CN114452447A - Mussel protein-polyamino acid coating and preparation method and application thereof - Google Patents
Mussel protein-polyamino acid coating and preparation method and application thereof Download PDFInfo
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- CN114452447A CN114452447A CN202110885488.0A CN202110885488A CN114452447A CN 114452447 A CN114452447 A CN 114452447A CN 202110885488 A CN202110885488 A CN 202110885488A CN 114452447 A CN114452447 A CN 114452447A
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- mussel protein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/08—Materials for coatings
- A61L29/085—Macromolecular materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
- A61L29/16—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/02—Methods for coating medical devices
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/06—Coatings containing a mixture of two or more compounds
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
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- Molecular Biology (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a mussel protein-polyamino acid coating, which comprises a mussel protein layer which is contacted with the surface of a device to be coated and a polyamino acid layer which is crosslinked with the mussel protein layer, wherein the preparation method comprises the steps of providing a base material to be coated, pretreating the surface of the base material, then putting the base material into a mussel protein solution for soaking at room temperature, taking out the base material, and uniformly drying the base material under a nitrogen blowing instrument to obtain a mussel protein modified base material; and (3) immersing the obtained mussel protein modified base material into polyglutamic acid solution for self-assembly reaction, then taking out and uniformly drying the base material under a nitrogen blowing instrument, and obtaining the mussel protein-polyamino acid coating on the surface of the base material to be coated. The invention enriches the polyelectrolyte self-assembly method, expands the application of biological macromolecules on medical materials, and provides an integrated solution for the antibacterial and lubricating requirements of medical catheters in practice.
Description
Technical Field
The invention relates to medical equipment and surface treatment, in particular to a preparation method of a hydrophilic lubricating antibacterial integrated composite catheter coating, belonging to the technical field of high polymer materials.
Background
With the development of modern medical technology, interventional catheters have found widespread use in clinical and postoperative recovery stages. However, the associated infections caused by them emerge endlessly. If the pathogenic bacteria adhere to the base material of the catheter and then generate a biological film, the deterioration of the injury of the patient is aggravated, the drug resistance of the organism is further formed, and the treatment of the disease is delayed.
The commonly used medical catheter is mostly made of hydrophobic polymer materials such as silica gel, polyvinyl chloride and polyurethane. Although they can provide good mechanical properties and biocompatibility, due to the nature of their own structure, there is a certain resistance in the use process, which causes many limitations, such as large friction force during intubation, easy damage to tissues and strong pain to patients, besides causing strong pain and burning to patients, also damages to human tissues, causing a series of complications such as inflammation and bacterial infection, and causing various physical and mental traumas. In addition, although the catheter is sterilized before use, secondary pollution is inevitably caused in the use process, bacterial infection is formed in the body, and serious harm is brought to postoperative recovery.
Therefore, how to avoid bacteria adhesion and improve the lubrication performance of the catheter surface, thereby reducing secondary infection, has become one of the urgent challenges in the field of medical devices today. At present, relevant scholars and enterprises do not consider the integration of antibacterial and lubricating functions, and a method for constructing a functional coating is adopted to respectively carry out single modification on the lubricating or antibacterial aspects, so that the comprehensive performance is greatly limited.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects of the prior art, starting from the optimization of comprehensive performance and simultaneously solving the problem of how to realize the integration of the hydrophilic performance and the antibacterial performance of the surface of the medical catheter, the invention provides the mussel protein-polyamino acid coating and the preparation method thereof. Therefore, the hydrophilic lubricating antibacterial composite coating with good biocompatibility can be formed by a simple method, and the surface of the common medical catheter is endowed with the characteristics of hydrophilic lubrication and active antibacterial.
To achieve the above objects, the present invention provides a mussel protein-polyamino acid coating comprising a mussel protein layer in contact with the surface of the device to be coated and a polyamino acid layer cross-linked with the mussel protein layer, the mussel protein layer being composed of a mussel protein-like substance.
The invention further provides a preparation method of the mussel protein-polyamino acid coating, which comprises the steps of providing a base material to be coated, pretreating the surface of the base material, soaking the base material in a mussel protein substance solution at room temperature, taking out the base material, and uniformly drying the base material under a nitrogen blowing instrument to obtain a mussel protein substance modified base material; and (3) immersing the substrate modified by the mussel protein substance into polyglutamic acid solution for self-assembly reaction, taking out the substrate, uniformly drying the substrate under a nitrogen blowing instrument, and obtaining the mussel protein-polyamino acid coating on the surface of the substrate to be coated.
