An autologous protein anticoagulant coating and its preparation method and application

Abstract

The application discloses a self-derived protein anticoagulant coating and a preparation method and application thereof. The coating utilizes the oxidation of hydrogen peroxide generated by the reaction of an oxidizing agent or a reducing agent with oxygen in the air, is based on the endogenous active center (such as hematin) removal mechanism of self-extracted proteins (such as hemoglobin), induces the oxidative self-assembly of the proteins, and loads an anticoagulant in the assembly process to form a protein-anticoagulant composite nanofilm. The application utilizes the self-proteins to avoid the transmission risk of pathogens such as HIV, PERVs and prions from the source; the preparation method is simple and rapid, and the obtained coating has excellent adhesion stability, good biocompatibility and anti-biofouling capacity on the surface of various substrates. In-vitro and in-vivo experiments prove that the coating has superior anticoagulant performance, reduces the risk of bleeding and liver and kidney toxicity, and can be used for the surface modification of blood-contacting medical devices such as a heart stent, an artificial blood vessel, a central venous catheter and an extracorporeal oxygenator.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and surface modification technology for medical devices, specifically relating to an autologous protein anticoagulant coating, its preparation method, and its application. Specifically, this invention relates to a technique for constructing a two-dimensional protein nanofilm on the surface of a medical device by inducing oxidative self-assembly of an autologous protein through direct oxidation by an oxidant or indirect oxidation by hydrogen peroxide generated from the reaction of a reducing agent with oxygen in the air. This nanofilm can load active biomolecules such as anticoagulants to form a composite coating with excellent anticoagulant function. Background Technology

[0002] Biomedical devices that come into contact with blood, such as interventional catheters, vascular stents, mechanical heart valves, extracorporeal circulation tubing, hemodialysis machines, and drug delivery systems, are indispensable tools in modern medical practice. These devices play a crucial role in saving lives and improving patient outcomes. However, when these foreign materials come into contact with the complex blood environment, they immediately trigger a series of host responses, with thrombosis and infectious complications being the most critical challenges. On the one hand, non-specific protein adsorption on the material surface can trigger a coagulation cascade, leading to thrombosis, which may not only cause device malfunction but also trigger life-threatening embolic events. On the other hand, the device surface provides a breeding ground for bacterial adhesion and biofilm formation, leading to difficult-to-treat local or systemic infections. Therefore, surface functionalization modification of blood-contact devices to construct coatings with both anticoagulant and antibacterial functions has been a core goal pursued in this field for a long time.

[0003] Among numerous coating materials, protein-based two-dimensional materials have attracted considerable attention due to their inherent biocompatibility, well-defined biological functions (such as anticoagulation and anti-adhesion), and ability to mimic the natural extracellular matrix. However, current research and applications largely rely on proteins of xenogeneic or allogeneic origin. This dependence on animal-derived (e.g., bovine, swine) or human-derived (donated) proteins poses significant biosafety risks: the potential transmission of pathogens. Studies have confirmed that bovine spongiform encephalopathy (BSE) prions, porcine endogenous retroviruses (PERVs), and human immunodeficiency virus (HIV) can potentially be introduced into the human body through these materials. The stringent physical or chemical treatments used to inactivate these pathogens often damage the higher-order structure and physiological activity of proteins, creating a dilemma of "inactivation equals inactivation." Especially after the World Health Organization introduced the concept of "Disease X" (a global pandemic caused by an unknown pathogen), the limitations of existing detection methods in the face of unknown pathogens further highlight the potential risks of exogenous protein materials.

