A natural bio-driven long stable anti-thrombus and endothelialization improved blood flow guiding device and preparation method

Abstract

The application belongs to the field of medical devices and surface functional modification of biological materials, and particularly relates to a natural biological driving long-stable anti-thrombus and endothelialization improved blood flow guiding device and a preparation method. The specific biological functional molecule layer provided by the application can realize natural anti-blood coagulation in the initial implantation stage, natural biological driving endothelialization progress, and long-stable anti-thrombus formation. The specific biological functional molecule layer combines endothelial progenitor cells and endothelial cells to realize specific endothelial progenitor cell capture, differentiation, endothelial cell adhesion, migration and proliferation, so as to maximize effective re-endothelialization and ensure natural biological characteristics, realize natural remodeling of the inner wall of a blood vessel to achieve long-stable anti-thrombus, and is a new functional concept that is difficult to achieve by other solutions in the prior art. The application is a surface improved blood guiding device with natural anti-coagulation and natural biological driving endothelialization progress to obtain long-stable anti-thrombus.

Description

Technical Field

[0001] The present invention belongs to the field of surface functional modification of medical devices and biomaterials, and specifically relates to a naturally biologically driven, long-stable anti-thrombotic and endothelialized improved blood flow guiding device and a preparation method thereof. Background Art

[0002] Blood flow redirecting devices (also known as dense mesh stents) are a new approach to endovascular treatment of intracranial aneurysms. By redirecting the impinging blood flow within the parent vessel to the distal normal vessel, they effectively reduce the blood flow velocity and volume within the aneurysm, thereby inducing thrombosis within the aneurysm. They also guide endothelial growth at the aneurysm site, achieving vascular remodeling and ultimately excluding the aneurysm from the circulatory system, ultimately achieving complete occlusion. This technology is becoming a first-line treatment option for intracranial aneurysms that are difficult to treat with traditional therapies, and is particularly suitable for complex aneurysms (such as giant aneurysms, wide-necked aneurysms, fusiform aneurysms, and dissecting aneurysms).

[0003] With the gradual large-scale application of blood flow guiding devices in clinical practice, some key problems have also been exposed, the most notable of which is acute and long-term thrombosis, which may significantly increase the risk of ischemic stroke. Therefore, patients need to receive long-term dual antiplatelet therapy (DAPT) during and after device implantation to reduce the risk of thrombosis. However, long-term DAPT treatment may lead to the occurrence of hemorrhagic complications, which are extremely risky, directly endanger the patient's life, and bring great mental and economic burdens, becoming the biggest problem currently faced by the implantation of blood flow guiding devices. Therefore, improving the anticoagulant properties of the surface of the blood flow guiding device and reducing thrombosis are the biggest needs in clinical applications.

[0004] At present, relevant studies at home and abroad have explored the use of surface functional modification to improve the anti-coagulation performance of blood flow guidance devices. These studies include the construction of biomimetic anti-coagulation functional coatings, anti-coagulation material coatings, and hydrophilic polymer coatings. However, these methods more reflect a relatively passive anti-coagulation strategy, and are still far from achieving the satisfactory results required in clinical practice in terms of achieving long-term anti-thrombosis. In fact, endothelial remodeling plays a vital role in long-term anti-thrombosis. However, the existing endothelialization-promoting coatings have a single component or a passive mechanism of action, which makes it difficult to achieve a more natural endothelialization process, thereby achieving a long, efficient and stable natural anti-coagulation-type remodeling of the vascular wall, which greatly limits its actual utility and translational application in clinical practice.

[0005] In response to the above problems, the present invention proposes an improved blood flow guiding device with the core strategy of combining natural anticoagulation with natural biologically driven endothelialization progress. Specifically, through molecular autonomous assembly technology, an interface coating with biologically active functions is constructed to effectively inhibit the coagulation reaction in the early stage of device implantation, thereby realizing a natural anticoagulant function. At the same time, the biological functional molecular layer further captures endothelial progenitor cells and calls endothelial cells respectively through a natural biological driving mechanism, promoting the natural progression of endothelialization, thereby achieving an efficient and long-lasting anti-thrombotic effect. This innovative design can better meet the key clinical needs for long-term antithrombotic effects on the surface of blood flow guiding devices, avoiding the high risks and burdens brought about by dual-antibody therapy. Summary of the Invention