Wherein the polyglutamic acid is gamma-polyglutamic acid pure product or gamma-polyglutamate, such as polyglutamic acid sodium salt, polyglutamic acid potassium salt, and polyglutamic acid calcium salt; among them, gamma-polyglutamic acid has a molecular weight of 50-3000kDa, preferably 2000 kDa. The polyglutamic acid (gamma-PGA) is an anionic polypeptide polymer formed by polymerizing L-glutamic acid and D-glutamic acid monomers through gamma-amido bonds by microorganisms, is a self-protection substance secreted by part of microorganisms, and has excellent hydrophilic lubricating property.
The mussel protein is mussel byssus protein which is also called mussel adhesive protein.
The stable coating formed by the mutual combination of the gamma-polyglutamic acid and the mussel protein can utilize a compound with a positive charge functional group on the mussel protein to be mutually attracted with a carboxyl phosphate with negative charge on the surface of a cell membrane, so that the cell membrane of germs is damaged and loses physiological functions, and finally the germs die, thereby achieving the purpose of actively inhibiting bacteria; the side chain of the gamma-polyglutamic acid contains a large amount of carboxyl, so that the hydrophilic effect is good, the contact angle of the surface of the catheter is reduced, and the lubricating effect is achieved.
The invention uses the gamma-PGA and mussel protein composite system as the basis of hydrophilic lubrication antibacterial, and utilizes the self-polymerization in-situ assembly of the gamma-PGA and the mussel protein composite system to construct the hydrophilic lubrication antibacterial coating with good biocompatibility on the surface of the base material. The reaction is carried out on the surface of the base material, so that the uniformity of the coverage of the base material is ensured to a great extent, the accurate analysis and research on the related characteristics of the guide pipe are facilitated, the reaction can be initiated only under the condition of weak base, and the damage of the violent chemical reaction to the product is avoided.
The base material is made of polyurethane, polyvinyl chloride or silica gel and other polymer materials.
Preferably, the substrate surface is pretreated by activating the substrate surface by plasma treatment, alkaline cooking or grafting. In some embodiments, the plasma treatment is a treatment in a plasma machine for 5 to 10 minutes; the alkali boiling method comprises the steps of soaking a base material in NaOH solution with the concentration of about 5mol/L, keeping the base material in a water bath kettle at about 60 ℃ for about 6 hours, taking out the base material, washing the base material with a large amount of deionized water, and naturally drying the base material in the air for later use; the grafting method comprises the steps of soaking a base material in an organic solution (0.1g/mL) of benzophenone serving as an initiator, standing for 30-60min, taking out, then soaking in a polyethylene glycol monomer solution (1.0-1.5mol/L) with hydrophilic lubricating property, irradiating for 45-60min by using ultraviolet light, forming a grafting layer with the lubricating effect on the surface, and grafting a monomer with a reactive functional group to a molecular chain of the base material through polymerization reaction.
In a preferred embodiment, the substrate is subjected to an ultrasonic cleaning treatment prior to activating the substrate surface. In one embodiment, the substrate is a catheter substrate, the catheter substrate is cut into a suitable length, such as 5-10cm, then a cleaning agent absolute ethyl alcohol is added, ultrasonic treatment is performed, deionized water is replaced, ultrasonic cleaning is continued for three times to completely remove impurities on the substrate, the substrate is placed into an oven for drying, and then the dried tube is treated in a plasma machine for standby. Preferably, the two times of ultrasonic treatment are both 10-30min, the power used by ultrasonic treatment is set to be 80-100w, the drying temperature of the oven is set to be 60 ℃, and the treatment time in the plasma machine is 5-10 min.
Preferably, the solvent of the mussel protein solution is Tris-HCl solution or PBS alkaline buffer solution (0.01M), and the concentration of the Tris-HCl solution is 0.02-0.08 g/100 mL; the concentration of the mussel protein solution is 2-10mg/mL, and the pH value is 8-9. The alkaline conditions favor activation of the functional groups of the mussel protein for further reaction with the substrate and polyglutamic acid.
The polyglutamic acid solution is prepared by the following method: adding polyglutamic acid into deionized water, magnetically stirring at normal temperature for 0.5-1h, and ultrasonically treating for 15-30min to promote dissolution until the solution is colorless and transparent.
Preferably, the concentration of the mussel protein solution is 2-10mg/mL, and the pH value is 8-9; the concentration of the polyglutamic acid solution is 0.2g/mL-2 g/mL.
