Disclosure of Invention
The embodiment of the invention provides a preparation method of a medical functional coating, which comprises the steps of 1) providing a substrate, forming a polydopamine coating containing acrylamide on the surface of the substrate, and 2) contacting a hydrophilic monomer containing carbon-carbon unsaturated bonds with the polydopamine coating containing acrylamide, and reacting and crosslinking to form a hydrophilic polymer coating.
In some embodiments, the method of preparing further comprises the step of 3) immobilizing functional molecules on the surface of the hydrophilic polymer coating.
In some embodiments, the functional molecules include one or more of extracellular matrix protein molecules, polysaccharide molecules, and antimicrobial peptide molecules.
The polysaccharide comprises one or two of heparin and hyaluronic acid.
In some embodiments, in step 3), the functional molecule is formed on the surface of the hydrophilic polymer coating by at least one of coupling fixation and electrostatic adsorption.
In some embodiments, in the step 3), the coupling and fixing step includes contacting the substrate with the hydrophilic polymer coating formed on the surface with an aqueous solution or a physiological buffer solution of the functional molecule, adding a coupling agent, and reacting for 2-8 hours to form the functional molecule on the surface of the hydrophilic polymer coating, wherein the mass of the coupling agent is 1/20-1/10 of the mass of the functional molecule.
In some embodiments, the concentration of the functional molecule in the aqueous solution or physiological buffer solution is 0.1-10 mg/mL.
In some embodiments, the functional molecule is an extracellular matrix protein molecule or an antimicrobial peptide molecule and the coupling agent is a carbodiimide and an N-hydroxysuccinimide.
In some embodiments, the functional molecule is a polysaccharide molecule and the coupling agent is carbonyl diimidazole.
In some embodiments, the hydrophilic monomer comprises one or more of acrylamide, polyethylene glycol acrylate, polyethylene glycol dimethacrylate, 1-vinyl-2-pyrrolidone, 2-methacryloxyethyl phosphorylcholine, thiobetaine and carboxybetaine.
In some embodiments, the hydrophilic monomer comprises acrylamide, and further comprises one or more of polyethylene glycol acrylic ester, polyethylene glycol dimethacrylate, 1-vinyl-2-pyrrolidone, 2-methacryloyloxyethyl phosphorylcholine, thiobetaine and carboxybetaine, wherein the mass ratio of the acrylamide to other hydrophilic monomers is 1:5-5:1.
In some embodiments, in step 2), the aqueous solution of the hydrophilic monomer containing carbon-carbon unsaturated bond is coated on the surface of the polydopamine coating containing acrylamide, and the hydrophilic monomer containing carbon-carbon unsaturated bond is reacted and crosslinked on the surface of the polydopamine coating containing acrylamide by ultraviolet irradiation for 1-10 minutes.
In some embodiments, in step 2), the mass fraction of the hydrophilic monomer in the aqueous solution of the hydrophilic monomer is 1wt% to 10wt%.
In some embodiments, in step 1), the substrate is contacted with an aqueous solution of dopamine and acrylamide, and the reaction is carried out for 12 to 48 hours at room temperature to form the polydopamine coating containing acrylamide.
In some embodiments, the aqueous solution of dopamine and acrylamide has a pH of 8.5 to 9.0.
In some embodiments, the concentration of the dopamine in the aqueous solution of the dopamine and the acrylamide is 0.1-2 mg/mL, and the concentration of the acrylamide is 0.1-2 mg/mL.
In some embodiments, the substrate comprises an intravascular implant comprising a vascular stent, a central venous catheter, a thrombus filter, a hemodialysis catheter, a medical balloon, and a medical balloon catheter.
The embodiment of the invention also provides a medical functional coating, which is prepared according to the preparation method of any medical functional coating.
Embodiments of the present invention also provide an implantable interventional medical device having at least a portion of its surface provided with a medical functional coating of any of the above.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
The preparation method comprises the steps of forming a polydopamine coating containing acrylamide on the surface of a substrate, contacting a hydrophilic monomer containing carbon-carbon unsaturated bonds with the polydopamine coating containing acrylamide, and reacting and crosslinking to form a hydrophilic polymer coating, wherein the hydrophilic polymer coating has good hydrophilic performance and anticoagulation performance.
