A biomedical coating and its preparation method and application

Abstract

The present invention discloses a biomedical coating and its preparation method and application. The coating comprises a polymer of formula I, the coating preparation method is simple, the coating is not easy to fall off and has excellent blood compatibility, and can be applied to the surface of a large number of substrate materials, so that the surface of the medical catheter has anticoagulant, antibacterial and bactericidal properties at the same time, avoiding the occurrence of thrombosis, blood vessel blockage and bacterial infection after implantation in the body.

Description

Technical Field

[0001] The present invention relates to the technical field of medical implant / intervention materials, and in particular to a biomedical coating and a preparation method and application thereof. Background Art

[0002] As an important tool in modern medicine, medical catheters are widely used as access channels in drainage, perfusion, drug administration, blood collection, and blood transmission, providing a strong technical guarantee for prolonging patients' lives and improving their quality of life. Among them, medical catheters that come into contact with blood often face complications such as thromboembolism and postoperative infection in clinical applications. These complications not only affect the service life of medical catheters, but also increase the physiological pain and economic burden of patients.

[0003] At present, the main strategy used in clinical treatment of complications is systemic administration of antibiotics and anticoagulants, but the use of antibiotics can easily lead to drug resistance in the body, and the use of anticoagulants such as heparin may cause side effects such as massive bleeding and multiple organ dysfunction. Therefore, solving the problem of medical catheters easily inducing thrombosis and infection during use is of great clinical significance.

[0004] The related art provides a method for preparing an antibacterial coating on the surface of medical metal using dopamine monomers and zwitterionic monomers containing double bonds in the presence of a small amount of cross-linking agent. The polydopamine-zwitterionic antibacterial coating prepared by this method has good antibacterial properties and biocompatibility. However, the coating prepared by this method only has the function of inhibiting bacterial adhesion, and has no bactericidal and anticoagulant properties, and faces the problems of bacterial infection and thromboembolism during clinical use. Relevant scholars have also studied the formation of Ag-polydopamine / tannic acid antibacterial coating on titanium through dopamine / tannic acid, and explored the antibacterial activity and toxicity of different silver ion concentrations. This type of antibacterial coating has the disadvantages of toxic accumulation and poor blood compatibility. Moreover, the coating only has a bactericidal function and no anticoagulant properties, and still faces the problems of bacterial adhesion and thromboembolism during clinical use. There is also a related technology that provides a method for preparing an antibacterial and anticoagulant coating, in which dopamine and polyethyleneimine are used to prepare a dopamine coating, and chitosan and heparin are used to prepare an anticoagulant and antibacterial composite coating. EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) are used to covalently bond the hydroxyl and quinone groups of the dopamine coating with the amino and carboxyl groups of the anticoagulant and antibacterial composite layer to achieve chemical composite of the two layers. However, the anticoagulant and antibacterial composite coating needs to react with a large number of groups of the chitosan and heparin molecules themselves, resulting in a decrease in the activity of the antibacterial and anticoagulant groups of the chitosan and heparin molecules in the coating, and the antibacterial and anticoagulant effects become worse. In addition, heparin has the risk of causing massive bleeding; the preparation of the coating also requires the addition of EDC and NHS amidation reagents, and the steps are cumbersome. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a biomedical coating and a preparation method and application thereof. The coating preparation method is simple, the coating is not easy to fall off and has excellent blood compatibility, and can be applied to the surface of a large number of substrate materials, so that the surface of the medical catheter has anticoagulant, antibacterial and bactericidal properties at the same time, avoiding the occurrence of thrombosis, vascular occlusion and bacterial infection after implantation in the body.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] According to a first aspect of the present invention, a polymer of formula I is provided:

[0008]

[0009] Among them, R1 is selected from a single bond, C1~C5 alkylene, substituted C1~C5 alkylene, and C1~C5 amide; R2 is selected from a single bond or C1~C5 alkyl or substituted C1~C5 alkyl; R3 is selected from a single bond or C1~C5 alkyl or substituted C1~C5 alkyl; R4 is selected from a single bond, C1~C5 alkylene or substituted C1~C5 alkylene, and C1~C5 acyloxy; R5 is a zwitterionic group; R6 is selected from a single bond, C1~C5 alkyl or substituted C1~C5 alkyl; R7 is selected from a single bond, a double bond, C1~C5 alkylene or substituted C1~C5 alkylene, C1~C5 acyloxy, and phenyl; a, b, and c are independently selected from any integer from 1 to 5.