Preferably, the base material is soaked in the mussel protein solution for 5-30min, then taken out and uniformly dried under a nitrogen blowing instrument to obtain the mussel protein modified base material, then the base material is soaked in the polyglutamic acid solution for 5-30min, and then taken out and uniformly dried under the nitrogen blowing instrument to obtain the mussel protein-polyamino acid antibacterial lubricating medical coating.
The invention further provides application of the mussel protein-polyamino acid coating to an antibacterial lubricating medical coating.
The invention further provides a medical catheter, the surface of which is coated with the mussel protein-polyamino acid coating. The mussel protein-polyamino acid coating is suitable for being applied to medical catheters, such as catheters, dialysis tubes, in-vivo interventional catheters, digestive tract tubes, respiratory tract tubes, drainage tubes and the like.
The invention starts from two aspects of lubrication and antibacterial performance of medical catheters, and prepares an MFP-PGA functional coating on the surface of PU (polyurethane) by means of the function that mussel protein (MFP) can be deposited on the surface of most materials and combining multiple interactions of MFP and gamma-polyglutamic acid (gamma-PGA). Through a series of characterization and performance tests, it was found that: the PU/MFP-PGA surface has good antibacterial lubricating property. The invention enriches the polyelectrolyte self-assembly method, expands the application of biological macromolecules on medical materials, and provides an integrated solution for the antibacterial and lubricating requirements of medical catheters in practice.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the invention starts from two problems of antibiosis and lubrication which need to be solved urgently in the field of the medical catheter, creatively combines the antibiosis and the lubrication effects in a unified way, and is not possessed by a single modified catheter in the market;
(2) the preparation of the composite coating is based on the layer-by-layer self-assembly technology (the reaction is carried out on the basis of ionic bonds formed among materials), the invention utilizes the characteristics that mussel protein contains various antibacterial groups and can be deposited on the surfaces of materials such as plastics, ceramics, metals and the like, plays the role of an adhesion layer in the composite coating, further has the electrostatic assembly effect with polyglutamic acid, introduces carboxyl with excellent hydrophilic performance, realizes the integrated design of antibacterial lubricating performance, utilizes the property that mussel protein can form solid covalent bonds with the surface of a base material, and promotes the self-polymerization of the mussel protein on the surface of a catheter to form a stable adhesion layer and then has the electrostatic assembly with gamma-PGA under mild conditions, thereby achieving excellent stability and coverage uniformity which cannot be realized by a common physical coating method;
(3) the composite coating is tested for protein adsorption resistance and bacterial adhesion resistance, and the results show that the composite coating has good bacteriostatic effect and protein adsorption resistance under the co-culture of escherichia coli and staphylococcus aureus, and has good application prospect in the field of biomedical materials;
(4) the raw materials used by the coating all have good biocompatibility and almost have no toxic or harmful effect on human bodies.
Drawings
The invention will be described in further detail with reference to the following drawings and detailed description:
FIG. 1 is a comparison of the hydrophilic properties of the composite coating prepared in example 1, wherein A is an untreated PU catheter substrate, B is a commercially available PU catheter substrate with an antibacterial surface, C is a commercially available PU catheter substrate with a lubricating surface, and D is a PU catheter substrate with a composite coating according to the present invention;
FIG. 2 is a comparison of antibacterial properties of the composite coating, wherein a represents Escherichia coli, b represents Staphylococcus aureus, 1 is an untreated PU catheter substrate, 2 is a commercially available PU catheter substrate with an antibacterial surface, and 3 is a PU catheter substrate treated by the composite coating of the present invention, and it can be observed that the contact part of 3 is more transparent, the bacterial growth is less, and the antibacterial effect is better;
FIG. 3 is a comparison of the anti-protein adhesion performance of the composite coating, wherein A is an untreated PU catheter substrate, B is a commercially available PU catheter substrate with an antibacterial surface, and C is a PU catheter substrate treated with the composite coating of the present invention, and in a sample diluted in the same gradient, the number of colonies formed on the surface of C is the least, i.e., the anti-protein adsorption performance is the most excellent;
fig. 4 is a quantitative analysis of the anti-protein adhesion performance of the composite coating, wherein a is an untreated PU catheter substrate, B is a commercially available PU catheter substrate with an antibacterial surface, and C is a PU catheter substrate with a composite coating, according to the present invention, when the MFP-PGA composite coating is introduced on the PU surface, the number of live bacteria adhered to the surfaces of e.coli and s.aureus is respectively reduced to 15% and 52% of the surface of the original PU substrate, and the anti-bacterial adhesion performance is verified;
fig. 5 is a biocompatibility analysis of the composite coating, wherein a is an untreated PU catheter substrate, B is a commercially available PU catheter substrate, and C is a composite coated treated PU catheter substrate of the present invention.