In the preparation method, ultraviolet light is adopted to polymerize one or more different hydrophilic monomers containing carbon-carbon unsaturated bonds at the same time, a cross-linked hydrophilic polymer coating is formed on the surface of the substrate, further, a hydrogel coating is formed, and the hydrophilic polymer coating is covalently bonded with the polydopamine coating containing acrylamide on the surface of the substrate, so that the preparation method has good stability and hydrophilic performance, and therefore, the service time in vivo is long, and the use effect is good.
The hydrophilic polymer coating has excellent biocompatibility and good hydrophilicity, and biomolecules with different functions can be fixed or electrostatically adsorbed on the surface of the hydrophilic polymer coating according to requirements, so that the biological function or multifunctional biological property of the substrate material property is endowed. Furthermore, extracellular matrix protein molecules are fixed on the surface of the hydrophilic polymer coating, so that the medical functional coating capable of promoting cell growth is prepared and obtained, and the medical functional coating has good anticoagulation performance, hydrophilic performance and biocompatibility.
According to the preparation method, the self-polymerization of dopamine is adopted to firstly form the transition layer containing double structures, the transition layer can form the transition layer with excellent binding force on the surface of most of matrix materials, and the existence of double bonds is beneficial to further modification on the surfaces of different matrix materials.
The technical scheme of the invention does not relate to organic solvents, has no organic solvent residue and has good application prospect.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
Since the physiological reaction between the implant material or the device and the host mainly occurs on the surface of the implant interventional medical device, the inventor prepares a surface modification layer with excellent performance on the surface of the implant interventional medical device to improve the biological performance of the surface, and prepares different modification layers according to different biological performance requirements. The inventor researches and discovers that the hydrogel coating with biological function is prepared on the surface of the implanted interventional medical device, so that the matrix material can be endowed with good biological function.
The specific embodiment of the invention provides a preparation method of a medical functional coating, which comprises the steps of 1) providing a substrate, forming a polydopamine coating containing acrylamide on the surface of the substrate, and 2) contacting a hydrophilic monomer containing carbon-carbon unsaturated bonds with the polydopamine coating containing acrylamide, and reacting and crosslinking to form a hydrophilic polymer coating.
In some embodiments, in the preparation method of the medical functional coating of the embodiment of the invention, in the step 1), the formation of the polydopamine coating containing acrylamide on the surface of the substrate comprises the steps of contacting the substrate with an aqueous solution of dopamine and acrylamide, and reacting for 12-48 hours at room temperature to form the polydopamine coating containing acrylamide on the surface of the substrate.
In some embodiments, the aqueous solution of dopamine and acrylamide has a pH of 8.5-9.0. In some specific embodiments, in the aqueous solution of dopamine and acrylamide, the concentration of the dopamine is 0.1-2 mg/mL, and the concentration of the acrylamide is 0.1-2 mg/mL. In some embodiments, the aqueous solution of dopamine and acrylamide is prepared by mixing separately configured aqueous solutions of dopamine and acrylamide, and in some embodiments, the concentration of dopamine configured is the same as the concentration of acrylamide.
According to the preparation method of the medical functional coating, the polydopamine coating containing the acrylamide is formed on the base material, and the polydopamine coating is tight and well adhered to the base material, so that the physical and chemical properties of the base material are not changed. The polydopamine can be adhered to various base materials, and has good adhesion, so that the polydopamine can be used as a transition layer for modifying the surfaces of various medical instrument products. In a specific embodiment of the invention, the polydopamine coating containing acrylamide contains double bonds, which is beneficial to further polymerization reaction.
According to the preparation method of the medical functional coating, a very complex structure is formed in the self-polymerization process of the dopamine, coordination bonding (such as metal ions) or chemical bonding (such as amino or sulfhydryl-containing substances) can be formed with other substances in the process of forming the polydopamine coating, so that the coating carrying bioactive factors can be formed on the surface by codeposition with other substances, and the coating still has the capability of further fixing bioactive molecules, so that the multifunctional bioactive surface can be constructed for regulating and controlling physiological microenvironment response around implantation, and the biocompatibility or other properties of a material or an instrument can be enhanced.
