Layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and application thereof

Abstract

The invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and application thereof, and relates to the technical field of medical equipment. According to the composite antibacterial stainless steel coating, medical 316L stainless steel is used as a substrate material, a dopamine solution is used as a raw material, Tris salt is added to initiate a polymerization reaction of dopamine, and a polydopamine film is formed on the surface of the stainless steel; the preparation method comprises the following steps: carrying out Michael addition reaction on an easily oxidized catechol group and lysozyme containing amino and sulfydryl in an aerobic environment to form stable covalent linkage, thereby preparing the lysozyme-loaded polydopamine composite membrane. In the invention, the antibacterial composite coating aims to effectively kill biofilm embedding bacteria and prevent bacterial adhesion and biofilm formation on the surface of an implant under the condition of not hurting an organism, so that the antibacterial property of the implantable stent is improved, and the problem of clinical postoperative explosive infection of the implant is solved; and more thoughts and means and beneficial exploration are provided for treatment of chronic refractory inflammation caused by biological membranes.

Description

Technical Field

[0001] The invention relates to the technical field of medical equipment, in particular to a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and application thereof. Background Art

[0002] In recent years, explosive infection after implant surgery has been a problem that has plagued clinical practice, and there are few implants with antibacterial coatings on the market. Secondary revision surgery to deal with postoperative infection puts patients under tremendous financial pressure and provides a poor treatment experience.

[0003] Faced with the dilemma of increasing clinical threats posed by drug-resistant bacteria, designing various types of antibacterial medical materials is an urgent problem that needs to be solved. In the process of modifying ordinary medical materials, the corrosion resistance, antibacterial properties, material hardness and other properties of the materials can be greatly improved. Although coatings have great potential to improve the performance of medical devices, the ideal coating design is still subject to various conditions, such as drug release kinetics, induced antibiotic resistance, organ toxicity, wear resistance, stability between coating layers, etc. A single coating is difficult to meet the needs of multiple coating designs, and cleverly constructing a coating model is a problem that current researchers need to solve.

[0004] At present, the emergence of bacterial resistance to vancomycin makes the treatment of drug-resistant bacteria more difficult. Bacterial colonies with biofilms will show stronger drug tolerance. Although gallium ions have significant antibacterial properties, they are still limited in their practical application in antibacterial and anti-inflammatory applications. The first is the toxicity problem that is common in metal ion antibacterial drugs. Gallium at high concentrations has a strong bactericidal effect on bacteria, but it also poses a threat to mammalian cells. Second, due to the hydrolysis of gallium ions themselves, Ga 3+ It is easily hydrolyzed in solution to form Ga(OH)3 precipitate, which greatly weakens the antibacterial effect of gallium itself and reduces its bioavailability.

[0005] Therefore, how to better apply gallium ions to antibacterial coatings and develop new antibacterial implants, requiring them to have better biocompatibility, stronger biofilm dispersion, and more suitable physical and chemical properties, is a very urgent issue in the field of medical science. The relevant research on biofilm inhibition is in its infancy. How to modify the surface of implants to make them antibacterial so that they can continuously release antibacterial substances in the body and ensure that they can exert efficient inhibition on bacteria, so that antibacterial stent materials can meet clinical applications, is a problem that must be considered.

[0006] Therefore, those skilled in the art provide a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and its application to solve the problems raised in the above background technology. Summary of the invention

[0007] (1) Technical problem to be solved

[0008] In view of the deficiencies of the prior art, the present invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and its application, constructs a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating, which continuously releases antibacterial metal ions in the body and inhibits bacteria along with lysozyme, can play an efficient inhibitory effect on bacteria at the implant site, effectively kill biofilm-embedded bacteria without harming the body, prevent bacteria adhesion and biofilm formation on the implant surface, improve the antibacterial performance of the implantable stent, solve the problem that explosive infections are prone to occur after implantation of implants, which has troubled the clinic, and provide more ideas, means and beneficial explorations for the treatment of chronic refractory inflammation caused by biofilms.

[0009] (2) Technical solution

[0010] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0011] A layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating uses medical 316L stainless steel as the base material, uses dopamine solution as the raw material, adds Tris salt to initiate the polymerization reaction of dopamine, and forms a polydopamine film on the stainless steel surface;

[0012] The easily oxidized catechol groups undergo a Michael addition reaction with lysozyme containing amino and mercapto groups in an aerobic environment to form a stable covalent bond, and a polydopamine composite film loaded with lysozyme is prepared;

[0013] Then, using the characteristic that metal ion gallium chelates with phenolic hydroxyl groups on the surface of polydopamine, it is uniformly covered on the surface of the polydopamine film by the immersion method, a composite antibacterial coating implant is prepared, and its formulation and preparation process are optimized.

[0014] Furthermore, the characterization analysis method of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating includes the following methods:

[0015] 1) Use X-ray photoelectron spectroscopy to analyze its chemical composition. The anode target of the XPS spectrometer used is magnesium, the charge potential is calibrated as the C1 s peak (284.8 eV), under the monochromatic Al Ka (1486.6 eV) light source, the vacuum degree is 12 Kv × 15 mA, and it works under the condition of 2 × 10 -7 Pa, adopt a scanning energy band of 0 - 1400 eV and a take-off angle of 30°. The front surface needs to be cleaned by Ar + ion sputtering, and then the surface of the GLDS coating is measured to determine the connection of lysozyme and the model construction of the coating;

[0016] 2) Analyze its surface elements using energy-dispersive X-ray spectroscopy, specifically analyze the distribution of C, N, S, O, and Ga elements on the coating surface;

[0017] 3) Observe its surface morphology using a FE-SEM field emission scanning electron microscope, and use a Gemini SEM 500 scanning electron microscope to characterize the surface morphology at different stages during the preparation of the antibacterial stainless steel. Its working voltage is 5 kV, and the magnification multiples are 2000 times and 20000 times respectively;

[0018] 4) Detect its surface morphology and roughness using an atomic force microscope. Use the tapping mode with a Si cantilever beam to measure two samples, GDS and GLDS, analyze the changes in their surface morphology. The test conditions are room temperature, air phase, the sampling frequency is 1 Hz, select a single-crystal silicon tip, the probe elastic coefficient is 40 N / m, and randomly select a 5×5 μm 2 area on the same sample surface for sampling 3 times, and calculate the average value of the surface roughness of the coating;

[0019] 5) Conduct contact angle analysis of the composite antibacterial coating implant. To evaluate the surface wettability of the specimen, randomly select 5 different positions on the specimen surface and drop 3 μL of deionized water respectively. Use a contact angle measuring instrument to obtain the average contact angle and contact angle hysteresis of the specimen. Contact angle hysteresis is the difference between the contact advancing angle and the contact receding angle. All samples in the experiment are set with 3 groups of replicate samples.

[0020] Furthermore, the characterization and analysis method of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating also includes the following methods:

[0021] Conduct a hemolysis experiment to evaluate the blood compatibility of the coating material using a hemolysis experiment. The process is as follows:

[0022] Draw 10 mL of whole blood from a healthy volunteer, add it to an anticoagulant tube, and conduct a coagulation test. Dilute it with 10 mL of physiological saline for every 8 mL of anticoagulated whole blood for standby;

[0023] Cut the sample into pieces of 2.5×1 cm 2 each section, rinse it several times with distilled water, soak it in physiological saline for 24 hours, pour out the soaking solution, add 10 mL of physiological saline again, then add 0.2 mL of diluted human blood, and vertically soak it in a roller device for 60 min (15 rpm / min);

[0024] Take out the coating material, centrifuge it at 2000 rpm / min for 10 min, suck out the supernatant and transfer it to a cuvette, and measure the absorbance at a wavelength of 545 nm using a UV spectrophotometer. The positive control uses 316L stainless steel plus double-distilled water, and the negative control uses 316L stainless steel plus 0.9% physiological saline;

[0025] The calculation formula for hemolysis degree is as follows:

[0026]

[0027] According to the national standard, a material hemolysis rate < 5% indicates that the material meets the hemolysis test requirements for medical materials;

[0028] The study on the release of gallium ions from the GLDS coating was carried out as follows:

[0029] Accurately cut a GLDS gallium-containing stainless steel sample with a size of 4×2 cm 2 and place it in a 15 mL centrifuge tube. Add 10 mL of PBS (pH = 7.4) release medium, and perform low-vacuum degassing to fully immerse the material in the release medium. Place it in a constant-temperature shaker at 37°C and 50 rpm for uniform oscillation;

[0030] At 3 h, 8 h, 18 h, 24 h, 48 h, and 72 h, take 500 μL of the supernatant for detecting the gallium ion concentration, then supplement 500 μL of PBS release medium, and continue the constant-temperature uniform oscillation. Use an atomic absorption spectrometer to measure the gallium ion concentration, and set three parallel controls for the samples;

[0031] Evaporate 500 μL of the solution sample to dryness and place it in the digestion inner tank. Add 3 ml of nitric acid solution, heat it in an oven at 100°C for 1 h and at 140°C for 5 h;

[0032] After digestion is completed, take it out after cooling, transfer the digestion tank into the fume hood, slowly open the tank, heat it in a water bath to volatilize the acid, rinse the inner cover with a small amount of ultrapure water, and wait until it is volatilized to 1 ml, then take it out and cool it to room temperature;

[0033] Transfer the digestion solution to a 10 ml volumetric flask, make up the volume to the mark with ultrapure water, and conduct a reagent blank experiment at the same time. Then, measure the absorption peak area of gallium according to the working conditions of the analytical instrument respectively, and calculate the gallium content in the sample based on the standard curve and dilution factor.

