CN114432489A

CN114432489A - Method for preparing polypeptide coating with long-acting antibacterial effect on metal surface

Info

Publication number: CN114432489A
Application number: CN202210115321.0A
Authority: CN
Inventors: 李贺杰
Original assignee: Sunshine Biotechnology Co ltd
Current assignee: Sunshine Biotechnology Co ltd
Priority date: 2022-01-29
Filing date: 2022-01-29
Publication date: 2022-05-06

Abstract

The invention discloses a method for preparing a polypeptide coating with a long-acting antibacterial effect on a metal surface, which comprises the following steps: (1) metal pretreatment: (a) polishing the metal; (b) ultrasonic cleaning in 80% ethanol at room temperature for 5 min; (c) ultraviolet inactivation treatment; (2) preparation of polypeptide coating: (a) mixing polycaprolactone/polyurethane particles with a chloroform solution according to the proportion of 3% W/V; (b) adding the polypeptide F3 into the solution prepared in the step (a) to form a homogeneous mixed solution; (c) and (2) immersing the metal treated in the step (1) into the homogeneous mixed solution, and ventilating for 2-4 hours to form a polypeptide coating layer on the metal surface. The magnesium-based biomaterial prepared by the method has stable and long-acting good antibacterial and anti-inflammatory functions. The coating has good biocompatibility and safety, and degradation products have no toxicity. The preparation method is simple and easy to implement, low in cost, low in energy consumption, free of environmental pollution and easy for large-scale production.

Description

Method for preparing polypeptide coating with long-acting antibacterial effect on metal surface

Technical Field

The invention relates to the manufacture of metal materials, in particular to a method for preparing a polypeptide coating with a long-acting antibacterial effect on a metal surface.

Background

With the development of human society and the increase of human activities, the damage of human bone tissues and hard tissues is more and more frequent, and thus the requirements for fixing, repairing and replacing corresponding bone tissues as well as biomaterials are more and more demanding. The traditional bone fixing and replacing materials such as titanium alloy, stainless steel and other metal materials have great difference with the elastic modulus of human bone tissues and the mechanical property of human bones, so that once the materials are implanted into a body, a plurality of problems are easy to occur: stress shielding, localized PH changes due to the release of metal ions, and thus, an intermediate infectious or inflammatory response. Thus, the biocompatibility is poor, and it is difficult to adapt to the bone healing process. The polymer material is difficult to be widely applied as a bone substitute material because of poor mechanical properties, particularly poor plasticity, toughness and radial mechanical properties.

As a typical light alloy, the magnesium alloy has almost the same elastic modulus with human bones, so the magnesium alloy is very close to the mechanical property of the human bones and is an ideal human bone substitute material. In addition, magnesium is an essential component for human metabolism and biological reaction, and magnesium has a good promoting effect on bone growth and strengthening through the combination with bone cells. Magnesium has good biocompatibility as a bone substitute material.

However, because magnesium is very active in chemical property, the magnesium and magnesium alloy transplantation material has a fast degradation rate due to the existence of various ions in the human body environment, so that the pH value of the local body fluid environment is obviously increased, thereby possibly causing alkalosis, causing local inflammatory reaction and cell death. Therefore, the control of the degradation rate of magnesium and magnesium alloy in vivo becomes a key problem for the application of magnesium alloy as bone graft material.

In addition, when the magnesium alloy is used as a substitute material in vivo and in vitro, the magnesium alloy does not have obvious bacteriostatic and anti-inflammatory effects, so that bacterial proliferation is easily caused in vivo and in vitro, and additional inflammatory reaction is caused. This further limits the wide application of magnesium alloys.

In order to solve the problem of too rapid degradation of magnesium alloys in vivo, many methods have been used to improve the corrosion resistance of magnesium, and various physical and chemical methods have been used to strengthen the surface. At present, in order to make the material have more biological activity and biocompatibility, various coatings with biological functions are generated; however, due to the defects of the magnesium alloy material, the functionality of the material surface coating is reduced, the activity is reduced, and the use of the material is influenced. And the existing biological functional coating is difficult to give consideration to the antibacterial and anti-inflammatory effects while improving the corrosion resistance of the material. Therefore, at present, a biological functional coating which can improve the corrosion resistance of the magnesium alloy and has anti-inflammatory and bacteriostatic functions is urgently needed to be prepared.

