An endothelium-promoting zwitterionic polymer coating and methods of making and using the same

Abstract

The application provides an endothelium-promoting zwitterionic polymer coating and a preparation method and application thereof, and relates to the field of medical coating materials.The zwitterionic monomer, hydrophobic alkyl chain monomer and functional monomer are used to synthesize a zwitterionic polymer containing a reactive functional group, and then the zwitterionic polymer is covalently grafted with a polypeptide, so that the polypeptide is uniformly distributed on the coating; the hydration layer formed by the zwitterionic polymer can resist non-specific protein adsorption and platelet adhesion (anti-coagulation); the polypeptide side chain enriched on the surface promotes endothelial cell adhesion (endothelium promotion), and the contradiction between the anti-fouling of the zwitterionic polymer and the promotion of cell adhesion is overcome; the application has the advantages of mild reaction conditions, simple post-treatment, strong repeatability, and the polypeptide is anchored to the polymer skeleton through a stable chemical bond, and the polypeptide is not prone to rapid loss in a physiological environment after film formation.

Description

Technical Field

[0001] This invention relates to the field of medical coating materials, and more specifically, to an endothelialization-promoting zwitterionic polymer coating, its preparation method, and its application. Background Technology

[0002] Cardiac occluders are medical devices used for transcatheter interventional treatment of congenital heart diseases (such as atrial septal defects, ventricular septal defects, and patent foramen ovale) and prevention of cardiac stroke. With the development of minimally invasive interventional techniques, cardiac occluders have become an important means of treating structural heart disease due to their advantages of minimal trauma and rapid recovery. Currently, the cardiac occluders used clinically are mainly made of nickel-titanium alloy, biodegradable polylactic acid (PLA), and biodegradable polydioxanone. Nickel-titanium alloy has the advantage of good shape memory properties, but its disadvantages are that it is a metallic material, and after implantation, it will remain permanently in the body, posing long-term risks such as metal ion release, wear, and conduction block. Moreover, some patients may be allergic to metals. Biodegradable occluders, on the other hand, can biodegrade within a suitable time while achieving effective occlusion, achieving non-implantable intervention and becoming the mainstream trend in the development of cardiac occluders. However, both biodegradable and non-biodegradable occluders face two major challenges in the long-term efficacy after implantation: device-related thrombosis and endothelialization failure.

[0003] Zwitterionic polymers (such as polyphosphorylcholine PMPC, polysulfobetaine PSBMA, polycarboxybetaine PCBMA, and polytrimethylamine N-oxide PTMAO) contain equal amounts of cationic quaternary ammonium groups and anionic carboxylate, phosphate, or sulfonate groups in their side chains, making them electrically neutral overall. They can form a dense and stable hydration layer with water molecules through strong electrostatic interactions, exhibiting excellent anti-protein adsorption and anti-platelet adhesion properties, making them ideal platforms for long-acting anticoagulation. However, while the inert surface and dense hydration layer of zwitterionic polymers resist non-specific protein adsorption and platelet adhesion, the lack of biorecognition sites also prevents endothelial cell adhesion. Their excellent antifouling ability also hinders endothelial cell adhesion, thus failing to promote rapid endothelialization of the occluder. Summary of the Invention

[0004] The present invention aims to provide an endothelialization-promoting zwitterionic polymer coating, its preparation method, and its application. The method involves synthesizing a zwitterionic polymer containing reactive functional groups from zwitterionic monomers, hydrophobic alkyl chain monomers, and functional group monomers. Then, peptides are covalently grafted onto the zwitterionic polymer, and the coating is sprayed onto a substrate material to obtain the coating. The peptide-zwitterionic polymer synthesized in this invention can resist non-specific protein adsorption and platelet adhesion (anticoagulation) through zwitterions; simultaneously, the surface-enriched peptide side chains promote endothelial cell adhesion (endothelialization), overcoming the contradiction between the antifouling and cell adhesion-promoting properties of zwitterionic polymers.

[0005] The technical problem solved by this invention is achieved by the following technical solution.

