CN114796611B

CN114796611B - Implantable antibacterial coating, preparation method and application thereof

Info

Publication number: CN114796611B
Application number: CN202110112193.XA
Authority: CN
Inventors: 请求不公布姓名
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2023-06-30
Anticipated expiration: 2041-01-27
Also published as: CN114796611A

Abstract

The invention provides an implantable antibacterial coating, a preparation method and application thereof, wherein the preparation method comprises the following steps: s1: providing an implantable material, mixing calcium into the surface of the implantable material, forming a composite surface on the implantable material, the mixing amount of calcium in the composite surface being 5-30 at%; s2: preparing a fibrinogen solution with the concentration of 0.1-50 mg/L by adopting phosphate buffer salt solution without calcium; s3: and (3) soaking the composite surface prepared in the step (S1) in the fibrinogen solution prepared in the step (S2), preserving the temperature for a certain time at 30-45 ℃, cleaning, and drying to obtain the implantable antibacterial coating. According to the research of the invention, the implantable material which is mixed with calcium and adsorbs fibrinogen has obvious antibacterial effect, and the antibacterial coating can be used on the surface of an implantable device with tissue integration requirement, and has very wide application prospect.

Description

Implantable antibacterial coating, preparation method and application thereof

Technical Field

The invention relates to the field of biological material surface modification, in particular to an implantable antibacterial coating, a preparation method and application thereof.

Background

Bacterial infection is one of the primary complications of failure of implantable medical devices, such as dental implants, internal fixation systems, artificial joints, and the like. To reduce the risk of infection, one often uses local release or immobilization of antibiotics, antimicrobial peptides, antimicrobial metals and their compounds (e.g., silver, copper, zinc), etc., to design an antimicrobial surface directed against bacteria. However, since mammalian cells and bacteria have many similar adhesion mechanisms, these designs have difficulty meeting both antimicrobial and long-term safety (including tissue integration) requirements of implantable medical device surfaces, and thus such techniques are largely incapable of meeting clinical application requirements.

Studies have shown that infection associated with implantation of biological materials is not only associated with bacterial contamination, but also with adverse reactions caused by the implanted material, compromising the host's inherent immunity against bacterial attack. Therefore, the antibacterial surface is designed aiming at the inherent immune reaction process of the repair of the human tissue injury, and the long-acting antibacterial performance is more hopefully obtained, so that the clinical application is realized. Fibrinogen plays a key role in the acute phase response of wound healing and is the first physical barrier against microbial invasion. During the implantation of the medical device, fibrinogen is immediately adsorbed on the surface of the device in contact with the liquid and affects the interaction and subsequent reaction of the device with immune cells of the human body. However, adsorption of fibrinogen to biological materials (including metals and polymeric materials) tends to promote bacterial adhesion, thereby adversely reducing implantation-related infections. Although the kinetics and related mechanisms of fibrinogen adsorption on the surface of biological materials have been known systematically over the past decades, direct regulation of fibrinogen adsorption has been reported to impart antimicrobial function to implantable medical devices.

Calcium-doped biomaterials (such as titanium and its alloys) have excellent biocompatibility that can promote tissue integration (especially bone tissue). However, calcium doping alone is not sufficient to prevent bacterial colonization of the material surface, and may even promote bacterial proliferation on the material surface, and is also detrimental to reducing infection associated with implantation of biological materials. In order to give consideration to the antibacterial function and the biocompatibility of the surface of the material, the calcium doping and the antibacterial agent are required to be used in a compounding way, for example, the calcium doping and the silver doping are required to be compounded in a patent CN 201210537119.3. This would increase the cost of mass production of the relevant surface treatment techniques, adverse implant device safety long-term management and clinical application. In addition, although calcium ions have been reported to bind to fibrinogen and thereby alter the three-dimensional conformation of the protein [ Biochimica et Biophysica Acta 1989,995,70-74] [ Biophysical Chemistry 2004,112,131-140], the use of calcium to react with fibrinogen for antibacterial purposes has not been reported.