CN 111494704A discloses a method for preparing magnesium-based alloy biomaterial with small peptide coating and application thereof, wherein magnesium-based alloy is respectively subjected to ultrasonic cleaning to remove impurities on the surface of magnesium-based alloy AZ31, chloroform is used for dissolving polyurethane, then the treated magnesium-based alloy AZ31 is put into chloroform liquid in which the polyurethane is dissolved, so that the magnesium-based alloy AZ31 is completely coated in the solution, the magnesium-based alloy AZ31 is taken out, the solution is kept still until the solution on the surface is solidified, then the polyurethane coated on the surface of the magnesium-based alloy AZ31 is activated by click reaction in a plasma reactor, and finally the polyurethane coated magnesium-based alloy AZ31 with the activated surface is put into sodium phosphate solution in which the polypeptide is dissolved for oscillation, so that the two react fully to form a corresponding polypeptide coating. The invention forms a stable biological coating, improves the antibacterial property and the corrosion resistance of the magnesium alloy, has good biological activity and human compatibility of the magnesium-based alloy biomaterial with the small peptide coating, and is applied to the preparation of hard tissue defect repair materials.

The above method has problems or disadvantages in that:

(1) the selected high molecular biological material is polyurethane;

(2) the method is characterized in that a coating is generated by a chemical click method, namely a polyurethane layer coated on the metal surface is generated by plasma click reaction to obtain functional groups (such as peroxy groups, hydroxyl groups and the like) on the metal surface, so that the polyurethane layer is activated and can be combined with polypeptide, and a polypeptide coating is generated on the polyurethane surface. The polypeptides in the coating are only present on the surface of the polyurethane.

(3) The metabolic/degradation products of the chemical click reaction to form the coating (polyurethane) are carbon dioxide, water and ammonia.

(4) The effective antibacterial effect of the coating generated by the click reaction can only be maintained for about 96 hours (four days). The maximum antibacterial effect is that the effective antibacterial area is about 380mm at 24 hours²And the antibacterial effect is rapidly attenuated to 70mm in 96 hours²The following.

(5) The antibacterial effect of the coating is determined by the release process of the polypeptide; the slow release of the click reaction coating polypeptide is only that of one layer on the surface, and the slow release process is continuously and greatly attenuated due to the limited amount of the polypeptide bound on the surface. The antimicrobial time is also short. The antimicrobial effect is also continuously attenuated by a large margin.

The research relates to the technical field of metal material science, in particular to a preparation method for coating a magnesium alloy surface with a coating made of polypeptide extracted from Rana temporaria chensinensis David by a convenient physical method. Experiments prove that the magnesium-based biomaterial prepared by the method has stable and long-acting good antibacterial and anti-inflammatory functions. The coating has good biocompatibility and safety, and degradation products have no toxicity. The preparation method is simple and easy to implement, low in cost, low in energy consumption, free of environmental pollution and easy for large-scale production.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for preparing a polypeptide coating with a long-acting antibacterial effect on a metal surface. The coating has good biocompatibility and safety, and degradation products have no toxicity. The preparation method is simple and easy to implement, low in cost, low in energy consumption, free of environmental pollution and easy for large-scale production.

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

a method for preparing a polypeptide coating with a long-acting antibacterial effect on a metal surface, wherein the metal is any one of magnesium, a cold-drawn magnesium alloy and an annealed magnesium alloy, and the method comprises the following steps:

(1) metal pretreatment:

(a) polishing the metal by 400-mesh silicon carbide paper for 1-3 minutes to remove an original oxide layer, and then polishing by 800-2400-mesh silicon carbide paper for 2-5 minutes to improve the surface quality of a sample and obtain uniform roughness; rotating the metal by 90 degrees to grind and polish, and removing the scratches generated previously;

(b) respectively putting the metals into 80% ethanol, and ultrasonically cleaning for 5 minutes at room temperature;

(c) before preparing the coating, the metal is irradiated by an ultraviolet lamp for 30 minutes for inactivation treatment;

(2) preparation of polypeptide coating:

(a) selecting polycaprolactone and polyurethane as coating agents for the metal surface, mixing solid white polycaprolactone/polyurethane particles and a colorless transparent chloroform solution, wherein the purity of the chloroform solution is more than or equal to 99.5%, mixing the particles and the chloroform solution according to the proportion of 3% W/V, and oscillating and stirring at room temperature;

(b) adding the synthesized polypeptide F3 with the purity of more than 95% into the solution prepared in the step (a), and then stirring the solution at room temperature for about 1 to 3 hours by using a high-speed centrifuge until a homogeneous mixed solution is formed;

(c) immersing the metal treated in the step (1) into the sterile homogeneous mixed solution; and (3) placing the soaked metal in a fume hood for 2-4 hours to enable chloroform to be quickly volatilized, so that a uniform, compact and stable polyurethane/polycaprolactone coating layer is formed on the surface of the metal.

The further preferred technical scheme is as follows:

the duration of the irradiation was 30 minutes.

The method has the advantages that:

(1) the invention generates the coating by a physical combination method, and the used high molecular biological material is mainly polycaprolactone.

(2) The polypeptide coating generated by the physical mixing method is uniformly distributed in the whole high polymer material layer (mainly polycaprolactone), from the surface to the inside and the bottom.

(3) The metabolic/degradation products that physically combine to form the coating are carbon dioxide and water. Compared with the two, the physical bonding coating is more biocompatible and safer.

(4) The physical bond coating is significantly greater in either antimicrobial time and antimicrobial effect due to the click reaction coating. The maximum antibacterial effect of the physical bonding coating is 24 hours, and the effective antibacterial area of both the magnesium alloy and the titanium alloy after coating is larger than 550mm². The effective antibacterial time of the physical bonding coating is far longer than that of a click reaction coating: wherein the antibacterial effect of the coated titanium alloy can last up to 7 days (fig. 8A); the antibacterial time of the coated magnesium alloy also reaches 6 days.

(5) The antimicrobial effect of the coating is determined by the release process of the polypeptide. The polypeptide is physically combined with the coating, the slow release of the polypeptide is carried out from top to bottom, and as the polypeptide in the upper layer is released, the polypeptide in the adjacent layer is supplemented, so that supplementary slow release according to a polypeptide concentration gradient is formed. The slow release of the polypeptide in the physical bond coating is gradual and the antimicrobial effect is gradually reduced. So that the duration of the antibacterial effect is longer and the effect is better. The titanium alloy after being coated has a stable antibacterial effect area from 72 hours to 144 hours. Furthermore, the physical binding coating may contain a substantially higher amount of polypeptide than the click reaction coating. The effective antibacterial time of the physically combined patent is improved by nearly 80% compared with that of the click-to-respond patent, and reaches about 7 days, so that the acute infection phase and inflammatory phase reactions of nearly one week after graft transplantation can be effectively inhibited. In addition, the maximum antibacterial effect is improved by nearly 50 percent. Therefore, compared with the prior art, the coating prepared by the physical combination method has faster and better application prospect.

(6) The physical combination method is simple to generate the coating, is convenient to operate, has less pollution to the environment and lower cost, and is easier for large-scale batch production.

Drawings

FIG. 1 is a microstructure of a metal of the present invention, wherein A is a pure magnesium metal; FIG. B shows cold drawn metal AZ 31; panel C anneals the metal AZ 31.

FIG. 2 is a structural diagram of polycaprolactone.

FIG. 3 is a schematic representation of the polypeptide + polycaprolactone/polyurethane coating formed by the physical method of the present invention.

FIG. 4 is an analysis of the antimicrobial effect of different metals coated with the same coating, wherein panel A is a PC + F3 coating; panel B is a PU + F3 coating.

FIG. 5 is an analysis of the antimicrobial effect of the same metal coated with different coatings, wherein panel A is cold drawn AZ 31; panel B shows pure magnesium; panel C is annealed AZ 31.

FIG. 6 is a SEM and EDS image, wherein the A is PC + F3 coating; FIG. B is a PU + F3 coating; panel C is EDS analysis of PC + F3 coatings of different metals; panel D is EDS analysis of PU + F3 coatings of different metals.

