CN113546223A

CN113546223A - Method for compositely constructing anticoagulant surface coating by utilizing dopamine and recombinant hirudin

Info

Publication number: CN113546223A
Application number: CN202110691723.0A
Authority: CN
Inventors: 郑志雯; 杜昶; 戴欣; 李雪杨
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2021-10-26

Abstract

The invention relates to the technical field of biological materials, and discloses a method for compositely constructing an anticoagulant surface coating by utilizing dopamine and recombinant hirudin, which comprises dopamine modification and functional modification of the recombinant hirudin on the surface of a material; anticoagulant surface coatings and applications are also provided. The method is simple and easy to operate, the anticoagulation performance of the substrate surface is obviously improved, the adhesion of platelets is inhibited, and the constructed material surface has good biological performance and no toxicity; the invention provides theoretical and experimental basis for industrial upgrading and clinical application of medical materials, has wide application prospect and important social and economic values, and also has important significance for promoting development of medical instruments and biomedical material industry and cultivating strategic emerging industry.

Description

Method for compositely constructing anticoagulant surface coating by utilizing dopamine and recombinant hirudin

Technical Field

The invention relates to the technical field of biological materials, in particular to a method for compositely constructing an anticoagulant surface coating by utilizing dopamine and recombinant hirudin.

Background

The biological material is a natural or artificial special functional material which is used for contacting and interacting with a living system and can carry out diagnosis, treatment, replacement, repair or induced regeneration on cells, tissues and organs of the biological material, and is also called as a biomedical material. Biomaterials are the field of interdiffusion of a variety of developing disciplines in the field of material science, whose research content relates to disciplines of material science, life science, chemistry, biology, anatomy, pathology, clinical medicine, pharmacology, etc., as well as to the domains of engineering and management sciences. The biological material comprises an artificial synthetic material and a natural material, and comprises a single material, a composite material and a hybrid material formed by combining living cells or natural tissues with non-living materials.

Dopamine is a nerve conduction substance secreted by a human body per se, plays a role of transmitting pulses to cells in the human body, and in an aerobic alkaline environment, dopamine monomers can rearrange through intermolecular crosslinking to form a dopamine polymerization layer on the surface of a material through self-polymerization, so that a new idea is provided for modification of the surface of the material, wherein silicon serving as a good biological material is a good material. Hirudin is a direct thrombin inhibitor, can inhibit coagulation process by inhibiting thrombin activity, and can be used as anticoagulant for preventing and treating intravascular embolism or thrombus, and preventing apoplexy or other thrombotic diseases. It blocks the coagulation process by affecting certain coagulation factors in the coagulation process, has less bleeding side effects, and has no anaphylaxis and immunogenic non-toxic reaction.

Disclosure of Invention

Based on the problems, the invention provides a method for compositely constructing the anticoagulation surface coating by utilizing dopamine and recombinant hirudin, the method is simple, convenient and easy to operate, the anticoagulation performance of the substrate surface is obviously improved, the adhesion of platelets is inhibited, the hemolysis rate of the surface of the prepared anticoagulation surface coating material is lower than 5%, and the constructed material surface has good biological performance and no toxicity.

In order to solve the technical problems, the invention provides a method for compositely constructing an anticoagulant surface coating by utilizing dopamine and recombinant hirudin, which comprises the following specific steps:

s1: soaking the material substrate in acetone solution, ultrasonically cleaning for 15min, taking out, rinsing with deionized water for three times, ultrasonically cleaning with ethanol and deionized water for 15min, rinsing with deionized water for three times after each ultrasonic cleaning, and heating in a cleaning solution at 60 deg.C for 15 min; after all the operations are finished, washing the substrate with deionized water for three times, and finally drying the substrate with nitrogen for later use;

s2: adding 0.5ml of TRIS-HCl buffer solution with the concentration of 1.5mol/l into 74.5ml of deionized water, then adding 150mg of dopamine, and uniformly mixing to obtain 2mg/ml of dopamine solution for later use;