[0006] On one hand, embodiments of this application provide a method for preparing an endothelialization-promoting zwitterionic polymer coating, comprising the following steps: S1: After mixing zwitterionic monomers, hydrophobic alkyl chain monomers and functional group monomers in a molar ratio of 3-6:3-6:1, an initiator was added, and the mixture was reacted at 75°C in the dark for 10 hours. After the reaction was completed, the mixture was washed to obtain zwitterionic polymer PMLA containing reactive functional groups. S2: PMLA was dissolved in methanol solution, triethylamine was added and stirred until homogeneous; then the peptide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole were dissolved in DMSO / DMF mixed solvent and stirred until completely dissolved, and activated in an ice bath; then the PMLA solution was added dropwise to the activated mixed solution and reacted at room temperature for 24 h; after the reaction was completed, the mixture was purified by dialysis with deionized water and freeze-dried to obtain the zwitterionic polymer PMLA-peptide containing the peptide; S3: Select a substrate material, treat it with oxygen plasma for 1-5 minutes, then prepare the PMLA-peptide into a membrane solution and coat it onto the substrate material to obtain an amphoteric polymer coating.

[0007] Furthermore, in step S1, the zwitterionic monomer is any one of methacryloyloxyethyl phosphoric acid choline, sulfobetaine methacrylate, trimethylamine N-oxide, or carboxybetaine methacrylate; the hydrophobic alkyl chain monomer is lauryl methacrylate or n-butyl methacrylate; and the functional group monomer is aminoethyl methacrylate, N... (3) Any one of aminopropyl (aminopropyl) methacrylamide, methacrylic acid, or glycidyl methacrylate.

[0008] Furthermore, the initiator is 5‰-2% azobisisobutyronitrile or azobisisoheptanenitrile.

[0009] Furthermore, in step S2, the polypeptide is any one of RGD, REDV, GAG, RGDGGK, PHSRN, cRGDfK, or YIGSR.

[0010] Furthermore, the mass ratio of PMLA, peptide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole was 200-500: 30-70: 78-90: 55-70; the ice bath activation time was 30 min, and the dialysis purification time was 2 days.

[0011] Furthermore, in step S3, the substrate material is any one of cobalt-based alloy, NiTi alloy, medical stainless steel polylactic acid, polylactic acid-glycolic acid, polycaprolactone, or polydioxanone.

[0012] Furthermore, the concentration of the membrane solution is 5–100 mg / mL.

[0013] Secondly, embodiments of this application provide an endothelialization-promoting zwitterionic polymer coating, which is prepared using the above-described method.

[0014] Thirdly, embodiments of this application provide the application of the above-mentioned endothelialization-promoting zwitterionic polymer coating in the surface modification of cardiac occluders.

[0015] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. This invention synthesizes a zwitterionic polymer containing reactive functional groups by combining zwitterionic monomers, hydrophobic alkyl chain monomers, and functional group monomers. Then, peptides are covalently grafted onto the zwitterionic polymer. This method allows the peptides to be evenly distributed in the coating. The hydration layer formed by the zwitterionic polymer can resist non-specific protein adsorption and platelet adhesion (anticoagulation). The peptide side chains enriched on the surface promote endothelial cell adhesion (endothelialization), overcoming the contradiction that zwitterionic polymers cannot simultaneously achieve antifouling and cell adhesion promotion. 2. The reaction conditions of this invention are mild, the post-processing is simple and highly reproducible, and the peptides are anchored to the polymer backbone through stable chemical bonds, making them less likely to detach or diffuse in the physiological environment after film formation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1This is a statistical chart showing the static platelet count and number on the surface of different groups of coatings in this embodiment of the invention; Figure 2 These are the static whole blood adhesion results on the coating surfaces of different groups in the embodiments of the present invention; Figure 3 These are fluorescence images of endothelial cells after different groups of coating treatments in embodiments of the present invention; Figure 4 This is a schematic diagram showing the CCK-8 results of endothelial cells after different groups of coating treatments in an embodiment of the present invention; Figure 5 The figures show the experimental results of endothelial cell migration after different groups of coating treatments in the embodiments of the present invention; Figure 6 The figures show the results of endothelial cell scratch experiments after different groups of coating treatments in this embodiment of the invention. Figure 7 These are H&E staining images of rat fibrous capsules after different coating treatments in this embodiment of the invention. Figure 8 The figures show the statistical results of the thickness of rat fibrous capsules after different coating treatments in the embodiments of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0020] In all embodiments of the present invention, the general structural formula of the zwitterionic polymer containing reactive functional groups is shown in Formula I: Formula I.