Disclosure of Invention

The invention aims to provide an implantable antibacterial coating, a preparation method and application thereof, so as to solve the problems that in the prior art, the antibacterial coating is easy to cause antibiotic resistance, cause adverse reactions of hosts and repair or integration of unfavorable tissues due to the direct adoption of antibacterial agents.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention there is provided a method of preparing an implantable antimicrobial coating comprising the steps of: s1: providing an implantable material, mixing calcium into the surface of the implantable material, forming a composite surface on the implantable material, the mixing amount of calcium in the composite surface being 5-30 at%; s2: preparing a fibrinogen solution with the concentration of 0.1-50 mg/L by adopting phosphate buffer salt solution without calcium; s3: and (3) soaking the composite surface prepared in the step (S1) in the fibrinogen solution prepared in the step (S2), preserving the temperature for a certain time at 30-45 ℃, cleaning, and drying to obtain the implantable antibacterial coating.

Preferably, in step S1, the implantable material includes: titanium, iron, zirconium, tantalum, niobium, polyetheretherketone.

It should be understood that the calcium doping technique performed in step S1 of the present invention is a physical process, and the chemical composition of the substrate is not limited, and thus, the implant materials commonly used in the field of implantable medical devices are suitable for the present invention.

Further preferably, in step S1, the mixing amount of calcium is 5 to 15at.%.

Further preferably, in step S2, the concentration of fibrinogen is 0.1 to 30mg/L.

Preferably, in step S1, the method of mixing calcium into the surface of the implantable material is a vacuum plating technique.

However, it should be understood that, since the calcium metal is very reactive and is well controlled by the vacuum plating technique, the step S1 of the present invention is not limited to the vacuum plating technique.

Preferably, in step S1, the vacuum coating technique includes: ion implantation, and/or thermal evaporation, and/or electron beam evaporation.

According to the present invention, if pure metal is used as the calcium source, thermal evaporation can be used in combination with electron beam evaporation; if a calcium-containing compound or alloy is used as the calcium source, only thermal evaporation or electron beam evaporation may be used; if a mixture of calcium and titanium is used as the calcium source, electron beam evaporation alone is used to deposit the titanium substrate.

Preferably, the ion implantation process parameters are: vacuum degree 10 ^-2 Pa～10 ^-6 Pa; injecting voltage is 5-40 kV, and injecting time is 20-120 min; the thermal evaporation process parameters are as follows: the temperature is 300-800 ℃ and the vacuum degree is 10 ^-2 Pa～10 ^-6 Pa, the deposition time is 1-60 min; the electron beam evaporation process parameters are as follows: the voltage is 5-15 kV, the current is 50-150A, and the vacuum degree is 10 ^-2 Pa～10 ^-6 Pa, the deposition time is 1-60 min.

Preferably, in step S3, the temperature is kept at 30-40 ℃ for 1-3 hours.

According to a second aspect of the present invention there is provided an implantable antimicrobial coating prepared according to the above-described preparation method.

According to a third aspect of the present invention there is provided the use of an implantable antimicrobial coating in the manufacture of a medical consumable comprising: artificial bone, artificial joint, implant, vascular stent.

As described in the background, it has been reported that calcium ions can bind to fibrinogen, thereby altering the three-dimensional conformation of the protein, although CaCl is used in the report ₂ Providing calcium ions as a calcium source, utilizing the calcium ions and the C-terminus of the fibrinogen alpha chainThe reaction was carried out, but no antibacterial function was reported for this conformational change. Therefore, the primary technical difficulty of the present invention is how to induce fibrinogen to adsorb on the surface of a substrate and expose a fragment thereof having an antibacterial function, and if only the antibacterial fragment is adsorbed without exposure, there is no antibacterial function.

Unlike this report, the present invention releases calcium ions and provides an alkaline environment by adding pure metallic calcium to the surface of a substrate, and by reacting metallic calcium with oxygen and an aqueous solution in the air, it can react calcium ions with not only the C-terminal but also the N-terminal of the alpha chain of fibrinogen, thereby exposing the B15-42 fragment of fibrinogen and realizing an antibacterial function.

The core idea of the invention is that the composite surface of calcium and implantable material is prepared by adopting a vacuum coating technology, then the fibrinogen layer is pre-adsorbed, the adsorption conformation of the fibrinogen is regulated and controlled by releasing calcium, and further the antibacterial performance is obtained, thus preparing the implantable antibacterial coating.

