Natural biological driven long-stability anti-thrombosis and endothelialization improved blood flow guiding device and preparation method thereof

Abstract

The invention belongs to the field of surface function modification of medical instruments and biological materials, and particularly relates to a natural biological driven long-term stable anti-thrombus and endothelialization improved blood flow guiding device and a preparation method. The specific biological functional molecular layer provided by the invention can realize natural-like anticoagulation at the initial stage of implantation and natural biological driving endothelialization progress, so as to realize long-acting steady-state antithrombosis, and the specific biological functional molecular layer combines endothelial progenitor cells and endothelial cells, so that the endothelial progenitor cells and the endothelial cells are combined, and the endothelial progenitor cells and the endothelial cells are combined. The method realizes capture and differentiation of specific endothelial progenitor cells and adhesion, migration and proliferation of endothelial cells, improves effective re-endothelialization to the greatest extent, ensures natural biological characteristics of the endothelial progenitor cells, realizes natural remodeling of blood vessel inner wall tissues to achieve the purpose of stable growth and thrombosis resistance, is a new function idea which is difficult to realize by other schemes in the prior art, and has wide application prospects. The blood guiding device is a surface-improved blood guiding device which has nature-like anticoagulation and can drive endothelialization progress to obtain a long-acting steady state through natural biological property.

Description

Technical Field

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

[0002] Blood flow guidance devices (also known as dense mesh stents) are a new way of endovascular treatment of intracranial aneurysms. By guiding the impact blood flow in the tumor-bearing blood vessels to the distal normal blood vessels, the blood flow velocity and volume in the aneurysm are effectively reduced, thereby inducing thrombosis in the aneurysm, and guiding the endothelial growth of the aneurysm site to achieve vascular blood flow remodeling, excluding the aneurysm from the blood circulation system, and ultimately achieving the effect of complete occlusion of the aneurysm. This technology is gradually becoming a first-line treatment option for intracranial aneurysms that are difficult to treat with traditional therapies, especially for complex aneurysms (such as giant aneurysms, wide-necked aneurysms, fusiform and dissecting aneurysms, etc.).

[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 is extremely risky, directly endangers the patient's life, and brings 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 improvement of the anticoagulant performance of blood flow guidance devices through surface functional modification. These studies include the construction of bionic anticoagulant functional coatings, anticoagulant material coatings, and hydrophilic polymer coatings. However, these methods more reflect a more passive anticoagulant strategy, and are still far from achieving the satisfactory effect required in clinical practice in achieving long-term antithrombotic effects. In fact, endothelial remodeling plays a vital role in long-term antithrombotic effects. 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 anticoagulant 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 a core strategy of combining natural anticoagulation with natural biologically driven endothelialization progression. Specifically, through the molecular autonomous assembly technology, an interface coating with biologically active functions is constructed, which effectively inhibits the coagulation reaction in the early stage of device implantation and realizes the natural anticoagulation 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, promotes the natural progression of endothelialization, and thus achieves efficient and long-lasting anti-thrombotic effects. This innovative design can better meet the key clinical needs for long-term anti-thrombotic 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 method for preparing a natural biologically driven long-stable anti-thrombotic and endothelialized improved blood flow guiding device provided by the present invention comprises the following steps:

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

[0008] S2: The blood flow guiding device with 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 to the boric acid aqueous solution described in S1 is 1:1.

[0010] Furthermore, 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%.

[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 demand of anti-thrombotic on the surface of blood flow guiding device, the present invention innovatively constructs a specific biological functional molecular layer of anti-coagulation and endothelialization, and successfully achieves the goal of long-term steady-state anti-thrombotic through the synergistic effect of natural anticoagulation and natural biological driving endothelialization. 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 in the figure, phosphorylcholine (PC molecule) and specific biomacromolecules (such as CD146 antibody, REDV peptide and VEGF) form a stable self-assembled biological functional molecular layer through van der Waals force and hydrogen bonding. This molecular layer not only maintains good biocompatibility at the molecular interface, but also avoids the destruction of the activity of the biomolecule itself by chemical bonding, ensuring the integrity of the natural functionality of the biomacromolecule.