[0034] Furthermore, the characterization and analysis method for the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating also includes the following methods:

[0035] The production of the standard curve is carried out as follows:

[0036] Dilute the standard solution of gallium element (1000 μg / mL) with 2% HNO3 to a gallium standard stock solution with a concentration of 1.0 μg / mL;

[0037] Appropriately and precisely pipette from the Ga standard stock solution (1.0 μg / mL), and use 2% HNO₃ solution as the solvent to prepare gallium solutions of 10, 20, 40, 60, 80, 100, 120, and 200 μg / L. At the same time, prepare a blank solution in the same way;

[0038] Precisely pipette 200 μL of each standard solution, automatically inject 20 μL of the standard solution into the graphite furnace atomizer, use the measured absorbance value as the ordinate, and the concentration as the abscissa to make a standard curve;

[0039] Then use the AAS method to determine the linear range, regression equation, and correlation coefficient of the standard solution of the Ga metal element.

[0040] Furthermore, for the method of evaluating the in vitro antibacterial performance of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating, the antibacterial performance of the antibacterial stainless steel is measured by the plate culture medium method. Pre-culture Staphylococcus aureus and Escherichia coli, incubate the antibacterial coating sample with them, measure the distance between the colony and the edge of the sample, and detect its in vitro antibacterial performance. The specific process is as follows:

[0041] S1. Solution preparation and equipment sterilization

[0042] Take 1.25 g of LB broth in a 50 mL conical flask, add 50 mL of ultrapure water to dissolve it, and tightly stopper the bottle mouth with gauze and wrap it with tin foil;

[0043] Take 9.4 g of PCA plate count agar in a 500 mL conical flask, add 500 mL of ultrapure water and shake well. Similarly, tightly stopper the bottle mouth with gauze and wrap it with tin foil;

[0044] Dissolve the solid powder of PBS buffer in 2 L of ultrapure water to prepare a PBS buffer solution with a pH of 7.4;

[0045] Place several 5 mL centrifuge tubes, several 90 mm petri dishes, the above-mentioned LB broth, PCA solution, and 500 mL of PBS buffer solution in a high-pressure steam sterilizer, and set the parameters to sterilize at 121 °C for 20 min;

[0046] S2. Culture medium preparation and bacterial proliferation

[0047] Pour the sterilized PCA solution into the petri dish while it is hot and spread it flat in the purification workbench. Each petri dish contains about 16 - 17 mL of PCA, and let it cool before inverting it in the ultra-clean bench;

[0048] Use a pipette to take 20 μL of the original Escherichia coli solution or Staphylococcus aureus solution and add it to the LB broth, gently shake it, and culture it at 37 °C for 12 h;

[0049] S3. Bacterial pretreatment

[0050] The successfully proliferated LB broth of Escherichia coli was aliquoted into several centrifuge tubes and centrifuged at 5000 rpm for 3 min to deposit the bacteria at the bottom of the centrifuge tubes. The supernatant was discarded, and PBS buffer was added to resuspend the bacteria. The absorbance was measured at a wavelength of 600 nm, and after appropriate dilution, the absorbance value was adjusted to about 0.15 to obtain a bacterial suspension with an appropriate concentration.

[0051] Furthermore, the experimental process for measuring the antibacterial performance of the antibacterial stainless steel by the plate culture medium method is as follows:

[0052] S1. The agar medium was melted and cooled to 45 °C. 10 ml of freshly cultured bacterial suspension within 24 hours was measured and then inoculated into 150 mL of agar.

[0053] S2. 15 ml of the inoculated agar was poured into a petri dish with a diameter of 100 mm. After standing for 15 minutes, the sample was placed on it.

[0054] S3. The sample was gently pressed with sterile forceps to ensure full contact between the sample and the inoculated agar. The petri dish was placed in an incubator at 37 °C for 24 hours, and the width of the inhibition zone was measured.

[0055] S4. First, the bacteria were pre-cultured and diluted to 2×10 7 cfu·mL -1 with phosphate buffer, and 20 μL of the diluted bacterial suspension was evenly spread on the agar plate.

[0056] S5. Finally, the sample with the antibacterial coating containing gallium, lysozyme, etc. was placed face up on the agar plate and co-incubated at 37 °C for 24 hours. Each sample was tested at least three times.

[0057] S6. The distance between the colony and the edge of the sample was measured from the back of the petri dish and calculated according to the following formula:

[0058]

[0059] where: W—the width of the inhibition zone, unit: mm;

[0060] T—the total diameter (total width) of the inhibition zone of the sample, unit: mm;

[0061] D—the diameter (width) of the sample, unit: mm.

[0062] Furthermore, the application of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating in the study of antibacterial effect and biocompatibility in vivo includes the following process:

[0063] 1) Prepare a mixed bacterial solution of the standard strain of Staphylococcus aureus ATCC35923 and the standard strain of Escherichia coli ATCC25922 in a ratio of 1:1.

[0064] 2) Establish a rat spinal infection model. Implant the designed and prepared composite antibacterial stainless steel coating into the back of SD rats. Evaluate and observe the inhibitory effect of GLDS on the mixed infection of Staphylococcus aureus and Escherichia coli by using the changes in rat body weight, the healing of incisions, the severity and scope of deep tissue inflammatory reactions, and the infection conditions of the tissues under the wound as indicators.

[0065] Furthermore, the experimental process of the application of the composite antibacterial stainless steel coating in the in vivo antibacterial effect and biocompatibility research is as follows:

[0066] Step 1. Take 30 male SD rats weighing about 200 g and randomly divide them into 5 groups, namely A, B, C, D, and E, with 6 rats in each group according to the random number table method.

[0067] Step 2. Anesthetize the rats by inhaling isoflurane. After the rats are anesthetized, continue to maintain anesthesia with isoflurane at a concentration of 1 - 2.5%. Shave the back hair of the rats, disinfect routinely, locate the body surface according to the spinous processes, make a posterior median incision, and make an incision about 2 cm long between L4 - L6. Cut through the skin and subcutaneous tissues layer by layer, and bluntly separate the erector spinae muscles along both sides of the spinous processes close to the bone surface until the bilateral laminae are reached.

[0068] Step 3. Place the sterilized bare steel, polydopamine-coated stainless steel, gallium nitrate-coated stainless steel, lysozyme-coated stainless steel, and gallium nitrate-lysozyme co-loaded stainless steel coatings with a size of 2 cm 2 between the erector spinae muscles and the spinous processes of the rats. Place a piece of the same type of metal material on each side of the spinous process of each rat, and drop 25 μL of the Staphylococcus aureus - Escherichia coli mixed bacterial solution (bacterial amount: 1×107 CFU / mL) on both sides. Suture the muscles tightly and close the incision layer by layer.

[0069] Step 4. After the rats wake up from anesthesia, observe that the rats are active, in good spirits, and have normal diet. Then put them in the SPF animal room for feeding and observation. Observe the mental state and activity of the rats on the day, 3 days, 7 days, and 14 days after the injury, and measure their body weights.

[0070] Step 5. Sacrifice the rats by cervical dislocation at 7 days and 14 days after the operation. Observe the healing of the incisions and the severity of deep tissue inflammatory reactions, measure the scope of the inflammatory reactions, and evaluate them according to the surgical incision healing grades.

[0071] Step 6. Cut open the rat wounds along the original incisions, observe the infection conditions of the tissues under the wounds and record them. Take out the metal sheets placed in the body, gently soak and rinse them with physiological saline to remove the tissues adhered to the metal surfaces and the non-adhered bacteria. Put the five groups of metal sheets into 24-well plates for standby.