According to the invention, the regulation of fibrinogen adsorption conformation is mainly realized by controlling the content of calcium in the substrate, and the low concentration of calcium cannot effectively expose the antibacterial fragments of fibrinogen; too high a concentration of calcium may cause premature denaturation of fibrinogen and thus not be effectively adsorbed on the surface of the substrate, so that the preferred calcium mixing amount of the present invention is 5 to 30at.%, and neither too low nor too high a concentration of calcium achieves the optimal antibacterial purpose.

According to the studies of the present invention, it was found that neither the implantable material mixed with calcium alone nor the implantable material adsorbed with fibrinogen alone has a remarkable inhibitory effect on bacterial growth, while the implantable material mixed with calcium and adsorbed with fibrinogen has a remarkable inhibitory effect. The antibacterial coating provided according to the invention can be used on the surface of implantable devices with tissue integration requirements, such as artificial bones, artificial joints, dental implant systems, vascular stents and the like.

The implantable antibacterial coating, the preparation method and the application thereof provided by the invention have the following advantages compared with the prior art:

1) The two components existing in the human body, namely calcium and fibrinogen, are used as raw materials for preparing the antibacterial coating, and the antibacterial function is activated by controlling the reaction between the two components, so that the antibacterial purpose is realized, and the problem of poor safety caused by the fact that the prior art mostly adopts toxic components which are not existing in the human body is avoided;

2) At present, the antibacterial coating is mainly prepared from materials toxic to bacteria, and calcium or fibrinogen adopted by the invention has no inhibition effect on bacterial adhesion, but has obvious inhibition effect after the reaction of calcium and fibrinogen with proper concentration, so the antibacterial coating has unobvious property;

3) The research shows that the implantable material which is prepared according to the invention and is simultaneously mixed with calcium and adsorbs fibrinogen has obvious antibacterial effect, can be used on the surface of implantable instruments with tissue integration requirements, such as artificial bones, artificial joints, dental implant systems, vascular stents and the like, and has very wide application prospect.

Drawings

FIG. 1 shows the results of the depth distribution of calcium concentration on a smooth pure calcium and pure titanium composite surface in example 1;

FIG. 2 shows an image of the surface of the material obtained by adsorbing fibrinogen on a smooth pure titanium surface in example 1, as observed under an atomic force microscope;

FIG. 3 shows an image of the surface of the material obtained by adsorbing fibrinogen on a smooth composite surface of pure calcium and pure titanium of example 1, as observed under an atomic force microscope;

FIG. 4 shows an image of the surface of the material obtained by adsorbing fibrinogen on the surface of the coarse pure calcium and pure titanium composite in example 2, observed under an atomic force microscope;

FIG. 5 is a view showing an image of the surface of a material obtained by adsorbing fibrinogen on the composite surface when the calcium mixing concentration is too low in example 3, observed under an atomic force microscope;

FIG. 6 shows an image of the surface of a material obtained by adsorbing fibrinogen on the composite surface when the fibrinogen concentration is too high in example 4, observed under an atomic force microscope;

FIG. 7 shows images of the result of the "lump" adsorption observation of fibrinogen obtained by too high a calcium incorporation concentration in example 5;

FIG. 8 shows an image of fibrinogen concentration too low to form a "network" adsorption observation in example 6;

FIG. 9 shows the morphology of Pseudomonas aeruginosa in example 7 after 24h incubation at 37℃on pure titanium film surface;

FIG. 10 shows the morphology of Pseudomonas aeruginosa in example 7 after 24h incubation at 37℃on fibrinogen-adsorbed pure titanium film surface;

FIG. 11 shows the morphology of Pseudomonas aeruginosa in example 7 after 24 hours of culture at 37℃on the surface of a pure calcium and pure titanium composite film (thickness 50 nm);

FIG. 12 shows the morphology of Pseudomonas aeruginosa in example 7 after 24 hours of culture at 37℃on the surface of fibrinogen-adsorbed pure calcium and pure titanium composite film (thickness 50 nm);

FIG. 13 shows the morphology of murine mesenchymal stem cells of example 7 after 24h incubation at 37℃on fibrinogen-adsorbed material surface;

FIG. 14 shows the morphology of Pseudomonas aeruginosa in example 8 after 24 hours of culture at 37℃on the surface of a pure calcium and pure titanium composite film (thickness 100 nm);