In some embodiments, in step 2), a hydrophilic monomer containing a carbon-carbon unsaturated bond is contacted with the polydopamine coating containing acrylamide, and the hydrophilic monomer is reacted and crosslinked to form a hydrophilic polymer coating. In some embodiments, the hydrophilic monomer containing carbon-carbon unsaturated bonds contains carbon-carbon double bonds or/and carbon-carbon triple bonds, and simultaneously contains hydrophilic groups, double bonds among the carbon-carbon unsaturated bonds and between the carbon-carbon unsaturated bonds and acrylic ester groups of the polydopamine coating undergo polymerization reaction, and the hydrophilic polymer coating is formed by crosslinking on the surface of the polydopamine coating.
In some embodiments, the hydrophilic polymer coating formed in step 2) is a hydrogel coating, and the hydrogel coating has a three-dimensional network structure, can be swelled and can hold a large amount of water in a swelled state without dissolution, and has extremely strong hydrophilic performance. The hydrogel coating is formed on the surface of the implanted medical instrument, has good wettability and lubricity in a human body, can be well compatible with human tissues, reduces adverse reactions and immune reactions, and reduces friction and abrasion, thereby prolonging the service life of the medical instrument.
In some embodiments, the hydrophilic monomer containing carbon-carbon unsaturation comprises one or more of acrylamide, polyethylene glycol acrylate, polyethylene glycol dimethacrylate, 1-vinyl-2-pyrrolidone, 2-methacryloxyethyl phosphorylcholine, thiobetaine and carboxybetaine.
In some embodiments, in order to further enhance the reactivity with the polydopamine coating containing acrylamide, and introduce amine groups, the hydrophilic monomers comprise acrylamide, in some embodiments, the hydrophilic monomers comprise acrylamide, and further comprise other hydrophilic monomers, wherein the other hydrophilic monomers comprise one or more of polyethylene glycol acrylate, polyethylene glycol dimethacrylate, 1-vinyl-2-pyrrolidone, 2-methacryloxyethyl phosphorylcholine, thiobetaine and carboxybetaine, and in some embodiments, the mass ratio of the acrylamide to the other hydrophilic monomers is 1:5-5:1, and specifically can be, for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1, and the like.
In some specific embodiments, acrylamide and thiobetaine (SBMA) are used as hydrophilic monomers to contact the polydopamine coating containing the acrylamide, and the hydrophilic polymer coating is formed through reaction crosslinking, and in some specific embodiments, the mass ratio of the acrylamide to the thiobetaine (SBMA) is 1:3-3:1, and can be, for example, 1:3, 1:2, 1:1, 2:1 or 3:1, etc.
In some embodiments, in the preparation method of the medical functional coating according to the embodiment of the present invention, in the step 2), an aqueous solution of a hydrophilic monomer containing a carbon-carbon unsaturated bond is coated on the surface of the polydopamine coating containing acrylamide, and the hydrophilic monomer containing a carbon-carbon unsaturated bond is crosslinked on the surface of the polydopamine coating containing acrylamide by ultraviolet irradiation for 1 to 10 minutes. Different hydrophilic monomers are polymerized by ultraviolet light, the formed hydrophilic polymer coating is covalently combined with the surface of the polydopamine containing acrylamide, and the structure is stable, so that the hydrophilic polymer coating has good hydrophilic performance and anticoagulation performance when being applied to the coating implanted on the surface of a medical instrument, and has long service time in vivo and good use effect.
In the preparation method of the medical functional coating of the specific embodiment of the invention, in some specific embodiments, in the step 2), the power of ultraviolet light is 400-500W, and the wavelength of the ultraviolet light is 360nm.
In the preparation method of the medical functional coating of the specific embodiment of the invention, in some specific embodiments, in the step 2), the ultraviolet irradiation time is 1-10 minutes.