[0072] Step 7. Strictly configure the live / dead bacterial fluorescent stain according to the instructions of the Live / Dead LIVE / DEAD BacLight Bacterial Viability Kits (L13152). Take two tubes of fluorescent dyes respectively, dissolve them in 10 mL of distilled water, mix well thoroughly, store at 4 °C, and use immediately after preparation;

[0073] Step 8. Add 400 μL of fluorescent stain solution to the 24-well plate containing five groups of metal sheets, stain at room temperature for 15 minutes, then gently rinse in 2.5 mL of physiological saline to remove the excess fluorescent stain solution, and then place a glass slide on it and observe under a laser confocal microscope;

[0074] Step 9. Observe whether there is pus, the degree and scope of inflammatory tissue and necrotic tissue in the taken pathological tissue, and investigate the pathological changes of the muscle tissue in contact with the metal surface of each group of rats by H&E staining method;

[0075] Step 10. Randomly sacrifice the animals on the 7th and 14th days after the operation, 3 animals in each group, and perform autopsy on the five groups of animals A, B, C, D, and E. Collect the main organs of the heart, liver, spleen, lung, and kidney in each group and fix them in 4% paraformaldehyde, and perform histopathological examination.

[0076] (III) Beneficial effects

[0077] The present invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and its application. It has the following beneficial effects:

[0078] 1. The present invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and its application. Lysozyme and gallium ions are co-loaded onto the polydopamine coating. Dopamine can spontaneously polymerize on the surface of the stainless steel material to form a thin polydopamine film. When the catechol groups in PDA are in the active quinone structure, they can undergo Michael addition reactions with the amino and sulfhydryl groups of lysozyme. At the same time, the catechol groups also act as chelating agents to ensure strong connection with gallium ions.

[0079] 2. The present invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and its application. Aiming at the problem of the general toxicity of metal ions and the phenomenon that gallium ions are easily decomposed, which limits their application in antibacterial implants, the antibacterial composite coating designed in the present invention solves the hydrolysis problem of gallium itself through the chelation of the polydopamine coating and gallium ions, slows down its release rate, and improves its utilization rate in vivo.

[0080] 3. The present invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and its application. Through the hydrogen bond adsorption performance between PDA and lysozyme, the lysozyme is firmly loaded in the coating, which not only ensures the firmness of the coating but also increases the antibacterial efficacy of the coating material. It can effectively kill biofilm-embedded bacteria without harming the body, improve the antibacterial performance of the stent, reduce the occurrence of bacterial drug resistance, and form an adhesive multifunctional film on the stainless steel surface by polydopamine to provide protection for the stainless steel substrate and improve its corrosion resistance.

[0081] 4. The present invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating and its application. The antibacterial composite coating aims to effectively kill biofilm-embedded bacteria without harming the body, prevent bacterial adhesion and biofilm formation on the surface of the implant, improve the antibacterial performance of the implantable stent, solve the problem of explosive infection after implantation in clinical practice, and provide more ideas, means and beneficial explorations for the treatment of chronic intractable inflammation caused by biofilms. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It is a flowchart of the design and preparation of the antibacterial stainless steel coating of the present invention;

[0083] Figure 2 It is a view of the appearance of the SS, PDS, GDS, and GLDS samples of the present invention;

[0084] Figure 3 It is an XPS energy spectrum diagram of the surface of the GLDS coating of the present invention;

[0085] Figure 4 It is an elemental scanning result diagram of the GDS and GLDS coatings of the present invention;

[0086] Figure 5 It is a scanning electron microscope image of the surfaces of the PDS (a), GDS (b), and GLDS (c) coatings of the present invention and an atomic force microscope image of the GDS (d) and GLDS (e) coatings (the scale of the main figure is 2 μm; the scale of the attached figure is 200 nm);

[0087] Figure 6 It is a diagram of the contact angle measurement results of the SS, PDS, GDS, and GLDS coatings of the present invention ( n = 3);

[0088] Figure 7 It is a curve diagram of the cumulative release of gallium ions from the GLDS coating of the present invention;

[0089] Figure 8 It is a hemolysis degree test diagram of the PDS, LDS, GDS, and GLDS coatings of the present invention (A: the soaking process of the hemolysis test; B: taking the supernatant after soaking and centrifugation);

[0090] Figure 9 Results of the rat spinal cord infection model of the present invention ((a) surgical procedure; (b) body weight change; (c) incision scar area on the 7th day and (d) on the 14th day after surgery; (e) wound healing; (f) local soft tissue infection after surgery.( n = 3)*P < 0.05, ***P < 0.01, ****P < 0.0005 vs. SS group);

[0091] Figure 10 Confocal laser scanning microscopy results of the mixed biofilm on different material surfaces at 7 and 14 days after surgery of the present invention (200× magnification, Scale bar = 25 μm) (Group A (control group), Group B (PDS group), Group C (GDS group), Group D (LDS group), Group E (GLDS group));

[0092] Figure 11 HE staining images of the soft tissues at the surgical site at 7 and 14 days after surgery of the present invention (20× and 40× magnifications, Scale bar = 100 μm) (Group A (control group), Group B (PDS group), Group C (GDS group), Group D (LDS group), Group E (GLDS group));

[0093] Figure 12 Results of the biocompatibility of rat organs at 7 and 14 days after surgery of the present invention (Group A (control group), Group B (PDS group), Group C (GDS group), Group D (LDS group), Group E (GLDS group)). Detailed implementation manners

[0094] Next, the technical solutions in the specific implementation manners of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the specific implementation manners of the present invention. Obviously, the described specific implementation manners are only a part of the specific implementation manners of the present invention, rather than all of the specific implementation manners. Based on the specific implementation manners of the present invention, all other specific implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0096] As Figure 1 shown, the specific implementation manner of the present invention provides a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating. Using medical 316L stainless steel as the substrate material, and using dopamine solution as the raw material, adding Tris salt to initiate the polymerization reaction of dopamine to form a polydopamine film on the stainless steel surface;

[0097] The easily oxidized catechol groups undergo a Michael addition reaction with lysozyme containing amino and mercapto groups in an aerobic environment to form a stable covalent bond, and a polydopamine composite film loaded with lysozyme is prepared;

[0098] Utilize the chelation property of metal ion gallium with phenolic hydroxyl groups on the surface of polydopamine, and through the immersion method, make it uniformly cover the surface of the polydopamine film to prepare a composite antibacterial coating implant;

[0099] As Figure 2 shown, the surface of untreated 316L stainless steel is bright silver on the SS surface, bright yellow on the PDS surface, bright yellow on the GDS surface, and dark purple on the GLDS surface.

[0100] Characterization and analysis methods for the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating include the following methods:

[0101] 1) Use X-ray photoelectron spectroscopy to analyze its chemical composition. The anode target of the XPS spectrometer used is magnesium, and the charge potential is calibrated with the C1 s peak (284.8 eV). Under the monochromatic Al Ka (1486.6 eV) light source, the vacuum degree is 12 Kv×15 mA, and it works under the condition of 2×10 -7 Pa. Adopt a scanning energy band of 0 - 1400 eV and a takeoff angle of 30°. The front surface needs to be cleaned by Ar + ion sputtering before detection, and then measure the surface of the GLDS coating to determine the connection of lysozyme and the model construction of the coating;

[0102] Lysozyme undergoes Michael addition / Schiff base reaction with the surface of polydopamine, so that lysozyme is immobilized on the polydopamine film. In order to confirm the presence of gallium and lysozyme on the GLDS surface, use XPS to characterize the elemental composition of the GLDS film surface, and the results are as Figure 3 shown;

[0103] Figure 3 (a) is the elemental composition diagram of the GLDS surface, Figure 3 (b - f) are the sub-peaks of N, Ga, S, C, and O respectively. The presence of signals of carbon, nitrogen, and oxygen proves the formation of PDA on the stainless steel surface. The 284.8 eV at the C1 s peak is for the C - C single bond, 286.3 eV is for the C - N single bond, and 288 eV is for the C - OOH bond.

[0104] The appearance of the Ga 3d peak and the S2p peak indicates the chelation of Ga ions and the grafting of lysozyme with polydopamine. Among them, the binding energies of the Ga 3d peak are 19.3 eV and 25.9 eV.

[0105] The two peaks can be respectively attributed to Ga 3d3 / 2 and Ga 3d1 / 2. The binding energies of the S2p peak are 163.2 eV and 164.8 eV. Among them, 163.2 eV is for the mercapto peak, and 164.8 eV is for the covalent bonding peak of the mercapto group with the polydopamine surface.

[0106] This result proves that gallium and lysozyme are immobilized on the surface of the polydopamine coating, and the GLDS coating is successfully prepared.

[0107] 2) Analyze the surface elements using energy-dispersive X-ray spectroscopy, specifically analyzing the distribution of elements C, N, S, O, and Ga on the coating surface;

[0108] The scanning results of the surface elements of GDS are as Figure 4 shown. Randomly select the GDS coating area for area scanning EDS energy spectrum analysis, which shows the presence of gallium element, proving the successful coating of gallium ions.

[0109] 3) Observe the surface morphology using a FE-SEM field emission scanning electron microscope. Use a Gemini SEM 500 scanning electron microscope to characterize the surface morphology at different stages during the preparation of antibacterial stainless steel. Its working voltage is 5 kV, and the magnification multiples are 2000 times and 20000 times respectively;

[0110] Figure 5 (a) The result shown is the surface morphology of the coating after PDA coverage under a 2k-fold scanning electron microscope. The upper left is the result of magnified observation under a 20k-fold microscope. Analyzed by Nano Measure software, it can be known that the average particle size is 0.14 μm. Except for a few scratches left after polishing, the stainless steel surface is basically flat, and polydopamine particles are evenly distributed on the surface.