FIG. 15 shows the morphology of Pseudomonas aeruginosa in example 8 after 24 hours of culture at 37℃on the surface of fibrinogen-adsorbed pure calcium and pure titanium composite film (thickness 100 nm);

FIG. 16 shows the morphology of Pseudomonas aeruginosa in example 9 after 24h incubation at 37℃on fibrinogen-adsorbed pure calcium and pure titanium composite film (thickness 25 nm) surface.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1

Polishing the surface of pure titanium block, and ion implantation (vacuum degree 10) ^-2 Pa～10 ^-6 Pa; injecting voltage of 5-40 kV and injecting time of 20-120 min), mixing pure calcium into smooth pure titanium surface to obtain composite surface, and measuring its surface by X-ray photoelectron spectroscopy (XPS)The flour calcium content may be 16.9at.% (fig. 1). A fibrinogen solution having a concentration of 0.1 to 1mg/L is prepared with a Phosphate Buffered Saline (PBS) containing no calcium. The titanium material mixed with calcium is soaked in the fibrinogen solution, the temperature is kept for 1 hour at 37 ℃, then PBS and deionized water are used for cleaning, and after the titanium material is placed for natural drying, an atomic force microscope is used for observing the surface of the material. It can be seen that fibrinogen is "granular" adsorbed on the surface of pure titanium (fig. 2), while it is "net" adsorbed on the surface of calcium-mixed titanium (fig. 3). This result indicates that calcium mixing can regulate the adsorption conformation of fibrinogen on the titanium surface.

Example 2

The surface of the block-shaped pure titanium is firstly treated by sand blasting and acid etching, and then the ion implantation technology (vacuum degree 10 is adopted ^-2 Pa～10 ^- ⁶ Pa; the injection voltage is 5-40 kV, the injection time is 20-120 min), pure calcium is mixed into the rough pure titanium surface to prepare a composite surface, and the surface calcium content can be 5.0-15.6 at.% measured by XPS. A fibrinogen solution having a concentration of 30-50 mg/L is prepared with Phosphate Buffered Saline (PBS) containing no calcium. The titanium material mixed with calcium is soaked in the fibrinogen solution, the temperature is kept for 1 hour at 37 ℃, then PBS and deionized water are used for cleaning, and after the titanium material is placed for natural drying, an Atomic Force Microscope (AFM) is used for observing the surface of the material. Fibrinogen is seen to be "network" adsorbed on the coarse titanium surface mixed with calcium (fig. 4).

Example 3

Polishing the surface of pure titanium block, and ion implantation (vacuum degree 10) ^-2 Pa～10 ^-6 Pa; injection voltage of 5-40 kV and injection time of 20-120 min), mixing pure calcium into a smooth pure titanium surface to prepare a composite surface, wherein the content of the surface calcium can be 2.7-4.6 at.% measured by X-ray photoelectron spectroscopy (XPS). A fibrinogen solution having a concentration of 0.1 to 1mg/L is prepared with a Phosphate Buffered Saline (PBS) containing no calcium. The titanium material mixed with calcium is soaked in the fibrinogen solution, the temperature is kept for 1 hour at 37 ℃, then PBS and deionized water are used for cleaning, and after the titanium material is placed for natural drying, an atomic force microscope is used for observing the surface of the material. Fibrinogen was not seen to be adsorbed "in the form of a net" as shown in example 1 on a smooth pure titanium surface mixed with calcium (figure 5).

Example 4

Polishing the surface of pure titanium block, and ion implantation (vacuum degree 10) ^-2 Pa～10 ^-6 Pa; injection voltage of 5-40 kV and injection time of 20-120 min), mixing pure calcium into a smooth pure titanium surface to prepare a composite surface, wherein the content of the surface calcium can be 15.1-21.9 at.% measured by X-ray photoelectron spectroscopy (XPS). A fibrinogen solution having a concentration of 50-60 mg/L is prepared with Phosphate Buffered Saline (PBS) containing no calcium. The titanium material mixed with calcium is soaked in the fibrinogen solution, the temperature is kept for 1 hour at 37 ℃, then PBS and deionized water are used for cleaning, and after the titanium material is placed for natural drying, an Atomic Force Microscope (AFM) is used for observing the surface of the material. Fibrinogen is not seen to be adsorbed on the calcium-blended titanium surface in the form of a "net" as shown in example 2, but rather in the form of a fibrinogen coating (fig. 6).

