Functional integrated blood flow diversion device mimicking endothelial exosomes and its preparation method

Abstract

This invention discloses a functionally integrated blood flow guiding device and its preparation method that mimics endothelial-derived exosomes. The device includes a blood flow guiding component and a functionalized coating integrated on the blood flow guiding component. The functionalized coating includes a base layer capable of catalytically releasing endogenous nitric oxide and an outer layer loaded on the surface of the base layer. The outer layer and the base layer together form a biofunctionally integrated surface similar to exosomes. This invention, on the one hand, can exert natural anti-acute coagulation function; on the other hand, through anti-inflammatory effects, the function of enhancing endothelial cell activity, and the continuous release of endogenous nitric oxide gas signaling molecules, it synergistically promotes the endothelialization process on the surface of the device and maintains a local anti-inflammatory microenvironment. This endows the device with excellent long-term antithrombotic function and the ability to inhibit vascular intimal hyperplasia. This allows the invention to adapt to higher and more complex vascular microenvironments and clinical treatment needs, ultimately achieving the therapeutic goal of efficiently ensuring long-term patency of tumor-bearing vessels.

Description

Technical Field

[0001] This invention relates to the field of surface functional modification technology for medical devices and biomaterials, and particularly to a functionally integrated blood flow guiding device that mimics endothelial-derived exosomes and its preparation method. Background Technology

[0002] Flow diverting devices (also known as mesh stents) are novel instruments used in the clinical treatment of intracranial aneurysms. Through their unique woven structure with high metal coverage, they guide most of the impulsive blood flow towards the aneurysm to distal normal vessels, while a small portion of weakened blood flow stagnates within the aneurysm, forming a thrombus and achieving aneurysm occlusion. Simultaneously, the device provides structural support for endothelial cell adhesion and growth, ultimately remodeling the lesion site, i.e., the carrier vessel. However, when using flow diverting devices for interventional treatment, patients face the core risks of acute coagulation and long-term thrombosis. Currently, the standard clinical protocol requires patients undergoing this treatment to receive long-term or even lifelong dual antiplatelet therapy (DAPT) during the perioperative period. However, this protocol significantly increases the risk of bleeding and imposes a heavy psychological and economic burden on patients and their families. Therefore, improving the anticoagulant / antithrombotic capabilities of flow diverting devices is a key technological direction for alleviating these clinical burdens. Functionalized coatings are an efficient technique for improving the biocompatibility of flow diverters and enhancing their anticoagulation / antithrombotic performance. However, existing flow diverter products (such as PED-Shield and P64 / P48-MW-HPC) rely solely on the physicochemical properties of a single component to achieve anticoagulation / antithrombotic functions. This single, passive functional mode easily leads to the risk of thrombosis due to insufficient anticoagulation in the early stages, and the risk of in-stent restenosis due to delayed endothelialization in the later stages. In addition, although current domestic and international research has focused on constructing ideal surfaces that can promote endothelialization of flow diverters, most coatings are simply stacks of functional components, failing to achieve multi-level, multi-functional synergistic effects, resulting in poor long-term application results. Furthermore, the vascular inflammatory microenvironment plays a crucial regulatory role in the repair process after device implantation: inflammation not only delays endothelial cell growth and endothelialization but may also induce abnormal proliferation of smooth muscle cells, further exacerbating the risk of in-stent restenosis; however, existing related studies generally lack targeted regulatory designs for the vascular inflammatory microenvironment after flow diverter implantation. Summary of the Invention

[0003] To address the above-mentioned problems, the present invention aims to provide a functionally integrated blood flow guiding device and its preparation method that mimics endothelial exosomes.

[0004] The technical solution of the present invention is as follows: On the one hand, a functionally integrated blood flow guiding device that mimics endothelial-derived exosomes is provided, comprising a blood flow guiding device and a functional coating integrated on the blood flow guiding device. The functional coating comprises a base layer capable of catalytically releasing endogenous nitric oxide and an outer layer loaded on the surface of the base layer, wherein the outer layer and the base layer can together form a biofunctionally integrated surface similar to exosomes.

[0005] Preferably, the base layer is polydopamine that chelates copper ions.

[0006] Preferably, the outer layer is a phosphoric choline lipid bilayer containing bioactive factors / molecules.

[0007] Preferably, the bioactive factors / molecules include Chemerin 15 peptide, REDV peptide, and recombinant VEGF protein.

[0008] Preferably, the outer layer is loaded onto the surface of the base layer by electrostatic self-assembly.

