Antibacterial and anticoagulant multifunctional material based on polydopamine as well as preparation method and application of antibacterial and anticoagulant multifunctional material

Abstract

The invention relates to an antibacterial and anticoagulant multifunctional material based on polydopamine and a preparation method and application thereof.The preparation method comprises the steps that a base material is cleaned, the base material is soaked in a modification solution, dried after being cleaned for the first time and then soaked in a reduction solution, and dried again after being cleaned for the second time; and then adding a heparin salt solution for incubation, and performing third cleaning to obtain the product. The modification liquid comprises dopamine hydrochloride, copper salt and a buffer solution; the reducing liquid comprises AgNO3 (silver nitrate). The polydopamine antibacterial and anticoagulant multifunctional material prepared by the invention has excellent antibacterial property and anticoagulant property and low cytotoxicity, and can be applied to the surfaces of various blood contact biological materials.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical interventional materials, and particularly relates to an antibacterial and anticoagulant multifunctional material based on polydopamine, and a preparation method and application thereof. Background Art

[0002] Thrombosis and infection are two major clinical complications of indwelling medical devices and implanted / interventional biomedical materials, often leading to the failure of indwelling devices, increased patient morbidity, mortality, and medical costs. In clinical practice, to solve these problems, the combined injection of anticoagulants (such as heparin, argatroban, etc.) and antibiotics to patients is a commonly used treatment method in clinics. However, the abuse of drugs will cause inevitable harm to the human body. Long-term injection of anticoagulants will bring the risk of massive bleeding and may lead to life-threatening heparin-induced thrombocytopenia (HIT). At the same time, the use of antibiotics may trigger the generation of bacterial drug resistance and cause symptoms such as fever, thrombophlebitis, and epidermal necrolysis, which may be worse than the infection itself and will bring great trouble to patients. Therefore, there is an urgent need for new treatment methods to combat thrombosis and infection. Starting from the inherent properties of the material itself and enhancing its anticoagulant and antibacterial properties is a more novel, simple, and harmless solution. Currently, there are mainly two approaches to the above solution: one is the modification of the material body, and the other is the modification of the material surface. The modification of the body is to adjust the composition and structure of the material itself and perform anticoagulant and antibacterial modification on the material itself. This method is complex in operation and high in implementation difficulty. While the modification of the material surface is simple in operation and low in cost, it can not only retain the inherent advantages of the matrix material itself but also greatly improve the comprehensive performance of the material.

[0003] When biomaterials come into contact with blood, the first step is the adsorption of serum proteins, followed by the activation of the coagulation pathway, which leads to thrombus formation. Surface modification has become a common method to improve the blood compatibility of biomaterials. Methods of surface modification include enhancing the hydrophilicity of the biomaterial surface, grafting zwitterionic polymers, designing microphase separation structures, and immobilizing anticoagulant biomacromolecules. Since heparin can inhibit thrombus formation by increasing the affinity of antithrombin III (AT-III) for thrombin, surface heparinization is considered a simple and effective method. Another promising anticoagulant substance is the bio-signaling molecule nitric oxide (NO), which is synthesized and secreted by endothelial cells and has properties such as inhibiting platelet adhesion and activation, anticoagulation, and activating the fibrinolytic system, playing an important role in the human cardiovascular system. Studies have shown that NO inhibits platelet adhesion and activation by upregulating the expression of cyclic guanosine monophosphate (cGMP), and thus is crucial for preventing thrombus formation. Although these surfaces have significantly improved the antithrombotic properties of implanted materials by immobilizing heparin or NO-generating catalytic substances, there are few reports on the synergistic improvement of blood compatibility or multifunctional surfaces by the two, and the antibacterial properties of the surfaces have not been evaluated.

[0004] In addition to thrombus formation, microbial invasion is also a challenge faced by blood-contact devices. Due to the lack of antibacterial properties that may lead to postoperative infections, various attempts have been made to add antibacterial drugs to biomaterials in contact with blood to improve their performance. These materials include natural antibacterial agents (such as chitosan and sorbic acid), organic antibacterial agents (such as antibiotics and quaternary ammonium salts), and inorganic antibacterial agents (such as silver nanoparticles (AgNPs) and others such as metal oxide nanoparticles). However, there is currently no medical material that combines anticoagulation and antibacterial properties to obtain dual functions.