[0111] Figure 5 (b) shows the GDS scanning electron microscope result and its magnified image. The average particle size is 0.182 μm. Compared with the PDS group, the particle size is slightly increased, and more evenly distributed dark micropores are formed on the stainless steel surface, which is beneficial for the deposition of antibacterial gallium ions on the PDS surface.

[0112] Figure 5 (c) shows the GLDS scanning electron microscope result and its magnified image. The average particle size is 0.171 μm. Compared with GDS, the particle size difference is not obvious. There are more dark micropores on the coating surface, which is beneficial for the loading of gallium ions and lysozyme.

[0113] Detect the surface morphology and roughness of the GDS and GLDS coatings using an atomic force microscope, as Figure 5 (d, e) shown. Among them, the average roughness of the GDS surface is 177 nm, and the average roughness of the LDS surface is 300 nm.

[0114] The results show that they are basically consistent with the scanning electron microscope results. The GLDS surface is rougher than the GDS surface, which is related to the small aggregation of lysozyme, indicating that the loading of lysozyme increases the roughness of the coating surface.

[0115] 4) The surface topography and roughness were detected using an atomic force microscope. The tapping mode with an Si cantilever was used to measure two types of samples, GDS and GLDS, and analyze the changes in their surface topography. The test conditions were room temperature, air phase, a sampling frequency of 1 Hz, a single-crystalline silicon tip was selected, the elastic coefficient of the probe was 40 N / m, and three samplings were performed in a randomly selected 5×5 μm area on the same sample surface to obtain the average surface roughness of the coating. 2 The average value of the surface roughness of the coating was calculated.

[0116] To investigate the hydrophilicity of the coating and the adhesion ability of dirt on the coating surface, the hydrophilicity and hydrophobicity of the sample surface were measured using a contact angle experiment. The results are shown as Figure 6 follows, and the results are expressed as the mean ± standard deviation.

[0117] The contact angle of the 316L stainless steel surface was 74.5 ± 3.0°, and the water contact angles of the PDS, GDS, and GLDS surfaces were 39.4 ± 2.4°, 46.4 ± 2.8°, and 49.4 ± 1.4°, respectively. The smaller the contact angle, the greater the hydrophilicity. This indicates that the coating model improves the hydrophilicity of the substrate, and PDS has better hydrophilicity than GDS and GLDS because a large number of hydroxyl groups on the surface of polydopamine endow it with hydrophilic properties.

[0118] 5) Contact angle analysis of the composite antibacterial coating implant was carried out. To evaluate the surface wetting performance of the specimen, 3 μL of deionized water was dropped at five different random positions on the specimen surface, and the average contact angle and contact angle hysteresis of the specimen were obtained using a contact angle measuring instrument. Contact angle hysteresis is the difference between the contact advancing angle and the contact receding angle. Three replicate samples were set for all test samples.

[0119] The gallium ion release amount of the gallium-loaded stainless steel implant under physiological conditions was determined by AAS, and three parallel experiments were performed for each sample.

[0120] Operating conditions of the instrument: Element Ga; analysis method, graphite furnace atomic absorption spectrometry; lamp current 6 mA; wavelength 287.4 nm; slit width 0.7 nm.

[0121] Other conditions: Drying (temperature 100 °C for 15 s) → Ashing (temperature 800 °C for 0 s) → Atomization (temperature 2600 °C for 3 s) → Cleaning (temperature 2700 °C for 2 s).

[0122] The in vitro gallium ion cumulative release curve of the composite antibacterial coating implant is shown as Figure 7 follows, and the controlled release effect of the coating model on gallium ions was analyzed through the release curve of the material gallium ions.

[0123] The results showed that within 12 hours of the initial release, the concentration of gallium ions increased rapidly, and gallium ions were able to be quickly released from the surface of the implant. Within 12-48 hours, the release curve of gallium ions gradually slowed down. Within 48-72 hours, the release of gallium ions stabilized and remained at a high level. This suggests that when the gallium-loaded stainless steel implant comes into contact with the wound surface in the early stage, it can quickly release a large amount of gallium ions, thereby quickly killing bacteria. The subsequent slow release of gallium ions is also conducive to consolidating the anti-inflammatory and antibacterial results and maintaining a good antibacterial effect.

[0124] 6) Conduct a hemolytic test to evaluate the blood compatibility of the coating material. The process is as follows:

[0125] Draw 10 mL of whole blood from healthy volunteers, add it to an anticoagulant tube, and perform a coagulation test. For every 8 mL of anticoagulated whole blood, add 10 mL of normal saline to dilute it for later use;

[0126] The samples were cut into 2.5 × 1 cm 2 For each section, rinse several times with distilled water, soak in saline for 24 hours, discard the soaking solution, add 10 mL of saline again, then add 0.2 mL of diluted human blood, and soak vertically in a drum device for 60 min (15 rpm / min);

[0127] The coating material was taken out and centrifuged at 2000 rpm / min for 10 min. The supernatant was transferred into a colorimetric cup and the absorbance was measured at a wavelength of 545 nm using an ultraviolet spectrophotometer. The positive control was 316L stainless steel plus double distilled water, and the negative control was 316L stainless steel plus 0.9% saline.

[0128] The calculation formula of hemolysis is as follows:

[0129]

[0130] According to national standards, a material hemolysis rate of <5% indicates that the material meets the hemolysis test requirements for medical materials;

[0131] Testing the hemolytic property of materials helps to evaluate the potential risks of biological materials that come into direct contact with blood in clinical applications, thereby improving the biosafety of the materials. Figure 8 This is a diagram of the hemolysis test experiment. A is the hemolysis test soaking process, and B is the centrifuge of the soaking liquid and the supernatant of each group for testing. The positive control uses 316L stainless steel plus double distilled water, and the hemolysis rate is 100%. The negative control uses 316L stainless steel plus 0.9% saline, and the hemolysis rate is 0%.

[0132] Table 1 shows the hemolysis test results of the four materials compared with bare steel, using mean ± standard deviation To express.

[0133] Table 1 Hemolysis test results of PDS, LDS, GDS, and GLDS coatings ( n = 3)

[0134]

[0135] According to the SO 10993 standard, when the material is in direct contact with blood, the hemolysis rate should be < 5%.

[0136] The results showed that when the experimental group was compared with the negative control, the hemolysis rates were all significantly < 5%, with no significant difference, meeting the international standard and satisfying the experimental conditions.

[0137] 7) Conduct the study on the release of gallium ions from the GLDS coating, and the process is as follows:

[0138] Accurately cut a GLDS gallium-containing stainless steel sample with a size of 4×2 cm2, put it into a 15 mL centrifuge tube, add 10 mL of PBS (pH = 7.4) release medium, and perform low-vacuum degassing to fully immerse the material in the release medium, and place it in a constant-temperature shaker at 37°C and 50 rpm for uniform oscillation;

[0139] At 3 h, 8 h, 18 h, 24 h, 48 h, and 72 h, take 500 μL of the supernatant for detecting the gallium ion concentration, then supplement 500 μL of PBS release medium, continue the constant-temperature uniform oscillation, and use an atomic absorption spectrometer to measure the gallium ion concentration. Three parallel controls are set for the samples;

[0140] Evaporate 500 μL of the solution sample to dryness and place it in the digestion inner tank, add 3 ml of nitric acid solution, heat it in an oven at 100°C for 1 h and at 140°C for 5 h;

[0141] After the digestion is completed, take it out after cooling, transfer the digestion tank into the fume hood, slowly open the tank, heat it in a water bath to volatilize the acid, rinse the inner cover with a small amount of ultrapure water, and wait until it volatilizes to 1 ml, then take it out and cool it to room temperature;

[0142] Transfer the digestion solution to a 10 ml volumetric flask, make up the volume to the scale with ultrapure water, and conduct a reagent blank experiment at the same time. Then, measure the absorption peak area of gallium according to the working conditions of the analytical instrument respectively, and calculate the gallium content in the sample according to the standard curve and dilution factor.

[0143] 8) Preparation of the standard curve, and the process is as follows:

[0144] Dilute the standard solution of gallium element (1000 μg / mL) with 2% HNO3 to a gallium standard stock solution with a concentration of 1.0 μg / mL;

[0145] Appropriately and precisely pipette from the Ga standard stock solution (1.0 μg / mL), and use 2% HNO₃ solution as the solvent to prepare gallium solutions with concentrations of 10, 20, 40, 60, 80, 100, 120, and 200 μg / L. At the same time, prepare a blank solution in the same way;

[0146] Precisely pipette 200 μL of each standard solution, automatically inject 20 μL of the standard solution into the graphite furnace atomizer, measure the absorbance value as the ordinate, and use the concentration as the abscissa to make a standard curve;

[0147] Then use the AAS method to determine the linear range, regression equation, and correlation coefficient of the standard solution of the Ga metal element.