In some embodiments, in the preparation method of the medical functional coating according to the embodiment of the present invention, in step 2), the hydrophilic monomer is coated on the surface of the polydopamine coating containing acrylamide in the form of an aqueous solution, and the total mass fraction of the hydrophilic monomer in the aqueous solution of the hydrophilic monomer is 1wt% to 10wt%, specifically, for example, may be 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, or the like.
The preparation method of the medical functional coating of the specific embodiment of the invention further comprises 3) fixing functional molecules on the surface of the hydrophilic polymer coating.
According to the preparation method of the medical functional coating, specific functional molecules can be fixed on the surface of the hydrophilic polymer coating according to application requirements, meanwhile, the hydrophilic polymer coating capable of fixing the functional molecules is firstly formed on a base material according to the characteristics of the functional molecules, and the stability of each prepared coating and the stability of the performance of the coating are required to be considered in the preparation method, so that the coating with the medical biological function is obtained.
In some embodiments, the functional molecules comprise one or more of extracellular matrix protein molecules, polysaccharide molecules and antibacterial peptide molecules.
In some embodiments, the functional molecules immobilized on the surface of the hydrophilic polymer coating are extracellular matrix protein molecules, which have the effect of promoting cell growth and can be used for a surface modification layer of an in vivo implantation interventional instrument which needs to promote cell growth.
In some embodiments, the extracellular matrix protein molecule is fibronectin.
In some embodiments, the functional molecules immobilized on the surface of the hydrophilic polymer coating are polysaccharide molecules, and the polysaccharide molecules have an anticoagulant effect and can be used for blood implantation interventions such as vascular stents and the like.
In some embodiments, the polysaccharide molecule comprises one or both of heparin and hyaluronic acid.
In some embodiments, the functional molecules immobilized on the surface of the hydrophilic polymer coating are antibacterial peptide molecules, and the antibacterial peptide molecules have antibacterial effect, so that the risk of bacterial infection of the implantation medical instrument can be reduced. Other antibacterial molecules may be used as the functional molecule for antibacterial action, and are not limited thereto.
In some embodiments, in the preparation method of the medical functional coating according to the embodiment of the present invention, in step 3), the functional molecule is formed on the surface of the hydrophilic polymer coating by at least one of coupling fixation and electrostatic adsorption.
In some embodiments, the coupling and fixing method of the medical functional coating comprises the steps of contacting a substrate with a hydrophilic polymer coating formed on the surface with an aqueous solution or a physiological buffer solution of functional molecules, adding a coupling agent, and reacting for 2-8 hours to form the functional molecules on the surface of the hydrophilic polymer coating.
In some embodiments, the physiological buffer solution of the functional molecule is a Tris-HCl buffer solution with a pH of 8-9.
In some embodiments, the concentration of the functional molecule is 0.1-10 mg/mL in an aqueous solution or a physiological buffer solution.
In some embodiments, the mass of the coupling agent is 1/20 to 1/10 of the mass of the functional molecule, and may be, for example, 1/20, 1/19, 1/18, 1/17, 1/16, 1/15, 1/14, 1/13, 1/12, 1/11, or 1/10, etc.
According to the preparation method of the medical functional coating, the coupling agent is selected according to the structure of the functional molecule, in some specific embodiments, the functional molecule is an extracellular matrix protein molecule or an antibacterial peptide molecule, the coupling agent is carbodiimide and N-hydroxysuccinimide, the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 2:1-6:1, and the coupling agent can be, for example, 2:1, 5:2, 3:1, 7:2, 4:1, 9:2, 5:1, 11:2, 6:1 and the like. In some embodiments, the functional molecule is a polysaccharide molecule and the coupling agent is carbonyl diimidazole.
In some embodiments, the hydrophilic polymer coating formed on the surface of the polydopamine coating containing acrylamide has positive or negative polarity due to hydrophilic groups or other groups, and part of groups of functional molecules have electronegativity or electropositivity, so that positive and negative charge attraction is generated between the hydrophilic polymer coating and the functional molecules, and the functional molecules are adsorbed on the surface of the hydrophilic polymer coating through charge force. The electrostatic adsorption does not need to add a coupling agent, and the functional molecules have relatively complete groups and good biological performance because no chemical reaction occurs.