[0009] Preferably, during electrostatic self-assembly, the concentration of the Chemerin 15 peptide is 0.05-1 mg / ml, the concentration of the REDV peptide is 0.05-1 mg / ml, the concentration of the VEGF recombinant protein is 0.1-2 ug / ml, and the concentration of phosphocholine is 3-7 mg / ml.

[0010] Preferably, the thickness of the outer layer is 5-500 nm.

[0011] On the other hand, a method for preparing the functionally integrated blood flow diversion device based on endothelial-derived exosomes as described in any one of the above claims is also provided, comprising the following steps: S1: Prepare the blood flow diversion device, and clean and dry it to obtain the processed blood flow diversion device; S2: Prepare the first raw material solution of the base layer, and place the treated blood flow guiding device into the first raw material solution for room temperature deposition to obtain a blood flow guiding device with a base layer; S3: Prepare the second raw material solution for the outer layer, and place the blood flow guiding device with the base layer into the second raw material solution for room temperature deposition and drying to obtain the functional integrated blood flow guiding device of the endothelial-derived exosome.

[0012] Preferably, in step S2, the time for room temperature deposition is 6-24 hours.

[0013] Preferably, in step S3, the time for room temperature deposition is 12-24 hours.

[0014] The beneficial effects of this invention are: This invention can exert an acute anticoagulant effect on the one hand, and enhance endothelial cell activity through anti-inflammatory effects on the other hand. By continuously releasing endogenous nitric oxide gas signaling molecules, it synergistically promotes the endothelialization process on the surface of the device and maintains a local anti-inflammatory microenvironment, thereby endowing the device with excellent antithrombotic properties and the ability to inhibit vascular intimal hyperplasia. This invention can adapt to the more complex diseased vascular microenvironment and clinical treatment needs, and ultimately achieve the treatment goal of efficiently ensuring long-term vascular patency. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram illustrating the mechanism of the functionalized coating of the present invention; Figure 2 This is a schematic diagram of the preparation process of the functionalized coating in Example 1; Figure 3 These are schematic diagrams of the surface morphology of the functionalized coatings in Example 1 and Comparative Examples 1-3; Figure 4 The image shows the coating morphology of the nickel-titanium alloy sheet and the nickel-titanium alloy blood flow guiding device in Example 1. Figure 5 The images show fluorescence of the bioactive factors / molecules in the functionalized coating of Example 1, XPS fine spectrum of Cu 2p, and atomic force microscopy (AFM) step pattern of the modified coating. Figure 6 The electron micrographs and platelet fluorescence images of whole blood after immersion in whole blood or platelet-rich plasma before and after modification of the nickel-titanium alloy sheet and nickel-titanium alloy blood flow guiding device in Example 1 are shown. Figure 7 The images show fluorescence images of the nickel-titanium alloy sheet before and after modification, and after direct culture of endothelial cells in Example 1. Figure 8 The images show fluorescence images of the nickel-titanium alloy sheet before and after modification, and after direct culture of smooth muscle cells in Example 1. Figure 9 The images show fluorescence images of the nickel-titanium alloy sheet before and after modification, and after direct culture of macrophages in Example 1. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and technical features described in this application can be combined with each other. It should also be pointed out that, unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terms "comprising" or "including" and similar words used in this invention refer to elements or objects preceding the word that encompass the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0018] On one hand, the present invention provides a functionally integrated blood flow guiding device that mimics endothelial-derived exosomes, including a blood flow guiding device and a functional coating integrated on the blood flow guiding device. The functional coating includes a base layer capable of catalytically releasing endogenous nitric oxide and an outer layer loaded on the surface of the base layer, and the outer layer and the base layer can together form a biofunctionally integrated surface similar to exosomes.

[0019] Exosomes are a class of microvesicles secreted by cells, with a diameter of 50-100 nanometers. Through endocytosis, their microscopic structural domains induce the aggregation of various bioactive molecules (proteins, RNA) on the cytoplasmic side surface, which are then released extracellularly via regulated exocytosis, coordinating intercellular communication. The outer layer and the base layer described in this invention can together form an exosome-like biofunctional integrated surface, thus enabling the realization of functions similar to exosomes using the structure and components of exosome-like molecules.