[0148] For the in vitro antibacterial performance evaluation method of this layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating, the antibacterial performance of the antibacterial stainless steel is measured by the plate culture medium method. Pre-culture Staphylococcus aureus and Escherichia coli, incubate the antibacterial coating sample with them, measure the distance between the colony and the edge of the sample, and detect its in vitro antibacterial performance. The specific process is as follows:

[0149] S1. Solution preparation and equipment sterilization

[0150] Take 1.25 g of LB broth in a 50 mL conical flask, add 50 mL of ultrapure water to dissolve it, and tightly stopper the bottle mouth with gauze and wrap it with tin foil;

[0151] Take 9.4 g of PCA plate count agar in a 500 mL conical flask, add 500 mL of ultrapure water and shake well. Similarly, tightly stopper the bottle mouth with gauze and wrap it with tin foil;

[0152] Dissolve the solid powder of PBS buffer in 2 L of ultrapure water to prepare a PBS buffer solution with a pH of 7.4;

[0153] Place several 5 mL centrifuge tubes, several 90 mm culture dishes, the above-mentioned LB broth, PCA solution, and 500 mL of PBS buffer solution in a high-pressure steam sterilizer, and set the parameters to 121 °C for 20 min for sterilization;

[0154] S2. Culture medium preparation and bacterial proliferation

[0155] Pour the sterilized PCA solution into the culture dish while it is still hot in the purification workbench and spread it flat. Each culture dish contains about 16 - 17 mL of PCA. After cooling, invert it in the ultra-clean bench;

[0156] Use a pipette to take 20 μL of the Escherichia coli stock solution or Staphylococcus aureus stock solution and add it to the LB broth. After gently shaking, incubate it at 37 °C for 12 h;

[0157] S3. Bacterial pretreatment

[0158] The successfully proliferated LB broth of Escherichia coli was aliquoted into several centrifuge tubes and centrifuged at 5000 rpm for 3 min to deposit the bacteria at the bottom of the centrifuge tubes. The supernatant was discarded, and PBS buffer was added to resuspend the bacteria. The absorbance was measured at a wavelength of 600 nm. After appropriate dilution, the absorbance value was adjusted to about 0.15 to obtain a bacterial suspension with an appropriate concentration.

[0159] The experimental process for measuring the antibacterial performance of antibacterial stainless steel by the plate culture medium method is as follows:

[0160] S1. Melt the agar medium and cool it to 45 °C. Measure 10 ml of freshly cultured bacterial suspension within 24 hours and then inoculate it into 150 mL of agar.

[0161] S2. Pour 15 ml of the inoculated agar into a petri dish with a diameter of 100 mm. After placing it for 15 minutes, put the sample on it.

[0162] S3. Gently press the sample with sterile forceps to ensure full contact between the sample and the inoculated agar. Place the petri dish in an incubator at 37 °C for 24 hours and measure the width of the inhibition zone.

[0163] S4. First, pre-culture the bacteria, dilute them to 2×107 cfu·mL-1 with phosphate buffer, and evenly cover 20 μL of the diluted bacterial suspension on the agar plate.

[0164] S5. Finally, place the antibacterial coating samples containing gallium, lysozyme, etc. face up on the agar plate and co-incubate them at 37 °C for 24 hours. Each sample should be tested at least three times.

[0165] S6. Measure the distance between the edge of the colony and the sample from the back of the petri dish and calculate according to the following formula:

[0166]

[0167] Where: W—the width of the antibacterial zone, unit: mm;

[0168] T—the total diameter (total width) of the antibacterial zone of the sample, unit: mm;

[0169] D—the diameter (width) of the sample, unit: mm.

[0170] After the implant device has antibacterial ability, during the stage of rapid bacterial reproduction, an obvious antibacterial zone can be formed around it. The presence of the inhibition zone also confirms that the material can release gallium ions that inhibit bacterial growth and kill the bacteria around the implant together with the loaded lysozyme. The larger the inhibition zone, the better the antibacterial performance of the material. The width (mm) of the inhibition zone of each sample was expressed as mean ± standard deviation For representation, as shown in Table 2 and Table 3:

[0171] Table 2 Antibacterial effects of four coating systems against Escherichia coli( n = 3)

[0172]

[0173] Table 3 Antibacterial effects of four coating systems against Staphylococcus aureus( n = 3)

[0174]

[0175] Note: — indicates that no antibacterial effect was observed.

[0176] The antibacterial effect of this composite antibacterial coating mainly comes from the fact that when the implant contacts bacteria, a large amount of gallium ions are first released, which can effectively penetrate the bacterial cell membrane in the initial stage, enter the cell to participate in the bacterial biochemical process, and disrupt the normal redox process of bacteria; at the same time, the lysozyme loaded on the implant surface can effectively inhibit the adsorption and reproduction of bacteria on the implant surface for a long time.

[0177] The application of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating in the study of antibacterial effect and biocompatibility in vivo implantation includes the following processes:

[0178] 1) Prepare a mixed bacterial solution of the standard strain of Staphylococcus aureus ATCC35923 and the standard strain of Escherichia coli ATCC25922 in a ratio of 1:1;

[0179] 2) Establish a rat spinal infection model, implant the designed and prepared composite antibacterial stainless steel coating into the back of SD rats, and evaluate and observe the inhibitory effect of GLDS on the mixed infection of Staphylococcus aureus and Escherichia coli by taking the changes in rat body weight, incision healing, severity of deep tissue inflammatory reaction, scope of inflammatory reaction, and infection situation of the tissue under the wound as indicators.

[0180] The specific experimental process is as follows:

[0181] Step 1. Take 30 male SD rats weighing about 200 g, and randomly divide them into 5 groups, namely A, B, C, D, and E, with 6 rats in each group according to the random number table method;

[0182] Step 2. Anesthetize the rats by inhaling isoflurane. After the rats are anesthetized, continue to maintain anesthesia with isoflurane at a concentration of 1 - 2.5%. Shave the back hair, disinfect routinely, perform surface positioning according to the spinous process, make a posterior median incision, cut about 2 cm long between L4 - L6, and cut through the skin and subcutaneous tissue layer by layer. Along both sides of the spinous process, bluntly separate the erector spinae muscles close to the bone surface to the bilateral laminae;

[0183] Step 3. Disinfect and use those with a size of 2 cm2 The bare steel, polydopamine-coated stainless steel, gallium nitrate-coated stainless steel, lysozyme-coated stainless steel, and gallium nitrate-lysozyme co-loaded stainless steel coatings were placed between the erector spinae muscles and the spinous processes of rats. One piece of the same type of metal material was placed on each side of the spinous process of each rat, and 25 μL of a mixed Staphylococcus aureus-Escherichia coli bacterial solution (bacterial count: 1×107 CFU / mL) was dropped on each side. The muscles were sutured tightly, and the incision was closed layer by layer.

[0184] Step 4. After the rats recovered from anesthesia and were observed to have normal activities, spirits, and diets, they were placed in a SPF animal room for feeding and observation. On the day of injury, and on the 3rd, 7th, and 14th days after injury, the spirits and activities of the rats were observed, and their weights were measured.

[0185] Step 5. The rats were sacrificed by spinal dislocation on the 7th and 14th days after surgery. The healing of the incision and the severity of the inflammatory reaction in the deep tissues were observed, the scope of the inflammatory reaction was measured, and the incision healing was evaluated according to the surgical incision healing grade.

[0186] Step 6. The rat wounds were incised along the original incision, the infection condition of the tissues under the wound was observed and recorded. The metal pieces placed in the body were taken out, gently soaked and rinsed with physiological saline to remove the tissues adhered to the metal surface and the non-adhered bacteria. The five groups of metal pieces were placed in 24-well plates for standby.

[0187] Step 7. The live / dead bacterial fluorescent staining agent was prepared strictly according to the instructions of Live / Dead LIVE / DEAD BacLight Bacterial Viability Kits (L13152). Two tubes of fluorescent dyes were taken and dissolved in 10 mL of distilled water, mixed well and stored at 4°C for immediate use.

[0188] Step 8. 400 μL of the fluorescent staining solution was added to the 24-well plates containing the five groups of metal pieces, stained at room temperature for 15 minutes, then gently rinsed in 2.5 mL of physiological saline to remove the excess fluorescent staining solution, and then a glass slide was placed on it and observed under a laser confocal microscope.

[0189] Step 9. Whether there was pus, the degree and scope of inflammatory tissues and necrotic tissues in the removed pathological tissues were observed, and the pathological changes of the muscle tissues in contact with the metal surface of each group of rats were investigated by H&E staining.