In some embodiments, the preparation method of the medical functional coating according to the embodiment of the present invention, in step 3), the step of forming the functional molecule on the surface of the hydrophilic polymer coating by electrostatic adsorption includes contacting a substrate having the hydrophilic polymer coating formed on the surface with an aqueous solution or a physiological buffer solution of the functional molecule, and electrostatic adsorbing for 2 to 24 hours to form the functional molecule on the surface of the hydrophilic polymer coating.
In some embodiments, the physiological buffer solution of the functional molecule is a Tris-HCl buffer solution with a pH of 8-9.
In some embodiments, the concentration of the functional molecule is 0.1-10 mg/mL in an aqueous solution or a physiological buffer solution.
In the preparation method of the medical functional coating of the specific embodiment of the invention, in the step 2), the hydrophilic polymer coating is formed on the surface of most of the polydopamine coating containing acrylamide, in the step 3), the functional molecules are possibly formed on the surface of the hydrophilic polymer coating through coupling fixation and electrostatic adsorption, the surface of the polydopamine coating containing acrylamide, which is not covered by the hydrophilic polymer coating, is provided with amino groups, the functional molecules are also coupled fixation or electrostatic adsorption with the amino groups, and finally the medical functional coating is prepared on a substrate.
In some embodiments, the substrate comprises an intravascular implant including a vascular stent, a central venous catheter, a thrombus filter, a hemodialysis catheter, a medical balloon catheter, and the like. The substrate may be a substrate on which a coating layer has been formed, or may be a substrate on which a coating layer has not been formed by exposure, but is not limited thereto.
In some embodiments, the material of the substrate is a metal material, such as stainless steel, titanium alloy, aluminum alloy, etc., for manufacturing surgical instruments, implantation instruments, etc., in some embodiments, the material of the substrate is a polymer material, such as polyurethane, polyethylene, polypropylene, etc., for manufacturing medical instruments, such as heart valves, hemodialyzers, etc., in some embodiments, the material of the substrate is a composite material, such as carbon fiber reinforced polymer, glass fiber reinforced polymer, etc., for manufacturing medical instruments, such as artificial joints, dental implants, etc.
The specific embodiment of the invention also provides a medical functional coating, which is prepared according to any of the preparation methods.
The specific embodiment of the invention also provides a planting intervention medical device, at least part of the surface of which is provided with the medical functional coating.
In some embodiments, the implantable interventional medical device includes a vascular stent, a central venous catheter, a thrombus filter, a hemodialysis catheter, a medical balloon catheter, and the like.
The technical scheme of the invention is further described by the following specific examples.
Examples
Description of the test methods
Water contact angle measurement was performed using a water contact angle measuring instrument (DSA 25, kruss GmbH, germany);
Platelet adhesion experiments, namely observing the adhesion quantity and morphology of platelets through a scanning electron microscope;
Measuring absorbance value by a spectrophotometer, and calculating protein absorption according to a standard curve;
and (3) performing endothelial cell adhesion growth experiments, namely taking fluorescent pictures of cells under a light-proof environment through a fluorescent microscope, and observing the adhesion condition of endothelial cells on the surface of a sample.
The detailed procedure of the above measurement method is shown in the following examples.
Example 1
(1) Preparing a polydopamine coating containing acrylamide, namely cleaning a polyurethane substrate (PU) by using an ultrasonic cleaner for 10 minutes, flushing with deionized water, drying for later use, preparing dopamine and acrylamide solutions with the concentration of 1mg/mL respectively, and mixing according to the volume ratio of 1:1;
(2) Preparing a hydrophilic polymer coating, namely preparing an aqueous solution of thiobetaine (SBMA) with the concentration of 5wt% and an aqueous solution of acrylamide (AAM) with the concentration of 5wt%, mixing according to the volume ratio of 1:1 to form a mixed solution, dripping 1mL of the mixed solution on the surface of a PUM sample, covering the surface with a cover glass, reacting for 2 minutes under the irradiation of ultraviolet light with the power of 400W and the wavelength of 360nm, and flushing the sample with deionized water after the reaction to prepare the hydrophilic polymer coating (PUH) on the surface of the sample;
(3) Preparing a medical functional coating, namely preparing 5mL of extracellular matrix protein (fibronectin) solution with the concentration of 1mg/mL, adding 1mL of EDC/NHS solution (prepared by mixing carbodiimide solution with the concentration of 10mM and N-hydroxysuccinimide solution with the concentration of 10mM according to the volume ratio of 3:1) into the solution, activating for 1 hour, dripping the activated extracellular matrix protein solution on the surface of PUH, fully reacting for 4 hours, and then cleaning and drying to prepare the coating (PUF) immobilized with extracellular matrix protein molecules.