[0016] In the early stage of implantation, the biological functional molecular layer, because its basic components are similar to the phosphoester structure of natural red blood cell membranes and prevents non-specific protein adhesion, effectively inhibits the early violent coagulation reaction on the device surface and provides a natural anticoagulant effect. At the same time, the molecular layer also provides specific functional bioactive molecules and a good microenvironment, promoting the natural biologically driven endothelialization process. Specifically, the multi-component and multifunctional characteristics of the molecular layer are manifested in the specific capture of endothelial progenitor cells: through precise recognition and adhesion, it effectively adsorbs endothelial progenitor cells in the circulation, and drives the differentiation of endothelial progenitor cells: provides a suitable microenvironment, promotes the natural differentiation of endothelial progenitor cells into mature endothelial cells, and then adheres to, migrates and proliferates endothelial cells: supports the adhesion and stable migration of differentiated endothelial cells on the surface of the device, while stimulating the proliferation of endothelial cells, further covering the surface of the device, and maintaining the natural endothelial structure and function to the greatest extent.

[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 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 steady-state anti-thrombotic surface. Relatively and specifically speaking, the present invention uniquely adopts a non-covalent bond connection method between PC molecules and biomacromolecules, 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 biomolecules - the non-covalent connection method avoids changes in the chemical structure of biomolecules and retains the natural activity of 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 biomolecules (such as REDV peptides, VEGF, CD146 antibodies) work synergistically, which can not only achieve a natural 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 uses a specific biological functional molecular layer to highly call on key functions and work in synergy with nature, so that the blood flow guidance device can form a continuous optimization and efficient long-term treatment process from the natural anticoagulation in the early stage of implantation to the subsequent natural biological driven endothelialization. This creation of a 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 schemes, including natural anticoagulation: through the biocompatibility of PC molecules, simulating the natural anticoagulation barrier and inhibiting early coagulation reactions; and natural biological driven endothelialization progress: through precise capture, inducing differentiation, stable adhesion and promoting proliferation, supporting the orderly reconstruction of endothelial progenitor cells and endothelial cells on the device surface; and finally achieving long-term steady-state antithrombosis: the efficient completion of the natural endothelialization process ensures the long-term natural biocompatibility of the blood flow guidance 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 embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0020] Figure 1 This is a 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 the coating regulating early anticoagulation and high-efficiency endothelial promotion to achieve long-term antithrombotic effect according to Example 1 of the present invention;

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

[0023] Figure 4 This is a schematic diagram of the hydrophilicity of the coating of the blood flow guiding device of Example 1 of the present invention before and after modification;

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

[0025] Figure 6 It is a schematic diagram of the blood circulation results of a half body of an animal before and after the modification of the nickel-titanium-based blood flow guiding device of Example 1 of the present invention;

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

[0027] The following will be combined with Example 1 of the present invention and the attached Figures 1 to 7 , the technical solution of the present invention is clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 taken out, cleaned and dried to obtain a sample with 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), 5 μL / mL CD146 antibody, 0.2 mg / mL REDV peptide, and 0.2 μg / mL vascular endothelial growth factor (VEGF), and deposited at room temperature for 12 hours. After drying at room temperature, an anticoagulant and pro-endothelial blood flow guiding device was obtained.

[0031] Combination 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 assembled through van der Waals forces and hydrogen bonds, which not only maintained a good biological interface, but also did not affect the activity of the biological macromolecules themselves, thus maintaining their natural biological functionality.

[0032] Combination 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 force for 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 can be maximized, and vascular tissue remodeling can be achieved to achieve long-term anti-thrombotic effect.

[0033] Combination 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 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.

[0034] Combination Figure 4 , Figure 4 The hydrophilicity of the sample was determined by water contact measurement. The modified blood flow guide device showed better hydrophilicity, which is also one of the indirect evidences that the active biomolecules maintain their natural structure or conformation. Its surface hydrophilic physicochemical properties can also directly improve its anticoagulant performance.