s3: soaking the substrate processed in the step S1 into the dopamine solution prepared in the step S2, leaving the substrate open and standing for 24 hours, taking the substrate out, ultrasonically cleaning the substrate for 10 minutes by using deionized water, and drying the substrate by using nitrogen for later use;

s4: diluting 10 times PBS buffer solution by 10 times, then diluting the hirudin solution with the concentration of 10mg/ml to 1mg/ml by the diluted PBS buffer solution, putting the diluted hirudin solution into a glass bottle, and storing the hirudin solution in a refrigerator at 4 ℃ for later use;

s5: and (4) putting the substrate processed in the step S3 into 1ml of the hirudin solution prepared in the step S4, standing for 24 hours, taking out, washing with deionized water for three times, and drying with nitrogen.

Further, the cleaning solution in step S1 includes the following components by volume ratio: 25% ammonia water: 30% of hydrogen peroxide: deionized water 1:1: 5.

Further, the substrate is a pure silicon wafer, and the number of the pure silicon wafers in the step S3 is 15.

In order to solve the technical problems, the invention also provides an anticoagulant surface coating.

In order to solve the technical problems, the invention also provides application of the anticoagulation surface coating in medical instruments or biomedical materials.

Compared with the prior art, the invention has the beneficial effects that: the method is simple and easy to operate, the anticoagulation performance of the substrate surface is obviously improved, the adhesion of platelets is inhibited, the hemolysis rate of the surface of the prepared anticoagulation surface coating material is lower than 5 percent, and the constructed material surface has good biological performance and no toxicity; the invention provides theoretical and experimental basis for industrial upgrading and clinical application of medical materials, has wide application prospect and important social and economic values, and also has important significance for promoting development of medical instruments and biomedical material industry and cultivating strategic emerging industry.

Drawings

FIG. 1 is a graph showing the results of the adhesion of an anticoagulant surface coating to platelets in an embodiment of the present invention;

FIG. 2 is an AFM three-dimensional perspective view of the dopamine modified front and back surface contrast in an embodiment of the invention;

FIG. 3 is an AFM three-dimensional view of the surface of the material grafted with hirudin following dopamine modification in an embodiment of the present invention;

FIG. 4 is a graph showing the adsorption quality of fibronectin (left) and albumin (right) on the surface of the modified material as a function of time according to an example of the present invention;

FIG. 5 is a graph showing the change in hemolysis rate in accordance with an embodiment of the present invention;

FIG. 6 is a graph of endothelial cell proliferation on the surface of the material for 1 day and 5 days in accordance with an embodiment of the present invention;

FIG. 7 is a graph of activated partial thromboplastin time for an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example (b):

in this example, a method for compositely constructing an anticoagulant surface coating by using dopamine and hirudin is studied, and the details are described below.

Treating a pure silicon wafer: will be 10X 5mm²Soaking the pure silicon wafer into an acetone solution, carrying out ultrasonic cleaning for 15min, taking out, rinsing with deionized water for three times, then respectively carrying out ultrasonic cleaning for 15min with ethanol and deionized water, rinsing with deionized water for three times after each ultrasonic cleaning, and finally putting into a cleaning solution and heating in a 60 ℃ water bath for 15 min; to accomplish all the above operationsAnd then, washing the pure silicon wafer three times by using deionized water, and finally blowing and drying by using nitrogen for later use. The cleaning solution used in this embodiment includes the following components by volume: 25% ammonia water: 30% of hydrogen peroxide: deionized water 1:1: 5.

Preparing a dopamine solution: adding 0.5ml of TRIS-HCl buffer solution with the concentration of 1.5mol/l into 74.5ml of deionized water, then adding 150mg of dopamine, uniformly mixing to obtain dopamine solution with the concentration of 2mg/ml, and finally pouring all the prepared dopamine solution into a glass watch glass for later use, wherein the dopamine solution is ready to use.