Example 2
A total of 4 samples (hereinafter referred to as PU, PUM, PUH, PUF samples) were subjected to water contact angle measurement on the PU substrate, the PUM coating-forming substrate, the PUH coating-forming substrate, and the PUF coating-forming substrate in example 1 to characterize the hydrophilic properties of the sample surfaces.
The water contact angle of the surfaces of different samples is measured by adopting a water contact angle measuring instrument, PU, PUM, PUH, PUF samples are respectively placed on an operation table, 20 microliters of deionized water is dripped on the surface of each sample, the water contact angle is measured by photographing after standing for 5 seconds, 3 parallel samples are measured for each sample, and the average value is obtained.
The water contact angle measurement results of different samples are shown in fig. 1, it can be seen from fig. 1 that the water contact angle of the unmodified polyurethane substrate reaches more than 80 degrees, the hydrophilicity is relatively poor, for a PUM sample, the water contact angle is greatly reduced due to the existence of hydrophilic groups due to the formation of an acrylamide-containing polydopamine coating on the surface, the PUH sample obtained after the PUM sample reacts and crosslinks thiobetaine (SBMA) and acrylamide (AAM) under ultraviolet light has more hydrophilic groups to form a hydrogel coating, the water contact angle of the surface of the sample reaches less than 20 degrees, and the surface water contact angle of a PUH sample is slightly increased and still has good hydrophilic performance after the PUH sample is further coupled with a PUH sample obtained by fixing fibronectin. It can be seen that the modified coating prepared in the examples of the present invention has excellent hydrophilic properties.
Example 3
Platelet adhesion experiments were performed on 4 samples (hereinafter referred to as PU, PUM, PUH, PUF samples) of the PU substrate, the PUM-coated substrate, the PUH-coated substrate, and the PUF-coated substrate of example 1 to characterize their anticoagulation performance.
Firstly, whole blood of a healthy person is centrifuged for 10 minutes at 1200r/min to obtain Platelet Rich Plasma (PRP), PU, PUM, PUH, PUF samples are respectively placed in 24 pore plates, 200 mu L of PRP is added into each sample pore, the mixture is incubated for 2.5 hours in an environment of 37 ℃, the samples are washed twice by normal saline, the platelets adhered to the surfaces are fixed for 3 hours at 4 ℃ by 2.5% glutaraldehyde, and finally, the platelets are dehydrated by gradient of 50%, 70%, 90% and 100% absolute ethyl alcohol sequentially, 15 minutes each time, and the platelets are dried at room temperature. The surface of the sample was observed for platelet adhesion by SEM.
Fig. 2 shows scanning electron micrographs of platelet adhesion on different sample surfaces, and as can be seen from fig. 2, the number of platelet adhesion on the surface of the unmodified Polyurethane (PU) substrate is the greatest, aggregation is the most serious, which indicates poor anticoagulation performance, compared with the PU sample, the number of platelet adhesion on the surface of the PUM sample forming the polydopamine coating containing acrylamide is obviously reduced, which indicates improved anticoagulation performance, and the number of platelet adhesion on the surface of the PUH sample forming the hydrophilic polymer coating and the PUF sample forming the fibronectin-immobilized coating is further reduced, so that the morphology is more complete, which indicates good improvement of anticoagulation performance, and the hydrophilic polymer coating and the fibronectin-immobilized hydrophilic polymer coating of the embodiment of the invention are proved to have good anticoagulation performance.