[0021] Example 1 This embodiment provides a detailed method for preparing an endothelialization-promoting zwitterionic polymer coating, with the following specific steps: S1: Accurately weigh 0.03 mol of methacryloxyethyl phosphorylcholine (MPC), 0.06 mol of lauryl methacrylate (LMA), and 0.01 mol of methacrylamide (APMA), add them to a 50 mL round-bottom flask, mix, and then add sufficient 5‰ azobisisobutyronitrile (AIBN). Stir well and react at 75 °C in the dark for 10 h. After the reaction is complete, precipitate the product three times with acetone to obtain a white powder, which is the zwitterionic polymer containing reactive functional groups, named PMLA.

[0022] S2: Dissolve 200 mg PMLA in methanol solution, then add an appropriate amount of triethylamine and stir until homogeneous. Set aside. Then, dissolve 70 mg RGD peptide, 78 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 55 mg 1-hydroxybenzotriazole (HOBT) in an appropriate amount of 1:1 DMSO / DMF mixed solvent and stir until completely dissolved. Activate in an ice bath for 30 min, then slowly add the PMLA methanol solution dropwise to the activated mixed solution and react at room temperature for 24 h. After the reaction, purify by dialysis with deionized water for two days and freeze-dry to finally obtain the zwitterionic polymer containing the peptide, named PMLA-RGD.

[0023] S3: Using NiTi sheet as substrate material, place it in a sealed cavity, evacuate to sub-atmospheric pressure, introduce oxygen to generate low-temperature plasma, and clean and activate the substrate surface for 1 minute; then dissolve PMLA-RGD in methanol solution to prepare a film solution, and then use dip coating, spray coating or other methods to coat the treated substrate material to obtain an endothelialization-promoting zwitterionic polymer coating.

[0024] Example 2 This embodiment provides another method for preparing an endothelialization-promoting zwitterionic polymer coating, the specific steps of which are as follows: S1: Accurately weigh 0.03 mol of carboxymethyl methacrylate (CBMA), 0.06 mol of lauryl methacrylate, and 0.01 mol of glycidyl methacrylate (GMA), add them to a 50 mL round-bottom flask, mix, add 2% azobisisobutyronitrile, stir well, and then react at 75 °C in the dark for 10 h. After the reaction is complete, precipitate three times with acetone to obtain a white powder, which is the zwitterionic polymer containing reactive functional groups, named PMLG.

[0025] S2: Dissolve 200 mg PMLG in sufficient ethanol solution and set aside; then add 70 mg RGDGGK in DMF solution to the PMLG solution, adjust the pH to 8.0-8.5, and react at 50℃ for 12 h. After the reaction, purify by dialysis with deionized water for two days, freeze-dry, and finally obtain the zwitterionic polymer containing peptides, named PMLG-RGDGGK.

[0026] S3: Using PLA as the substrate material, place it in a sealed cavity, evacuate to sub-atmospheric pressure, introduce oxygen to generate low-temperature plasma, and clean and activate the substrate surface for 5 minutes; then dissolve PMLG-RGDGGK in an ethanol solution to prepare a film solution, and then use dip coating, spray coating or other methods to coat the film solution onto the treated substrate material to obtain the film.

[0027] Performance testing: The performance of the coatings obtained in Examples 1-2 was verified, including the following methods: (a) Static platelet adhesion.