[0005] Therefore, how to find a simple and effective means of dual-functional surface has become an urgent problem to be solved currently. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a poly-dopamine-based antibacterial and anticoagulant multifunctional material, its preparation method and application. The poly-dopamine-based antibacterial and anticoagulant multifunctional material prepared by the present invention has excellent antibacterial and anticoagulant properties and low cytotoxicity, and can be applied to the surfaces of various blood-contact biomaterials.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a poly-dopamine-based antibacterial and anticoagulant multifunctional material. The steps of the preparation method include cleaning a substrate, soaking the substrate in a modification solution, drying after the first cleaning, then soaking in a reduction solution, drying again after the second cleaning, and then incubating in a heparin salt solution, and obtaining the product after the third cleaning;

[0009] The modification solution includes dopamine hydrochloride, a copper salt, and a buffer solution;

[0010] The reduction solution includes AgNO 3 、AgNO 3 、 any one or a combination of at least two of AgF, AgCl, AgBr, or AgI.

[0011] Existing invention technologies that simultaneously achieve two functions are difficult or involve relatively complex sample pretreatment and cumbersome chemical processes, and the stability of the coating is often poor. The preparation method of the present invention mainly forms a coating by solution soaking, with relatively simple operations. The reagents used are easily obtained and are all non-toxic and harmless, causing no pollution to the environment, and do not involve excessive complex operations, which is more conducive to application in actual production.

[0012] Preferably, the cleaning agent for cleaning the substrate includes any one or a combination of at least two of deionized water, acetone, or absolute ethanol.

[0013] Preferably, the substrate includes any one or a combination of at least two of stainless steel, pure titanium, or a titanium alloy.

[0014] Preferably, the titanium alloy includes Ti-6Al-4V and / or Ti-3Al-5Mo-4.5V.

[0015] Preferably, the copper salt includes any one or a combination of at least two of copper chloride, copper carbonate, copper sulfate, copper nitrate, copper iodide, copper acetate, or cupric acetate.

[0016] Preferably, the concentration of dopamine hydrochloride in the modification solution is 0.5 - 5 mg / mL, for example, it can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5 mg / mL, etc.

[0017] Preferably, the concentration of the copper salt in the modification solution is 0.1 - 1 mg / mL, for example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, etc.

[0018] Preferably, the buffer solution includes Tris-HCl buffer solution.

[0019] Preferably, the concentration of the buffer solution is 10-100 mM, for example, it can be 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, etc.

[0020] Preferably, the pH value of the buffer solution is 8-9, for example, it can be 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9, etc.

[0021] Preferably, the soaking time of the substrate in the modification solution is 12-24 h, and the temperature is 20-30 °C. The 12-24 h can be, for example, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h or 24 h, etc. The 20-30 °C can be, for example, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C or 30 °C, etc.

[0022] Preferably, the cleaning agent for the first cleaning includes any one or a combination of at least two of deionized water, acetone or absolute ethanol.

[0023] Preferably, the environmental condition for drying is a vacuum environment.

[0024] Preferably, the drying temperature is 40-60 °C, and the time is 4-8 h. The 40-60 °C can be, for example, 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C or 60 °C, etc. The 4-8 h can be, for example, 4 h, 5 h, 6 h, 7 h or 8 h, etc.

[0025] Preferably, the concentration of the reducing solution is 0.1-1 mg / mL, for example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1 mg / mL, etc.

[0026] Preferably, the soaking time of the substrate in the reducing solution is 8-16 h, and the temperature is 20-30 °C. The 8-16 h can be, for example, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h or 16 h, etc. The 20-30 °C can be, for example, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C or 30 °C, etc.

[0027] In the present invention, the method for connecting silver nanoparticles is relatively simple. Only immersion reduction is required to obtain the connected multifunctional material, without the need for too many complex preparation methods. It can be prepared only by immersion incubation. Moreover, the properties of silver nanoparticles are relatively stable, with low cytotoxicity and high safety.

[0028] The cleaning agent for the second cleaning includes any one or a combination of at least two of deionized water, acetone or absolute ethanol.

[0029] Preferably, the environmental condition for the re-drying is a vacuum environment.

