled water, and store it for later use;

D) Immerse the sample obtained in step C) into a 2 mg/ml avidin solution and react at 37°C for 2 hours; synthesize a biotinylated enzyme-responsive polypeptide whose structure is Biotin-PRQITAG-NH ₂ . Soak the above samples in 30 μg/ml polypeptide solution, react at 37°C for 2 hours, rinse the samples with double distilled water, and store them for later use;

E) Add dropwise the EDC/NHS/MES crosslinker solution with a molar ratio of 2:1:1 to the SDF-1α solution with a concentration of 300 ng/ml, and immediately soak the sample obtained in step D) in it after mixing evenly , After 2 hours of reaction at 37°C, the sample was washed with double distilled water.

A further improvement scheme of the present invention is that, in the step C), the molecular weight of polyethyleneimine is in the range of 60-75kDa.

A further improvement scheme of the present invention is that, in the step E), MES is 0.05mol.

A further improvement scheme of the present invention is, in the step E), in the EDC/NHS/MES crosslinking agent solution,

V _SDF-1 /V _crosslinker = 10/1.

Compared with Example 3, in Example 1, in the process of biochemical reaction, a reasonable shortening of the reaction time is conducive to maintaining the biological activity of biomolecules, so as to better exert its biological function; Example 2 is relative to Example 3. In other words, according to the mechanism of biochemical reaction, the loading density of surface biomolecules is increased to the greatest extent, which is beneficial to the longer-term and effective display of surface functions. It can be seen that the effect of the nano-coating obtained by Example 1 and Example 2 is better than that of the nano-coating obtained by Example 3. Therefore, if the nano-coating obtained by Example 3 with the worst analysis effect can solve the technical problem to be solved by this application, then Embodiment 1 and Embodiment 2 can also absolutely solve the technical problem to be solved by this application.

It can be seen from Figure 2 that when the material does not contain MMP9 in the environment, the release of SDF-1 on the surface of the material quickly tends to be gentle, showing good stability, while when the environment contains MMP9, the surface nanocoating can be induced. The peptides of the layer are degraded, thereby controlling the sustained release of SDF-1. Therefore, the constructed nanocoating can release SDF-1 under the stimulation of the signaling enzyme MMP9 released by vascular injury in the blood flow environment in vivo, and promote the wound healing.

It can be seen from Figure 3 that the constructed nano-coating can effectively reduce the adhesion and activation of platelets, thereby avoiding coagulation or thrombus on the surface.

It can be seen from Figure 4 that the constructed nanocoatings also have a certain effect of inducing the homing and aggregation of endothelial progenitor cells in the absence of MMP9, but it is not obvious; in the presence of MMP9, the nanocoatings show significant induction. The effect of endothelial progenitor cell aggregation, which facilitates rapid healing after vascular injury.

Claims (6)

1. A construction method of an enzyme response type multifunctional nano coating is characterized by comprising the following steps:

A) polishing and cleaning the surface of 316L stainless steel, immersing the surface into a dopamine solution with the concentration of 2 mg/ml for reaction for 12 hours, carrying out ultrasonic cleaning by double distilled water, repeating the steps for 2 times to obtain the surface deposited with 3 layers of polydopamine coatings, and drying at 37 ℃;

B) soaking the sample deposited with the polydopamine coating in the step A) in 0.1 ~ 0.5.5 mg/ml avidin solution, standing and reacting for 8 ~ 24 hours at 37 ℃, then cleaning the sample with double distilled water, and storing for later use;

C) the method comprises the following steps of (1) blending a biotinylated heparin sodium solution with the concentration of 10 ~ 20 mg/ml and a polyethyleneimine solution with the concentration of 1 ~ 2 mg/ml in an equal volume manner, and performing ultrasonic treatment for 5 minutes at room temperature to obtain a nanoparticle suspension;

immersing the sample obtained in the step B) in the nanoparticle suspension, carrying out oscillation reaction for 8 ~ 24 hours at 37 ℃, rinsing the sample by using double distilled water, and storing for later use;

D) immersing the sample obtained in step C) into 1 ~ 3 mg/ml avidin solution, reacting at 37 deg.C for 1 ~ 3 hr, and synthesizing biotinylated enzyme-responsive polypeptide with structure of Biotin-PRQITAG-NH₂；

Soaking the sample in 10 ~ 50 mu g/ml polypeptide solution, reacting for 1 ~ 3 hours at 37 ℃, rinsing the sample with double distilled water, and storing for later use;

E) and (2) dropwise adding an EDC/NHS/MES crosslinking agent solution with the molar ratio of 2:1:1 into an SDF-1 alpha solution with the concentration of 100 ~ 500 ng/ml, uniformly mixing, immediately soaking the sample obtained in the step D) in the solution, reacting for 1 ~ 3 hours at 37 ℃, and cleaning the sample with double distilled water to obtain the product.

2. The method for constructing an enzyme-responsive multifunctional nano-coating according to claim 1, wherein the method comprises the following steps: and B), C), D) and E), wherein the solvent of the biomolecule solution in the steps B), C), D) and E) is phosphate buffer solution.

3. The method for constructing an enzyme-responsive multifunctional nano-coating according to claim 1, wherein the method comprises the following steps: and in the steps B), C), D) and E), the sample is refrigerated and stored at the temperature of 4 ℃ under the condition of surface wetting.

4. The method for constructing an enzyme-responsive multifunctional nano-coating according to claim 1, wherein in the step C), the molecular weight of polyethyleneimine is in the range of 60 ~ 75 kDa.

5. The method for constructing an enzyme-responsive multifunctional nano-coating according to claim 1, wherein the method comprises the following steps: in the step E), MES is 0.05 mol.

6. The method for constructing an enzyme-responsive multifunctional nano-coating according to claim 1, wherein the method comprises the following steps: in the step E), in EDC/NHS/MES cross-linking agent solution, V_SDF-1/V_{Crosslinking agent}=10/1。

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