CN109731137B - Preparation method of albumin coating with biological antifouling function and material with biological antifouling function - Google Patents
Preparation method of albumin coating with biological antifouling function and material with biological antifouling function Download PDFInfo
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Abstract
The invention discloses a preparation method of an albumin coating with a biological anti-fouling function and a material with the biological anti-fouling function, and relates to the technical field of medical materials. The preparation method of the albumin coating with the biological anti-fouling function comprises the following steps: mixing albumin, a solvent and a protein structure conversion agent to form dip-coating mixed liquor; soaking the material to be modified in the dip-coating mixed solution; wherein the protein structure conversion agent is a strong oxidant or a strong reducing agent. The material with the biological anti-fouling function comprises a modified substrate and an albumin coating formed on the modified substrate, wherein the albumin is in a beta-sheet structure. The preparation method of the material is simple and easy, the bonding strength of the coating and the base material is high, the long-term anticoagulation performance and the antibacterial performance are realized, and the adhesion of blood vessel cells and tissues can be inhibited.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a preparation method of an albumin coating with a biological anti-fouling function and a material with the biological anti-fouling function.
Background
Devices such as artificial hearts, pacemakers, ventricular assist devices, etc. which come into contact with blood of a human body are accompanied by problems of thrombosis and infection in clinical application, which directly results in increase of morbidity and mortality of patients. In clinical practice, the means for preventing infection and thrombosis mainly adopts heparin or antibiotics for drug treatment, and the administration increases the bleeding of patients and is easy to cause the occurrence of heparin-induced thrombocytopenia. In addition, antibiotics also cause a plurality of serious adverse reactions after being applied to human bodies.
In order to reduce the clinical application complications, the material surface functionalization mode is carried out at the same time, and the method is an effective means for solving the problems of infection, thrombus and the like. Surface modification methods such as polyethylene glycol (PEG) grafting/coating, super-hydrophobic surface treatment, surface heparinization, surface micro-nano structure treatment and the like are mostly complex to operate, materials are often required to be treated to introduce functional groups, so that functional molecules are fixed, and the operation process is very complex.
The albumin is mainly present in blood and whey of mammals, and if the albumin is pre-adsorbed on the surface of the material, the albumin can effectively prevent the adsorption of other proteins, thereby having the functions of anticoagulation and antibiosis. However, the method of modifying the surface of the material by using albumin mainly adopts a covalent grafting mode, and the effect of the method is often limited by the fixed amount of the surface albumin.
Disclosure of Invention
The invention aims to provide a preparation method of an albumin coating with a biological anti-fouling function, which is used for conveniently fixing albumin molecules by adopting a one-step dip coating mode.
The invention also aims to provide a material with a biological anti-fouling function, and the preparation method is simple and easy to implement and has good biological anti-fouling performance.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
forming a coating on the material to be modified by using the dip-coating mixed solution;
wherein, the dip-coating mixed solution is formed by mixing an albumin solution and a protein structure conversion agent, and the protein structure conversion agent is a strong oxidant or a strong reducing agent.
The invention also provides a material with a biological anti-fouling function, which comprises a modified substrate and an albumin coating formed on the modified substrate, wherein the albumin has a beta-folded structure;
preferably, the material with the biological anti-fouling function is prepared by the preparation method.
The embodiment of the invention provides a preparation method of an albumin coating with a biological anti-fouling function, which has the beneficial effects that: by mixing albumin and a protein structure conversion agent, a disulfide bond of the albumin is broken by a strong oxidant or a strong reducing agent, and the albumin is converted from an alpha helical structure to a beta folded structure, so that the albumin has the adhesion property with materials, can be modified on the surfaces of almost all shapes and types of materials, and has good bonding strength with the surface of a base material.
The invention also provides a material with a biological anti-fouling function, which comprises a modified base material and an albumin coating formed on the modified base material, wherein the albumin is in a beta-folded structure.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a comparison of stainless steel materials before and after modification in an example of the present invention;
FIG. 2 is a comparison of materials before and after modification of a silica gel material in an example of the present invention and after modification in a comparative example;
FIG. 3 is a diagram showing the bacteriostatic effect of the modified material prepared in the examples and comparative examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The preparation method of the albumin coating with the anti-fouling function and the material with the anti-fouling function provided by the embodiment of the invention are specifically described below.