Dopamine-modified silicon surface: and soaking the processed pure silicon wafers into the prepared dopamine solution, standing for 24 hours in an open manner, taking out, ultrasonically cleaning for 10min by using deionized water, and blow-drying by using nitrogen for later use, wherein the number of the pure silicon wafers processed in the step is 15.

In this embodiment, after the surfaces of the cleaned silicon wafer and the processed silicon wafer are modified with dopamine, the contact angle, the surface morphology, the surface element groups, and the like of the silicon wafer are characterized. C spectrum of the modified surface can be fitted with a C-O/C-N characteristic peak, N spectrum can be fitted with a C-N/-NH3+ characteristic peak, O spectrum can be fitted with a C ═ O/O-H characteristic peak, and XPS result shows that the dopamine modified surface is successfully prepared; AFM results show that the surface roughness after modification is increased from 0.82nm to 1.289nm, and particularly shown in figure 2; the contact angle results show that the surface hydrophilicity after dopamine modification is slightly reduced, and the water contact angle is increased from 43 degrees to 49.7 degrees.

Preparing a hirudin solution by using a Tris-HCl buffer solution: diluting Tris-HCl buffer solution with the concentration of 1.5mol/L by 150 times, then diluting hirudin solution with the concentration of 10mg/ml to 1mg/ml by using the diluted Tris-HCl buffer solution, putting the diluted hirudin solution into a glass bottle, and storing the hirudin solution in a refrigerator at 4 ℃ for later use.

Preparing a hirudin solution by using a PBS buffer solution: diluting 10 times PBS buffer solution, diluting hirudin solution with concentration of 10mg/ml to 1mg/ml with diluted PBS buffer solution, placing the diluted hirudin solution into a glass bottle, and storing in refrigerator at 4 deg.C for use.

And (3) taking two pure silicon wafers after dopamine modification treatment, respectively putting the two pure silicon wafers into 1ml of hirudin solution prepared by Tris-HCl buffer solution and 1ml of hirudin solution prepared by PBS buffer solution, standing for 24 hours, taking out the pure silicon wafers, washing the pure silicon wafers with deionized water for three times, and drying the pure silicon wafers with nitrogen.

And (3) characterization: and (3) carrying out surface energy state analysis on the sample (Si-DA-rH) before and after hirudin modification by using an X-ray photoelectron spectrometer. The optimal condition of modification is determined mainly according to the content change of N, O, Si three elements, and then the hydrophilicity, surface morphology, roughness and the like of the sample before and after modification under the optimal condition are characterized by a standard contact angle meter and an atomic force microscope.

In this example, the experiment of bonding hirudin to dopamine-modified silicon wafer was carried out by X-ray photoelectron spectroscopy, water contact angle apparatus, atomic force microscope. The result shows that the dopamine modified silicon surface successfully bonds hirudin in the hirudin solution prepared by PBS, the hirudin cannot be bonded in the hirudin solution prepared by Tris-HCl, and the bonding effect of the hirudin solution prepared by PBS at 1mg/ml is better than that of the hirudin solution prepared by PBS at 0.25mg/ml and the hirudin solution prepared by PBS at 0.5 mg/ml.

The pure silicon wafer selected in this embodiment is only one of the substrate materials, the pure silicon wafer of this embodiment may be replaced by any substrate, and the dopamine of this embodiment may undergo self-polymerization on any substrate.

After soaking in 1mg/ml hirudin solution in PBS buffer at pH 7.4, the surface showed N-C ═ O and the content of C-N, -NH3+ decreased. The hirudin is bonded on the dopamine polymerization layer, so that the originally smooth and round surface is rougher, and a plurality of fine protrusions appear, so that the surface roughness is increased from 1.289nm to 2.907nm, specifically shown in figure 3; after the dopamine polymerization layer is grafted with hirudin, the water contact angle is reduced by about 3 degrees, and the surface hydrophilicity is increased.

