CN107376036B

CN107376036B - Construction method of enzyme response type multifunctional nano coating

Info

Publication number: CN107376036B
Application number: CN201710487281.1A
Authority: CN
Inventors: 刘涛; 罗逊; 刘诗卉; 龚韬; 汤潇涵; 潘长江; 丁红燕
Original assignee: Shenzhen Kerry Rehabilitation Medical Research Institute; Huaiyin Institute of Technology
Current assignee: Shenzhen Kerry Rehabilitation Medical Research Institute; Huaiyin Institute of Technology
Priority date: 2017-06-23
Filing date: 2017-06-23
Publication date: 2020-01-07
Anticipated expiration: 2037-06-23
Also published as: CN107376036A

Abstract

The invention discloses a construction method of an enzyme response type nano coating, which comprises the steps of firstly fixing avidin molecules on a stainless steel surface coated with dopamine, secondly fixing biotinylated heparin/PEI nano particles on the surface of a material by utilizing the specific recognition and combination action between avidin and biotin, and further combining the high-concentration avidin molecules with the residual biotin on the surface of the nano particle coating so as to introduce new biotin combination sites; then, the interaction between biotin and avidin is continuously utilized to assemble biotinylated enzyme response polypeptide to the surface of the nanoparticle, and finally, EDC/NHS/MES coupling agent is used to covalently fix SDF-1 alpha to the amino terminal of the polypeptide, so that the multifunctional nano coating with the matrix metalloproteinase 9 response characteristic is constructed. The invention constructs a multifunctional layer with anticoagulation and endothelial regeneration induction capability on the titanium surface, and obviously improves the blood compatibility and damaged endothelial repair capability of the material.

Description

Construction method of enzyme response type multifunctional nano coating

Technical Field

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

Background

Cardiovascular diseases such as coronary heart disease seriously threaten the life health of human beings. At present, the blood circulation condition of patients with coronary heart disease is improved clinically mainly through metal vascular stent interventional therapy. However, the traditional metal bare stent and the drug eluting stent often cause thrombosis and vascular intimal hyperplasia at middle and late stages after implantation due to insufficient biocompatibility, thereby causing implantation failure and even endangering the life safety of patients.

The method for improving the biocompatibility of the material is commonly used at present by biologically modifying the surface of the material to endow the material with good anticoagulation capacity and endothelial regeneration induction capacity. However, in complex environments in vivo, many biological coatings are difficult to maintain for long periods of time, but rapidly lose their biological activity in dynamic environments in vivo. In order to achieve a long-term effective functioning of the bio-modification layer, it is necessary to design the bio-modification layer specifically for the biological environment in which it is located.

Heparin is an anticoagulant drug widely used clinically and is also a commonly used substance for surface modification of cardiovascular materials. However, because the heparin molecules lack sites for direct reaction with the surface of the material, the invention firstly introduces avidin molecules on the surface of the material, then carries out biotinylation modification on the heparin molecules, and introduces the heparin molecules on the surface of the material by utilizing the specific binding action between biotin and avidin. In order to improve the loading capacity of surface heparin, polycation electrolyte polyethyleneimine rich in amino is introduced, and the nanoparticles are fixed on the surface of the material by utilizing the characteristic that biotinylated heparin can perform electrostatic interaction with the polyethyleneimine to form the nanoparticles.

The mesenchymal derived factor-1 alpha (SDF-1 alpha) is a chemotactic factor which has strong chemotactic effect on bone marrow CXCR4+ stem cells and Endothelial Progenitor Cells (EPCs), and simultaneously has the functions of stimulating the growth of endothelial cells and inducing the endothelial progenitor cells to differentiate into endothelial cells. Research shows that SDF-1 alpha can accelerate vascular injury repair and endothelial regeneration by inducing endothelial progenitor cells to gather and differentiate to vascular injury sites.