[0004] Given the above risks, developing autologous biomaterials has become a promising and safer strategy. The human blood environment itself is a rich protein library, with high concentrations and well-defined functions of albumin, fibrinogen, etc. Using the patient's own blood components to construct coatings can theoretically eliminate the risk of cross-infection of pathogens at the source, and the safe blood donation volume (200-400 mL) is sufficient to meet clinical preparation needs. At present, the main methods for constructing coatings using autologous blood components include: (1) Physical adsorption method: using the weak interaction forces (such as van der Waals forces and electrostatic interactions) between the material surface and proteins to achieve spontaneous fixation of proteins. This method is simple to operate and has mild conditions, but the resulting coating has poor stability and weak binding force, and is prone to desorption and detachment under blood flow. It can usually only form a single layer of protein coverage, making it difficult to achieve multifunctional and long-term stable modification effects. (2) Chemical grafting method: through the formation of stable covalent bonds between the amino and carboxyl functional groups of proteins and the active groups (such as -NHS and -epoxy groups) on the material surface. This method significantly improves the stability of the coating, but it requires that the material surface must have specific functional groups available for reaction, and the reaction conditions may affect the native conformation and activity of the protein, thus requiring high process specifications. (3) Layer-by-layer self-assembly method: Polyelectrolytes with opposite charges (including proteins, polysaccharides, etc.) are alternately deposited on the substrate to form a multilayer coating. Its advantage is that the thickness, structure and function of the coating can be precisely controlled by adjusting the number of layers and components, and a variety of functional molecules can be introduced. However, this method is cumbersome and time-consuming, and the long-term stability of the multilayer structure in vivo, especially the resistance of the interlayer interface to blood flow erosion, is still a key issue restricting its clinical application.

[0005] Therefore, an urgent challenge facing the field of biomaterial coatings is how to develop a two-dimensional multifunctional biomaterial coating that is both efficient and simple to prepare, and can fundamentally avoid the risk of pathogen transmission, in order to address the shortcomings of existing biomedical devices that come into contact with blood in terms of antithrombosis and anti-infection. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of high pathogen transmission risk, complex preparation process, and insufficient coating stability of the anticoagulant coating used in existing blood contact biomedical devices, and to provide an autologous protein anticoagulant coating with universality, adhesion stability, good biocompatibility, and on-the-spot preparation, as well as its preparation method and application.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides an autologous protein anticoagulant coating, wherein the coating is a nanofilm formed on the surface of a medical device by the self-assembly of autologous protein and anticoagulant under oxidation; the oxidation is provided by hydrogen peroxide generated by the reaction of a reducing agent with oxygen in the air, or by the oxidizing agent directly.

[0009] Secondly, the present invention provides a method for preparing the autologous protein anticoagulant coating, the method comprising: obtaining a protein from a target patient, premixing the protein with an anticoagulant, contacting it with an oxidant solution or a reducing agent solution on the surface or interface of a medical device, and forming an autologous protein-anticoagulant composite nanofilm in situ through an oxidation-reduction triggered reaction, wherein the composite nanofilm constitutes the anticoagulant coating.

[0010] As a specific embodiment of the method of the present invention, the preparation method includes the following steps:

[0011] The anticoagulant was dissolved in a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution containing autologous protein to obtain solution A;

[0012] Dissolve the oxidizing or reducing agent in HEPES buffer solution, and adjust the pH to 3-11 with NaOH or HCl to obtain solution B;

[0013] Solution A and solution B are mixed evenly at a volume ratio of 1:1 to obtain a reaction mixture.

[0014] The reaction mixture is applied to the surface of a medical device and allowed to stand for reaction, thereby forming an autologous protein-anticoagulant composite nanofilm coating in situ on the device surface.

[0015] Furthermore, the aforementioned proteins are selected from any one or more of hemoglobin, human serum albumin, immunoglobulins, fibrinogen, transferrin, ceruloplasmin, lactoferrin, ferritin, myoglobin, prothrombin, lactate dehydrogenase, transaminase, catalase, glutathione peroxidase, and insulin.