[0030] Preferably, the temperature for the re-drying is 40 - 60 °C and the time is 4 - 8 h. The 40 - 60 °C can be, for example, 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C or 60 °C, etc. The 4 - 8 h can be, for example, 4 h, 5 h, 6 h, 7 h or 8 h, etc.

[0031] Preferably, the heparin salt solution includes sodium heparin.

[0032] Preferably, the concentration of the heparin salt solution is 1 - 10 mg / mL. It can be, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL, etc.

[0033] Preferably, the preparation method of the heparin salt solution includes dissolving the heparin salt in PBS buffer solution;

[0034] Preferably, the pH value of the PBS buffer solution is 7 - 9. It can be, for example, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8 or 9, etc.

[0035] Preferably, the incubation time is 1 - 24 h and the temperature is 20 - 30 °C. The 1 - 24 h can be, for example, 1 h, 5 h, 10 h, 15 h, 20 h or 24 h, etc. The 20 - 30 °C can be, for example, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C or 30 °C, etc.

[0036] Preferably, the cleaning agent for the third cleaning includes any one or a combination of at least two of PBS buffer solution, deionized water, acetone or absolute ethanol.

[0037] In the second aspect, the present invention provides a poly-dopamine-based antibacterial and anticoagulant multifunctional material, which is prepared according to the preparation method of the poly-dopamine-based antibacterial and anticoagulant multifunctional material described in the first aspect.

[0038] In the present invention, heparin and metal nanoparticles are respectively connected on the basis of polydopamine. Among them, heparin, as an anticoagulant biopolymer, can significantly improve the antithrombotic effect of the material, while metal nanoparticles have excellent bactericidal effects. The combined use of the above two materials can achieve the dual functions of antibacterial and anticoagulation.

[0039] In the experiments of the present invention, the silver nanoparticles used belong to natural antibacterial agents. Compared with organic antibacterial agents, inorganic antibacterial agents have more stable and lasting antibacterial ability. Silver nanoparticles (AgNPs) are clusters of silver atoms with diameters ranging from 1 to 100 nanometers. AgNPs and Ag+ have antibacterial effects on a variety of microorganisms, including Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and many fungi. More importantly, AgNPs do not cause bacterial resistance like other antibiotics.

[0040] In the present invention, polydopamine fixes copper ions through reversible coordination bonds to form metal-phenolic networks (MPNs) with copper ions. And silver nanoparticles are formed by reducing silver ions in silver nitrate to silver nanoparticles by the reducing groups (such as phenolic hydroxyl groups) in polydopamine.

[0041] In a third aspect, the present invention provides an application of the polydopamine-based antibacterial and anticoagulant multifunctional material according to the second aspect in the preparation of anticoagulant and antibacterial biomedical implant materials.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] 1. The present invention provides a polydopamine-based antibacterial and anticoagulant multifunctional material. The polydopamine coating can provide stable adhesion, and at the same time, a catalyst for generating nitric oxide (copper ions) and heparin can be secondarily loaded and fixed on the coating, and silver nanoparticles can be in-situ reduced on its surface to obtain an antibacterial and anticoagulant multifunctional material.

[0044] 2. The preparation process adopted in the present invention mainly forms a coating by solution immersion. The operation is relatively simple, the reagents used are easily obtained, and they are all non-toxic and harmless and will not cause environmental pollution, which is suitable for subsequent production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the implementation roadmap of Example 1;

[0046] Figure 2 It is the result diagram of scanning electron microscope test;

[0047] Figure 3 It is the column chart of the water contact angle test results;

[0048] Figure 4 Experimental result graph for nitric oxide release test;

[0049] Figure 5 Scanning electron microscope image for anti - platelet adhesion test;

[0050] Figure 6 Columnar result graph for anti - platelet adhesion rate;

[0051] Figure 7 Experimental result graph for APTT test;

[0052] Figure 8 Colony growth result graph for antibacterial test;

[0053] Figure 9 Inhibition efficiency result graph against Escherichia coli;

[0054] Figure 10 Inhibition efficiency result graph against Staphylococcus aureus;

[0055] Figure 11 Experimental result graph for cytotoxicity test;

[0056] Figure 12 Experimental result graph for coating thickness change rate;

[0057] Figure 13 Experimental result graph for water contact angle change within one week. Detailed implementation mode

[0058] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation modes. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the patent protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0059] Sources of reagents and instruments used in the following examples:

[0060] Scanning electron microscope: FESEM, JSM - IT800, JEOL, Japan;

[0061] Contact angle system: DSAHT1600, KRUSS GmbH, Germany;

[0062] Nitric oxide assay kit: Total nitric oxide detection kit, S0024, Beyoinme, China;

[0063] Laser three - dimensional imaging microscope system: VKX150K, KEYENCE, Japan.