The PRQITAG polypeptide is a polypeptide with special response to matrix metalloproteinase 9 (MMP-9), and can be decomposed in the presence of MMP-9. MMP-9 is an important functional protein in the process of repairing vascular injury, and can rapidly degrade vascular basement membrane and extracellular matrix components, so that the components can release various proteins and growth factors to stimulate the migration and proliferation of vascular cells. Thus, MMP-9 may also be considered a signaling protein for vascular injury repair. By utilizing the characteristic, the invention takes the PRQITAG polypeptide with MMP-9 response characteristic as a connecting molecule, one end of the connecting molecule is fixed on the surface of the nano-particle coating, and the other end is connected with the chemotactic factor SDF-1 alpha. When a large amount of MMP-9 is secreted due to the damaged vascular tissue, the surface of the material is induced to release SDF-1 alpha molecules, so that the repair of the vascular damage is accelerated.

The biological function layer with the enzyme response characteristic can improve the blood compatibility of the material surface, and can release the biological molecules as required according to the change of the environment in blood vessels, thereby effectively improving the utilization rate and action time of the biological molecules and promoting the repair of blood vessel injury. However, no report about the surface modification of the enzyme-responsive nano-coating cardiovascular material exists at present.

Disclosure of Invention

The invention aims to provide a construction method of an enzyme response type multifunctional nano coating, which can realize the release of biological factors according to requirements by performing biochemical modification on the surface of a cardiovascular material, effectively improve the biocompatibility of the material and promote the damage repair capacity; creatively selects a construction mode of a multi-functional layer, and utilizes specific interaction among various biomolecules under a certain biomolecule concentration proportion to realize ordered assembly of functional biomolecules on the surface of the material, thereby achieving the characteristics that in a specific biological environment, the biomolecules on the surface of the material are released as required, and the biological function of the surface is continuously and stably exerted; the construction process and the fixing method of the biological microenvironment are simple and easy to operate, expensive and complex equipment is not needed, the process cost is low, the controllability is strong, and the effect is obvious; the fixation of various biomolecules on the surface of the stainless steel is carried out in a soaking mode, so that the biomolecules can be uniformly fixed on each part of the material, the functional modification of the surfaces of various cardiovascular implantation instruments with complex structures can be realized, and the application range is wide.

The technical scheme adopted by the invention for realizing the aim is that the construction method of the enzyme response type multifunctional nano coating comprises 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 biotinylated heparin sodium solution with the concentration of 10 ~ 20 mg/ml and the polyethyleneimine solution with the concentration of 1 ~ 2 mg/ml are mixed in the same volume, and subjected to ultrasonic treatment for 5 minutes at room temperature to obtain nanoparticle suspension, the sample obtained in the step B is immersed in the nanoparticle suspension, oscillation reaction is performed for 8 ~ 24 hours at 37 ℃, and then the sample is rinsed by double distilled water and stored 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-NH₂Soaking the sample in 10 ~ 50 mug/ml polypeptide solution, reacting at 37 ℃ for 1 ~ 3 hours, 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.

In a further improvement of the invention, the solvents of the biomolecule solutions in the steps B), C), D) and E) are phosphate buffer solutions.

In a further improvement of the invention, in the steps B), C), D) and E), the samples are all stored in a refrigerated manner at a temperature of 4 ℃ under the condition of wet surface.

In a further development of the invention, in step C), the polyethyleneimine has a molecular weight in the range of 60 ~ 75 kDa.

In a further development of the invention, in step E), the amount of MES is 0.05 mol.

In a further development of the invention, in step E), the EDC/NHS/MES crosslinker solution is added,

V_SDF-1/V_{crosslinking agent}=10/1。

Referring to the attached figure 1 of the specification, the reaction process of the invention comprises the following steps, wherein the first step is grafting avidin on the surface of stainless steel. Firstly, a polydopamine coating is deposited on the surface of 316L stainless steel, and avidin is fixed on the surface of a material by utilizing the characteristic that quinone groups on the surface of dopamine can perform Schiff base reaction with amino groups in avidin molecules; secondly, synthesizing biotinylated heparin/polyethyleneimine nanoparticles by utilizing intermolecular electrostatic interaction; thirdly, fixing the nanoparticles on the surface of the material by utilizing the characteristic that the biotin molecules on the surface of the nanoparticles can be specifically identified and combined with the avidin molecules on the surface of the material; fourthly, using high-concentration avidin molecules to combine the rest biotin molecules on the surface of the nano coating, and simultaneously introducing avidin combination sites; fifthly, synthesizing biotinylated matrix metalloproteinase 9 response type polypeptide, and fixing the polypeptide on the surface of the material by utilizing the specific recognition function of the binding site of biotin molecules and avidin on the surface of the material; and sixthly, activating carboxyl in the SDF-1 alpha molecule by using an EDC/NHS/MES crosslinking agent, and performing dehydration condensation reaction with amino at the tail end of the polypeptide molecule on the surface of the material, thereby fixing the SDF-1 alpha on the surface of the material.