[0016] Furthermore, the oxidant is selected from any one of hydrogen peroxide, sodium hypochlorite, dilute nitric acid, bromine water, peracetic acid, potassium permanganate, and chromic acid cleaning solution; the reducing agent is selected from any one of ascorbic acid, tris(2-carboxyethyl)phosphine, cysteine, oxalic acid, citric acid, glycyrrhetinic acid, glutathione, uric acid, gallic acid, lipoic acid, mercaptoethanol, dithiothreitol, glucose, and fructose.

[0017] Furthermore, the aforementioned anticoagulant is selected from any one or more of heparin, sodium citrate, warfarin, rivaroxaban, apixaban, aspirin, clopidogrel, and ticagrelor.

[0018] Furthermore, the aforementioned medical devices are selected from any one of the following: cardiac stents, artificial blood vessels, vascular stents, heart valves, central venous catheters, dialysis catheters, extracorporeal circulation oxygenators, extracorporeal membrane oxygenators, ventricular assist devices, and hernia patches.

[0019] Furthermore, the materials of the aforementioned medical devices include metals and alloys, polymers, or bioprosthetic valve materials; the metals and alloys include any one of stainless steel, titanium alloys, cobalt-chromium alloys, nickel-titanium alloys, magnesium alloys, and zinc alloys; the polymers include any one or copolymers of polylactic acid, polyethylene, polyurethane, silicone rubber, polyester, polyvinyl chloride, polycaprolactone, polyvinylidene fluoride, polybutyl methacrylate, polylactic acid-glycolic acid copolymer, polyvinylpyrrolidone, polypropylene, polymethylpentene, and polyglycolic acid; the bioprosthetic valve materials include any one of porcine aortic valves, porcine small intestinal submucosa, porcine dermis, and bovine pericardium.

[0020] Furthermore, in the above preparation method, the concentration of protein in solution A is 0.5–50 mg / mL, and the concentration of anticoagulant is 0.5–50 mg / mL; the concentration of oxidant or reducing agent in solution B is 0.5–50 mg / mL.

[0021] Furthermore, in the above preparation method, it is preferred that the concentration of protein in solution A is 5-20 mg / mL and the concentration of anticoagulant is 5-20 mg / mL, and it is preferred that the concentration of oxidant or reducing agent in solution B is 5-20 mg / mL, and the pH of solution B is adjusted to 5-10.

[0022] Furthermore, in the above preparation method, the mass ratio of the protein to the anticoagulant is 1:0.5-2, and the mass ratio of the protein to the oxidant or reducing agent is 1:0.5-2.

[0023] Furthermore, in the above preparation method, the reaction mixture is applied in any of the following ways: the reaction mixture is dropped onto the surface of the medical device to form a nanofilm at the gas-liquid interface; or the medical device is inverted onto the surface of the reaction mixture to form a nanofilm at the solid-liquid interface; or the reaction mixture is injected into the interior of the medical device, and after the reaction is completed, it is rinsed to form a uniform nanofilm on the inner wall of the medical device.

[0024] Thirdly, the present invention provides a blood contact medical device, wherein the surface of the medical device comprises the above-mentioned autologous protein-anticoagulant composite nanofilm coating.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. This invention uses autologous proteins as the main building material for the coating, which completely avoids the risk of transmission of pathogens (such as HIV, PERVs, prions, etc.) that may be carried by animal-derived or allogeneic human proteins from the source. This is in line with the development trend of personalized medicine and biosafety, and is especially suitable for dealing with the potential threat of unknown pathogens ("X disease").

[0027] 2. This invention directly initiates protein oxidative self-assembly through oxidizing or reducing agents, with mild reaction conditions (room temperature, aqueous phase), requiring no complex equipment or harsh chemical reaction conditions. The preparation process can be completed within minutes, making it particularly suitable for immediate clinical use. Personalized anticoagulant coatings can be rapidly prepared using patients' preoperative autologous blood donation.