[0006] The present invention provides a method for preparing a naturally biologically driven, long-stable, anti-thrombotic and endothelialized improved blood flow guiding device, comprising the following steps:

[0007] S1: Clean and dry a blood flow diversion device (cobalt-chromium, nickel-titanium alloy, or other metal substrate), mix an aqueous solution of ammonium hexafluorotitanate and an aqueous solution of boric acid to obtain a mixed solution A, and immerse the blood flow diversion device in the mixed solution to obtain a blood flow diversion device with a TiO2 base layer;

[0008] S2: The blood flow guiding device with a TiO2 base layer of S1 is immersed in a mixed solution B of phosphorylcholine, CD146 antibody, REDV peptide, and vascular endothelial growth factor, and liquid deposition is performed at room temperature. After drying, a naturally biologically driven, long-stable anti-thrombotic and endothelialized improved blood flow guiding device is obtained.

[0009] Furthermore, the volume ratio of the ammonium hexafluorotitanate aqueous solution and the boric acid aqueous solution described in S1 is 1:1.

[0010] Furthermore, the concentration of phosphorylcholine in S2 is 1-10 g / L, the concentration of REDV peptide is 0.1-1 mg / ml, the concentration of vascular endothelial growth factor is 0.1-1 μg / ml, and the dilution ratio of CD146 antibody is 0.5% to 2%.

[0011] Furthermore, the liquid phase deposition time described in S2 is 1-24 hours.

[0012] A naturally biologically driven, long-stable, anti-thrombotic and endothelialized improved blood flow guiding device, wherein the naturally biologically driven, long-stable, anti-thrombotic and endothelialized improved blood flow guiding device is a biological functional molecular layer.

[0013] Furthermore, the thickness of the biological functional molecule layer of the blood flow guiding device is 5-100 nm.

[0014] Beneficial effects

[0015] Aiming at the key clinical need of anti-thrombotic effect on the surface of blood flow guidance devices, the present invention innovatively constructs a specific biological functional molecular layer that is anti-coagulant and promotes endothelialization. Through the synergistic effect of natural anticoagulant and natural biological driving endothelialization, the goal of long-term steady-state anti-thrombotic effect is successfully achieved. The biological functional molecular layer in the present invention is constructed based on the principle of non-covalent self-assembly, such as Figure 1 As shown, phosphorylcholine (PC) molecules interact with specific biomacromolecules (such as CD146 antibodies, REDV peptides, and VEGF) through van der Waals forces and hydrogen bonds to form a stable self-assembled biofunctional molecular layer. This molecular layer not only maintains good biocompatibility at the molecular interface, but also avoids the destruction of the biomolecule's own activity by chemical bonding, ensuring the integrity of the biomacromolecule's natural functionality.

[0016] During the initial implantation phase, the biofunctional molecular layer, due to its basic components being similar to the phosphoester structure of natural red blood cell membranes and by preventing nonspecific protein adhesion, effectively inhibits the early, intense coagulation reaction on the device surface, providing a natural-like anticoagulant effect. At the same time, the molecular layer also provides specific functional bioactive molecules and a favorable microenvironment, promoting a natural, biologically driven endothelialization process. Specifically, the multi-component and multifunctional properties of the molecular layer are manifested in the specific capture of endothelial progenitor cells: through precise recognition and adhesion, it effectively adsorbs circulating endothelial progenitor cells, and drives endothelial progenitor cell differentiation: providing a suitable microenvironment, promoting the natural differentiation of endothelial progenitor cells into mature endothelial cells, and then the adhesion, migration, and proliferation of endothelial cells: supporting the adhesion and stable migration of differentiated endothelial cells on the device surface, while stimulating the proliferation of endothelial cells, further covering the device surface, and maintaining the natural endothelial structure and function to the greatest extent possible.