The preparation method of the albumin coating with the biological anti-fouling function provided by the embodiment of the invention comprises the following steps:
s1 preparation of dip-coating mixed solution
Mixing albumin, a solvent and a protein structure conversion agent to form dip-coating mixed liquor; wherein the protein structure conversion agent is a strong oxidant or a strong reducing agent. The disulfide bond in the albumin is broken by using a strong oxidant or a strong reducing agent, so that the albumin is converted from an alpha helical structure to a beta sheet structure, and the beta sheet structure albumin has the performance of one-step self-assembly.
Specifically, the concentration of albumin in the dip-coating mixture is 0.002 to 90mg/mL, preferably 0.1 to 30mg/mL, and more preferably 0.5 to 15 mg/mL. The amount of albumin used will affect the anticoagulant properties of the coating and also the strength of the bond between the coating and the material.
Generally, the albumin includes any one or more of bovine serum albumin, human serum albumin, whey albumin. The albumin is modified by a strong reducing agent or a strong oxidizing agent, and the self-assembly capacity of the albumin is endowed by the generated beta-sheet structure.
Specifically, the strong reducing agent includes one or more of mercaptoethanol, dithiothreitol, and reduced glutathione. The concentration of the strong reducing agent in the dip-coating mixed liquid is 0.002-20mg/mL, preferably 0.1-10 mg/mL. The selection of the strong reducing agent can convert the alpha helical structure of the albumin into the beta folded structure, and the reaction rate is high, so that the coating can be quickly formed. The dosage of the strong reducing agent needs to be controlled, so that the albumin is prevented from being denatured due to excessive dosage, and the albumin is prevented from being insufficiently reacted due to insufficient dosage.
Specifically, the strong oxidant comprises any one or more of trivalent cobalt salt, chlorate, potassium permanganate, persulfate, potassium dichromate, concentrated sulfuric acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, perchloric acid, ozone, hydrogen peroxide, fluorine gas, chlorine gas, sodium bismuthate, periodic acid, sodium ferrate and lead dioxide. The strong oxidants can transform the structure of albumin, and the transformation efficiency is fast.
Further, in the dip-coating mixed solution, the concentration of trivalent cobalt salt, potassium permanganate, potassium dichromate, sodium bismuthate, persulfate, periodic acid, sodium ferrate and lead dioxide is 0.001-20 mg/mL; preferably 0.2-10 mg/mL. In the dip-coating mixed liquid, the mass fraction of concentrated sulfuric acid, chlorate, hydroiodic acid, nitric acid, hydrobromic acid, perchloric acid, hydrochloric acid, chlorine, hydrogen peroxide, fluorine gas and ozone is 0.02-50%; preferably 0.1 to 30%. Also, the amount of strong oxidizing agent used needs to be strictly controlled to prevent denaturation of albumin or insufficient reaction of albumin.
Preferably, the dip-coating mixed solution is prepared by mixing an aqueous albumin solution with a protein structure conversion agent, and the raw materials can be more fully mixed by adopting a solution mixing mode to prevent the local concentration from being too high.
S2, soaking and modifying materials
Soaking the material to be modified in the dip-coating mixed solution for 2 seconds to 8 days at the reaction temperature of 0 to 100 ℃. Preferably, the soaking time is 10-240min, and the reaction temperature is 15-45 ℃. The material to be modified can form a functional coating on the surface of the material after being soaked for a short time, and the bonding strength between the coating and the material is high, so that the method is suitable for industrial application.
Preferably, after the material to be modified is reacted in the dip-coating mixed solution, the modified sample is washed and dried. The washing process is favorable for removing unreacted raw materials adhered to the surface of the material, and the purity of the product is improved.
The preparation method in the embodiment of the invention has the advantages that: (1) the method is convenient to operate, does not need complex reaction process, expensive special equipment, chemical reagents and the like, does not need pretreatment to introduce reaction functional groups, and has low process cost. (2) The beta-sheet structure can make albumin adhere well to the surface of the material, is suitable for materials with almost all shapes and materials, and is suitable for popularization and application. (3) The albumin has uniform coating, easily controlled thickness and high bonding strength between the coating and the material, can be used as long-term anticoagulant material and antibacterial material, and also has the function of inhibiting adhesion of blood vessel cells and tissues.