[0028] 3. This invention induces the formation of a covalent cross-linked network between protein molecules through oxidation, and these molecules synergistically assemble with anticoagulant molecules through weak interactions. The resulting nanofilm adheres firmly to the surface of medical devices, significantly superior to traditional physical adsorption methods. The coating remains stable over a long period under blood flow conditions, preventing detachment and ensuring the durability of the anticoagulant effect. Furthermore, the coating is composed entirely of autologous proteins and clinically recognized anticoagulants, without any added chemical cross-linking agents or synthetic polymers, thus avoiding cytotoxic or immunogenic reactions. In vitro and in vivo experiments have demonstrated that this coating effectively resists non-specific protein adsorption, platelet adhesion, bacterial adhesion, and biofilm formation, exhibiting excellent blood compatibility and tissue compatibility.

[0029] 4. The protein nanofilms prepared by this invention have adjustable thickness at the nanoscale and transparency exceeding 90%, without affecting the optical properties of the medical devices themselves. They are suitable for medical devices requiring visualization during operation (such as interventional catheters and endoscopic instruments). Furthermore, the size and morphology of the film can be precisely controlled through reaction conditions (such as concentration, pH, and reaction time) to meet the needs of different application scenarios.

[0030] 5. The coating formation mechanism of the present invention does not rely on specific functional groups on the surface of medical devices. It can form a uniform film on the surface of various medical device materials such as metals, inorganic non-metals, and polymers through self-assembly at the gas-liquid interface or solid-liquid interface, which greatly expands its universality in the surface modification of various medical devices.

[0031] 6. The coating of this invention can be compounded with a variety of clinical anticoagulant drugs (such as heparin, aspirin, etc.), and utilize the loading and sustained-release effect of the protein matrix to achieve local and long-lasting anticoagulant effect, reduce the bleeding risk caused by systemic medication, and provide a safer surface solution for cardiovascular disease treatment, blood purification, extracorporeal circulation, etc. It is expected to solve the core problems of existing blood contact biomedical devices in clinical applications, and has important scientific research value and broad industrialization prospects. Attached Figure Description

[0032] Figure 1 This is a gel electrophoresis (SDS-PAGE) image of extracted hemoglobin and commercially available hemoglobin.

[0033] Figure 2 It is an optical photograph and shows the transparency of a hemoglobin nanofilm.

[0034] Figure 3 This is an atomic force microscope image of a hemoglobin nanofilm.

[0035] Figure 4 These are fluorescence microscope comparison images used to verify the risk of virus transmission.

[0036] Figure 5 This is a graph showing the results of a qPCR experiment to verify the risk of virus transmission.

[0037] Figure 6 This is an atomic force microscope image of a cytochrome C nanofilm.

[0038] Figure 7 This is an atomic force microscope image of a ferritin nanofilm.

[0039] Figure 8 This is an atomic force microscope image of a catalase nanofilm.

[0040] Figure 9 This is an atomic force microscope image of a superoxide dismutase nanofilm.

[0041] Figure 10 This is a diagram illustrating the effect of the gold chip in Example 1, which has an autologous hemoglobin-heparin composite nanofilm formed on its surface, on resisting the adsorption of proteins and sugars.

[0042] Figure 11 This is a scanning electron microscope image of the silicon wafer with an autologous hemoglobin-heparin composite nanofilm formed on its surface in Example 1, showing its antiplatelet adhesion properties.

[0043] Figure 12 This is a scanning electron microscope image of the silicon wafer with an autologous hemoglobin-heparin composite nanofilm formed on its surface in Example 1, showing its antibacterial adhesion properties.

[0044] Figure 13 This is a scanning electron microscope image of the silicon wafer with an autologous hemoglobin-heparin composite nanofilm formed on its surface in Example 1, showing its resistance to red blood cell adhesion.

[0045] Figure 14 This is an optical photograph of the autologous hemoglobin-heparin composite nanofilm formed on the inner wall of the capillary and the PP mesh in Example 2 after being stained with Congo red.

[0046] Figure 15This is an optical photograph of the thrombosis situation of the medical PVC pipe with an autologous hemoglobin-heparin composite nanofilm formed on its surface in Example 3 after blood circulation.