[0017] Blood flow guidance devices modified with MPC polymers or other coatings are beginning to appear on the market. Although they can achieve a certain anticoagulant effect, their single function and the chemical bonding between molecules will greatly weaken their natural activity. Even considering the combination with biological macromolecules, it is difficult to ensure the natural existence of their chemical structure and spatial conformation. Therefore, it is difficult to achieve a more natural initial anticoagulant surface and the subsequent natural endothelialization process to obtain a long-term stable anti-thrombotic surface. Relatively and specifically speaking, the present invention uniquely adopts a non-covalent bond connection method between PC molecules and biological macromolecules, and independently forms a stable molecular layer based on van der Waals forces and hydrogen bonds, which has the following significant advantages: protecting the activity of biological molecules - the non-covalent connection method avoids changes in the chemical structure of biological molecules and retains the natural activity of the molecules to the maximum extent; improving the stability of the molecular layer: the molecular layer formed by self-assembly has excellent physical stability and can maintain surface anticoagulation and endothelialization functions for a long time; functional component diversity integration and synergistic enhancement: different biological molecules (such as REDV peptide, VEGF, CD146 antibody) work synergistically, which can not only achieve a natural-like anticoagulant effect, but also biologically drive the natural, step-by-step and coordinated promotion of endothelialization progress, which is difficult to achieve with existing coatings, especially chemical bonding construction methods.

[0018] The present invention, through the construction of a specific biological functional molecular layer, highly mobilizes key functions and works in synergy with nature, enabling the blood flow guiding device to achieve a continuous optimization and efficient long-term treatment process from natural anticoagulation in the initial stage of implantation to subsequent natural biologically driven endothelialization. This creative biological molecular layer not only fills the limitations of single-functional materials, but also opens up a new direction for material biological surface modification technology. In short, the specific biological functional molecular binding layer of the present invention realizes the following innovative functional concepts that are difficult to achieve with other solutions, including natural anticoagulation: through the biocompatibility of PC molecules, it simulates the natural anticoagulant barrier and inhibits early coagulation reactions; and natural biologically driven endothelialization progress: through precise capture, induction of differentiation, stable adhesion and promotion of proliferation, it supports the orderly reconstruction of endothelial progenitor cells and endothelial cells on the device surface; ultimately achieving long-term steady-state antithrombosis: the efficient completion of the natural endothelialization process ensures the long-term natural biocompatibility of the blood flow guiding device and reduces the risk of thrombosis and restenosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following is a brief introduction to the drawings required for use in the examples. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be considered as limiting the scope. A person of ordinary skill in the art can also derive other relevant drawings based on these drawings without inventive effort. In the drawings:

[0020] Figure 1 Schematic diagram of preparing a biomolecular self-assembly coating according to Example 1 of the present invention;

[0021] Figure 2 This is a schematic diagram of Example 1 of the present invention, in which the coating adjusts early anticoagulation and high-efficiency endothelial promotion to achieve long-term antithrombotic effect;

[0022] Figure 3 Schematic diagram of the morphology and thickness (SEM and AFM) of the nickel-titanium alloy sheet and blood flow guidance device product before and after modification of the present invention;

[0023] Figure 4 Schematic diagram of the hydrophilicity of the coating of the blood flow guiding device before and after modification according to Example 1 of the present invention;

[0024] Figure 5 Fluorescence images and scanning electron microscopy images of platelet adhesion before and after modification of the nickel-titanium-based blood flow guiding device of Example 1 of the present invention;

[0025] Figure 6 Schematic diagram of animal half-body blood circulation results before and after modification of the nickel-titanium-based blood flow guiding device according to Example 1 of the present invention;

[0026] Figure 7 This is a fluorescence schematic diagram of the endothelial cell adhesion and proliferation results before and after modification of the nickel-titanium alloy sheet of Example 1 of the present invention. DETAILED DESCRIPTION

[0027] The following is a combination of Example 1 of the present invention and the accompanying Figures 1 to 7 The present invention clearly and completely describes the technical solution of the present invention. It is obvious that the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0028] Example 1

[0029] S1: The blood flow guide device is cleaned and dried for use, and then an aqueous solution of ammonium hexafluorotitanate ((NH4)2TiF6) and an aqueous solution of boric acid (H3BO3) are mixed in equal volumes to obtain a mixed solution to prepare a TiO2 base layer. The obtained sample is removed, cleaned, and dried to obtain a sample having a TiO2 base layer;

[0030] S2: The sample of S1 with a TiO2 base layer was immersed in a mixed solution of 2 g / L phosphorylcholine (PC) concentration, 5 μL / mL CD146 antibody concentration, 0.2 mg / mL REDV peptide concentration, and 0.2 μg / mL vascular endothelial growth factor (VEGF) concentration, and deposited at room temperature for 12 hours. After drying at room temperature, an anti-coagulant and pro-endothelial blood flow guiding device was obtained.