[0010] In some embodiments of the present invention, the molecular weight of the polymer is 10 KDa to 180 KDa; preferably 10 KDa to 50 KDa; preferably 12 KDa to 20 KDa.

[0011] In some preferred embodiments of the present invention, the zwitterionic group is selected from at least one of a phosphorylcholine group, a carboxylate betaine group or a sulfonate betaine group.

[0012] In some more preferred embodiments of the present invention, the substituents of the alkylene or alkyl groups include halogen.

[0013] In some more preferred embodiments of the present invention, the polymer of formula I is selected from:

[0014]

[0015]

[0016] Wherein, the definitions of a, b, and c are as described above.

[0017] According to a second aspect of the present invention, a method for preparing the polymer is provided, comprising the following steps:

[0018] In an inert atmosphere, an initiator is added to a mixed solution of a zwitterionic derivative modified with an active olefin group, a dopamine derivative modified with an active olefin group and a sulfonate modified with an active olefin group to carry out a free radical polymerization reaction to obtain the polymer.

[0019] In some embodiments of the present invention, the mass ratio of zwitterion derivatives, dopamine derivatives and sulfonates in the mixed solution is (1-5): (1-3): (1-5); preferably, the mixed solution comprises 30wt%-50wt% of zwitterion derivatives, 20wt% of dopamine derivatives, and 30wt%-50wt% of sulfonates; preferably, the mixed solution comprises 30wt% of zwitterion derivatives, 20wt% of dopamine derivatives, and 50wt% of sulfonates; preferably, the mixed solution comprises 40wt% of zwitterion derivatives, 20wt% of dopamine derivatives, and 40wt% of sulfonates; preferably, the mixed solution comprises 50wt% of zwitterion derivatives, 20wt% of dopamine derivatives, and 30wt% of sulfonates.

[0020] In some preferred embodiments of the present invention, the zwitterionic derivative includes at least one of 2-methacryloyloxyethyl phosphorylcholine, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt.

[0021] In some more preferred embodiments of the present invention, the dopamine derivative includes at least one of 3-methacryloyl dopamine, N-[2-(3,4-dihydroxyphenyl)ethyl]-2-acrylamide, and 3-(3,4-dihydroxyphenyl)-N-propylene-2-propionamide.

[0022] In some more preferred embodiments of the present invention, the sulfonate includes at least one of sodium p-styrene sulfonate, sodium vinyl sulfonate, and sodium allyl sulfonate.

[0023] In some more preferred embodiments of the present invention, the initiator includes at least one of azobisisobutyronitrile, azobisisoheptanenitrile and dimethyl azobisisobutyrate.

[0024] In some more preferred embodiments of the present invention, the solvent of the mixed solution includes at least one of methanol, ethanol, N,N-dimethylformamide, dimethyl sulfoxide or water.

[0025] In some more preferred embodiments of the present invention, the temperature of the free radical polymerization reaction is 60°C to 80°C, preferably 65°C.

[0026] According to a third aspect of the present invention, a coating is provided, comprising the polymer.

[0027] In some embodiments of the present invention, the mass concentration of the polymer in the coating is 0.5 mg / mL to 10 mg / mL.

[0028] In some preferred embodiments of the present invention, the coating further comprises nanosilver particles; preferably, the mass concentration of the nanosilver particles is 0.01 mg / mL to 1.0 mg / mL; preferably, the mass concentration of the nanosilver particles is 0.1 mg / mL to 0.8 mg / mL; preferably, the mass concentration of the nanosilver particles is 0.1 mg / mL to 0.5 mg / mL.