The embodiment of the invention also provides a material with a biological anti-fouling function, which comprises a modified substrate and an albumin coating formed on the modified substrate, wherein the albumin has a beta-folded structure; preferably, the material with the biological anti-fouling function is prepared by the preparation method. The material has good anticoagulation and antibacterial properties, and the bonding strength between the coating and the base material is high.
The modified substrate (material to be modified) in the embodiment of the present invention may be any material for film formation at present, and is hardly limited to the shape and material. The method specifically comprises the following steps: (1) metal material: stainless steel, titanium and its alloys, cobalt-based alloys, nickel-titanium alloys, magnesium and its alloys, zinc and its alloys, iron and its alloys, and the like. (2) Inorganic materials: and inorganic materials such as silicon dioxide, titanium dioxide, carbon materials (C), silicon, titanium dioxide, titanium oxide, and titanium nitride. (3) High polymer material: polyester (PET), polyvinyl alcohol (PVALC), Polyethylene (PE), Polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), Polystyrene (PS), Polyurethane (PU), polypropylene (PP), Polycarbonate (PC), polyacrylic acid (PAA) and derivatives thereof, polyethylene glycol and derivatives thereof, and the like. (4) Biomedical micro-nano particles: zinc oxide nanoparticles (quantum dots), silica nanoparticles (quantum dots), titanium oxide nanoparticles (quantum dots). (5) Natural biological material: plastic starch-based materials (PSM), sodium alginate (sodium alginate), collagen (collagen), fibrin (fibrin protein), sodium hyaluronate (sodium hyaluronate), gelatin (gelatin), and the like. (6) Artificially synthesizing a polypeptide hydrogel material: poly-L-glutamic acid, poly-L-lysine, and the like. The kind of the base material is not limited to the above materials, and may be a mixture of the above materials.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing a bovine serum albumin aqueous solution and concentrated sulfuric acid, adding water to adjust the concentration of the bovine serum albumin in the mixed solution to be 0.002mg/L, and the mass fraction of the concentrated sulfuric acid to be about 0.02%.
And (3) placing the polyvinyl chloride (PVC) extracorporeal circulation catheter to be modified into the mixed solution, and soaking for about 2s at the temperature of 0 ℃. And cleaning and drying the sample.
Example 2
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
after mixing the human serum albumin aqueous solution with perchloric acid, adding water to adjust the concentration of the human serum albumin in the mixed solution to be 90mg/L, wherein the mass fraction of the perchloric acid is about 50%.
The nickel-titanium alloy inferior vena cava filter to be modified is placed in the mixed solution and soaked for about 5 days at the temperature of 100 ℃. And cleaning and drying the sample.
Example 3
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing the whey albumin aqueous solution and potassium permanganate, adding water to adjust the concentration of the whey albumin in the mixed solution to be 0.1mg/L and the concentration of the potassium permanganate to be about 0.001 mg/L.
And (3) placing the central venous catheter of the silica gel to be modified in the mixed solution, and soaking for about 10min at the temperature of 15 ℃. And cleaning and drying the sample.
Example 4
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
after mixing the whey albumin aqueous solution with potassium dichromate, water was added to adjust the concentration of whey albumin in the mixed solution to 30mg/L and the concentration of potassium dichromate to about 20 mg/L.
And (3) placing the polysulfone hemodialyzer membrane to be modified into the mixed solution, and soaking for about 240min at the temperature of 15 ℃. And cleaning and drying the sample.
Example 5
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing whey albumin water solution and sodium bismuthate, adding water to adjust the concentration of whey albumin in the mixed solution to be 0.5mg/L and the concentration of sodium bismuthate to be about 0.2 mg/L.
And (3) placing the polysulfone hemodialyzer membrane to be modified into the mixed solution, and soaking for about 10min at the temperature of 45 ℃. And cleaning and drying the sample.
Example 6
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing the whey albumin aqueous solution with sodium ferrate, adding water to adjust the concentration of the whey albumin in the mixed solution to be 15mg/L and the concentration of the sodium ferrate to be about 10 mg/L.
And (3) placing the central venous catheter of the silica gel to be modified in the mixed solution, and soaking for about 15min at the temperature of 35 ℃. And cleaning and drying the sample.
Example 7
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing whey albumin water solution and mercaptoethanol, adding water to adjust the concentration of whey albumin in the mixed solution to 0.01mg/L and the concentration of mercaptoethanol to about 0.002 mg/L.