[0031] Combine Figure 1 , Figure 1 The self-assembly principle based on non-covalent forces was demonstrated. PC molecules and biological molecules (CD146 antibody, REDV peptide and VEGF) were evenly combined and assembled through van der Waals forces and hydrogen bonds, which not only maintained a good biological interface but also did not affect the self-activity of the biological macromolecules, thus maintaining their natural biological functionality.

[0032] Combine Figure 2 , Figure 2 The coating is functionalized and can effectively prevent nonspecific protein adhesion in the early stage of implantation, thereby inhibiting the early violent coagulation reaction of the material. At the same time, it cooperates with the bioactive molecular interface of the whole process to provide a good microenvironment driving the subsequent natural re-endothelialization process of the device surface. By mobilizing the adhesion and differentiation of endothelial progenitor cells and the adhesion, migration and proliferation of endothelial cells, the natural re-endothelialization process is maximized, and vascular tissue remodeling is achieved to achieve long-term anti-thrombotic effect.

[0033] Combine Figure 3 , Figure 3 The microscopic morphology of the prepared coating is shown, and it can be seen that the various components are successfully self-assembled and constructed on the sample surface to obtain a specific biological functional molecular layer with nanoscale structure and thickness. From the theoretical and material characterization results, it is inferred that its binding force is based on non-chemical bonding (such as covalent) interaction form.

[0034] Combine Figure 4 , Figure 4 The hydrophilicity of the sample was determined by water contact measurements. The modified blood flow diversion device exhibited improved hydrophilicity, which is indirect evidence that the active biomolecules maintain their natural structure or conformation. The surface hydrophilicity of the device also directly improves its anticoagulant performance.

[0035] Combine Figure 5 , Figure 5Platelet adhesion and activation tests yielded fluorescence images and scanning electron microscopy images of platelet adhesion before and after modification. The experimental procedure involved mixing fresh whole blood with sodium citrate in a 9:1 ratio. The mixed blood was centrifuged at 1500 rpm for 15 minutes to obtain a sample, the supernatant of which was platelet-rich plasma (PRP). 100 μL of PRP was dripped onto the sample surface and incubated statically at 37°C for 1 hour. The sample was rinsed with 0.9% NaCl solution to remove non-adherent platelets and fixed with 2.5% glutaraldehyde at room temperature for at least 4 hours. Platelet adhesion and activation were then evaluated using fluorescence staining and scanning electron microscopy. 70 μL of diluted rhodamine was dripped onto the sample surface and allowed to stand in the dark for 5 minutes before observation under a fluorescence microscope. The sample was then dehydrated in a graded ethanol series (30%, 50%, 70%, 90%, and 100%) for 10 minutes. After critical point drying, the sample was gold-sprayed and observed under a scanning electron microscope.

[0036] Fluorescence staining and scanning electron microscopy were used to evaluate platelet adhesion and activation. Representative fluorescence images and SEM images of platelet adhesion are shown in Figure 2. Figure 4 As shown in the figure, compared with the NiTi sample, the number of platelets adhering to the surface of the modified sample of the present invention is significantly reduced, which fully demonstrates that the modified coating has a good anticoagulant effect.

[0037] Combine Figure 6 , Figure 6 The results of the animal hemicycle blood flow analysis before and after modification were presented. All experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the institutional ethics committee. Adult New Zealand white rabbits weighing between 2.5 and 3.5 kg were anesthetized, and their carotid arteries and external jugular veins were dissected and connected to PVC tubing containing samples to create arteriovenous circuits. All arteriovenous circuits were terminated after 2 hours of circulation. Samples from the circuits were flushed with 0.9% NaCl, and cross-sectional photographs were taken. The samples were fixed with 2.5% glutaraldehyde for more than 4 hours, dehydrated with gradient ethanol, dried, and gold-sprayed for observation using scanning electron microscopy. The modified samples showed significantly reduced internal blockage, with no obvious thrombus formation, while the unmodified (NiTi) group exhibited severe thrombosis and embolism. SEM images revealed that porous spaces remained between the braided filaments in the modified samples, and the number of platelets and fibrinogen adhered was significantly lower than in the unmodified group.