[0029] According to a fourth aspect of the present invention, a medical material is provided, comprising a substrate and the coating loaded on the substrate.

[0030] In some embodiments of the present invention, the substrate includes at least one of PET, polycarbonate, silicone, silicone rubber, polyurethane, polystyrene, polyamide, polyether block polyamide, fluorinated ethylene-propylene copolymer, polymethyl methacrylate, thermoplastic elastomer, latex, stainless steel, polyethylene, polyvinyl chloride, polypropylene, polyurethane, polyurethane elastomer, polylactic acid, and polytetrafluoroethylene.

[0031] The beneficial effects of the present invention are:

[0032] 1. The dopamine in the polymer of the present invention is oxidized and self-polymerized, not only to form an active polydopamine thin layer on the surface of the substrate, making it an antifouling coating that is not easy to fall off, but also to enhance the overall stability of the coating; on the other hand, polydopamine reduces silver ions to nanosilver, and fixes the nanosilver on the surface of the substrate through the adhesion of polydopamine. The sulfonate structure monomer modified with active alkenyl in the polymer has an anticoagulant activity similar to that of heparin, and overcomes the shortcomings of high cost and easy inactivation of heparin.

[0033] 2. The coating of the present invention has high stability and has anticoagulant, antibacterial and bactericidal effects. The medical materials modified with the coating can not only resist protein adsorption, platelet and bacterial adhesion, but also kill bacteria. The anti-infection rate of bacteria is as high as over 98%, and the antibacterial effect is obvious. At the same time, the platelet adhesion is reduced by more than about 98%, the whole blood coagulation time is prolonged by more than double, the anti-thrombosis effect is obvious, and the anticoagulant and antibacterial functions are effectively realized.

[0034] 3. The coating of the present invention can modify a wide range of substrates, not limited to the modification of medical catheters, but can be widely used in the surface modification of equipment materials that come into contact with blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the infrared spectrum of PMDS, the product of S1 in Example 1 of the present invention.

[0036] Figure 2 This is the SEM image of the S3 coating in Example 1 of the present invention.

[0037] Figure 3 This is the SEM image of the S3 coating in Example 3 of the present invention.

[0038] Figure 4 The antibacterial effect diagram of the blank PET of the present invention and the coating prepared in Example 1.

[0039] Figure 5 The platelet adhesion SEM images of the blank PET of the present invention and the coating prepared in Example 1 are shown. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0041] Example 1

[0042] This embodiment prepares a coating, and the specific process is as follows:

[0043] S1: 0.5 g 2-methacryloyloxyethyl phosphorylcholine (MPC), 0.2 g 3-methacryloyl dopamine (DMA) and 0.3 g sodium p-styrene sulfonate (NaSS) were dissolved in ethanol, and then free radical polymerization was carried out under the action of 0.1 mmol azobisisobutyronitrile (AIBN) initiator. The reaction temperature of the free radical polymerization reaction was 65°C and the reaction time was 24 hours. After the reaction was completed, the polymer product PMDS was dialyzed using a dialysis bag with a molecular weight cutoff of 6000D to 8000Da, and then freeze-dried at -50°C to obtain a phospholipid polymer containing dopamine and sulfonate group structures.

[0044] S2: The polymer PMDS prepared in S1 is dissolved in Tris-HCl buffer solution to prepare a solution with a concentration of 2 mg / mL. The PET substrate is cleaned and immersed in the above solution. After 24 hours, it is taken out and cleaned to obtain a phospholipid antifouling coating on the surface of the PET film, which has anticoagulant and antibacterial functions.

[0045] S3: The substrate prepared in S2 is immersed in a silver nitrate solution with a concentration of 0.1 mg / mL, and after being protected from light for 24 hours, it is taken out and washed, and after drying, a nanosilver coating with bactericidal function is obtained on the surface of the PET film, and finally a phospholipid / nanosilver coating with anticoagulant and antibacterial function is prepared.

[0046] The infrared spectrum of S1 product PMDS is as follows Figure 1 shown.