[0064] Example 1

[0065] In this example, a multifunctional material with antibacterial and anticoagulant properties based on polydopamine was prepared.

[0066] (1) Substrate surface pretreatment

[0067] Using a cutting machine, commercially available pure titanium Ti substrates were pre-cut into rectangular samples of the following dimensions: 10 mm × 10 mm × 5 mm. The cut samples were successively polished and cleaned with metallographic sandpapers of specifications 200#, 2000#, and 5000#, and then polished to a mirror surface with a polishing solution. Then, the samples were ultrasonically cleaned with deionized water, acetone, and absolute ethanol for 20 min each to remove organic substances, oils, and impurities on the titanium surface. Finally, the surfaces of all samples were thoroughly rinsed with deionized water and placed in a vacuum drying oven at 45 °C until completely dry to obtain clean substrates.

[0068] (2) Connecting polydopamine to the substrate surface

[0069] The prepared substrate Ti was immersed in 10 mL of a Tris-HCl buffer solution (10.0 mM, pH = 8.5) containing 2 mg / mL dopamine hydrochloride (DA) and 0.2 mg / mL copper chloride at 25 °C for 12 h. After washing 3 times with deionized water, it was dried in a vacuum oven at 45 °C for 4 h to obtain a Ti substrate coated with polydopamine PDA.

[0070] (3) Connecting metal nanoparticles

[0071] Silver nitrate was dissolved in deionized water to prepare a 0.2 mg / mL AgNO 3 solution. The sample prepared in step (2) was immersed in 10 mL of the AgNO 3 solution at 25 °C for 16 h. Then, the sample was taken out, thoroughly rinsed 3 times with deionized water, and dried in a vacuum oven at 45 °C for 4 h.

[0072] (4) Grafting heparin

[0073] Heparin sodium solution was added to a PBS buffer solution with a pH value of 7.2 to prepare a heparin sodium solution with a concentration of 5 mg / mL. The sample prepared in step (3) was immersed in the heparin sodium solution and incubated at 25 °C for 60 min, rinsed three times with PBS, and dried in a vacuum oven at 45 °C for 4 h. The sample was named TiDA / Cu-Hep@Ag. The specific preparation process is as Figure 1 shown.

[0074] Comparative Example 1

[0075] In this comparative example, a multifunctional material with antibacterial and anticoagulant properties based on polydopamine was prepared. The difference from Example 1 was that steps (2) - (4) were not carried out, and the sample was named Ti, and the rest was the same as in Example 1.

[0076] Comparative Example 2

[0077] In this comparative example, a poly-dopamine-based antibacterial and anticoagulant multifunctional material was prepared. The difference from Example 1 was only that steps (3)-(4) were not carried out, and the sample was named TiDA / Cu, and the rest was the same as in Example 1.

[0078] Comparative Example 3

[0079] In this comparative example, a poly-dopamine-based antibacterial and anticoagulant multifunctional material was prepared. The difference from Example 1 was only that step (3) was not carried out, and the sample was named TiDA / Cu-Hep, and the rest was the same as in Example 1.

[0080] Comparative Example 4

[0081] In this comparative example, a poly-dopamine-based antibacterial and anticoagulant multifunctional material was prepared. The difference from Example 1 was only that step (4) was not carried out, and the sample was named TiDA / Cu@Ag, and the rest was the same as in Example 1.

[0082] Test Example 1

[0083] In this test example, the samples prepared in the above examples and comparative examples were characterized and tested.

[0084] The above samples were characterized and tested, including scanning electron microscopy and water contact angle, to verify the successful preparation of the coating and its hydrophilic properties. To study the hydrophilicity of the coating, the water contact angle (WCA) of 2 μL of ultrapure water droplets at room temperature (25 °C) was evaluated using a contact angle system. Among them, 3 parallel samples were measured for each sample, and the average value was taken and the standard deviation was calculated.