[0190] Step 10. The animals were randomly sacrificed on the 7th and 14th days after surgery, 3 animals in each group. Autopsies were performed on the five groups of animals A, B, C, D, and E, and the main organs of the heart, liver, spleen, lung, and kidney of each group were collected and fixed in 4% paraformaldehyde for histopathological examination.

[0191] ① Observation of the postoperative conditions of the animals

[0192] Figure 9 (a) shows the preparation process of the rat spinal implant surgery model. During the operation, observe the rat's breathing and heart rate at any time. Adjust the surgical progress appropriately according to the changes in the rat's physical signs to ensure the success of the operation.

[0193] Animal gross experiment observation:

[0194] On the day after surgery, in the five groups of animals in Group A (SS group), Group B (PDS group), Group C (GDS group), Group D (LDS group), and Group E (GLDS group), all showed slow movement, decreased ability to eat independently, red and swollen back skin, but no increase in secretions at the incision, and no secretions flowing from the mouth, nose, or urethra. There was no significant difference in the activity ability among the five groups of A, B, C, D, and E, and no death cases occurred.

[0195] Three days after surgery, the locomotor ability returned to normal and the ability to eat independently recovered. The water intake of Group A and Group B decreased significantly, while the water intake of Group C, Group D, and Group E increased. Group C had the highest activity level. Group D had larger scar tissue on the skin surface, abnormal secretions from the mouth and nose, sticky excrement, and back exudation and scar tissue formation. All five groups could climb over a 12-cm-high cardboard box, and no death cases occurred.

[0196] Seven days after surgery, the movement of all five groups was normal, and the diet and water intake were good. The activity levels of Group A and Group B were poor, while Group C, Group D, and Group E could freely climb over a 15-cm-high cardboard box.

[0197] Fourteen days after surgery, the movement of all five groups was normal. The activity levels of Group A and Group B were relatively poor, while the activity levels of Group C, Group D, and Group E were good. The surface scar of Group E was the smallest and almost invisible to the naked eye, and the recovery was the best. All of them could freely climb over a 15-cm-high cardboard box.

[0198] ② Weight change of rats after surgery

[0199] Analysis of variance was used for comparison between groups and within groups. The test level α = 0.05, and P < 0.05 indicated statistical significance; the test level α = 0.001, and P < 0.001 indicated a large statistical significance. The experimental data were expressed as mean ± standard deviation. There was no statistical significance in the within-group comparison; there was statistical significance in the between-group comparison. By comparing the weight of the five groups of rats at different time periods after surgery, no obvious difference in weight was found among the five groups, indicating that the material had no obvious effect on the weight of the rats. Among them, each group of rats had varying degrees of weight gain at different time periods within 14 days after surgery.

[0200] Table 4 is the weight statistics table of the five groups of rats. Figure 9 (b) is the weight change graph of the five groups of rats.

[0201] Table 4 Statistical table of weight changes of the five groups of rats on the day after surgery, 3 days, 7 days, and 14 days ( n = 6, 14 days after surgery, n = 3)

[0202]

[0203] ③ Analysis of the incision healing and infection conditions of animals after surgery

[0204] Analysis of the incision infection conditions at 3, 7, and 14 days after surgery:

[0205] At 3 days after surgery, five groups of rats were sacrificed, Figure 9 (e) It can be seen that on the surface of the skin tissue at the incision of Group A (control group / SS group), punctate scars appeared; in Group B (PDS group), scars appeared at the incision, with small - area scars extending around, and exudate formed at the incision; in Group C (GDS group), the local infection condition was relatively mild, no scars appeared temporarily, and the wound healing condition at the incision was better; in Group D (LDS group), no scars appeared temporarily, and the incision wound healing was incomplete; in Group E (GLDS group), the local infection was relatively mild, no scars appeared temporarily, and the wound recovery was better.

[0206] At 7 days after surgery, five groups of rats were sacrificed, Figure 8 (e) It can be seen that necrosis appeared on the surface of the skin tissue at the incision of Group A (control group / SS group); in Group B (PDS group), scars appeared at the incision and around the incision; in Group C (GDS group), the local infection condition was relatively mild, and the recovery condition was significantly better than that of Groups A and B, no scars appeared, and the wound healing condition at the incision was better; in Group D (LDS group), no scars appeared, and the incision wound healing was incomplete; in Group E (GLDS group), the local infection was the mildest, no scars appeared, and the wound recovery condition was the best.

[0207] At 14 days after surgery, five groups of rats were sacrificed, 9(e) It can be seen that the skin tissue at the incision of Group A (control group / SS group) did not heal completely; in Group B (PDS group), obvious cyst protrusions appeared around the incision, and the incision did not heal; in Group C (GDS group), the wound healed completely, the skin was flat, and the local infection condition was relatively mild; in Group D (LDS group), the wound healed completely, the skin was flat, and no scar necrosis and other conditions appeared; in Group E (GLDS group), the wound healed completely, the newly - grown hair almost completely covered the incision, the skin was flat, and no scar necrosis and other conditions appeared.

[0208] Open the rat wound along the original incision, as Figure 9(f) Observe the soft tissue infection under the wound 7 days after surgery: In group A, the soft tissue at the incision was severely eroded and necrotic. The necrotic tissue was light yellow, with a dull color, and there was obvious purulent serous exudate. In group B (PDS group), there was an obvious cystic protrusion around the incision. After incision, obvious pus was wrapped, and the surrounding tissue was dark red, with severe inflammatory manifestations. In group C (GDS group), serous exudate could be observed about 5 mm near the wound. The area of pus formation was significantly smaller than that in groups A and B, and the local inflammatory condition was milder. In group D (LDS group), the situation was roughly the same as that in group C. In group E (GLDS group), the performance was the best. Almost no pus was found to remain, the muscle color was bright, and the inflammatory condition was the mildest.

[0209] Size of the necrotic area: group A > group B > groups C / D > group E. According to the wound healing grade classification, the healing conditions were as follows: group A had a third-class healing, group B had a third-class healing, group C had a second-class healing, group D had a second-class healing, and group E had a first-class healing.

[0210] Observe the soft tissue infection under the wound 14 days after surgery: In group A (control group / SS group), dark red soft tissue structure could be seen at the incision, which was necrotic tissue, wrapping dark yellow and light yellow pus, with a dull color. In group B (PDS group), purulent serous exudate was found around the implant, and the surrounding tissue was dark red, with severe inflammatory manifestations. In group C (GDS group), cysts were found in the dermal layer of the wound, and no obvious pus was found at the implant implantation site. The necrotic area was relatively small, and the local inflammatory condition was milder. In group D (LDS group), there was a small amount of serous exudate at the implant implantation site. In group E (GLDS group), the performance was the best. No obvious pus was found to remain, the muscle color was bright, and the inflammatory condition was the mildest.

[0211] Size of the necrotic area: group A > group B > groups C / D > group E. Healing conditions: group A had a third-class healing, group B had a third-class healing, group C had a third-class healing, group D had a second-class healing, and group E had a first-class healing.

[0212] Use software such as Prism to process the data. Calculate the wound healing rate: [(initial wound area - wound area at a certain moment) / initial wound area]×100%.

[0213] Figure 9 (c) shows the wound healing rates of the five groups of rats 7 days after surgery. It can be seen that the healing rate of group E rats was the highest, followed by groups B, C, and D. Compared with the control group, there were statistical differences in groups B, C, D, and E, and the difference in group E was the most obvious. No statistical differences were found in pairwise comparisons among groups B, C, D, and E.

[0214] Figure 9 (d) shows the wound healing rates of the five groups of rats 14 days after surgery. It can be seen that the healing rate of group E rats was the highest, followed by groups C and D. Compared with the control group, there were statistical differences in groups C, D, and E, and the difference in group E was the most obvious, with the best healing effect. No statistical differences were found in pairwise comparisons among groups B, C, D, and E.

[0215] ④ Observation of the effect of GLDS coating on bacterial biofilm using laser confocal microscopy

[0216] Depend on Figure 10 It can be seen that 7 days after surgery, scattered green and red fluorescence signals in the shape of spots were visible in all five groups, and weak biofilm structures were formed in all five groups. During the experiment, the red and green fluorescence density of groups A and B was greater than that of groups C, D, and E; the red and green fluorescence signal intensities of groups A and B were almost the same; the green fluorescence signal of group C was stronger than the red fluorescence signal; the green fluorescence signal of group D was slightly stronger than the red fluorescence signal; the green fluorescence signal intensity of group E was significantly weaker than the red fluorescence. All five groups had a certain degree of biofilm formation, but group E had the strongest bactericidal effect.