And (3) placing the central venous catheter of the silica gel to be modified in the mixed solution, and soaking for about 15min at the temperature of 35 ℃. And cleaning and drying the sample.
Example 8
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing whey albumin aqueous solution and dithiothreitol, adding water to adjust the concentration of whey albumin in the mixed solution to 10mg/L and the concentration of dithiothreitol to about 20 mg/L.
And (3) placing the central venous catheter of the silica gel to be modified in the mixed solution, and soaking for about 15min at the temperature of 35 ℃. And cleaning and drying the sample.
Example 9
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
after mixing the whey albumin aqueous solution with the reduced glutathione, water was added to adjust the concentration of whey albumin in the mixture to 0.1mg/L and the concentration of reduced glutathione to about 0.1 mg/L.
And (3) placing the central venous catheter of the silica gel to be modified in the mixed solution, and soaking for about 15min at the temperature of 35 ℃. And cleaning and drying the sample.
Example 10
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
after mixing the whey albumin aqueous solution with the reduced glutathione, water was added to adjust the concentration of whey albumin in the mixed solution to 10mg/L and the concentration of the reduced glutathione to about 10 mg/L.
And (3) placing the central venous catheter of the silica gel to be modified in the mixed solution, and soaking for about 15min at the temperature of 35 ℃. And cleaning and drying the sample.
Example 11
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing whey albumin water solution and mercaptoethanol, adding water to adjust the concentration of whey albumin in the mixed solution to 0.1mg/L and the concentration of mercaptoethanol to about 0.05 mg/L.
And (3) placing the stainless steel material into the mixed solution, and soaking for about 5min at the temperature of 25 ℃. And cleaning and drying the sample.
Example 12
The embodiment provides a preparation method of an albumin coating with a biological anti-fouling function, which comprises the following steps:
mixing whey albumin water solution and mercaptoethanol, adding water to adjust the concentration of whey albumin in the mixed solution to 0.1mg/L and the concentration of mercaptoethanol to about 0.05 mg/L.
And (3) placing the silica gel material in the mixed solution, and soaking for about 5min at the temperature of 25 ℃. And cleaning and drying the sample.
Comparative example 1
The comparative example provides a method for introducing albumin on the surface of a material, and adopts a covalent grafting method, and comprises the following specific steps: (1) dissolving 1.2mg of Tris with 1mL of distilled water to obtain a buffer solution with the pH value of about 8.5, then adding 2mg of dopamine, adding a stainless steel sample after full dissolution, and reacting for 12 hours to obtain a polydopamine coating on the surface of the stainless steel sample. (2) Dissolving 5mg of albumin in Tris buffer solution (as above), adding the polydopamine coating modified stainless steel sample obtained in the step 1 after the albumin is sufficiently dissolved, carrying out surface albumin covalent grafting, and after 12 hours of grafting, sequentially washing and drying the sample by using phosphate buffer solution and distilled water to obtain a sample with albumin covalently fixed on the surface.
Test example 1
In example 11, the states before and after soaking of the stainless material were compared as shown in fig. 1. Illustrating that example 11 successfully deposits an albumin coating on the surface of stainless steel and the coating exhibits strong bonding to the substrate.
Test example 2
The materials prepared in example 12 and comparative example 1 were subjected to scanning electron microscopy characterization, and the results are shown in fig. 2.
As can be seen from fig. 2, the albumin coating deposited on the silica gel surface according to the embodiment of the present invention has a superior function of inhibiting platelet adhesion and activation compared to the surface using conventional covalently grafted albumin molecules. Through the surface of covalent grafting albumin, as the covalent grafting amount of the albumin is limited by the density of reactive functional groups introduced on the surface of the material, more platelets still adhere to the surface, part of the platelets still are activated, and some platelets even have the condition of content outflow. The albumin coating modified sample has small quantity of surface platelets and is in a resting spherical shape.
Test example 3
The antibacterial properties of the products obtained in example 12 and comparative example 1 were tested, and the test results are shown in fig. 3.
As can be seen from fig. 3, the albumin coating deposited on the surface of the silica gel in the embodiment of the present invention can inhibit the formation of plaque more effectively and has more excellent antibacterial function than the surface using conventional covalent grafting albumin molecules.
Test example 4
The materials prepared in examples 1-10 were tested for antimicrobial properties by the following test methods: and continuously soaking the modified material in PBS simulated body fluid for one month, and respectively testing the bacteriostatic rates of the modified material on staphylococcus aureus and escherichia coli.