[0047] from Figure 1 It can be seen that in the infrared spectrum of polymer PMDS, 3400-3500cm -1 The peak at 1714 cm corresponds to the phenolic hydroxyl group Ar-OH and the NH in the amide bond. -1 The stretching vibration peak is -C=O, 1488cm -1 The peak is -N + (CH3)3,1266cm -1 The peak corresponds to -SO3 - ,1086cm -1 The corresponding is -PO. The infrared spectrum of polymer PMDS proves the successful preparation of the polymer.

[0048] SEM images of S3 coating Figure 2 shown.

[0049] from Figure 2 It can be seen that Ag + The silver nanoparticles were complexed by the catechol groups in the polymer coating and successfully reduced to silver nanoparticles, which were deposited on the surface of the substrate.

[0050] Example 2

[0051] In this example, a coating was prepared. The specific process was the same as that in Example 1, except that the material feeding amounts in S1 were adjusted to 0.4 g MPC, 0.2 g DMA and 0.4 g NaSS.

[0052] Example 3

[0053] In this example, a coating was prepared. The specific process was the same as that in Example 1, except that the material feeding amounts in S1 were adjusted to 0.3 g MPC, 0.2 g DMA and 0.5 g NaSS.

[0054] Example 4

[0055] This embodiment prepares a coating, and the specific process is the same as that of Example 1, except that the concentration of the silver nitrate solution in S3 is adjusted to 0.25 mg / mL.

[0056] SEM images of S3 coating Figure 3 shown.

[0057] from Figure 3 It can be seen that Ag + The catechol groups in the polymer coating are complexed and successfully reduced to nanosilver particles, which are deposited on the surface of the substrate. Compared with the nanosilver particles prepared in Example 1, the nanosilver particles prepared in this example have a higher density.

[0058] Example 5

[0059] This embodiment prepares a coating, and the specific process is the same as that of embodiment 1, except that S3 is not performed.

[0060] Example 6

[0061] In this embodiment, a coating was prepared. The specific process was the same as that in Example 1, except that the feed of S1 was changed to 0.5 g 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), 0.2 g 3-methacryloyldopamine (DMA) and 0.3 g sodium p-styrenesulfonate (NaSS) for polymerization to prepare the polymer PCDS.

[0062] Example 7

[0063] In this embodiment, a coating is prepared. The specific process is the same as that in Example 1, except that the S1 feed is changed to 0.5 g 2-methacryloyloxyethyl phosphorylcholine (MPC), 0.2 g N-[2-(3,4-dihydroxyphenyl)ethyl]-2-acrylamide (DEP) and 0.3 g sodium p-styrenesulfonate (NaSS) for polymerization to prepare the polymer PMES.

[0064] Comparative Example 1

[0065] In this comparative example, a coating was prepared. The specific process was the same as that of Example 1, except that the feed of S1 was changed to 0.5 g MPC and 0.5 g DMA for polymerization to prepare the polymer P-MPC-DMA.

[0066] Comparative Example 2

[0067] In this comparative example, a coating was prepared. The specific process was the same as that of Example 1, except that the feed of S1 was changed to 0.5 g NaSS and 0.5 g DMA for polymerization to prepare the polymer P-NaSS-DMA.

[0068] Test example

[0069] This test example performs performance tests on blank PET, coatings prepared in the embodiments and comparative examples, and the specific process is as follows:

[0070] Contact angle test: carried out on the side of the film in contact with the air, distilled water, droplet volume: 2μL; temperature: 25℃; measure the contact angle value 1 minute after the water droplet contacts the surface, and take 5 points as the average value.

[0071] Cell activity test: Mouse fibroblast L929 was used as the material to determine the cytotoxicity of the coating. First, the sample was cut into 1cm x 1cm size, placed in a 12-well plate, and sterilized with UV light; then, the fibroblast suspension (density of 2x10 4 cells / cm 2 ) were inoculated onto the samples and incubated at 37°C in 5% CO2 for 1 day; the well plate without sample was used as blank control. Cell viability was determined by CCK-8 method.