Compared with the prior art, the invention has the beneficial effects that:

the first method for constructing the enzyme response type multifunctional nano coating creatively selects a construction mode of a multifunctional layer, and utilizes specific interaction among various biomolecules under a certain biomolecule concentration ratio to realize ordered assembly of functional biomolecules on the surface of a material, thereby achieving the characteristics that in a specific biological environment, the biomolecules on the surface of the material are released as required, and the biological function of the surface is continuously and stably exerted.

And secondly, the construction method of the enzyme response type multifunctional nano coating, the construction process and the fixing method of the biological microenvironment are simple and easy to operate, expensive and complicated equipment is not needed, the process cost is lower, the controllability is strong, and the effect is obvious.

And thirdly, the fixation of various biomolecules on the surface of the stainless steel is carried out in a soaking mode, so that the biomolecules can be uniformly fixed on all parts of the material, the functional modification of the surfaces of various cardiovascular implantation instruments with complex structures can be realized, and the application range is wide.

Drawings

The method of the present invention is further described in detail below with reference to the drawings and examples.

FIG. 1 is a schematic diagram of the steps of constructing a matrix metalloproteinase 9 responsive nano-coating in the method of the present invention. (1) Deposition of dopamine coating on the surface of 316L stainless steel and assembly of avidin; (2) preparing biotinylated heparin/polyethyleneimine nanoparticles; (3) fixing the nano particles on the surface of the material; (4) assembling avidin molecules on the surface of the nano-particle coating; (5) assembling biotinylation enzyme response type polypeptide on the surface of the material; (6) and (3) fixing the SDF-1 alpha on the surface of the material.

FIG. 2 is a graph showing the effect of MMP9 on the release kinetics of SDF-1 under conditions mimicking in vivo flow fields.

FIG. 3 shows the results of fluorescent staining of platelets on the surface of different samples after 2 hours of adhesion.

FIG. 4 is a graph showing the results of the enzyme-responsive nanocoating inducing homing of endothelial progenitor cells in the chemotaxis chamber model in the presence of MMP 9. Dopamine coated samples were blank controls.

Detailed Description

Example one

Referring to fig. 1, a first embodiment of the present invention is a method for constructing an enzyme-responsive nano-coating, comprising the steps of:

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

C) and (3) blending the biotinylated heparin sodium solution with the concentration of 10 mg/ml and the polyethyleneimine solution with the concentration of 1 mg/ml in an equal volume, and performing ultrasonic treatment for 5 minutes at room temperature to obtain the nanoparticle suspension. Immersing the sample obtained in the step B into the nanoparticle suspension, carrying out oscillation reaction for 8 hours at the temperature of 37 ℃, rinsing the sample by using double distilled water, and storing for later use;

D) immersing the sample obtained in the step C) into 1 mg/ml avidin solution, and reacting for 1 hour at 37 ℃; synthesizing biotinylated enzyme response type polypeptide with the structure of Biotin-PRQITAG-NH₂. Soaking the sample in 10 mu g/ml polypeptide solution, reacting for 1 hour 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 ng/ml, uniformly mixing, immediately soaking the sample obtained in the step D) in the solution, reacting for 1 hour at 37 ℃, and then washing the sample with double distilled water to obtain the product.