[0047] Figure 16 This is a high-resolution field emission scanning electron microscope image of the thrombosis situation of the medical PVC pipe with an autologous hemoglobin-heparin composite nanofilm formed on its surface in Example 3 after blood circulation.

[0048] Figure 17 This is a comparison chart showing the quality of thrombus formation in medical PVC pipes with an autologous hemoglobin-heparin composite nanofilm formed on the surface in Example 3 after blood circulation. Detailed Implementation

[0049] This invention uses autologous proteins as the main building material for the coating, and experiments have demonstrated that it can avoid the risk of virus transmission. Specific experiments are as follows:

[0050] 1. Extraction and purification of autologous hemoglobin

[0051] Freshly drawn human whole blood was centrifuged at 4 °C and 4000 rpm for 10 minutes. The supernatant (plasma and leukocyte layer) was carefully discarded, retaining the erythrocyte precipitate. The erythrocytes were resuspended in physiological saline (0.9% NaCl) and washed three times under the same conditions to remove residual plasma proteins. Four volumes of ice-cold hypotonic lysis buffer were added to the erythrocytes, and the cells were incubated on ice for 30 minutes to rupture the cell membranes and release hemoglobin. The lysis buffer was centrifuged at 4 °C and 9000 rpm for 20 minutes, and the hemoglobin-rich red supernatant was collected, discarding the precipitate fragments. The crude extract was placed in a pre-hydrated dialysis bag and subjected to gradient dialysis desalting at 4 °C and 100 rpm with continuous shaking. Purity was determined by SDS-PAGE gel electrophoresis, and the results are as follows. Figure 1 As shown, the extracted hemoglobin has a molecular weight of approximately 64.5 kDa and no visible contaminating protein bands, indicating that high-purity autologous hemoglobin was obtained.

[0052] 2. Preparation of autologous hemoglobin nanofilms

[0053] 50 mg of the autologous hemoglobin obtained above was dissolved in 5 mL of HEPES buffer solution to obtain solution A; 50 mg of ascorbic acid was dissolved in 5 mL of HEPES buffer solution, and the pH was adjusted to 7 with NaOH to obtain solution B; solution A and solution B were mixed evenly at a volume ratio of 1:1 to obtain a reaction mixture; the resulting reaction mixture was dropped onto the surface of a glass slide and allowed to stand for 4 hours to form a clear hemoglobin nanofilm at the gas-liquid interface. Figure 2a); After immersing a quartz sheet in the resulting reaction mixture and allowing it to stand for 4 hours, a hemoglobin nanofilm is formed on the surface of the quartz sheet. The visible light transmittance of the resulting hemoglobin nanofilm is approximately 90% as measured by a UV-Vis spectrophotometer. Figure 2 b) By placing the silicon wafer upside down over the resulting reaction mixture and allowing it to stand for 4 hours, a hemoglobin nanofilm will form on the surface of the silicon wafer, such as... Figure 3 As shown, the hemoglobin nanofilm is formed from dense, continuous oligomers, and the film can be stably adhered to the silicon wafer surface.

[0054] 3. Verification of the viral transmission risk of autologous hemoglobin nanofilms

[0055] Eight healthy SD rats were randomly divided into two groups (n=4): a control group and an experimental group. On day 8 of feeding, the rats in the experimental group were injected via tail vein with 0.2 mL of lentivirus expressing green fluorescent protein (GFP) (titer: 1×10⁻⁶). 8 The control group received no treatment (TU / mL). All rats continued to be fed under standard conditions. On day 13, tail vein blood (0.3–0.5 mL / rat) was collected from all rats, and hemoglobin solution was purified using the method described in Experiment 1 above. Hemoglobin nanofilms were prepared using the method described in Experiment 2, and smears were prepared from the PBS extract of the hemoglobin nanofilms for observation under a fluorescence microscope. The results showed that no fluorescence of green fluorescent protein was observed in the control group (TU / mL). Figure 4 a), while the experimental group showed bright green fluorescence, indicating that the use of heterologous proteins may lead to the introduction and retention of the virus (a). Figure 4 b). Simultaneously, using its extract as a DNA template, and employing primers specifically designed for the GFP sequence, PCR amplification was performed to detect viral signals. qPCR analysis further confirmed the risk of viral transmission; the amplification curves of the experimental group exhibited a typical S-shaped pattern (b). Figure 5 a). The corresponding threshold cycle (Ct) values ​​also indicated that high copy numbers of viral particles remained in the PBS eluent of the experimental group, in stark contrast to the control group, where no amplification signal was detected. Figure 5 b).