In this example, the interference adsorption behavior of pure silicon sheet modified to common Fibronectin (FN) and Bovine Serum Albumin (BSA) in blood was studied, specifically, as shown in fig. 4, the adsorption quality and adsorption rate of FN and BSA were increased on the surface of the material modified with dopamine, and the adsorption quality and adsorption rate of both proteins were decreased after further modification with hirudin, which indicates that further modification with hirudin inhibits the adsorption of FN and BSA to a certain extent, and the adsorption inhibition to BSA is more significant.

In this example, the adhesion of the anticoagulant surface coating prepared by the above method to platelets was studied, fresh rabbit blood was centrifuged at 1000rpm for 10min, and platelet-rich supernatant was collected. The sterilized silicon wafers were immersed in the platelet rich supernatant and incubated at 37 ℃ for 1h, after which the wafers were gently rinsed with sterile saline, then fixed in 2.5% glutaraldehyde at 37 ℃ for 4h, dehydrated with various concentrations of alcohol (25%, 50%, 75%, 100%) and observed for the number and morphology of platelets adhering to the sections using a Scanning Electron Microscope (SEM).

After incubating with the platelet-rich supernatant, the number and the form of platelets adhered to the silicon wafer are detected by a scanning electron microscope. The modification concentrations of hirudin grafted on the surface of the material after dopamine modification are 250 mug/ml, 500 mug/ml and 1mg/ml, and the samples are named as rH1-PDA-Si, rH2-PDA-Si and rH 3-PDA-Si. Referring to the attached figure 1, platelet adhesion and aggregation can be seen on the blank silicon wafer, and part of platelets are dendritic and have lower activity; in contrast, these platelets, modified with Polydopamine (PDA), hirudin, are round, indicating a healthy inactive phenotype. As mentioned above, PDA and hirudin coatings impart a certain anti-platelet adhesion to silicon wafers, which is important for preventing thrombosis and intimal hyperplasia.

This example collected fresh rabbit blood in a test tube, containing sodium citrate, and then diluted 5-fold with physiological saline. Soaking a sterile silicon wafer in a diluted blood sample, incubating for 1h at 37 ℃, respectively setting blood solutions diluted by physiological saline and ultrapure water as a negative control and a positive control, measuring absorbance (A) at 540nm of a centrifuged supernatant by using an enzyme-labeling instrument, and calculating the hemolysis rate according to the following formula: the hemolysis ratio (%) × (experimental group a-negative control group a)/(positive control group a-negative control group a) × 100%.

Since hemolysis is caused by rupture of erythrocytes due to various factors, the current mechanism is unknown, and therefore, materials for implantable applications must meet the criteria of ISO10993-4-4 with a hemolysis rate of less than 5%. Referring to fig. 5, the hemolysis test results of this example show that all samples have hemolysis rate below 1.2% and good blood compatibility. It is worth noting that the hemolysis rate of the silicon chip loaded with hirudin is obviously reduced to below 0.4%, which is mainly due to the protection effect of hirudin, which can prevent the interaction of red blood cells and the surface of the silicon chip.

This example also performed Human Umbilical Vein Endothelial Cell (HUVECs) proliferation experiments: human Umbilical Vein Endothelial Cells (HUVECs) in HyClone containing 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin^TMDulbecco's Modified Eagles Medium (DMEM) at 37 ℃ with 5% CO₂Culturing under an atmosphere; mixing 1X 1cm²The silicon wafers of (2) were placed in a 24-well plate with HUVECs at 2X 10⁴Inoculating the solution on the surface of a silicon wafer at a density of one ml, and culturing for 1d, 3d and 5d respectively; staining the cells by using a live/dead cytotoxicity kit, and finally taking a picture by using an inverted fluorescence microscope; detecting cell adhesion test and cell proliferation by CCK-8 method; specifically, after incubating with cells for 1d, 3d, and 5d, the aspirated medium was rinsed with PBS, 200. mu.l of CCK-8 solution (10% CCK-8 and 90% complete medium) was added to each sample, incubated at 37 ℃ for 2h, aspirated and transferred to a 96-well plate to detect absorbance values of 450nm using a microplate reader, and three replicates were set for each sample.