[0217] Fourteen days after surgery, scattered green and red fluorescent signals were visible in all five groups. During the experiment, the fluorescence density of groups A and B was greater than that of groups C, D, and E, and the fluorescence signal of group E was the weakest and sparsest; the intensity of the green fluorescent signal of groups A and B was much greater than that of the red signal, indicating that the number of dead bacteria in groups A and B was very small and the material did not have the ability to kill bacteria; the intensity of the green fluorescent signal and the red fluorescent signal of groups C and D was almost the same; the area of ​​the red and green fluorescent signals of group E was the smallest, and the red fluorescent signal was stronger than the green fluorescent signal, indicating that on the 14th day, the GLDS group still had effective bactericidal ability. All five groups had a certain degree of biofilm formation, and the formation area of ​​group E was the smallest, indicating that group E had certain characteristics of inhibiting biofilm formation.

[0218] ⑤H&E staining observation of wound soft tissue

[0219] 7 days: After HE staining, diffuse infiltration of inflammatory cells in the striated muscles of groups A, B, C, D, and E was observed, accompanied by varying degrees of muscle tissue necrosis. Groups A and B had the most severe inflammatory infiltration, severe cell necrosis, ruptured angiogenesis, and a large amount of extracellular matrix deposition; Groups C and D had better inflammatory infiltration and cell necrosis than groups A and B. In group C, obvious cell deformation, rupture, and dissolution, matrix deposition, angiogenesis, and large-scale granulation tissue proliferation and repair were observed; Group D was slightly worse than group C, with rhabdomyolysis, obvious inflammatory cell infiltration, and large-scale granulation tissue proliferation and repair; Group E had the best situation, with fewer inflammatory cells diffused in the striated muscles, slight cell deformation, and tight connections, such as Figure 11 shown.

[0220] 14 days: After HE staining, varying degrees of diffuse infiltration of inflammatory cells and varying degrees of muscle tissue necrosis were observed in the striated muscles of groups A, B, C, D, and E. In group A, there was severe inflammatory infiltration, severe cell necrosis, massive deposition of extracellular matrix, and granulation tissue formation. In group B, the inflammatory infiltration was the most severe, but the cell necrosis was less than that in group A, and a large area of granulation tissue was formed. In groups C and D, the inflammatory infiltration and cell necrosis were improved compared with those in groups A and B. In group C, cell deformation, rupture, and dissolution were significantly observed, matrix deposition occurred, new blood vessels were generated, and a large area of granulation tissue proliferated and repaired, and the area of granulation tissue was significantly larger than that in groups A and B. The situation in group D was slightly better than that in group C, with inflammatory cells diffusing near the striated muscles and the cell connections being relatively tight. Group E had the best situation, with a significant reduction in the diffusion of inflammatory cells in the striated muscles, slight cell deformation, and tight connections, as Figure 11 shown.

[0221] ⑥ In vivo biocompatibility investigation

[0222] Animals were randomly sacrificed at 7 days and 14 days after surgery, and autopsy was performed on the animals in groups A, B, C, D, and E. There were no abnormal secretions around their eyes, mouths, and noses. Histopathological examination of the main organs such as the heart, liver, spleen, lungs, and kidneys in each group, as Figure 12 shown, showed no obvious abnormalities, indicating that the composite antibacterial coating implant system had no organ toxicity and good biocompatibility.

[0223] In the present invention, lysozyme and gallium ions are co-loaded onto the polydopamine coating to prepare a stainless steel coating with a synergistic antibacterial effect. Dopamine can spontaneously polymerize on the surface of the stainless steel material to form a thin polydopamine film. When the catechol groups in PDA are in the active quinone structure, they can undergo Michael addition reactions with the amino and sulfhydryl groups of lysozyme, and at the same time, the catechol groups also act as chelating agents to ensure a strong connection with gallium ions. There is also a hydrogen bond adsorption property between PDA and lysozyme, ensuring the firmness of the coating. It can effectively kill biofilm-embedded bacteria without harming the body, improve the antibacterial performance of the stent, and reduce the occurrence of bacterial drug resistance.

[0224] In the present invention, polydopamine forms an adhesive and multifunctional thin film on the stainless steel surface, providing protection for the stainless steel substrate and improving its corrosion resistance. At the same time, the chelation of the polydopamine coating with gallium ions solves the hydrolysis problem existing in gallium itself, slows down its release rate, improves its utilization rate in the body, and the loaded lysozyme also increases the antibacterial efficacy of the coating material. The implementation of the present invention can provide more ideas and means for the treatment of chronic refractory inflammation caused by biofilms, provide more data support for clinical use, lay a foundation for the possible practical application of antibacterial implants, and provide a more marketable and clinically applicable application prospect.

[0225] Although specific embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions, and variations can be made to these specific embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating, characterized in that, Using medical 316L stainless steel as the base material and dopamine solution as the raw material, adding Tris salt to initiate the polymerization reaction of dopamine to form a polydopamine film on the stainless steel surface; The easily oxidized catechol groups undergo a Michael addition reaction with lysozyme containing amino and mercapto groups in an aerobic environment to form a stable covalent bond, preparing a polydopamine composite film loaded with lysozyme; Then, utilizing the chelation property of metal ion gallium with the phenolic hydroxyl groups on the surface of polydopamine, it is evenly covered on the surface of the polydopamine film by the immersion method to prepare a composite antibacterial coating implant, and its formulation and preparation process are optimized.

2. The characterization analysis method of a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating according to claim 1, characterized in that, Including the following methods: 1) Analyze its chemical composition using X-ray photoelectron spectroscopy. The anode target of the XPS spectrometer used is magnesium, and the charge potential is calibrated with the C1 s peak. Under the condition of a monochromatic Al Ka light source, a vacuum degree of 12 Kv × 15 mA, and 2 × 10 -7 Pa, work with a scanning energy band of 0 - 1400 eV and a take-off angle of 30°. The front surface needs to be cleaned by Ar + ion sputtering before detection, and then measure the surface of the GLDS coating to determine the connection of lysozyme and the construction of the coating model; 2) Use energy-dispersive X-ray spectroscopy to analyze the surface elements, specifically analyze the distribution of C, N, S, O, and Ga elements on the coating surface; 3) Use a FE-SEM field emission scanning electron microscope to observe the surface morphology, and use a Gemini SEM 500 scanning electron microscope to characterize the surface morphology at different stages during the preparation of antibacterial stainless steel. Its working voltage is 5 kV, and the magnification multiples are 2000 times and 20000 times respectively; 4) Detect its surface topography and roughness using an atomic force microscope. Use the tapping mode with an Si cantilever beam to measure two samples, GDS and GLDS, and analyze the changes in their surface topography. The test conditions are room temperature, air phase, a sampling frequency of 1 Hz, a single-crystal silicon tip is selected, the elastic coefficient of the probe is 40 N / m, and samples of 5×5 μm 2 are sampled three times on the surface of the same sample, and the average value of the surface roughness of the coating is obtained; 5) Conduct contact angle analysis of the composite antibacterial coating implant. To evaluate the surface wettability of the specimen, randomly select 5 different positions on the specimen surface and drop 3 μL of deionized water respectively. Use a contact angle measuring instrument to obtain the average contact angle and contact angle hysteresis of the specimen. The contact angle hysteresis is the difference between the contact advancing angle and the contact receding angle. All samples in the experiment are set with 3 groups of replicates.

3. The characterization analysis method of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating according to claim 2, characterized in that, It also includes the following methods: Conduct a hemolysis experiment to evaluate the blood compatibility of the coating material by the hemolysis experiment. The process is as follows: Draw 10 mL of whole blood from a healthy volunteer, add it to an anticoagulation tube, and conduct a coagulation test. Dilute it with 10 mL of normal saline for every 8 mL of anticoagulated whole blood for standby; Cut the sample into pieces of 2.5×1 cm 2 For each piece, rinse it several times with distilled water, add normal saline and soak for 24 hours, pour out the soaking solution, add 10 mL of normal saline again, then add 0.2 mL of diluted human blood, and soak vertically in the roller device for 60 min; Take out the coating material, centrifuge it at 2000 rpm / min for 10 min, suck the supernatant into a cuvette, and measure the absorbance at a wavelength of 545 nm with a UV spectrophotometer. The positive control uses 316L stainless steel plus double-distilled water, and the negative control uses 316L stainless steel plus 0.9% normal saline; The calculation formula for hemolysis degree is as follows: According to the national standard, if the hemolysis rate of the material < 5%, it indicates that the material meets the hemolysis experiment requirements for medical materials; Conduct a study on the release of gallium ions from the GLDS coating. The process is as follows: Accurately cut a 4×2 cm 2 sized GLDS gallium-containing stainless steel sample, place it in a 15 mL centrifuge tube, add 10 mL of PBS (pH = 7.4) release medium, perform low-vacuum degassing to fully immerse the material in the release medium, and place it in a constant-speed shaker at 37°C and 50 rpm; At 3 h, 8 h, 18 h, 24 h, 48 h, and 72 h, suck 500 μL of the supernatant for detecting the gallium ion concentration, then supplement 500 μL of PBS release medium, continue to oscillate at a constant temperature and speed, and use an atomic absorption spectrometer to measure the gallium ion concentration. The samples are set with three parallel controls; Evaporate 500 μL of the solution sample to dryness and place it in an inner digestion tank, add 3 ml of nitric acid solution, heat it in an oven at 100 °C for 1 h and at 140 °C for 5 h; After digestion is completed, take it out after cooling, move the digestion tank into a fume hood, slowly open the tank, heat it in a water bath to volatilize the acid, rinse the inner lid with a small amount of ultrapure water, wait until it volatilizes to 1 ml, and take it out and cool to room temperature; Transfer the digestive juice to a 10-ml volumetric flask, dilute it to the mark with ultrapure water, and conduct a reagent blank test simultaneously. Then, measure the absorption peak area of gallium according to the working conditions of the analytical instrument, and calculate the gallium content in the sample based on the standard curve and dilution factor.