[0085] The scanning electron microscopy detection results are as Figure 2 shown. The surface morphology of the original Ti prepared in Comparative Example 1 was smooth and flat, while there were abundant aggregates of polydopamine nanoparticles on the TiDA / Cu surface prepared in Comparative Example 2. By analyzing higher-resolution images, it could be clearly seen that the average diameter of the polydopamine nanoparticles was about 230 nm. Then, after immersing the metal surface in AgNO 3 solution, many new nanoparticles different from the polydopamine aggregates could be seen on the surface of the TiDA / Cu@Ag sample prepared in Comparative Example 4, confirming that the rich hydroxyl structure of the PDA layer could in-situ reduce silver ions to AgNPs. The size of the Ag nanoparticles was 100 nm, smaller than the aggregates of polydopamine nanoparticles, and the shape of the AgNPs was also different from that of the polydopamine nanoparticles, with spherical, oval, and rod-shaped.

[0086] From Figure 3It can be seen that the water contact angle of TiDA / Cu prepared in Comparative Example 2 is 63.8°, showing little change compared with that of pure Ti (66.23°) prepared in Comparative Example 1, and the hydrophilicity of both is weaker than that of the heparin-grafted surface. However, after heparin was grafted onto the PDA surface, that is, TiDA / Cu-Hep prepared in Comparative Example 3, the contact angle decreased from 66.23° to 35.8°, indicating enhanced hydrophilicity, which is due to the successful grafting of hydrophilic heparin. After the deposition of silver nanoparticles, the surface water contact angle remained stable at about 40°, indicating that the sample has good hydrophilicity and can partially inhibit platelet adsorption.

[0087] Test Example 2

[0088] This test example detects the anticoagulant performance of the samples prepared in the above-mentioned examples and test examples.

[0089] (1) Nitric oxide release detection

[0090] Using a nitric oxide assay kit, the release behavior of NO catalyzed by immobilized Cu ions was analyzed using Griess reagent. Before the experiment, the samples prepared in Example 1 were soaked in 1% SDS for 30 min, then soaked in DIW (5 times), and then thoroughly washed 3 times with phosphate buffered saline (PBS). The samples were then incubated in 1 mL of PBS solution containing 10 μM NO donor L-glutathione (GSH) and 10 μM S-nitrosoglutathione (GSNO) in the dark at 37 °C. The standard reagents (Griess reagent I and II) were diluted with PBS solution. 50 μL of the sample solution was added to a 96-well microplate, and then 50 μL of the diluted Griess reagent I and II were added to each well in sequence. After incubating the reaction for 15 minutes, the absorbance of the mixed solution at 540 nm was measured using a microplate reader (Multiskan FC, Thermo Scientific). Finally, the cumulative concentration of catalytic NO was calculated according to the calibration curve constructed with the NaNO 2 concentration provided in the kit, and the results were evaluated as a function of time.

[0091] It can be seen from Figure 4 that within the first 24 hours, in the presence of the drug, NO was rapidly released from the TiDA / Cu-Hep@Ag surface prepared in Example 1 in PBS, and the concentration was 18 μM after 24 hours. In addition, the release rate of NO from the modified surface remained relatively fast in the first 3 days and then slowed down in the following 4 days. After continuous release for 7 days, the NO concentration reached 94.2 μM. These results indicate that the immobilized Cu 2+It has good catalytic activity, and the prepared surface can effectively decompose endogenous donors to generate NO in a physiological environment. Therefore, we believe that the TiDA / Cu-Hep@Ag surface prepared in Example 1 has good potential for application as an anticoagulant material because it can stably release NO for 7 days.

[0092] (2) Anti-platelet adhesion experiment

[0093] Before the experiment, the samples prepared in the above examples, comparative examples, and glass were irradiated with ultraviolet light at 254 nm for 15 min, and 5 mL of PBS with a pH of 7.4 was added to a 10 mL centrifuge tube and equilibrated for 12 h. To obtain platelet-rich plasma (PRP), fresh rabbit whole blood containing anticoagulant citrate was centrifuged at 1000 rpm for 10 min. Considering chemical instability, 200 μL of PRP with additional NO donors (10 μM GSNO and 10 μM GSH) was added to the surface of the prepared samples, incubated at 37 °C for 120 min, then gently rinsed 3 times with NaCl solution, and fixed with 4% paraformaldehyde fixative at room temperature for 2 h. To observe the morphology of platelets, the samples were dehydrated, sputter-coated with gold, and subjected to scanning electron microscopy testing at an accelerating voltage of 15 kV. Finally, the number of platelets adhered to the samples was recorded using a laser three-dimensional imaging microscope system.