Detailed Description
The present invention will be better understood by further explanation with reference to examples. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
In the following examples, JYSP-360 contact angle measuring instrument was selected for the purpose of examining the hydrophilic property. Firstly, dripping a drop of water on the surfaces of different substrates by using a static liquid drop method, then measuring the contact angle of different coatings by using a three-point method, and taking the average value of the three measurements as the final result of the contact angle of the coating.
Example 1 preparation of a mussel protein-polyamino acid antibacterial lubricating medical coating.
Through the investigation of the formation of coatings by using mussel protein (mussel byssus protein) and polyglutamic acid solutions with different concentration gradients, the following findings are obtained: when the concentration of the mussel protein is less than 2mg/mL, the adhesion property is poor, the number of functional groups participating in the reaction is small, and thus the stability of the coating after being combined with polyglutamic acid is poor, while exceeding 10mg/mL may affect the uniformity of the surface of the coating.
Similarly, when the concentration of polyglutamic acid is less than 0.2g/mL, the carboxyl group playing a role in hydrophilic lubrication is also reduced due to the lower concentration, and there is no significant lubricating effect, while when the concentration reaches 2g/mL, the solubility is reduced, which is not favorable for practical operation. In summary, preferably, the concentration of mussel protein is 2-10mg/mL and the concentration of polyglutamic acid is 0.2-2 g/mL.
Therefore, the concentration of the mussel protein solution is 2-10mg/mL, and the concentration of the polyglutamic acid solution is 0.2-2 g/mL. A suitable coating is prepared below using a mussel protein solution of 2mg/mL and a polyglutamic acid solution of 0.2g/mL as an example. Coatings of other concentrations can be prepared in the same way, and the concentrations can be adjusted according to cost and demand.
The preparation method of the composite coating comprises the following steps:
(1) pretreatment of a base material: firstly cutting a catheter base material into a proper length, such as 5-10cm, adding a cleaning agent absolute ethyl alcohol, performing ultrasonic treatment, replacing deionized water, continuously performing ultrasonic alternative cleaning for three times to completely remove impurities on the base material, drying the base material in an oven, and then treating the dried pipe in a plasma machine for later use. Preferably, the two times of ultrasonic treatment are 15min, the power used for ultrasonic treatment is 100w, the drying temperature of the oven is 60 ℃, and the treatment time in the plasma machine is 8 min.
(2) Soaking the treated base material in a mussel protein solution (2mg/mL, Tris-HCl solution with a solvent of 0.05g/100mL to prepare 100mL) for 15min, taking out and uniformly drying under a nitrogen blowing instrument to obtain a mussel protein modified base material, then soaking in a polyglutamic acid solution (molecular weight: 2000kDa, concentration: 0.2g/mL to prepare 100mL) for 15min, taking out and uniformly drying under the nitrogen blowing instrument to obtain the mussel protein-polyamino acid antibacterial lubricating medical coating.
In this example, the hydrophilic performance of the prepared composite coating is examined, and the result is shown in fig. 1, where a is an untreated PU catheter substrate, B is a commercially available PU catheter substrate with a surface treated with an antibacterial agent (by adding an inorganic heavy metal additive such as silver ions to the substrate raw material), C is a commercially available PU catheter substrate with a surface treated with a lubricating agent (by coating a lubricant such as polyvinylpyrrolidone or paraffin on the surface), and D is a PU catheter substrate treated with the composite coating of the present invention, and it can be directly observed that the catheter surface treated with the hydrophilic lubricating antibacterial agent of the present invention has better hydrophilicity. These results fully demonstrate that the composite coatings of the present invention perform well in hydrophilic performance, indicating that the coatings of the present invention have good hydrophilic lubricating properties.
Example 2 activation of E.coli species (ATCC 25933) and S.aureus species (ATCC 6538).
The implementation is the preliminary preparation work of the embodiment 3 and the embodiment 4, mainly comprising the culture medium preparation and the strain activation, and the specific steps are as follows:
(1) preparation of culture medium
The liquid culture medium of the lysis Broth (Lysozyme Broth) used in the experiment is specifically prepared by the following steps: firstly, 1g of tryptone, 0.5 g of yeast extract and l g of NaC1 powder are weighed and dissolved in 100ml of distilled water, and after uniform mixing, the liquid is sterilized for 30 minutes in an autoclave at 121 ℃, and then the mixture is stored in a refrigerator at 4 ℃ for later use.