FIG. 7 is a schematic comparison and analysis of click reaction to polypeptide coating and physical mixing to polypeptide coating.

Fig. 8 is a graph showing the comparison of the in vitro antibacterial effect of the polypeptide coating produced by the click reaction and the polypeptide coating produced by the physical mixing and the analysis of the polypeptide sustained-release model.

FIG. 9 is a graph showing the in vivo antibacterial effect test.

FIG. 10 is a graph showing the results of in vitro antibacterial experiments after the magnesium alloy coated with the physical mixed coating is inhibited for 72 hours in vivo and tissues at the transplanted site are ground and diluted.

Detailed Description

The present invention will be further described with reference to the following examples.

Referring to fig. 1 to 3, the method for preparing a polypeptide coating with a long-lasting antibacterial effect on a metal surface according to the present invention, wherein the metal is any one of magnesium, a cold-drawn magnesium alloy and an annealed magnesium alloy, comprises the following steps:

(1) metal pretreatment:

(2) preparation of polypeptide coating:

The duration of the irradiation was 30 minutes.

Experiments and Performance tests

Preparation of Metal samples

Metal samples, metals including Pure Magnesium (PM), Cold drawn magnesium alloy AZ31(Cold extruded AZ31, CE AZ31), and annealed magnesium alloy AZ31(full annealed AZ31, FAAZ 31). All three magnesium alloys are rods with a diameter of 5.0 mm. The FA AZ31 sample was subjected to a complete recrystallization annealing heat treatment. They were heated to 330 ℃ and 350 ℃ in an inert gas atmosphere (argon), held for 3-5 hours, and then cooled in a furnace. All samples were processed into small disks (5 mm in diameter) having a thickness of 2 mm. The average grain sizes of the three magnesium alloy samples were: the average grain size of pure magnesium was 22 μm, the average grain size of the cold drawn AZ31 alloy was 18.2 μm, and the average grain size of the annealed AZ31 alloy was 15.9 μm. The hardness test results are as follows: the brinell hardness of the cold drawn AZ31 was 79 HV; the brinell hardness of annealed AZ31 was 67.1; whereas the brinell hardness of pure magnesium is 42.8 HV.

The test piece sample is polished by 400-mesh silicon carbide paper for 1-3 minutes to remove the original oxide layer, and then polished by 800-2400-mesh silicon carbide paper for 2-5 minutes to improve the surface quality of the sample and obtain uniform roughness. All metal samples were rotated 90 degrees to ensure that the next grinding and polishing step removed the previously created scratches. Then, the metal samples were respectively put into 80% ethanol for 5 minutes of ultrasonic cleaning at room temperature. Prior to the preparation of the coating, all sample materials were subjected to an inactivation treatment: the mixture was irradiated with an ultraviolet lamp for 30 minutes.

(II) preparation of the coating

(a) Selecting polycaprolactone and Selectophore^TMThe produced polyurethane (grade MQ100) is used as a coating agent for a metal surface, and solid white polycaprolactone/polyurethane particles are firstly mixed with colorless transparent chloroform solution (the purity is more than or equal to 99.5 percent) according to the proportion of 3 percent (W/V) and stirred under oscillation at room temperature.

(b) The synthesized high purity (> 95%) polypeptide F3 was then added to the well stirred solution of chloroform poly and caprolactone/polyurethane and stirred with a high speed centrifuge at room temperature for about 1-3 hours until a homogenous mixed solution was formed.

(c) Three magnesium-based alloy samples (short cylindrical, 5mm in diameter and 2mm in thickness) subjected to ultrasonic surface treatment are placed into a glass vessel subjected to aseptic treatment.

(d) Three metal samples are immersed in 200 mu L of polypeptide + chloroform + polyurethane/polycaprolactone solution, three magnesium alloy materials and glassware which are uniformly coated with polyurethane/polycaprolactone are placed in a fume hood for 2-4 hours, and chloroform is rapidly volatilized, so that a uniform, compact and stable polyurethane/polycaprolactone coating layer is formed on the metal surface.