[0072] The antibacterial rate test process is as follows: Use E. coli to test the antibacterial ability. 2 ) were placed in a 12-well plate, and each sample was treated with 500 μL of bacterial suspension for 10 6 CFU mL -1 Incubate at a constant temperature of 37°C for 24 hours. Subsequently, rinse the non-adherent bacteria on the sample with normal saline, place it in a sterile centrifuge tube, and add 4 mL of normal saline to the centrifuge tube. Oscillate the centrifuge tube evenly with a vortex mixer, and then dilute the separated bacteria with normal saline. Add the diluted bacterial suspension to Luria-Bertani agar, incubate at 37°C for 24 hours, and calculate the number of Escherichia coli colonies cultured on the agar after separation. Antibacterial rate (%) = (AB) / B×100%, where A is the number of bacterial colonies of blank material, and B is the number of bacterial colonies of coated material. The antibacterial effect diagram of blank PET and the coating prepared in Example 1 is shown in the figure below. Figure 4 shown.

[0073] Anti-protein (BSA and BFG) adhesion rate test: The samples to be tested were pre-equilibrated with PBS solution overnight, immersed in 1 mL of BSA solution (2 mg / mL) and BFG solution (2 mg / mL), respectively, and incubated at 37°C for 90 min. Subsequently, the non-adherent proteins on the samples were washed and removed. The adherent proteins were separated with 1% SDS for 1 hour. The protein concentration was detected using a micro-BCA protein detection kit.

[0074] The test process of antiplatelet adhesion rate is as follows: fresh rabbit blood is mixed with 3.8wt% sodium citrate in a ratio of 9:1, centrifuged at a centrifugal speed of 1500r / min in a centrifuge, and the supernatant is platelet-rich plasma (PRP) after centrifugation. 50μL PRP is added to the sample surface and spread evenly, and then placed in a 37°C constant temperature incubator for 3h. The sample is taken out, washed with phosphate buffer for more than 3 times to remove the unadhered platelets, and then immersed in a 2.5% glutaraldehyde aqueous solution for overnight fixation. After fixation, it is dehydrated with 50%, 75%, 90%, and 100% ethanol aqueous solution with a concentration gradient, and then dealcoholized with 50%, 75%, 90%, and 100% isoamyl acetate ethanol solution with a concentration gradient. After the sample is dried, the platelet adhesion on the sample surface is observed with a scanning electron microscope to analyze the platelet adhesion and denaturation degree. The platelet adhesion SEM images of the blank PET and the coating prepared in Example 1 are shown in Figure 5 shown.

[0075] Coagulation time (APTT and TT) test: fresh rabbit blood was mixed with 3.8 wt% sodium citrate in a ratio of 9:1, and centrifuged at 3000 r / min for 15 min to prepare anemic plasma (PPP). 2 ) were placed in a 12-well plate, 500 μL PPP was added to the surface of each sample, and then placed in a cell culture incubator at 37°C for 15 minutes, and then the plasma was separated. The APTT and TT of the cultured plasma were recorded on an automatic blood coagulation instrument. Each sample was measured three times in parallel. The results are shown in Table 1:

[0076] Table 1

[0077]

[0078] The water contact angle of the PET film of Example 1 after modification with the phospholipid polymer containing dopamine and sodium sulfonate structure and the nanosilver coating decreased from 72.0° (blank PET) to 37.2°, indicating that the hydrophilicity was improved, proving the successful preparation of the phospholipid coating. Figure 2 It can be seen that the scanning electron microscope test observed that the PET film surface was evenly distributed with particles of about 50nm in diameter, proving the successful preparation of nanosilver. After the PET film was modified with the phospholipid polymer nanosilver coating, the anti-infection rate of bacteria reached more than 98%, and the toxicity to mouse fibroblast L929 was low, and the cell activity was more than 95%. The protein adsorption of BSA and BFG on the unmodified PET film was 18.5μg / cm 2 and 15.5 μg / cm 2The protein adsorption of BSA and BFG on the PET membrane modified by phospholipid nanosilver coating decreased significantly, down to 2.7 μg / cm 2 and 2.4 μg / cm 2 The adhesion rates of anti-protein BSA and BFG were 85.0% and 84.5% respectively. The platelet adhesion capacity of the unmodified PET membrane was about 3.39×10 7 cell / cm 2 , the platelet adhesion of the PET membrane modified by coating decreased to 0.06×10 7 cell / cm 2 , the antiplatelet adhesion rate is 98.2%. The APTT time of the unmodified PET film is 40s, and the APTT time of the PET film modified with the phospholipid nano-silver coating is extended to 82s. The TT time of the PET film before and after the modification of the phospholipid nano-silver coating is increased from 20s to 24s. In summary, the phospholipid nano-silver coating of the present invention has good anticoagulation and bactericidal abilities.