Example 4
Plasma protein adsorption experiments were performed on 4 samples (hereinafter referred to as PU, PUM, PUH, PUF samples) of the PU substrate, the PUM-coated substrate, the PUH-coated substrate, and the PUF-coated substrate of example 1 to characterize their anticoagulation performance.
Washing PU, PUM, PUH, PUF samples, drying, placing into a 24-well plate, preparing 1mg/mL Bovine Serum Albumin (BSA) and Fibrinogen (FIB) solutions with 0.01mol/L PBS respectively, adding 2mL of albumin and fibrinogen solutions into each sample, fully adsorbing for 2.5 hours at 37 ℃, rinsing the samples with PBS for 3 times, immersing the samples into 2mL of sodium dodecyl sulfate (SDS, 1 wt%) solution, ultrasonically desorbing for 30 minutes, placing 100 mu L of mixed solution of SDS solution and 100 mu L of BCA working solution at 60 ℃ for reacting for 20 minutes, absorbing 150 mu L of mixed solution into a 96-well plate, measuring absorbance values at the wavelength of 562nm, testing 3 parallel samples, calculating the adsorption quantity of the bovine serum albumin and the fibrinogen according to a standard curve, and recording the calculation result in FIG. 3.
The results of plasma protein adhesion experiments on the surfaces of different samples are shown in fig. 3, and it can be seen that the surface of the unmodified Polyurethane (PU) substrate has less albumin adsorption, more fibrinogen adsorption and the effect of fibrinogen on promoting coagulation can result in poor anticoagulation performance, while the surface albumin adsorption amount of the PUH sample forming the hydrophilic polymer coating and the PUF sample forming the coating for fixing fibronectin is more than that of the PU sample and the PUM sample, the fibrinogen adsorption is less, the albumin adsorption promoting capacity is better, and the anticoagulation performance is better.
Example 5
Endothelial cell adhesion growth experiments were performed on 4 samples (hereinafter referred to as PU, PUM, PUH, PUF samples) of the PU substrate, the PUM-coated substrate, the PUH-coated substrate, and the PUF-coated substrate of example 1.
The samples are washed and dried and placed in 24-hole culture plates for sterilization under an ultraviolet lamp for 12 hours, 0.5mL of endothelial cell suspension with the concentration of 5 multiplied by 105/mL and 1.5mL of cell culture medium are added into each sample, the samples are placed in a 37 ℃ cell culture box with the concentration of 5% of CO2 for incubation for 6 hours and 24 hours respectively, after washing twice with normal saline, 100 mu L of rhodamine (prepared by PBS and 10 mu g/mL) is added into each sample for dyeing for 20 minutes, after washing twice with normal saline, 100 mu L of 4, 6-diamidino-2-phenylindole (prepared by DAPI with the concentration of 500 ng/mL) is added for dyeing for 10 minutes, washing twice with normal saline again, finally, fluorescent pictures of the cells are taken by adopting a fluorescent microscope, and the adhesion quantity and the morphology of the endothelial cells on the samples are observed.
Fig. 4 shows fluorescent fiber photographs of endothelial cell adhesion on the surfaces of different samples, and it can be seen that unmodified Polyurethane (PU) has better endothelial adhesion performance, a PUM sample with an acrylamide-containing polydopamine coating formed on the surface and a PUH sample with a hydrophilic polymer coating formed on the surface, the good hydrophilic performance prevents cell adhesion, resulting in a reduction in cell number, and a PUF sample with a fibronectin-immobilized coating formed on the surface has significantly increased cell number compared with the PUM sample and the PUH sample, indicating that the surface functional coating of the PUF sample can promote endothelial cell growth.
In conclusion, the hydrophilic polymer coating (PUF) and the hydrophilic polymer coating (PUH) with the immobilized functional molecules endow the surface of the substrate with good hydrophilic performance and anticoagulation performance, and the hydrophilic polymer coating (PUF) with the immobilized functional molecules has good hydrophilic performance and anticoagulation performance, also has the performance of promoting endothelial cell generation and has good biological functionality.
The foregoing description of the exemplary embodiments has been presented only for the purpose of illustrating the principles of the invention and is not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and the spirit of the invention, and these modifications and improvements are also within the scope of the invention.