Example 5

Polishing the surface of pure titanium block, and ion implantation (vacuum degree 10) ^-2 Pa～10 ^-6 Pa; injection voltage of 5-40 kV and injection time of 20-120 min), mixing pure calcium into a smooth pure titanium surface to prepare a composite surface, wherein the content of the surface calcium can be 30.7-41.6 at.% measured by X-ray photoelectron spectroscopy (XPS). A fibrinogen solution having a concentration of 0.1 to 1mg/L is prepared with a Phosphate Buffered Saline (PBS) containing no calcium. The titanium material mixed with calcium is soaked in the fibrinogen solution, the temperature is kept for 1 hour at 37 ℃, then PBS and deionized water are used for cleaning, and after the titanium material is placed for natural drying, an atomic force microscope is used for observing the surface of the material. Common fibrinogen is adsorbed in "lumps" on the smooth pure titanium surface mixed with calcium (fig. 7).

Example 6

The surface of the block-shaped pure titanium is firstly treated by sand blasting and acid etching, and then the ion implantation technology (vacuum degree 10 is adopted ^-2 Pa～10 ^- ⁶ Pa; the injection voltage is 5-40 kV, the injection time is 20-120 min), pure calcium is mixed into the rough pure titanium surface to prepare a composite surface, and the surface calcium content can be 5.0-15.6 at.% measured by XPS. A fibrinogen solution having a concentration of 0.01 to 0.1mg/L is prepared with a Phosphate Buffered Saline (PBS) containing no calcium. Will mix the calciumThe titanium material of (2) is soaked in the fibrinogen solution, the temperature is kept for 1 hour at 37 ℃, then PBS and deionized water are used for cleaning, and after the titanium material is placed for natural drying, an Atomic Force Microscope (AFM) is used for observing the surface of the material. Fibrinogen was not seen to be adsorbed on the calcium-blended titanium surface in the "net" shape shown in example 2 (fig. 8).

Example 7

Adopts a process (vacuum degree 10) of compounding thermal evaporation (calcium source, thermal evaporation temperature 300-800 ℃) and electron beam evaporation (titanium source, working voltage 5-15 kV and current 50-150A) ^-2 Pa～10 ^-6 Pa; deposition time was 4 min), a thin film (thickness about 50 nm) of pure calcium and pure titanium composite was deposited, and its surface calcium content was 11.1at.% as measured by X-ray photoelectron spectroscopy (XPS). Fibrinogen solution at a concentration of 10mg/L was prepared with Phosphate Buffered Saline (PBS) without calcium. Soaking pure calcium and pure titanium composite film in the fibrinogen solution, maintaining at 37deg.C for 1 hr, washing with PBS and deionized water, standing, naturally drying, and planting Pseudomonas aeruginosa (10) ⁸ cells/mL) and incubated at 37 ℃ for 24 hours, dehydrated and dried, and then observed by scanning electron microscopy for bacterial growth on the surface of the material. Bacteria were seen to grow normally on pure titanium surfaces (fig. 9), fibrinogen-adsorbed pure titanium surfaces (fig. 10) and calcium-only pure titanium surfaces (fig. 11), while their growth was significantly inhibited on fibrinogen-adsorbed calcium and titanium mixed film surfaces (fig. 12). Further examination showed that this inhibition was associated with calcium in the film changing the adsorbed conformation of fibrinogen. More importantly, mammalian cells (murine mesenchymal stem cells) can grow on the surface of the material with normal adhesion (fig. 13).