(III) bacteriostatic comparison experiment

The bacteriostatic comparison experiment specifically operates as follows:

(a) samples of the bacteriostasis test are prepared into small-size magnesium sheets with the diameter of 5mm and the thickness of 2mm, and the dimensional tolerance is 0.002 mm;

(b) the original magnesium alloy AZ31(CE AZ31, FAAZ31, PM) in three different processing states is respectively subjected to a physical method to form a polypeptide coating of polyurethane + F3 and polycaprolactone + F3 with polyurethane and polycaprolactone. And (3) carrying out surface cleaning on three magnesium alloy samples coated with the coatings: cleaning with purified water, and cleaning in an ultrasonic cleaner for 3-5 min to remove impurities and adhesion on the surface;

(c) and putting the cleaned various samples into a bacteriostasis experiment dish, and carrying out bacteriostasis test on drug-resistant staphylococcus aureus for 168 hours in a constant-temperature incubator at 37 ℃.

(d) Staphylococcus aureus (MRSA, GDM1.1263) with methicillin resistance was collected in log phase and MH medium was used to adjust the suspension concentration to 2.0X 105 CFU/ml. And (4) dipping bacteria liquid by using a sterile cotton swab, and rotating and extruding the tube wall for several times to remove excessive bacteria liquid. The whole M-H drug sensitive agar plate (Guangzhou teleomo) was spread evenly with swabs. 20ug of F3 polypeptide was added to each drug sensitive paper (OXOID, USA), and the paper was applied to M-H agar plates, which were inverted, incubated at 37 deg.C, and incubated overnight. 20ug piperacillin sodium tazobactam sodium (Tazocin) was used as a control with a blank drug sensitive tablet (BASD) and the original AZ31 magnesium alloy in both states. And measuring the size of the bacteriostatic zone by using a vernier caliper.

The results are shown in the following table:

TABLE I in vitro bacteriostasis test result of physically combined polypeptide coating (diameter of bacteriostasis zone)

In order to more effectively compare the antibacterial performance of different samples, the quantity of the effective inhibition area of the inhibition zone is introduced to measure the size of the antibacterial capability. The specific calculation can be referred to the following formula:

Ae＝At-As (1)

wherein, Ae is the effective area of the bacteriostatic ring, and At and As are the total area and the original area of the sample respectively. At and As can be obtained by the following formulas:

As＝πrs² (2)

At＝πrt² (3)

wherein rs is the original diameter of the sample, and rt is the total diameter (i.e. the average value of the results measured in the experiment) including the effective area of the antibacterial ring and the original area of the sample.

As can be seen from the part A and the table I in the graph of FIG. 4, for the coating of the polycaprolactone PC + F3 polypeptide, the annealed alloy AZ31 and pure magnesium coated with the coating have the best in vitro antibacterial effect, and the antibacterial effect can last for 6-7 days (between 144-168 hours); the antibacterial abilities of the magnesium alloy and the magnesium alloy are not much different, and the annealed magnesium alloy is better. The in vitro antibacterial effect of the cold-drawn AZ31 coated with the same coating is only maintained for 4-5 days (between 96-120 hours). And the antibacterial ability is obviously lower than the former two. As can be seen from the graph B and table one in fig. 4, for the polyurethane PU + F3 polypeptide coating, pure magnesium coated with the coating has a more significant antibacterial effect (this is slightly different from the previous research results, and the analysis reason is that there are more coated coatings). And the annealed magnesium alloy AZ31 and the cold-drawn magnesium alloy AZ31 have similar antibacterial effect after being coated with the PU + F3 coating. The antibacterial effect of the three metal materials coated with the PU + F3 coating can be maintained for 96-120 hours, about 4-5 days.

Fig. 5 compares the difference in antibacterial effect when the same metal is coated with different coatings: the effective antimicrobial time of the PC + F3 coatings of the two remaining metals (pure magnesium and annealed AZ31) was extended by about 2 days (48 hours) compared to PU + F3, except for cold drawn AZ 31. Moreover, compared with PU + F3, the antibacterial ability of the PC + F3 coating is obviously improved for the three metals, and the annealing AZ31 is most remarkable: the effective bacteriostatic area is increased by nearly one time from 300mm2 of PU + F3 to 560mm2 of PC + F3.

In conclusion, the PC + F3 coating and the PU + F3 coating of the three metals have long-term antibacterial capability. The antimicrobial capacity and antimicrobial time of the PC + F3 coating were better compared to the PU + F3 coating.