(2) Strain activation
Escherichia coli (ATCC 25933) and Staphylococcus aureus (ATCC 6538) stored in a refrigerator at-80 ℃ were taken out and thawed, 1 ml of the thawed solution was taken in a previously sterilized LB broth Erlenmeyer flask (operating in a clean bench), and then the Erlenmeyer flask was placed on a constant temperature shaker (temperature set at about 37 ℃) to culture for 12 hours, and then stored in a refrigerator at 4 ℃ for later use.
Example 3 testing of the antimicrobial properties of a mussel protein-polyamino acid antimicrobial lubricating medical coating.
15 g of agar powder was weighed and dissolved in 100ml of LB, and then placed in a vertical autoclave with parameters set as follows: sterilizing at 121 deg.C for 30 min. Immediately, the mixture is poured into a cell culture dish in a super clean bench while the mixture is hot, and air-dried for standby. Diluting the culture solution of Escherichia coli and Staphylococcus aureus to 10% with PBS buffer solution6CFU/mL. 100 microliter of diluted bacteria solution is sucked and evenly dropped into an LB culture plate, and a coating rod is used for evenly coating the bacteria solution until the bacteria solution is completely absorbed. Adhering three substrates (1 is an untreated PU catheter substrate, 2 is a commercially available PU catheter substrate with the surface subjected to antibacterial treatment, and 3 is a PU catheter substrate treated by the composite coating) 1, 2 and 3 to a solid culture plate, and placing the solid culture plate in a constant-temperature incubator at 37 ℃ for incubation for 12 hours. Observing the formation of the bacteriostatic circle at the bottom and around the material, and taking a picture and recording by using a camera. In this example, the antibacterial performance of the prepared composite coating was examined, and the results are shown in fig. 2. It can be observed that the contact part of the substrate treated by the composite coating is more transparent, the bacterial growth is less, and the antibacterial effect is better. These results fully demonstrate that the composite coatings of the present invention perform excellently against bacteria.
Example 4 testing of the anti-protein adsorption properties of mussel protein-polyamino acid antibacterial lubricious medical coatings.
Diluting the culture solution of Escherichia coli and Staphylococcus aureus to 10% with PBS buffer solution7CFU/mL. The A, B, C three substrates were clamped in a 24-well plate with sterilized forceps, and then 1 ml of diluted bacterial solution was dropped into the well, and the plate was left to stand and incubated in a 37 ℃ incubator for 4 hours. The well was aspirated and 1 ml of PBS buffer was slowly added to wash the substrate surface to remove loosely adhering bacteria, and this procedure was repeated three times. The washed sheet was taken out, placed in a centrifuge tube containing 3mL of PBS buffer solution, and subjected to ultrasonic treatment for 5 minutes to peel the surface of the sheet from the sheetAn attached bacterium. The bacterial solution peeled from the surface was diluted in a gradient at a constant rate, 100. mu.L of the diluted bacterial solution was sucked and dropped uniformly on an LB-agar plate, and then uniformly spread with a spreading bar until the bacterial solution was completely absorbed by the agar plate. Then, the LB-agar plate is placed in a constant temperature incubator (the temperature is set by simulating the normal human body temperature) for overnight incubation, the bacterial reproduction state on the LB-agar plate is observed, the colony number is counted, and the record is taken by a camera. In this example, the results of examining the protein adhesion resistance of the prepared composite coating are shown in FIGS. 3 to 5. It can be observed that in the same sample diluted in gradient, the number of colonies formed on the surface of the substrate treated by the composite coating is the least, and the number of live bacteria adhered on the surface of the E.coli and S.aureus is respectively reduced to 15% and 52% of the original PU substrate surface, so that the antibacterial adhesion performance is verified, and the results fully show that the composite coating of the invention is excellent in the protein adsorption resistance performance.
Example 5 biocompatibility testing of mussel protein-polyamino acid antibacterial lubricating medical coatings.
This example examines the biocompatibility of the prepared composite coating.