[0028] Fresh rabbit blood was collected using sodium citrate anticoagulant tubes. Whole blood was centrifuged at 1500 rpm for 15 min to obtain the supernatant platelet-rich plasma (PRP). Samples were placed in 24-well cell culture plates, with three replicates per group. 100 μL of PRP was evenly spread on the sample surface, and then transferred to a 37 ℃ incubator for 1 h. After incubation, unadhered platelets were gently rinsed with physiological saline, and the samples were fixed in 2.5% glutaraldehyde solution for 12 h. After fixation, residual glutaraldehyde was removed from the sample surface, and the samples were sequentially dehydrated using 50%, 75%, 90%, and 100% graded ethanol / water solutions. The dried samples were observed under a scanning electron microscope (SEM). The effect of the coating on platelet adhesion and activation was evaluated by analyzing the morphology and activation of platelets in the SEM images and counting the number of adhered platelets.

[0029] Experimental results are as follows Figure 1As shown in the SEM morphology, platelets adhered extensively and spread fully on the NiTi substrate surface, exhibiting well-developed pseudopodia and high platelet activation. After modification with PMLA and PMLG polymer coatings, the number of adhered platelets decreased significantly, and the cells were mostly round, with spreading and activation levels significantly suppressed. This is because PMLA and PMLG contain phosphorylcholine (PC) groups, forming a dense hydration layer that inhibits platelet adhesion and activation. Compared to the pure polymer coating, the grafted peptide (RGD / RGDGGK) slightly increased platelet adhesion. This is because RGD can specifically recognize and bind to integrin receptors on the platelet cell membrane surface, thereby inducing platelet aggregation. Quantitative statistical results were consistent with the morphological observations; the platelet adhesion density in the NiTi group was significantly higher than that in all coating modification groups, with the PMLG coating exhibiting the best anti-platelet adhesion performance. *** indicates extremely significant statistical differences between groups (p<0.001), n=3.

[0030] (ii) Whole blood adhesion.

[0031] The dried and cleaned samples were placed in 24-well plates, and 300 μL of collected blood was added to each well, ensuring that the blood evenly covered the sample surface. The plates were incubated at 37 ℃ for 1 h. After incubation, the sample surface was slowly rinsed three times with physiological saline, followed by treatment according to the fixation method for platelet adhesion experiments and gradient dehydration. After dehydration, the samples were dried, and SEM was used to observe and analyze the adhesion morphology and quantity of erythrocytes, platelets, and plasma proteins on the coating surface.

[0032] Experimental results are as follows Figure 2 As shown in the SEM results, a large number of erythrocytes and plasma proteins adhered to the unmodified NiTi substrate surface, and the erythrocytes exhibited aggregation and abnormal morphology, indicating poor blood compatibility. After modification with PMLA and PMLG polymer coatings, the amount of erythrocyte adhesion on the material surface decreased significantly, and the erythrocytes mostly maintained a biconcave disc shape. Both polymer coatings effectively improved the anticoagulant properties of the nickel-titanium alloy due to the ability of zwitterions to resist non-specific protein adsorption, with the PMLG coating showing the best effect in inhibiting platelet adhesion. After grafting peptides such as RGD and RGDGGK, the number of erythrocytes adhered increased slightly compared to the pure polymer coating group, but was still much lower than that of the bare NiTi group, indicating that the introduction of peptides into the polymer did not reduce the good blood compatibility of the polymer coating. In summary, the PMLA-RGD and PMLG-RGDGGK modification systems can introduce cell adhesion active sites while maintaining low platelet adhesion and low thrombosis risk, making them ideal surface modification schemes that balance anticoagulation and endothelial repair promotion.

[0033] (iii) Static endothelial cell culture.

[0034] After UV sterilization, the samples were inoculated into 24-well plates at a density of 1×10⁻⁶. 4 Endothelial cell suspension (cells / mL) was added to each well with 500 μL of cell suspension. Cell viability was assessed using a CCK-8 assay kit on days 1 and 3, and the absorbance (OD) of each well was measured at 450 nm using a multi-mode microplate reader. The samples were then washed twice with physiological saline, stained with acridine orange (AO) in the dark, and cell morphology was observed under a laser confocal microscope. Cell nuclei were counted using fluorescence imaging to quantify cell adhesion.