(IV) scanning electron microscope SEM and energy spectrum analysis EDS

The surface morphology and elemental composition of two coatings coated with three metallic materials were analyzed using energy dispersive X-ray spectroscopy (EDS) in combination with a scanning electron microscope JEOL 6010 SEM.

As can be seen from the graphs A and B in FIG. 6, the surface of the PC + F3 coating is full of obvious granular structures, and the surface undulation is obvious; the PU + F3 coating surface is relatively flat and has less fluctuation. As seen from the results shown in fig. 6, the wavy surface dense structure increases the contact area between the polypeptide and the surface, and significantly increases the amount of the polypeptide attached to the surface, thereby significantly improving the antibacterial ability of the material.

In addition, the fluctuated structure (large-scale cavity relative to the polypeptide, which is trapped by the polypeptide) can enable the polypeptide to be bound in certain areas to a certain extent, so that the polypeptide presents a structural gradient slow release when in contact with bacteria, thereby prolonging the antibacterial effect.

In addition, as seen from fig. C and D in fig. 6, when the two coatings coat three different magnesium alloy materials, there is no significant difference in the composition of the surfaces. It can be seen that the PU + F3 and PC + F3 coatings were uniform for all three materials. The method of preparing the coating by physical bonding is stable and feasible without causing significant fluctuations in the composition of the coating.

In conclusion, coatings prepared using this physical method are viable and effective. Especially, the antibacterial effect of the coating of PC + F3 is more obvious and has longer duration, and meanwhile, the degradation products are only carbon dioxide and water which are harmless to human bodies and can be absorbed.

Referring to fig. 7, a comparison and analysis of the principle of generating a polypeptide coating by a click reaction with the principle of generating a polypeptide coating by physical mixing is shown, wherein the click reaction method is shown as a principle of generating a coating; FIG. B illustrates the principle of the physical bonding method to produce a coating.

The coating is generated by a chemical click method, namely a polyurethane layer coated on the metal surface is generated by a plasma click reaction to obtain functional groups (such as peroxy groups, hydroxyl groups and the like) on the metal surface, so that the polyurethane layer is activated and can be combined with polypeptide, and a polypeptide coating is generated on the polyurethane surface. The polypeptides in the coating are only present on the surface of the polyurethane.

The polypeptide coating generated by a purposefully designed specific physical mixing method is uniformly distributed in the whole high polymer material layer polycaprolactone from the surface to the inside and the bottom.

Referring to fig. 8, a graph for comparing the in vitro antibacterial effect of the polypeptide coating generated by the click reaction and the polypeptide coating generated by the physical mixing and analyzing the polypeptide sustained-release model is shown.

Comparing the in vitro antibacterial effect of the polypeptide coating generated by the click reaction and the polypeptide coating generated by physical mixing and analyzing a polypeptide sustained-release model: FIG. A is an in vitro antibacterial experiment of polycaprolactone generated by a physical method and titanium alloy coated by a F3 polypeptide coating; FIG. B is an in vitro antibacterial experiment of a magnesium alloy coated with polycaprolactone and a F3 polypeptide coating and generated by a physical method; FIG. C is an in vitro antibacterial experiment of polyurethane generated by a click method and magnesium alloy coated with F3 polypeptide coating; FIG. D shows the polypeptide release process by physical binding to form a coating; figure E click-through should generate a polypeptide slow release process for the coating.

The antimicrobial effect and duration of the coatings produced by the two methods are very different: the effective antibacterial effect of the coating generated by the click reaction can only be maintained for about 96 hours (four days), as shown in the graph C. The maximum antibacterial effect is that the effective antibacterial area is about 380mm at 24 hours²And the antibacterial effect is rapidly attenuated to 70mm in 96 hours²The following. The physical bonding coating is obviously greatly due to the click reaction coating in the antibacterial time and the antibacterial effect. The maximum antibacterial effect of the physical bonding coating is 24 hours, and the effective antibacterial area of both the magnesium alloy and the titanium alloy is larger than 550mm²As shown in fig. A, C. The effective antibacterial time of the physical bonding coating is far longer than that of a click reaction coating: wherein the antibacterial effect of the coated titanium alloy can last for 7 days (fig. a); the antibacterial time of the coated magnesium alloy also reaches 6 days (figure C).