The cytotoxicity of A, B, C three substrates on L929 mouse fibroblasts was tested by the thiazole blue (MTT) method. A, B, C substrates were soaked in basal cell culture medium and stored in a 4 ℃ refrigerator for 1, 2 and 3 days. Cells were seeded per well in a 96-well plate and incubated in a 37 ℃ incubator for 36 hours, and the medium in the wells was aspirated and replaced with the substrate-pretreated medium, with DMEM medium without any treatment as a control group. After 24 and 48 hours incubation in the 37 ℃ incubator, respectively, the medium was aspirated and each well was gently rinsed with PBS buffer and this procedure was repeated three times. 0.2 ml of MTT in DMEM (0.5 mg/ml) was added to the wells and incubated at 37 ℃ for 4 hours in an incubator. The medium was gently aspirated and 0.2 ml of dimethyl sulfoxide was added to the wells to dissolve the blue-violet crystalline formazan. The absorbance of the solution at 570 nm was measured after 15 minutes using a microplate reader, and the results were expressed as percentages relative to the control. The results are shown in FIG. 5. The results show that: the survival rate of the mouse cells treated by the composite coating for 72h is as high as 90%, and the coating has almost no toxic effect on mouse fibroblasts.
The hydrophilic lubricating antibacterial composite coating prepared by the method disclosed by the invention is uniform in coverage and strong in stability. The preparation process is simple and quick, can realize catheter functionalization, can be applied to industrial production, meets the requirement of medical catheter on antibiosis, and avoids bacterial secondary infection in vivo; the pain of intubation of a patient is reduced; and has good market prospect in the aspects of optimizing the operation process, promoting the postoperative recovery of patients and the like.
Claims (10)
1. A mussel protein-polyamino acid coating is characterized by comprising a mussel protein layer which is in contact with the surface of a device to be coated and a polyamino acid layer which is crosslinked with the mussel protein layer, wherein the mussel protein layer is made of mussel protein substances.
2. The method for preparing a mussel protein-polyamino acid coating according to claim 1, wherein a substrate to be coated is provided, the surface of the substrate is pretreated, the substrate is then immersed in a mussel protein solution at room temperature, and then the substrate is taken out and uniformly dried under a nitrogen blower to obtain a mussel protein substance-modified substrate; and (3) immersing the substrate modified by the mussel protein substance into polyglutamic acid solution for self-assembly reaction, taking out the substrate, uniformly drying the substrate under a nitrogen blowing instrument, and obtaining the mussel protein-polyamino acid coating on the surface of the substrate to be coated.
3. The method according to claim 2, wherein the polyglutamic acid is a pure gamma-polyglutamic acid or a gamma-polyglutamate; the molecular weight of the gamma-polyglutamic acid is 50-3000 kDa.
4. The method according to claim 2, wherein the mussel protein is mussel byssus protein.
5. The method of claim 2, wherein the substrate is polyurethane, polyvinyl chloride, or silica gel.
6. The method according to claim 2, wherein the pretreatment of the surface of the substrate is to activate the surface of the substrate by plasma treatment, alkaline cooking or grafting.
7. The preparation method according to claim 1, wherein the solvent of the mussel protein solution is Tris-HCl solution or PBS alkaline buffer solution, and the concentration of the Tris-HCl solution is 0.02-0.08 g/100 mL; the concentration of the mussel protein solution is 2-10mg/mL, and the pH value is 8-9.
8. The method according to claim 2, wherein the polyglutamic acid solution is prepared by: adding polyglutamic acid into deionized water, magnetically stirring at normal temperature for 0.5-1h, and ultrasonically treating for 15-30min to promote dissolution, wherein the concentration of polyglutamic acid solution is 0.2-2g/mL when the solution is colorless and transparent.
9. Use of the mussel protein-polyamino acid coating of claim 1 for antimicrobial lubricating medical coatings.
10. A medical catheter having a surface coated with the mussel protein-polyamino acid coating of claim 1.
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CN115181169A (en) * | 2022-05-13 | 2022-10-14 | 南京工业大学 | Method for improving adhesion of recombinant mussel protein |
CN115177793A (en) * | 2022-07-20 | 2022-10-14 | 南京工业大学 | Method for modifying mussel protein-polyglutamic acid antibacterial lubricating medical hydrogel coating on surface of base material |
CN115777774A (en) * | 2022-11-11 | 2023-03-14 | 南京工业大学 | Carboxymethyl cellulose-based edible film containing mussel foot protein and preparation method and application thereof |
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CN115777774A (en) * | 2022-11-11 | 2023-03-14 | 南京工业大学 | Carboxymethyl cellulose-based edible film containing mussel foot protein and preparation method and application thereof |
CN117159813A (en) * | 2023-09-08 | 2023-12-05 | 浙江海圣医疗器械股份有限公司 | Medical catheter with hydrophilic coating and preparation method thereof |
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