[0035] Experimental results are as follows Figure 3 and Figure 4 The results of 1-day and 3-day culture of each sample with endothelial cells are shown. It can be seen that after 1 day of co-culture, the number of endothelial cells on the PMLA and PMLG coating surfaces is low due to the anti-adhesion effect of zwitterions and the lack of specific sites, while the adhesion of cells in the peptide-modified coating is somewhat improved. After 3 days of culture, no significant proliferation of cells was observed in the PMLA and PMLG polymer coatings, while the peptide-modified PMLA-RGD and PMLG-RGDGGK coatings showed a promoting effect on endothelial cell proliferation. This is because the RGD peptide (Arg-Gly-Asp) interacts with integrin receptors on the endothelial cell surface (such as α5β1, α...). v β3) has a high selective affinity, which makes up for the lack of bioactive sites in pure PMLA, thereby promoting the spread and proliferation of endothelial cells.

[0036] The quantitative results of CCK-8 were completely consistent with the trends observed in the qualitative fluorescence analysis. At 1 day of culture, the absorbance at 450 nm in the PMLA and PMLG groups was significantly lower than that in the NiTi control group; at 3 days of culture, the absorbance in the PMLA and PMLG groups only increased slightly, with a statistically significant difference between the two groups (*p<0.05); the cell viability in the blank NiTi group was significantly higher than that in the PMLA and PMLG groups, but lower than that in the PMLA-RGD and PMLG-RGDGGK groups. Statistical analysis showed that the cell viability in the PMLG-RGDGGK group was significantly higher than that in the NiTi group (***p<0.001), and also had a significant advantage over the PMLA-RGD group (**p<0.01). The above results confirm that pure PMLA and PMLG polymers have poor cell compatibility, which hinders cell adhesion and proliferation. Grafted peptides can effectively improve the bioactivity of polymer coatings, and the PMLG-RGDGGK coating is better than the PMLA-RGD coating in promoting cell adhesion and proliferation. The PMLA-RGD and PMLG-RGDGGK modified coatings have excellent cell affinity, indicating their application potential in the field of surface modification of implantable medical devices.

[0037] (iv) Endothelial cell migration experiment.

[0038] Nickel-titanium alloy foil sheets measuring 1 cm × 3 cm × 0.1 mm were cut and folded at a 90° angle to prepare a coating with one side of bare nickel-titanium alloy and the other side of peptide-grafted zwitterionic polymer. The sterilized samples were placed uncoated side down in the wells of a 24-well culture plate, and 500 μL of a 2 × 10⁻⁶ m³ / h solution was added to each well. 4 Endothelial cell suspensions of cells / mL were cultured for 24 h, then the samples were inverted 90° so that the coated side was facing down, and then cultured for another 24 h. The samples were then washed, fixed and stained in sequence. After processing, the samples were observed under a laser confocal microscope, and the migration distance of the endothelial cells was calculated.

[0039] Experimental results are as follows Figure 5 As shown, the crease method can simulate the physiological process of endothelial cell migration to the occluder surface, and intuitively evaluate the guiding and promoting effect of the coating on the directional migration of cells. The results directly reflect the actual efficacy of the coating in accelerating endothelialization of the occluder surface. Statistical data show that the migration distance of endothelial cells on the nickel-titanium sample surface is 464.3±9.7 μm, the migration distances of cells on the PMLA and PMLG coating surfaces are 204.7±7.5 μm and 237.6±9.4 μm, respectively, and the migration distances of cells on the PMLA-RGD and PMLG-RGDGGK coating surfaces reach 900.6±19.2 μm and 981.6±14.7 μm, respectively. Compared with the nickel-titanium sample, the PMLA-RGD and PMLG-RGDGGK coatings showed a significant promoting effect on endothelial cell migration, with migration distances increased by 94.0% and 111.4%, respectively; while the PMLA and PMLG samples showed a significant migration inhibition effect, with migration distances decreased by 55.9% and 48.8%, respectively, compared with the nickel-titanium sample. The results of endothelial cell culture and migration experiments showed that although the dense hydration layer formed by the phosphorylcholine (PC) groups of the PMLA and PMLG coatings has good blood compatibility, it significantly inhibits the non-specific adhesion, proliferation and migration of endothelial cells, making it difficult to meet the clinical need for rapid endothelialization after occluder implantation.