Furthermore, the antimicrobial effect of the coating is determined by the release process of the polypeptide. However, the polypeptide release process of the two coatings is distinct. The polypeptide is released from the upper layer, and the polypeptide in the adjacent layer is supplemented, so that the supplementary slow release according to the polypeptide concentration gradient is formed. The slow release of the polypeptide in the physical bond coating is gradual, and thus the antibacterial effect is gradually attenuated. So that the duration of the antibacterial effect is longer and the effect is better. The titanium alloy after being coated has a stable antibacterial effect zone from 72 hours to 144 hours. Furthermore, the physical binding coating may contain a substantially higher amount of polypeptide than the click reaction coating.

The slow release of the click reaction coating polypeptide is only that of one layer on the surface, and the slow release process is continuously and greatly attenuated due to the limited amount of the polypeptide bound on the surface. The antimicrobial time is also short. The antimicrobial effect is also continuously attenuated by a large margin.

In conclusion, the effective antibacterial time of the physically combined patent is improved by nearly 80% compared with that of the click-to-answer patent, and reaches about 7 days, so that the acute infection phase and inflammatory phase reactions of nearly one week after graft transplantation can be effectively inhibited. In addition, the maximum antibacterial effect is improved by nearly 50 percent. Therefore, compared with the prior art, the coating prepared by the physical combination method has faster and better application prospect.

FIG. 9 is a graph showing in vivo antibacterial effect tests.

The click reaction coating is not transplanted in a mouse body, and in-vivo antibacterial experimental research is carried out. And the physical bonding coating is transplanted into the body, and in-vivo observation and antibacterial experimental analysis are carried out. The specific operation is that the magnesium alloy coated with polycaprolactone and F3 polypeptide is kept around the tibia of SD rat for 72 hours, and 106CFU of staphylococcus aureus resistant bacteria are dripped into the magnesium alloy during the transplantation process. Tissues around the graft were then ground and diluted by different fold: 10, 100, 103, 104, 105, 106. Then the tissues diluted to different times are placed in a bacterial culture dish, the same amount of staphylococcus aureus resistant bacteria is dripped in the tissues, and the tissues are cultured for 24 hours at room temperature and observed.

By analyzing fig. 9, it can be seen that no significant abnormal change or significant bubble generation was observed in the tissue surrounding the graft at 72 hours in the graft, indicating that the 7 groups of materials were stable, no corrosion occurred, and no inflammatory reaction occurred during this procedure.

FIG. 10 is a graph showing the results of in vitro antibacterial experiments after grinding and diluting tissues at a transplanted site 72 hours after the magnesium alloy is coated by the physical mixed coating: a control group; B1E; C2P; D3A; E1E + PC + F3; F2P + PC + F3; G3A + PC + F3.

The result shows that when 72 hours in vivo inhibition is carried out, the metal coated with the polycaprolactone and the F3 polypeptide coating is extracted from surrounding tissues after 24 hours for observation, and the antibacterial effect is obvious. Comparison of the three pure metals as coating shows that the tissue from the annealed AZ31 (3A) graft site has better antibacterial effect than the tissue surrounding the other two gold species. Similarly, the tissue surrounding 3A coated with polycaprolactone and F3 polypeptide by physical means also had better antibacterial effect. This indicates that the polypeptides in the coating enter the body tissue surrounding the graft 72 hours after implantation, thereby providing the tissue with a significant antimicrobial effect. Therefore, the metal coated with the physical combination coating still has antibacterial effect for 72 hours after being transplanted into a body. This directly demonstrates that the physical bonding process produces the in vivo antimicrobial effect of the coating.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a polypeptide coating with a long-acting antibacterial effect on a metal surface, wherein the metal is any one of magnesium, a cold-drawn magnesium alloy and an annealed magnesium alloy, and the method is characterized by comprising the following steps of:

(1) metal pretreatment:

(2) preparation of polypeptide coating:

2. The method for preparing a polypeptide coating with long-acting antibacterial effect on a metal surface according to claim 1, wherein the method comprises the following steps: the duration of the irradiation was 30 minutes.