The results show that the bacteriostatic rates of the materials obtained in examples 1 to 10 against staphylococcus aureus are as follows: 91.3%, 96.2, 99.6, 99.8, 99.4, 99.8, 92.2, 98.2, 99.3 and 99.4. The bacteriostasis rate to the escherichia coli is as follows in sequence: 89.2%, 94.3, 99.4, 99.7, 99.6, 99.4, 93.7, 99.1, 99.5 and 99.8.
Therefore, the modified material with the albumin coating, which is prepared by the preparation method in the embodiment of the invention, has good antibacterial performance, the dosage of each component in the reaction process has certain influence on the antibacterial effect, and the antibacterial effect can be further increased by adopting the optimal dosage of the components.
In summary, according to the preparation method of the albumin coating with the biological anti-fouling function provided by the invention, the albumin and the protein structure conversion agent are mixed, the disulfide bond of the albumin is broken by using the strong oxidant or the strong reducing agent, and the albumin is converted from the alpha helical structure to the beta folded structure, so that the albumin is endowed with the adhesion property with the material, and has good bonding strength with the surface of the substrate.
The invention also provides a material with a biological anti-fouling function, which comprises a modified base material and an albumin coating formed on the modified base material, wherein the albumin is in a beta-folded structure, and the material has high bonding strength between the coating and the base material, has long-term anticoagulation performance and antibacterial performance, and can inhibit adhesion of vascular cells and tissues.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A preparation method of an albumin coating with a biological anti-fouling function is characterized by comprising the following steps:
forming a coating on the material to be modified by using the dip-coating mixed solution;
the dip-coating mixed solution is formed by mixing an albumin solution and a protein structure conversion agent, wherein the protein structure conversion agent is a strong oxidant or a strong reducing agent.
2. The method for preparing albumin coating with biological anti-fouling function according to claim 1, wherein the concentration of albumin in the dip-coating mixed solution is 0.002-90 mg/mL.
3. The method for preparing albumin coating with biological anti-fouling function according to claim 2, wherein the albumin comprises any one or more of bovine serum albumin, human serum albumin and whey albumin.
4. The method for preparing albumin coating with biological anti-fouling function according to claim 1, characterized in that the strong reducing agent comprises one or more of mercaptoethanol, dithiothreitol and reduced glutathione.
5. The method for preparing albumin coating with biological anti-fouling function according to claim 4, wherein the concentration of the strong reducing agent in the dip-coating mixed solution is 0.002-20 mg/mL.
6. The method of claim 1, wherein the strong oxidant comprises any one or more of trivalent cobalt salt, chlorate, potassium permanganate, persulfate, potassium dichromate, concentrated sulfuric acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, perchloric acid, ozone, hydrogen peroxide, fluorine gas, chlorine gas, sodium bismuthate, periodic acid, sodium ferrate and lead dioxide.
7. The method for preparing albumin coating with biological anti-fouling function according to claim 6, wherein when said strong oxidant is any one or more selected from the group consisting of said trivalent cobalt salt, said potassium permanganate, said potassium dichromate, said sodium bismuthate, said persulfate, said periodic acid, said sodium ferrate and said lead dioxide, the concentration of said strong oxidant is 0.001-20 mg/mL;
when the strong oxidant is any one or more of the concentrated sulfuric acid, the chlorate, the hydroiodic acid, the nitric acid, the hydrobromic acid, the perchloric acid, the hydrochloric acid, the chlorine, the hydrogen peroxide, the fluorine gas and the ozone, the mass fraction of the strong oxidant is 0.02-50%.
8. The method for preparing albumin coating with biological anti-fouling function according to claim 1, wherein the dip-coating mixed solution is prepared by mixing albumin aqueous solution and protein structure conversion agent.
9. The method for preparing the albumin coating with the biological anti-fouling function according to claim 1, characterized in that the material to be modified is soaked in the dip-coating mixed solution to form a coating;
the soaking time is 2 seconds to 8 days;
the reaction temperature is 0-100 ℃.
10. The material with the biological anti-fouling function is characterized by comprising a modified substrate and an albumin coating formed on the modified substrate, wherein the albumin has a beta-sheet structure; the material with the biological anti-fouling function is prepared by the preparation method of any one of claims 1-9.
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