[0040] (v) Endothelial cell scratch test.

[0041] HUVECs were inoculated onto the sample surface at a density of 6 × 10⁻⁶. 4Cells / mL were cultured until cell confluence reached over 90%. Then, using a 10 μL pipette tip, two parallel scratches were made at an angle perpendicular to the sample surface, creating a blank area without endothelial cell coverage. The culture medium was immediately replaced with fresh medium, and cultured for another day. After acridine orange AO staining in the dark, cell migration was observed under a laser confocal microscope. The scratch area at each time point was measured using ImageJ software, and the scratch healing rate was calculated to quantitatively evaluate the migration performance of endothelial cells.

[0042] Experimental results are as follows Figure 6 As shown, the scratch healing method can simulate the physiological scenario of endothelial repair after vascular injury and evaluate the promoting effect of the surface coating of the cardiac occluder on endothelial cell migration. The results showed that after 24 h of culture, endothelial cells on the surfaces of NiTi, PMLA-RGD, and PMLG-RGDGGK samples migrated to the scratch area (the area in front of the white parallel lines), while endothelial cells on the PMLA and PMLG surfaces did not migrate. Quantitative analysis of the fluorescence images showed that the endothelial cell re-coverage ratios in the scratch area of ​​the PMLA-RGD and PMLG-RGDGGK groups were 64.4% and 72.0%, respectively, significantly higher than the 37.2% in the NiTi blank group, while the PMLA group showed almost no cell re-coverage in the scratch area. The experimental results indicate that the peptide-modified PMLA-RGD and PMLG-RGDGGK coatings can promote endothelial cell migration to the scratch area.

[0043] (vi) Subcutaneous implantation experiment in rats.

[0044] A 30 mg / mL sodium pentobarbital hydrochloride solution was prepared using physiological saline. Rats from the same growing environment were selected, weighed, and divided into groups. Each rat was then injected intraperitoneally at a dose of 1 mL / kg. After injection, the backs of the rats were shaved and the skin disinfected. Three 1.5 cm incisions were made in the center of the rat's back, and the fascia on both sides was carefully separated. The sample was implanted into the separated fascial space, ensuring contact between the sample and the skin. The incisions were sutured after implantation. To prevent postoperative infection, the rats were injected with penicillin for three consecutive days postoperatively. After being fed to the preset time point, rats in each group were anesthetized, and the fibrous capsule tissue encapsulating the sample was separated. The fibrous capsule was fixed with 4% paraformaldehyde, and the sample was removed after fixation. The capsule tissue was then embedded in paraffin and sectioned using a rotary microtome. After H&E staining, the growth of the fibrous capsule was observed and photographed using an optical microscope.

[0045] Experimental results are as follows Figure 7 and Figure 8As shown, the pure NiTi group had the largest fibrous capsule thickness. PMLA and PMLG treatments significantly reduced the fibrous capsule thickness and alleviated inflammatory cell infiltration. Further grafting with RGD and RGDGGK active peptides further thinned the fibrous capsule. Quantitative statistical results of fibrous capsule thickness showed that the average thickness of the fibrous capsules in each group was as follows: NiTi group (68.6±7.1 μm), PMLA group (52.6±4.6 μm), PMLG group (48.9±7.2 μm), PMLA-RGD group (39.8±5.4 μm), and PMLG-RGDGGK group (33.6±6.9 μm). Statistical analysis indicated that the fibrous capsule thickness of the bare NiTi group was significantly higher than that of all other coated modification groups (P<0.001). Compared with the pure NiTi sample, the PMLA-RGD and PMLG-RGDGGK groups showed reductions in fibrous capsule thickness of 42.0% and 51.0%, respectively. This confirms that zwitterionic polymer coatings can effectively inhibit the fibrotic reaction of foreign bodies after implantation.

[0046] Therefore, the zwitterionic polymer coating that promotes endothelialization provided by the present invention has excellent anticoagulant properties and also promotes endothelial repair. It can also reduce foreign body fibrosis and inflammatory response. These characteristics indicate that the coating is suitable for surface modification of implantable medical devices (such as cardiac occluders) to make them have better therapeutic effects.