After 24h incubation, ECs in 24-well plates were stained with live and dead staining solution and live cells showed green fluorescence. Referring to FIG. 6, ECs are shown in growth state, similar to the blank sample, indicating that the silicon wafer modified with hirudin has a certain cell compatibility. After the cells are incubated for 5 days, the silicon slice loaded with hirudin with lower concentration (250 mu g/ml and 500 mu g/ml modification groups) has no obvious promotion effect on the proliferation of endothelial cells, when the concentration of the hirudin solution reaches 1mg/ml, the modified silicon slice has obvious promotion effect on the proliferation of the endothelial cells, and the experimental result shows that the concentration of the hirudin solution required for promoting the rapid endothelialization reaches 1mg/ml at least. The CCK-8 results are consistent with the above results.

Activated partial thromboplastinTime is mainly used to test the specific changes of the intrinsic coagulation system, which refers to the system in which coagulation factors from plasma participate in coagulation. In this example, 30.0mL of anticoagulated human whole blood was centrifuged at 1500rpm for 10min to obtain ischemic platelet plasma (PPP). The APTT test procedure is as follows: firstly, 1.0X 1.0cm²The silicon wafers were placed in a clean 24-well cell culture plate, followed by adding 0.2mL of PPP to the surface of each of the above samples until the samples were completely infiltrated, incubating at 37 ℃ for 1h, then removing plasma, and measuring APTT using a full-automatic coagulometer.

Referring to fig. 7, the APTT test results of the pure Si group and the PDA-Si group were 24.8s and 25.2s, respectively, and thus the hydrophilicity of the membrane did not greatly affect the APTT. The APTT value tested by the silicon chip loaded with hirudin can reach 38s, and the APTT is prolonged along with the increase of the hirudin concentration. The results show that the silicon chip for modifying hirudin has good anticoagulant property, mainly because hirudin can form a stable non-covalent stoichiometric compound with thrombin without active enzyme, and plasma thrombin is inactivated quickly after contacting with hirudin, thereby inhibiting the process of converting fibrinogen into fibrin, which is the main reason for raising APTT.

In conclusion, the coating prepared by the embodiment obviously improves the anticoagulation performance of the substrate surface, inhibits the adhesion of platelets, ensures that the hemolysis rate of the surface of the coating material meets the standard specification of ISO10993-4-4, and has good biological performance and no toxicity on the surface of the material.

The dopamine polymer film used in the embodiment endows the surface of the material with good biological performance, and provides abundant positively charged groups such as quinone groups and amino groups, and the negative electricity groups of the hirudin molecules and the dopamine polymer film are stably combined on the surface through hydrogen bonds. The anticoagulant surface coating prepared by the embodiment can be applied to medical instruments or biomedical materials, provides theoretical and experimental basis for industrial upgrading and clinical application of the medical materials, has wide application prospect and important social and economic values, and has important significance for promoting development of medical instruments and biomedical material industries and breeding strategic emerging industries.

The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.

Claims

1. The method for compositely constructing the anticoagulation surface coating by utilizing the dopamine and the recombinant hirudin is characterized by comprising the following specific steps of:

2. The method for compositely constructing an anticoagulant surface coating by using dopamine and recombinant hirudin according to claim 1, wherein the cleaning solution in the step S1 comprises the following components by volume ratio: 25% ammonia water: 30% of hydrogen peroxide: deionized water 1:1: 5.

3. The method for compositely constructing an anticoagulant surface coating by using dopamine and recombinant hirudin according to claim 1, wherein the substrate is a pure silicon wafer, and the number of the pure silicon wafers in the step S3 is 15.

4. An anticoagulant surface coating constructed by the method of any one of claims 1 to 3.

5. Use of the anticoagulant surface coating of claim 4 in a medical device or biomedical material.