[0079] On the one hand, the present invention utilizes the dopamine adhesion mechanism to prepare an antifouling coating on the surface of a substrate by a dip coating method containing a phospholipid polymer containing dopamine and sodium styrene sulfonate, thereby realizing the functions of anticoagulation and antibacterial adhesion; on the other hand, polydopamine after oxidation of dopamine is used to reduce silver ions to nanosilver particles to prepare a coating with a bactericidal function. The hydrophilicity of the coating prepared by the method of the present invention is significantly improved, and it can effectively prevent the adsorption of fibrinogen, platelets, bacteria, etc., and it also has bactericidal properties. The coating has excellent anticoagulant effect and long-term stability, and strong antibacterial properties. Compared with the blank group, the coating group can reduce the anti-protein adsorption by about 85%, the platelet adhesion can be reduced by up to 98.2%, the whole blood coagulation time is extended by more than one time, the antibacterial rate is as high as more than 98%, the cytotoxicity is low, the biocompatibility is good, and the anti-thrombosis and anti-bacterial infection effects are obvious. In addition, the preparation method provided by the present invention has a simple process, strong operability, and is suitable for large-scale production and application.

[0080] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polymer of formula I: ； The polymer of formula I is selected from: 、 、 ； in, a, b, and c are independently any integers; the molecular weight of the polymer is 10KDa to 50KDa; The preparation method of the polymer comprises the following steps: Under an inert atmosphere, an initiator is added to a mixed solution of a zwitterionic derivative modified with an active olefin group, a dopamine derivative modified with an active olefin group and a sulfonate modified with an active olefin group to carry out a free radical polymerization reaction to obtain the polymer; the mass ratio of the zwitterionic derivative, the dopamine derivative and the sulfonate in the mixed solution is (1-5):(1-3):(1-5).

2. A method for preparing the polymer as claimed in claim 1, Features: The following steps are involved: Under an inert atmosphere, an initiator is added to a mixed solution of a zwitterionic derivative modified with an active olefin group, a dopamine derivative modified with an active olefin group and a sulfonate modified with an active olefin group to carry out a free radical polymerization reaction to obtain the polymer; the mass ratio of the zwitterionic derivative, the dopamine derivative and the sulfonate in the mixed solution is (1-5):(1-3):(1-5).

3. The method for preparing a polymer according to claim 2, Features: The zwitterionic derivative includes at least one of 2-methacryloyloxyethyl phosphorylcholine, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt.

4. The method for preparing a polymer according to claim 2, Features: The dopamine derivative includes at least one of 3-methacryloyl dopamine, N-[2-(3,4-dihydroxyphenyl)ethyl]-2-acrylamide, and 3-(3,4-dihydroxyphenyl)-N-propylene-2-propionamide.

5. The method for preparing a polymer according to claim 2, Features: The sulfonate includes at least one of sodium p-styrene sulfonate, sodium vinyl sulfonate and sodium allyl sulfonate.

6. A coating comprising the polymer according to claim 1.

7. The coating according to claim 6, Features: The mass concentration of the polymer in the coating is 0.5 mg / mL to 10 mg / mL.

8. The coating according to claim 6, Features: The coating also includes nanosilver particles.

9. A medical material comprising a substrate and the coating according to claim 6 supported on the substrate.

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