[0035] Combination Figure 5 , Figure 5Through the platelet adhesion and activation test, the platelet adhesion fluorescence image and scanning electron microscopy image before and after modification can be obtained. The specific experimental operation is: fresh whole blood sodium citrate is mixed at a ratio of 9:1, and the mixed blood is centrifuged at 1500rpm for 15min to obtain a sample, wherein the supernatant in the sample is platelet-rich pulp (PRP); 100μL of PRP is dripped on the surface of the sample, incubated at 37℃ static conditions for 1h, and the sample is 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. Then the platelet adhesion and activation are evaluated by fluorescence staining and scanning electron microscopy results; 70μL of diluted rhodamine is dripped on the surface of the sample, and after being placed in a dark environment for 5 minutes, the sample is observed by fluorescence microscopy, and then the sample is dehydrated in a graded concentration ethanol series (30%, 50%, 70%, 90% and 100%) for 10 minutes. After critical point drying, gold spraying is performed and observed by scanning electron microscopy.

[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, 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] Combination Figure 6 , Figure 6 The results of animal hemibody blood circulation before and after modification are shown in the following table. The specific experimental operation is as follows: All procedures of animal experiments comply with the Guide for the Care and Use of Laboratory Animals and are approved by the Institutional Ethics Committee. After anesthetizing adult New Zealand white rabbits weighing between 2.5 and 3.5 kg, the carotid artery and external jugular vein of the rabbits were stripped and connected to the PVC tube with the built-in sample to form an arteriovenous loop. All arteriovenous loops of the rabbits were terminated after 2 hours of circulation; the samples from the loops were rinsed with 0.9% NaCl, and cross-sectional photos were taken; the samples were fixed with 2.5% glutaraldehyde for more than 4 hours, dried after gradient dehydration with ethanol, sprayed with gold, and observed by scanning electron microscopy. It can be seen that the internal blockage rate of the modified samples was significantly reduced, and no obvious thrombus embolism was formed, while the unmodified (NiTi) group showed severe thrombosis and embolism. In the SEM results, there were still porous spaces between the braided wires in the modified samples, and the number of platelets and fibrinogen adhesions was significantly lower than that of the unmodified group.

[0038] Combination Figure 7 , Figure 7 In order to directly culture the 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 an incubator for 2 hours to evaluate the cell adhesion results; 4 Cells were inoculated at a cell density of 10 cells / mL, and the cell proliferation results were evaluated after culturing for 1 day, 3 days, and 5 days; the samples were washed with PBS, fixed in 2.5% glutaraldehyde for more than 4 hours, stained with rhodamine and observed; the cells were dehydrated in ethanol gradient, critical point dried, sprayed with gold, and observed by scanning electron microscopy. This experiment was used to evaluate the molecular self-assembly coating in this example to promote endothelial adhesion and proliferation. The results showed that the modified material surface can significantly promote the adhesion and proliferation of endothelial cells, showing excellent endothelial functionalization.

[0039] Regarding the EPC capture strategy, CD146 antibody is a unique target for selectively capturing late endothelial progenitor cells (EPC), and can efficiently and specifically capture non-early and late endothelial cells (EC). This is also different from other cardiovascular stents coated with anti-CD34 antibodies, because CD34 + It is not a strict marker of EPC, that is, it also exists on hematopoietic stem / progenitor cells (HSPC), and it is also not found in fully differentiated endothelial cells (EC) and late endothelial progenitor cells (EPC) with a known higher degree of differentiation, resulting in insignificant clinical effects. At the same time, the synergistic effect of CD146 and vascular endothelial growth factor (VEGF) in the present invention can significantly promote the specific capture of EPC and the differentiation of captured EPC into EC; VEGF can also cooperate with REDV to act on the cell pathway of EC, thereby promoting EC adhesion, migration and proliferation. The coating gives full play to the natural biological drive of endothelialization progress, achieves long-term and stable anti-thrombotic, and also embodies the new concept of material biochemistry.

Claims

1. A method for preparing a naturally biologically driven long-stable anti-thrombotic and endothelialized improved blood flow guiding device, characterized in that: The following steps are involved: S1: cleaning and drying the blood flow guiding device, mixing an aqueous solution of ammonium hexafluorotitanate and an aqueous solution of boric acid to obtain a mixed solution A, and immersing the blood flow guiding device in the mixed solution to obtain a blood flow guiding device with a TiO2 bottom layer; S2: The blood flow guiding device with 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 to 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 natural biologically driven long-stable 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. A naturally biologically driven long-stable 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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