[0056] Furthermore, protein nanofilms were prepared using different proteins according to the method described in Experiment 2 above to verify the universality of the preparation method of this invention. 50 mg of cytochrome C, ferritin, catalase, or superoxide dismutase was dissolved in 5 mL of HEPES buffer solution to obtain solution A; 50 mg of ascorbic acid was dissolved in 5 mL of HEPES buffer solution, and the pH was adjusted to 7 with NaOH to obtain solution B; solutions A and B were mixed evenly at a volume ratio of 1:1 to obtain a reaction mixture; a silicon wafer was inverted and placed above the reaction mixture, and after standing for 4 hours, cytochrome C (…) was formed on the surface of the silicon wafer. Figure 6 ) or ferritin ( Figure 7 ) or catalase ( Figure 8 ) or superoxide dismutase ( Figure 9 The nanofilms are all formed from dense, continuous oligomers, and the films can stably adhere to the silicon wafer surface. This indicates that the above-mentioned method for preparing hemoglobin films is universally applicable to a variety of proteins.

[0057] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0058] Example 1

[0059] 50 mg of the autologous hemoglobin obtained above was dissolved in 5 mL of HEPES buffer solution, and 100 mg of heparin was added to obtain solution A; 50 mg of ascorbic acid was dissolved in 5 mL of HEPES buffer solution, and the pH was adjusted to 7 with NaOH to obtain solution B; solution A and solution B were mixed evenly at a volume ratio of 1:1 to obtain a reaction mixture; the obtained reaction mixture was dropped onto the surface of a sealing film, and then a gold chip and a silicon wafer were inverted on top of the reaction mixture. After standing for 2 hours, autologous hemoglobin-heparin composite nanofilms were formed on the surfaces of the gold chip and the silicon wafer, respectively. The composite nanofilms constitute an anticoagulant coating.

[0060] The anti-protein and anti-carbohydrate adsorption properties of blank gold chips and gold chips with autologous hemoglobin-heparin composite nanofilms formed on their surfaces were tested using a quartz crystal microbalance (QCM). Figure 10As shown, the gold chip with an autologous hemoglobin-heparin composite nanofilm on its surface exhibits significantly lower adsorption of proteins (horseradish peroxidase (HRP), fibrinogen, human serum albumin (HSA), fetal bovine serum albumin (FBS), bovine serum albumin (BSA), lysozyme, milk, α-lactalbumin, β-lactoglobulin, etc.) and carbohydrates (lactose, D-ribose, glucose) compared to the blank gold chip. Scanning electron microscopy was used to observe the adhesion of platelets, bacteria, and red blood cells on the surfaces of both the blank silicon wafer and the silicon wafer with the autologous hemoglobin-heparin composite nanofilm. Figures 11-13 The results showed that the silicon wafer with the formation of an autologous hemoglobin-heparin composite nanofilm exhibited significantly lower adhesion of platelets, bacteria, and erythrocytes compared to the blank silicon wafer. These results indicate that the autologous hemoglobin-heparin composite nanofilm can effectively resist the adsorption of non-specific proteins and carbohydrates, as well as the adhesion of platelets, erythrocytes, and bacteria, and the formation of biofilms.