Example 8

Adopts a process (vacuum degree 10) of compounding thermal evaporation (calcium source, thermal evaporation temperature 300-800 ℃) and electron beam evaporation (titanium source, working voltage 5-15 kV and current 50-150A) ^-2 Pa～10 ^-6 Pa; the deposition time is 8 min), a film (the thickness is about 100 nm) of pure calcium and pure titanium composite is deposited, and the surface calcium content can reach 10.1at.% as measured by X-ray photoelectron spectroscopy (XPS). Fibrinogen solution at a concentration of 10mg/L was prepared with Phosphate Buffered Saline (PBS) without calcium. Film of pure calcium and pure titaniumSoaking in the fibrinogen solution, maintaining at 37deg.C for 1 hr, washing with PBS and deionized water, standing, naturally drying, and culturing to obtain Pseudomonas aeruginosa (10) ⁸ cells/mL) and incubated at 37 ℃ for 24 hours, dehydrated and dried, and then observed by scanning electron microscopy for bacterial growth on the surface of the material. It can be seen that bacteria grew normally on pure titanium surfaces mixed with only calcium (fig. 14), whereas their growth was significantly inhibited on the surface of the fibrinogen-adsorbed calcium and titanium mixed film (fig. 15). Further examination showed that this inhibition was associated with calcium in the film changing the adsorbed conformation of fibrinogen.

Example 9

Adopts a process (vacuum degree 10) of compounding thermal evaporation (calcium source, thermal evaporation temperature 300-800 ℃) and electron beam evaporation (titanium source, working voltage 5-15 kV and current 50-150A) ^-2 Pa～10 ^-6 Pa; deposition time was 1.5 min), a thin film (thickness about 25 nm) of pure calcium and pure titanium was deposited, and its surface calcium content was 2.85at.% as measured by X-ray photoelectron spectroscopy (XPS). Fibrinogen solution at a concentration of 10mg/L was prepared with Phosphate Buffered Saline (PBS) without calcium. Soaking pure calcium and pure titanium composite film in the fibrinogen solution, maintaining at 37deg.C for 1 hr, washing with PBS and deionized water, standing, naturally drying, and planting Pseudomonas aeruginosa (10) ⁸ cells/mL) and incubated at 37 ℃ for 24 hours, dehydrated and dried, and then observed by scanning electron microscopy for bacterial growth on the surface of the material. It can be seen that bacteria can still grow normally on the surface of samples incubated with fibrinogen solution (fig. 16), which is associated with a low calcium content, which is not likely to cause a change in the adsorbed conformation of fibrinogen without exposing its antimicrobial fragments.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.

Claims

1. A method of preparing an implantable antimicrobial coating comprising the steps of:

s1: providing an implantable material, mixing pure calcium into the surface of the implantable material, wherein the method for mixing pure calcium into the surface of the implantable material is a vacuum coating technology, and the vacuum coating technology comprises the following steps: ion implantation or thermal evaporation combined with electron beam evaporation, forming a composite surface on the implantable material, wherein the mixing amount of calcium in the composite surface is 5-30 at%;

s2: preparing a fibrinogen solution with the concentration of 0.1-50 mg/L by adopting a phosphate buffer salt solution without calcium; and

s3: and (3) soaking the composite surface prepared in the step (S1) in the fibrinogen solution prepared in the step (S2), preserving the temperature for a certain time at 30-45 ℃, cleaning, and drying to obtain the implantable antibacterial coating.

2. The method of claim 1, wherein in step S1, the implantable material comprises: titanium, iron, zirconium, tantalum, niobium, polyetheretherketone.

3. The method according to claim 1, wherein in the step S1, the mixing amount of the calcium is 5 to 15 at%.

4. The method according to claim 1, wherein the concentration of fibrinogen in step S2 is 0.1-30 mg/L.

5. The method of claim 1, wherein the ion implantation process parameters are: vacuum degree 10 ^-2 Pa~10 ^-6 Pa; injecting voltage is 5-40 kV, and injecting time is 20-120 min; the thermal evaporation process parameters are as follows: the temperature is 300-800 ℃ and the vacuum degree is 10 ^-2 Pa~10 ^-6 Pa, the deposition time is 1-60 min; the electron beam evaporation process parameters are as follows: the voltage is 5-15 kV, the current is 50-150A, and the vacuum degree is 10 ^-2 Pa~10 ^-6 Pa, the deposition time is 1-60 min.

6. The preparation method according to claim 1, wherein in the step S3, the temperature is kept at 30-40 ℃ for 1-3 hours.

7. An implantable antimicrobial coating prepared according to the preparation method of any one of claims 1-6.

8. Use of the implantable antimicrobial coating according to claim 7 in the preparation of an implantable medical device comprising: artificial bone, artificial joint, implant, vascular stent.