[0094] It can be seen from Figure 5 that abundant platelets adhered to the original Ti surface prepared in Comparative Example 1, showing a spreading dendritic or branched shape with a high density; on the contrary, after fixing the metal phenolic network containing Cu ions, the number of adhered platelets decreased significantly because Cu ions can catalyze the decomposition of GSNO to generate NO to counteract platelet adhesion. The platelet morphology on TiDA / Cu prepared in Comparative Example 2 and TiDA / Cu@Ag prepared in Comparative Example 4 was round or oval with fewer pseudopods. In addition, the platelets on TiDA / Cu-Hep prepared in Comparative Example 3 and TiDA / Cu-Hep@Ag prepared in Example 1 were very dispersed due to the introduction of heparin, the platelet shape was very regular, with little deformation, and had good anti-platelet adhesion ability.

[0095] The results showed a trend similar to that of the APTT test. The number of platelets adhered to the samples was measured using a laser three-dimensional imaging microscope system, and the results are as Figure 6 shown. The quantitative results showed that the platelet adhesion rate on pure Ti was similar to that on glass, which was 91.5%, while the platelet adhesion rates of other samples decreased significantly, which were 30.8%, 32.2%, 21.8%, and 28.3% respectively.

[0096] (3) APTT test

[0097] The activated partial thromboplastin time (APTT) was evaluated by exposing platelet-poor plasma (PPP) to pure titanium / modified titanium surfaces. Fresh whole blood was collected from rabbits, and the citrate blood was centrifuged at 3500 rpm for 10 minutes in a test tube to separate PPP. The sterilized samples prepared in the examples, comparative examples, and glass were incubated with 0.5 ml of PPP at 37 °C for 3 hours. Then, 50 μL of the incubated PPP and 50 μL of APTT activating substance were thoroughly mixed and incubated at 37 °C for 3 minutes. Finally, the APTT values of different samples after incubation were detected using an automatic coagulation analyzer, and the APTT of fresh PPP was measured as the positive control group

[0098] As can be seen from Figure 7 that the coagulation time of the pure Ti substrate prepared in Comparative Example 1 was 28.2 ± 0.1 s, showing no significant change compared with the control group PPP (28.2 ± 0.2 s). In contrast, the APTT times of TiDA / Cu prepared in Comparative Example 2 and TiDA / Cu@Ag prepared in Comparative Example 4 were significantly prolonged, being 47.1 ± 1.6 s and 42.0 ± 0.9 s, respectively, indicating that the generation of NO by the PDA coating enhanced the anticoagulant performance. Among them, the TiDA / Cu-Hep group prepared in Comparative Example 3 and the TiDA / Cu-Hep@Ag group prepared in Example 1 had the longest coagulation times, being 61.0 ± 1.6 s and 58.6 ± 1.3 s, respectively. The results of APTT were completely consistent with the results of NO generation, reflecting that the modified samples had good blood compatibility and could inhibit the formation of thrombus after clinical implantation

[0099] Test Example 3

[0100] The antibacterial ability of the samples prepared in the above examples and test examples was tested in this test example

[0101] To evaluate the antibacterial properties of different samples, Gram-negative Escherichia coli (E. coli, ATCC25922) and Gram-positive Staphylococcus aureus (S. aureus, ATCC25922) were used. LB liquid and solid media, bacterial suspensions, and sterilized samples prepared in the examples and comparative examples were prepared in advance. After diluting the bacterial suspension to 1.0×10 6 CFU / mL, 200 μL of the bacterial suspension was added and placed in a 24-well plate. After incubating at 37 °C in a shaker for 24 h, the bacterial suspension was eluted with 1 mL of sterile PBS solution. The collected bacterial suspension was inoculated on Luria-Bertani (LB) agar plates, and serial dilutions of 10-fold were made for counting. Finally, the colony formation and the number of bacteria were recorded, and the relative survival rate and antibacterial performance of the bacteria were analyzed