4. The characterization analysis method of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating according to claim 2, characterized in that, It also includes the following methods: The preparation of the standard curve is as follows: Dilute the standard solution of gallium element with 2% HNO3 to prepare a gallium standard stock solution with a concentration of 1.0 μg / mL. Precisely take appropriate amounts from the Ga standard stock solution, use 2% HNO3 solution as the solvent to prepare gallium solutions with concentrations of 10, 20, 40, 60, 80, 100, 120, and 200 μg / L, and prepare a blank solution in the same way simultaneously. Precisely measure 200 μL of each standard solution, automatically inject 20 μL of the standard solution into the graphite furnace atomizer, use the measured absorbance value as the ordinate and the concentration as the abscissa to prepare the standard curve. Then, use the AAS method to determine the linear range, regression equation, and correlation coefficient of the standard solution of Ga metal element.

5. The in vitro antibacterial performance evaluation method of a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating according to claim 1, characterized in that, The antibacterial performance of antibacterial stainless steel is measured by the plate culture medium method. Pre-culture Staphylococcus aureus and Escherichia coli, incubate the antibacterial coating sample with them, measure the distance between the colonies and the edge of the sample, and detect its in vitro antibacterial performance. The specific process is as follows: S1. Solution preparation and equipment sterilization Take 1.25 g of LB broth in a 50-mL conical flask, add 50 mL of ultrapure water to dissolve it, tightly plug the bottle mouth with gauze and wrap it with tin foil. Take 9.4 g of PCA plate count agar in a 500-mL conical flask, add 500 mL of ultrapure water and shake well, and also tightly plug the bottle mouth with gauze and wrap it with tin foil. Dissolve the solid powder of PBS buffer in 2 L of ultrapure water to prepare a PBS buffer solution with a pH of 7.

4. Place several 5-mL centrifuge tubes, several 90-mm culture dishes, the above-mentioned LB broth, PCA solution, and 500 mL of PBS buffer solution in a high-pressure steam sterilizer, and set the parameters for sterilization at 121 °C for 20 min. S2. Medium preparation and bacterial proliferation Pour the sterilized PCA solution into the culture dish while it is still hot in a clean bench and spread it flat. Each culture dish contains about 16 - 17 mL of PCA. After cooling, invert it in the ultra-clean bench. Use a pipette to take 20 μL of the original Escherichia coli solution or Staphylococcus aureus solution and add it to the LB broth. After gently shaking, incubate it at 37 °C for 12 h. S3. Bacterial pretreatment Dispense the LB broth with successfully proliferated Escherichia coli into several centrifuge tubes, centrifuge at 5000 rpm for 3 min to deposit the bacteria at the bottom of the centrifuge tube, discard the supernatant, add PBS buffer to resuspend the bacteria, measure the absorbance at a wavelength of 600 nm, and appropriately dilute it to make the absorbance value around 0.15, that is, obtain a bacterial solution with an appropriate concentration.

6. The in vitro antibacterial performance evaluation method of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating according to claim 5, characterized in that, The experimental process for measuring the antibacterial performance of antibacterial stainless steel by the plate culture medium method is as follows: S1. Melt the agar medium and cool it to 45 °C. Measure 10 ml of fresh bacterial solution cultured within 24 hours, and then inoculate it into 150 mL of agar. S2. Pour 15 ml of the inoculated agar into a 100 mm diameter culture dish and place it on the sample after 15 minutes. S3. Use sterile tweezers to gently press the sample to make it fully contact with the inoculated agar, place the culture dish in an incubator at 37°C for 24 hours, and measure the width of the inhibition ring; S4. First, pre-culture the bacteria and dilute them to 2×10 7 cfu·mL -1 with phosphate buffer, and evenly cover 20 μL of the diluted bacterial suspension on the agar plate; S5. Finally, the antimicrobial coating samples containing gallium, lysozyme, etc. were spread face up on the agar plate and incubated at 37°C for 24 hours. Each sample was tested at least three times. S6. Measure the distance between the colony and the edge of the sample from the back of the culture dish and calculate it according to the following formula: Where: W is the width of the antibacterial zone, in mm; T—the total diameter of the antibacterial zone of the sample, in mm; D—diameter of the sample, in mm.

7. Use of a layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating in the study of antibacterial effects and biocompatibility in vivo implantation, characterized in that, The process includes: 1) Prepare a mixed bacterial solution by mixing Staphylococcus aureus standard strain ATCC35923 and Escherichia coli standard strain ATCC25922 at a ratio of 1:1; 2) A rat spinal infection model was established, and the designed and prepared composite antibacterial stainless steel coating was implanted into the back of SD rats. The changes in rat weight, wound healing, the severity of deep tissue inflammatory response, the range of inflammatory response, and the infection of subwound tissues were used as indicators to evaluate the inhibitory effect of GLDS on mixed infection of Staphylococcus aureus and Escherichia coli.

8. Use of the layer-by-layer self-assembled gallium-lysozyme composite antibacterial stainless steel coating according to claim 7 in the research on antibacterial effect and biocompatibility of in vivo implantation, characterized in that, The experimental process is as follows: Step 1. Take 30 male SD rats weighing about 200 g and randomly divide them into 5 groups, A, B, C, D, and E, with 6 rats in each group. Step 2. The rats were anesthetized by inhalation of isoflurane. After the rats were anesthetized, 1-2.5% isoflurane was continued to be used to maintain anesthesia. The back hair was trimmed, routine disinfection was performed, and the body surface was located according to the spinous process. A posterior midline incision was made, and a 2-cm long incision was made between L4 and L6. The skin and subcutaneous tissue were cut layer by layer, and the erector spinae muscles were bluntly separated to the bilateral lamina along both sides of the spinous process and close to the bone surface; Step 3. Place the sterilized bare steel, polydopamine-coated stainless steel, gallium nitrate-coated stainless steel, lysozyme-coated stainless steel, and gallium nitrate-lysozyme co-loaded stainless steel coating with a size of 2 cm 2 between the erector spinae muscle and the spinous process of the rat. Place a piece of the same type of metal material on each side of the spinous process of each rat, and drop 25 μL of the Staphylococcus aureus-Escherichia coli mixed bacterial solution on both sides. Suture the muscle tightly and close the incision layer by layer; Step 4. After the rats recover from anesthesia, observe their activities, spirits, and diet, and place them in the SPF animal room for observation. On the day, 3 days, 7 days, and 14 days after injury, observe the spirits, activities, and measure their body weights. Step 5. The rats were killed by spinal dislocation 7 and 14 days after surgery to observe the wound healing and the severity of deep tissue inflammation, measure the range of inflammation, and evaluate according to the surgical wound healing grade; Step 6. Cut the rat wound along the original incision, observe and record the infection of the tissue under the wound, remove the metal pieces placed in the body, gently soak and rinse with saline to remove the tissue adhered to the metal surface and the bacteria that are not adhered, and put the five groups of metal pieces into 24-well plates for standby use; Step 7. Prepare live / dead bacterial fluorescent stain strictly according to the instructions of Live / Dead LIVE / DEAD BacLight Bacterial Viability Kits. Take two tubes of fluorescent dye respectively, dissolve them in 10 mL of distilled water, mix thoroughly and store at 4°C. Prepare and use immediately. Step 8. Add 400 μL of fluorescent dye solution to the 24-well plate containing five groups of metal sheets, stain at room temperature for 15 minutes, then place it in 2.5 mL of physiological saline and gently rinse to remove the excess fluorescent dye solution, and then place a glass slide on it and observe under a laser confocal microscope; Step 9. Observe whether there is pus in the removed pathological tissue, and the degree and scope of inflammatory tissue and necrotic tissue, and investigate the pathological changes of the muscle tissue in contact with the metal surface of each group of rats by H&E staining method; Step 10. Randomly sacrifice the animals on the 7th and 14th days after the operation, 3 animals in each group, and perform autopsy on the five groups of animals A, B, C, D, and E. Collect the main organs of the heart, liver, spleen, lung, and kidney of each group and fix them in 4% paraformaldehyde, and perform histopathological examination.

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