[0038] Combine Figure 7 , Figure 7 In order to compare the results of direct culture of endothelial cells before and after modification, human umbilical vein endothelial cells (HUVECs) were used to evaluate the cell compatibility of the samples. The specific experimental operation process was as follows: the samples were sterilized and placed in a 24-well cell culture plate. 4Cells were seeded in the well plate at a cell density of 1×10 cells / mL and placed in the incubator for 2 hours to evaluate the cell adhesion results; 4 Cells were seeded at a density of 100 cells / mL and cultured for 1, 3, and 5 days to assess cell proliferation. The samples were washed with PBS and fixed in 2.5% glutaraldehyde for at least 4 hours, then stained with rhodamine for observation. The cells were then dehydrated in an ethanol gradient, critical point dried, and gold-sprayed for observation using a scanning electron microscope. This experiment evaluated the ability of the molecularly self-assembled coating in this example to promote endothelial adhesion and proliferation. The results showed that the modified material surface significantly promoted endothelial cell adhesion and proliferation, demonstrating excellent endothelial functionalization.

[0039] Regarding the EPC capture strategy, CD146 antibody serves as a unique target for selectively capturing late endothelial progenitor cells (EPCs), and can efficiently and specifically capture non-early and late endothelial cells (ECs). This is also different from other cardiovascular stents coated with anti-CD34 antibodies, because CD34 + It is not a strict marker for EPCs; it is also present on hematopoietic stem / progenitor cells (HSPCs) and is absent from fully differentiated endothelial cells (ECs) and late endothelial progenitor cells (EPCs), which are known to have a higher degree of differentiation. This results in a limited clinical effect. Furthermore, the synergistic effect of CD146 and vascular endothelial growth factor (VEGF) in the present invention significantly promotes the specific capture of EPCs and their differentiation into ECs. VEGF can also synergize with REDV to act on EC cellular pathways, thereby promoting EC adhesion, migration, and proliferation. This coating fully leverages the natural biological drive for endothelialization, achieving long-term, stable antithrombotic effects and embodying the new concept of biomaterials.

Claims

1. A method for preparing a naturally biologically driven, long-lasting, anti-thrombotic and endothelialized improved blood flow guiding device, characterized in that: The following steps are involved: S1: The blood flow guiding device is cleaned and dried, and an aqueous ammonium hexafluorotitanate solution and an aqueous boric acid solution are mixed to obtain a mixed solution A. The blood flow guiding device is immersed in the mixed solution to obtain a blood flow guiding device with a TiO2 bottom layer; S2: The blood flow guiding device with a TiO2 base layer of S1 is immersed in a mixed solution B of phosphorylcholine, CD146 antibody, REDV peptide, and vascular endothelial growth factor, and liquid deposition is performed at room temperature. After drying, a naturally biologically driven, long-stable anti-thrombotic and endothelialized improved blood flow guiding device is obtained.

2. The method for preparing a naturally biologically driven, long-stable, anti-thrombotic and endothelialized improved blood flow guiding device according to claim 1, characterized in that: The volume ratio of the ammonium hexafluorotitanate aqueous solution and the boric acid aqueous solution described in S1 is 1:

1.

3. The method for preparing a naturally biologically driven, long-stable, anti-thrombotic and endothelialized improved blood flow guiding device according to claim 1, characterized in that: The concentration of phosphorylcholine described in S2 is 1-10 g / L, the concentration of REDV peptide is 0.1-1 mg / mL, the concentration of vascular endothelial growth factor is 0.1-1 μg / mL, and the dilution ratio of CD146 antibody is 0.5% to 2%.

4. The method for preparing a naturally biologically driven, long-stable, anti-thrombotic and endothelialized improved blood flow guiding device according to claim 1, characterized in that: The liquid phase deposition time described in S2 is 1-24 hours.

5. A naturally biologically driven, long-lasting, anti-thrombotic and endothelialized improved blood flow guiding device obtained by the preparation method according to any one of claims 1 to 4, characterized in that: The naturally biologically driven long-stable anti-thrombotic and endothelialized improved blood flow guiding device is a biological functional molecular layer.

6. The naturally biologically driven, long-lasting, anti-thrombotic and endothelialized improved blood flow guiding device according to claim 5, characterized in that: The thickness of the biological functional molecule layer of the blood flow guiding device is 5-100 nm.

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