[0102] Figure 8 、 Figure 9 and Figure 10shows the formation and number of colonies after culturing with different samples, as well as the corresponding quantitative antibacterial efficiency of each group. From Figure 8 , it can be seen that according to the colony images, compared with the sample prepared in Comparative Example 1, the number of colonies on the surfaces of TiDA / Cu prepared in Comparative Example 2 and TiDA / Cu-Hep prepared in Comparative Example 3 did not decrease significantly. On the contrary, after culturing with TiDA / Cu@Ag prepared in Comparative Example 4 and TiDA / Cu-Hep@Ag prepared in Example 1, the number of colonies of the two bacteria on the LB agar plates decreased significantly. According to the quantitative analysis, as Figure 9 and Figure 10 show, the number of colonies of Escherichia coli and Staphylococcus aureus on the surface of TiDA / Cu@Ag prepared in Comparative Example 4 decreased significantly by 2 logarithmic values, by 99.99% and 98.14% respectively. The heparin-grafted surface TiDA / Cu-Hep@Ag prepared in Example 1 also showed similar results to TiDA / Cu@Ag prepared in Comparative Example 4, with antibacterial efficiency against Escherichia coli (99.95%) and Staphylococcus aureus (97.74%). These results indicate that the modified surface has excellent antibacterial properties against both Escherichia coli and Staphylococcus aureus, which is attributed to the role of AgNPs as antibacterial agents.

[0103] Test Example 4

[0104] This test example conducts a cytotoxicity experiment to characterize biocompatibility

[0105] The cell compatibility of the samples was detected by the CCK-8 method and the Live / Dead Cell Staining Kit for human umbilical vein endothelial cells (HUVECs). For this purpose, HUVECs were first harvested and seeded in 24-well plates at a density of 1×10 4 cells / well and allowed to adhere for 12 hours. After autoclaving, the sterilized samples were placed in the well plates. HUVECs in the logarithmic growth phase were seeded in 24-well plates at a density of 4×10 4 cells / well and cultured in a 37°C, 5% CO 2 incubator for 24 and 72 hours. Then, the culture medium was removed, and each well was washed 3 times with PBS. A culture medium containing 10% CCK-8 and 5% CO 2 was added at 1000 μL / well and cultured in a 37°C incubator for 2 hours. 100 μL / well of the supernatant was transferred to a new 96-well plate, and the absorbance at 450 nm was detected by an enzyme-linked immunosorbent assay analyzer. Finally, live / dead staining was observed with an inverted fluorescence microscope.

[0106] The test results are as Figure 11 shown. From Figure 11It can be seen that the toxicity of TiDA / Cu@Ag prepared in Comparative Example 4 and TiDA / Cu-Hep@Ag prepared in Example 1 is not obvious. Compared with the pure Ti sample prepared in Comparative Example 1, after culturing with HUVECs for 24 h, TiDA / Cu-Hep prepared in Comparative Example 3 has a slight promoting effect on cell proliferation. Among them, after culturing with TiDA / Cu, TiDA / Cu@Ag, TiDA / Cu-hep and TiDA / Cu-Hep@Ag for 24 h, the cell survival rates are 98.01, 95.42, 100.53 and 93.09%, respectively. The above results show that although AgNPs have antibacterial properties, our samples still maintain good cell compatibility with HUVECs.

[0107] Test Example 5

[0108] In this test example, a stability test was carried out to characterize the practicability of the coating structure.

[0109] To evaluate the durability of the surface coating, the TiDA / Cu-Hep@Ag sample prepared in Example 1 was immersed in 5 ml of phosphate buffered saline (PBS) with a pH of 5.5 and shaken at a speed of 120 revolutions per minute at room temperature. The changes in the water contact angle and film thickness of the TiDA / Cu-Hep@Ag substrate were monitored within one week.

[0110] The test results are as Figure 12 、 Figure 13 shown. From Figure 12 the results show that after incubation in the oscillating acidic environment for 7 days, the thickness of the prepared coating only decreased to 83.2 ± 1.4%. From Figure 13 it can also be seen that the water contact angle WCA of the TiDA / Cu-Hep@Ag coating does not change significantly with time. It shows that the preparation of polydopamine and AgNPs on the Ti substrate is stable, and the TiDA / Cu-Hep@Ag coating has good stability, which is crucial for the application of this coating as an anticoagulant / antibacterial surface.