V_SDF-1/V_{crosslinking agent}=10/1。

Example two

A construction method of an enzyme response type nano coating comprises the following steps:

B) soaking the sample deposited with the polydopamine coating in the step A) in 0.5 mg/ml avidin solution, standing and reacting for 8 ~ 24 hours at 37 ℃, then washing the sample with double distilled water, and storing for later use;

C) and (3) blending the biotinylated heparin sodium solution with the concentration of 20 mg/ml and the polyethyleneimine solution with the concentration of 2 mg/ml in an equal volume, and performing ultrasonic treatment for 5 minutes at room temperature to obtain the nanoparticle suspension. Immersing the sample obtained in the step B into the nanoparticle suspension, carrying out oscillation reaction for 24 hours at the temperature of 37 ℃, rinsing the sample by using double distilled water, and storing for later use;

D) immersing the sample obtained in the step C) into 3 mg/ml avidin solution, and reacting for 3 hours at 37 ℃; synthesizing biotinylated enzyme response type polypeptide with the structure of Biotin-PRQITAG-NH₂. Soaking the sample in 50 mu g/ml polypeptide solution, reacting for 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 500 ng/ml, uniformly mixing, immediately soaking the sample obtained in the step D) in the solution, reacting for 3 hours at 37 ℃, and then washing the sample with double distilled water to obtain the product.

V_SDF-1/V_{crosslinking agent}=10/1。

EXAMPLE III

B) soaking the sample deposited with the polydopamine coating in the step A) in 0.3 mg/ml avidin solution, standing and reacting for 12 hours at 37 ℃, then cleaning the sample with double distilled water, and storing for later use;

C) and (3) blending the biotinylated heparin sodium solution with the concentration of 15 mg/ml and the polyethyleneimine solution with the concentration of 1.5 mg/ml in an equal volume, and performing ultrasonic treatment for 5 minutes at room temperature to obtain the nanoparticle suspension. Immersing the sample obtained in the step B into the nanoparticle suspension, carrying out oscillation reaction for 12 hours at the temperature of 37 ℃, rinsing the sample by using double distilled water, and storing for later use;

D) immersing the sample obtained in the step C) into 2 mg/ml avidin solution, and reacting for 2 hours at 37 ℃; synthesizing biotinylated enzyme response type polypeptide with the structure of Biotin-PRQITAG-NH₂. Soaking the sample in 30 mu g/ml polypeptide solution, reacting for 2 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 300 ng/ml, uniformly mixing, immediately soaking the sample obtained in the step D) in the solution, reacting for 2 hours at 37 ℃, and then washing the sample with double distilled water to obtain the product.

V_SDF-1/V_{crosslinking agent}=10/1。

Compared with the example 3, in the biochemical reaction process, the reasonable shortening of the reaction time is beneficial to maintaining the biological activity of the biological molecules, so that the biological functions can be better exerted; compared with the embodiment 3, the embodiment 2 has the advantages that the loading density of surface biomolecules is improved to the greatest extent according to the mechanism of biochemical reaction, and the surface function can be effectively exerted for a long time. It can be seen that the effect of the nanocoatings obtained in examples 1 and 2 is superior to that of the nanocoating obtained in example 3. Therefore, the nanolayered coating obtained by example 3, which has the worst analytical effect, can solve the technical problems to be solved by the present application, and that can also be absolutely solved by example 1 and example 2.

As can be seen from FIG. 2, when the material is not in the environment containing MMP9, the release of SDF-1 on the surface of the material quickly tends to be flat and shows better stability, and when the material is in the environment containing MMP9, the polypeptide of the surface nano coating can be induced to degrade, so that the sustained release of SDF-1 is controlled. Therefore, the constructed nano coating can release SDF-1 under the stimulation of a signaling enzyme MMP9 released by vascular injury in an in-vivo blood flow environment, and the injury healing is promoted.

As can be seen from FIG. 3, the constructed nano-coating can effectively reduce the adhesion and activation of platelets, thereby avoiding the coagulation or thrombosis on the surface.

As can be seen from fig. 4, the constructed nanocoating has a certain but not obvious effect of inducing homing and aggregation of endothelial progenitor cells in the absence of MMP 9; when MMP9 is present, the nanocoating exhibits a significant effect of inducing endothelial progenitor cell aggregation, which facilitates rapid healing after vascular injury.

Claims

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

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;

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。