[0061] Example 2

[0062] 50 mg of the autologous hemoglobin obtained above was dissolved in 5 mL of HEPES buffer solution, and 100 mg of heparin was added to obtain solution A; 50 mg of ascorbic acid was dissolved in 5 mL of HEPES buffer solution, and the pH was adjusted to 7 with NaOH to obtain solution B; solutions A and B were mixed evenly at a volume ratio of 1:1 to obtain a reaction mixture; the obtained reaction mixture was dropped onto the surface of a sealing film, and then a capillary glass tube and a PP mesh were immersed in the reaction mixture. After standing for 12 hours, autologous hemoglobin-heparin composite nanofilms were formed on the surfaces of the capillary glass tube and the PP mesh, respectively. The composite nanofilms constituted an anticoagulant coating. After rinsing the nanofilms with ultrapure water, they were then soaked in a specific staining solution for amyloid protein (1 mg / mL Congo red solution) for 2 hours for staining. The obvious difference in red color before and after the reaction indicated that the inner wall of the capillary ( Figure 14 a) and PP mesh ( Figure 14 Both b) and c) have uniform and continuous nanofilms formed on their surfaces.

[0063] Example 3

[0064] 50 mg of the autologous hemoglobin obtained above was dissolved in 5 mL of HEPES buffer solution, and 100 mg of heparin was added to obtain solution A; 50 mg of ascorbic acid was dissolved in 5 mL of HEPES buffer solution, and the pH was adjusted to 7 with NaOH to obtain solution B; solutions A and B were mixed evenly at a volume ratio of 1:1 to obtain a reaction mixture; the resulting reaction mixture was dropped onto the surface of a sealing film, and then the reaction mixture was introduced into a medical PVC pipeline. After standing for 24 hours, an autologous hemoglobin-heparin composite nanofilm was formed on the inner wall of the medical PVC pipeline, and the composite nanofilm constituted an anticoagulant coating. The medical PVC pipeline was rinsed with ultrapure water and then dried. Figure 15 As shown, after blood circulation, a large number of thrombi formed on the surface of blank medical PVC tubing, almost completely blocking the tubing; while thrombi hardly formed on the surface of medical PVC tubing with an anticoagulant coating. According to... Figure 16 High-resolution field emission scanning electron microscopy images show that blank medical PVC tubing has a large number of blood cells, platelets, and fibrinogen networks adhering to its surface after blood circulation, while medical PVC tubing with an anticoagulant coating ( Figure 16 b) Only a small amount of red blood cells are deposited on the surface. For example... Figure 17 As shown, the mass of the thrombus formed by the blank medical PVC tubing (Control) is approximately 20 times that of the thrombus formed by the medical PVC tubing (Coated) with the anticoagulant coating. These experimental results demonstrate that the anticoagulant coating of the present invention exhibits excellent anticoagulant properties both in vivo and in vitro.

Claims

1. A method for preparing an autologous protein anticoagulant coating, characterized in that, include: Proteins are obtained from the target patient, and the proteins are premixed with an anticoagulant. The mixture is then brought into contact with an oxidant solution or a reducing agent solution on the surface or interface of a medical device. An in-situ autologous protein-anticoagulant composite nanofilm is formed through an oxidation-reduction triggered reaction. The composite nanofilm constitutes the anticoagulant coating.

2. The method for preparing an autologous protein anticoagulant coating according to claim 1, characterized in that, The protein is selected from any one or more of hemoglobin, human serum albumin, immunoglobulin, fibrinogen, transferrin, ceruloplasmin, lactoferrin, ferritin, myoglobin, prothrombin, lactate dehydrogenase, transaminase, catalase, glutathione peroxidase, and insulin.

3. The method for preparing an autologous protein anticoagulant coating according to claim 1, characterized in that, The oxidizing agent is selected from any one or more of hydrogen peroxide, sodium hypochlorite, dilute nitric acid, bromine water, peracetic acid, potassium permanganate, and chromic acid cleaning solution; the reducing agent is selected from any one or more of ascorbic acid, tris(2-carboxyethyl)phosphine, cysteine, oxalic acid, citric acid, glycyrrhetinic acid, glutathione, uric acid, gallic acid, lipoic acid, mercaptoethanol, dithiothreitol, glucose, and fructose.