[0111] In summary, the antibacterial and anticoagulant multifunctional material of polydopamine prepared by the present invention has excellent antibacterial and anticoagulant properties and low cytotoxicity, and can be applied to the surfaces of various blood-contact biomaterials.

[0112] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for preparing an antibacterial and anticoagulant multifunctional material based on polydopamine, characterized in that: The preparation method comprises the steps of washing the substrate, soaking the substrate in a modification solution, drying after a first washing, soaking the substrate in a reducing solution, washing the substrate for a second time, drying the substrate again, adding a heparin salt solution for incubation, and washing the substrate for a third time to obtain the substrate; The modification solution comprises dopamine hydrochloride, copper salt and buffer; The reducing solution includes any one of AgNO3, AgNO3, AgF, AgCl, AgBr or AgI, or a combination of at least two thereof.

2. The method for preparing the antibacterial and anticoagulant multifunctional material based on polydopamine according to claim 1, characterized in that: The cleaning agent for cleaning the substrate includes any one of deionized water, acetone or anhydrous ethanol, or a combination of at least two of them.

3. The method for preparing the antibacterial and anticoagulant multifunctional material based on polydopamine according to claim 1 or 2, characterized in that: The substrate includes any one of stainless steel, pure titanium or titanium alloy, or a combination of at least two thereof; Preferably, the titanium alloy includes Ti-6Al-4V and / or Ti-3Al-5Mo-4.5V.

4. The method for preparing the antibacterial and anticoagulant multifunctional material based on polydopamine according to any one of claims 1 to 3, characterized in that: The copper salt includes any one of copper chloride, copper carbonate, copper sulfate, copper nitrate, copper iodide, copper acetate or copper acetate, or a combination of at least two thereof; Preferably, the concentration of dopamine hydrochloride in the modification solution is 0.5-5 mg / mL; Preferably, the concentration of copper salt in the modification solution is 0.1-1 mg / mL; Preferably, the buffer comprises a Tris-HCl buffer solution; Preferably, the concentration of the buffer is 10-100 mM; Preferably, the pH value of the buffer solution is 8-9.

5. The method for preparing the antibacterial and anticoagulant multifunctional material based on polydopamine according to any one of claims 1 to 4, characterized in that: The substrate is immersed in the modification solution for 12-24 hours at a temperature of 20-30°C; The cleaning agent for the first cleaning includes any one of deionized water, acetone or anhydrous ethanol, or a combination of at least two thereof; Preferably, the drying environment condition is a vacuum environment; Preferably, the drying temperature is 40-60° C. and the drying time is 4-8 hours.

6. The method for preparing the antibacterial and anticoagulant multifunctional material based on polydopamine according to any one of claims 1 to 5, characterized in that: The concentration of the reducing solution is 0.1-1 mg / mL; Preferably, the substrate is immersed in the reducing solution for 8-16 hours at a temperature of 20-30°C; Preferably, the cleaning agent for the second cleaning includes any one of deionized water, acetone or anhydrous ethanol, or a combination of at least two thereof; Preferably, the environmental condition for the secondary drying is a vacuum environment; Preferably, the re-drying temperature is 40-60° C. and the time is 4-8 hours.

7. The method for preparing the antibacterial and anticoagulant multifunctional material based on polydopamine according to any one of claims 1 to 6, characterized in that: The heparin salt solution includes heparin sodium; Preferably, the concentration of the heparin salt solution is 1-10 mg / mL; Preferably, the method for preparing the heparin saline solution comprises dissolving the heparin saline solution in PBS buffer; Preferably, the pH value of the PBS buffer is 7-9.

8. The method for preparing the antibacterial and anticoagulant multifunctional material based on polydopamine according to any one of claims 1 to 7, characterized in that: The incubation time is 1-24h, and the temperature is 20-30°C; Preferably, the cleaning agent for the third cleaning includes any one of PBS buffer, deionized water, acetone or anhydrous ethanol, or a combination of at least two thereof.

9. An antibacterial and anticoagulant multifunctional material based on polydopamine, characterized in that: The multifunctional material is prepared according to the method for preparing an antibacterial and anticoagulant multifunctional material based on polydopamine according to any one of claims 1-8.

10. Use of the antibacterial and anticoagulant multifunctional material based on polydopamine according to claim 9 in the preparation of anticoagulant and antibacterial biomedical implant materials.

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