[0047] In summary, the embodiments of the present invention provide an endothelialization-promoting zwitterionic polymer coating, its preparation method, and its application. The present invention synthesizes a zwitterionic polymer containing reactive functional groups by combining zwitterionic monomers, hydrophobic alkyl chain monomers, and functional group monomers. Then, peptides are covalently grafted onto the zwitterionic polymer, which allows for uniform distribution of the peptides in the coating. The hydration layer formed by the zwitterionic polymer resists non-specific protein adsorption and platelet adhesion (anticoagulation). The surface-enriched peptide side chains promote endothelial cell adhesion (endothelialization), overcoming the contradiction between the zwitterionic polymer's antifouling and cell adhesion-promoting properties. The present invention features mild reaction conditions, simple post-processing, and high reproducibility. Furthermore, the peptides are anchored to the polymer backbone through stable chemical bonds, making them less prone to loss in the physiological environment after film formation.

[0048] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing an endothelialization-promoting zwitterionic polymer coating, characterized in that, The steps include the following: S1: After mixing zwitterionic monomers, hydrophobic alkyl chain monomers and functional group monomers in a molar ratio of 3-6:3-6:1, an initiator was added, and the mixture was reacted at 75°C in the dark for 10 hours. After the reaction was completed, the mixture was washed to obtain zwitterionic polymer PMLA containing reactive functional groups. S2: Dissolve the PMLA in methanol solution, add triethylamine and stir until homogeneous; then dissolve the peptide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole in a DMSO / DMF mixed solvent, stir until completely dissolved, and activate in an ice bath; then add the PMLA solution dropwise to the activated mixed solution and react at room temperature for 24 h; after the reaction is complete, purify by dialysis with deionized water and freeze-dry to obtain the zwitterionic polymer PMLA-peptide containing the peptide; S3: Select a substrate material, treat it with oxygen plasma for 1-5 minutes, then prepare the PMLA-peptide into a membrane solution and coat it onto the substrate material to obtain a zwitterionic polymer coating.

2. The preparation method according to claim 1, characterized in that, In step S1, the zwitterionic monomer is any one of methacryloyloxyethyl phosphoric acid choline, sulfobetaine methacrylate, trimethylamine N-oxide, or carboxybetaine methacrylate; the hydrophobic alkyl chain monomer is any one of ethyl methacrylate, methyl methacrylate, octyl methacrylate, decyl methacrylate, lauryl methacrylate, n-butyl methacrylate, ethyl acrylate, methyl acrylate, decyl acrylate, and n-butyl acrylate; the functional group monomer is aminoethyl methacrylate, N... (3) Any one of the following: aminopropyl)methacrylamide, methacryloyloxypropyltrimethoxysilane, methacryloyloxyethyl isocyanate, N-allylethylenediamine, allylamine, hydroxyethyl methacrylate, 4-vinylbenzyl azide, methacrylic acid, or glycidyl methacrylate.

3. The preparation method according to claim 2, characterized in that, The initiator is 5‰-2% azobisisobutyronitrile or azobisisoheptanenitrile.

4. The preparation method according to claim 1, characterized in that, In step S2, the polypeptide is any one of RGD, REDV, GAG, RGDGGK, PHSRN, cRGDfK, or YIGSR.

5. The preparation method according to claim 4, characterized in that, The mass ratio of PMLA, polypeptide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole is 200-500: 30-70: 78-90: 55-70; the ice bath activation time is 30 min; and the dialysis purification time is 2 days.

6. The preparation method according to claim 1, characterized in that, In step S3, the substrate material is any one of cobalt-based alloy, NiTi alloy, medical stainless steel, polylactic acid, polylactic acid-glycolic acid, polycaprolactone, or polydioxanone.

7. The preparation method according to claim 6, characterized in that, The concentration of the membrane solution is 5-100 mg / mL.

8. An endothelialization-promoting zwitterionic polymer coating prepared by the preparation method according to any one of claims 1-7.

9. The application of the zwitterionic polymer coating for promoting endothelialization as described in claim 8 in the surface modification of cardiac occluders.

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