4. The method for preparing an autologous protein anticoagulant coating according to claim 1, characterized in that, The anticoagulant is selected from any one or more of heparin, sodium citrate, warfarin, rivaroxaban, apixaban, aspirin, clopidogrel, and ticagrelor.

5. The method for preparing an autologous protein anticoagulant coating according to claim 1, characterized in that, The medical device is selected from any one of the following: cardiac stent, artificial blood vessel, vascular stent, heart valve, central venous catheter, dialysis catheter, extracorporeal circulation oxygenator, extracorporeal membrane oxygenator, ventricular assist device, and hernia patch.

6. The method for preparing an autologous protein anticoagulant coating according to claim 5, characterized in that, The materials of the medical device include metals and alloys, polymers, or bioprosthetic valve materials; the metals and alloys include any one of stainless steel, titanium alloys, cobalt-chromium alloys, nickel-titanium alloys, magnesium alloys, and zinc alloys; the polymers include any one or copolymers of polylactic acid, polyethylene, polyurethane, silicone rubber, polyester, polyvinyl chloride, polycaprolactone, polyvinylidene fluoride, polybutyl methacrylate, polylactic acid-glycolic acid copolymer, polyvinylpyrrolidone, polypropylene, polymethylpentene, and polyglycolic acid; the bioprosthetic valve materials include any one of porcine aortic valves, porcine small intestinal submucosa, porcine dermis, and bovine pericardium.

7. The method for preparing an autologous protein anticoagulant coating according to claim 1, characterized in that, The specific steps include the following: The anticoagulant was dissolved in a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution containing autologous protein to obtain solution A; Dissolve the oxidizing or reducing agent in a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, and adjust the pH to 3-11 with NaOH or HCl to obtain solution B; Solution A and solution B are mixed evenly at a volume ratio of 1:1 to obtain a reaction mixture. The reaction mixture is applied to the surface of a medical device and allowed to stand for reaction, thereby forming an autologous protein-anticoagulant composite nanofilm coating in situ on the device surface.

8. The method for preparing the autologous protein anticoagulant coating according to claim 7, characterized in that, The concentration of protein in solution A is 0.5–50 mg / mL, and the concentration of anticoagulant is 0.5–50 mg / mL; the concentration of oxidant or reducing agent in solution B is 0.5–50 mg / mL.

9. The method for preparing the autologous protein anticoagulant coating according to claim 8, characterized in that, The concentration of protein in solution A is 5–20 mg / mL, the concentration of anticoagulant is 5–20 mg / mL, and the concentration of oxidant or reducing agent in solution B is 5–20 mg / mL; the pH of solution B is adjusted to 5–10.

10. The method for preparing an autologous protein anticoagulant coating according to claim 7, characterized in that, The mass ratio of the protein to the anticoagulant is 1:0.5 to 2, and the mass ratio of the protein to the oxidant or reducing agent is 1:0.5 to 2.

11. The method for preparing an autologous protein anticoagulant coating according to claim 7, characterized in that, The reaction mixture can be applied in any of the following ways: drop the reaction mixture onto the surface of the medical device to form a nanofilm at the gas-liquid interface; or invert the medical device onto the surface of the reaction mixture to form a nanofilm at the solid-liquid interface; or inject the reaction mixture into the interior of the medical device, rinse after the reaction is complete, and form a uniform nanofilm on the inner wall of the medical device.

12. The autologous protein-anticoagulant composite nanofilm coating prepared by the method according to any one of claims 1 to 11.

13. A blood contact medical device, characterized in that, The surface of the medical device comprises the autologous protein-anticoagulant composite nanofilm coating as described in claim 12.

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