[0020] In one specific embodiment, the substrate is polydopamine chelated with copper ions. In this embodiment, the polydopamine in the substrate has high natural biocompatibility, high substrate adhesion, and weak electrical properties. The chelated copper ions in the substrate can act as a substitute for endothelial nitric oxide synthase (eNOS), continuously catalyzing the release of endogenous nitric oxide gas. Nitric oxide (NO) is a small molecule gaseous signal that can act directly on target cells without the mediation of traditional receptors, and is known as a vasoactive factor or reactive nitrogen mediator. Exosomes can carry and release NO or NO-related bioactive factors, thus playing a NO-mediated role in various physiological and pathological processes such as vasodilation, anti-inflammation, immune defense, and tissue repair.

[0021] In one specific embodiment, the outer layer is a phosphocholine lipid bilayer containing bioactive factors / molecules. Accordingly, the bioactive factors / molecules include Chemerin 15 peptide, REDV peptide, and recombinant VEGF protein.

[0022] Endothelial cell-derived exosomes, with a lipid bilayer as their "backbone," carry a variety of bioactive molecules, including endothelial nitric oxide synthase (eNOS) as mentioned above, as well as typical vascular endothelial growth factors (such as recombinant VEGF protein) and anti-inflammatory active factors such as IL-10. Under physiological conditions, they maintain healthy vascular homeostasis and extend into areas such as tissue repair, immune regulation, and metabolic regulation. Phosphocholine (PC), as a major component of the endothelial cell-derived exosome "backbone," provides anchoring sites for membrane proteins (such as eNOS) and easily targeted "surface molecules," regulating their activity.

[0023] In the above embodiments, a phosphocholine lipid bilayer containing bioactive factors / molecules is used, which can work together with the base layer to simulate endothelial-derived exosomes. Among them, REDV peptide and Chemerin 15 peptide, as artificially modified cell-targeting "surface molecules", accurately recognize mature endothelial cells and macrophages respectively, enabling them to specifically enhance endothelial cell vitality and regulate the inflammatory phenotype of macrophages, thereby mediating their natural endothelialization process and inhibiting intimal hyperplasia.

[0024] In one specific embodiment, the outer layer is loaded onto the surface of the base layer via electrostatic self-assembly. During electrostatic self-assembly, the concentrations of the Chemerin 15 peptide, the REDV peptide, and the recombinant VEGF protein are 0.1-2 μg / ml, and the phosphoric acid choline concentration is 3-7 mg / ml.

[0025] It should be noted that electrostatic self-assembly is only a preferred method of the present invention, and other chemical deposition methods in the prior art, such as covalent bonding, can also be applied to the present invention.

[0026] In one specific embodiment, when using the present invention, such as Figure 1 As shown, in the initial stage of interventional treatment, the implantation of the flow diversion device can damage the vascular endothelium, causing tissue inflammation. Chemerin 15 peptide, which precisely binds to and influences macrophage phenotype, regulates the inflammatory microenvironment of the blood vessels. Endogenous nitric oxide gas released via copper ion catalysis can inhibit platelet adhesion activation, thus addressing acute coagulation issues. The phosphocholine (PC) molecular layer passively resists fouling, hindering the non-specific adsorption of erythrocytes, fibrin, etc. Subsequently, REDV adhesion peptide, which specifically recognizes endothelial cells, can capture mature endothelial cells, which then proliferate and migrate under the influence of vascular endothelial growth factor (VEGF), jointly achieving rapid endothelialization of the device. Simultaneously, the continuously released nitric oxide gas inhibits excessive proliferation of smooth muscle cells and can synergistically maintain the anti-inflammatory microenvironment with Chemerin 15 peptide, while VEGF promotes rapid endothelialization of the device.

[0027] Inspired by the structure and function of endothelial-derived exosomes, this invention combines bioactive polypeptides and proteins with the release of gaseous signaling molecules through electrostatic self-assembly and non-covalent interactions. This endows the blood flow diversion device with the ability to regulate the inflammatory microenvironment and possesses multi-level, multi-angle, specific, efficient, and synergistic bioactivity. Unlike blood flow diversion devices that utilize simple physicochemical properties and single-sided biological functions, this invention implements the biomimetic concept of endothelial-derived exosomes at both structural and functional levels. It overcomes the limitations of current blood flow diversion device research, which suffers from low specificity and short-term effects of single-sided biological functions. This provides a new approach for blood flow diversion devices to adapt to complex and challenging vascular microenvironments and maintain long-term vascular patency at lesion sites.

[0028] On the other hand, the present invention also provides a method for preparing the functionally integrated blood flow diversion device based on endothelial-derived exosomes as described in any one of the above claims, comprising the following steps: S1: Prepare the blood flow diversion device, and clean and dry it to obtain the processed blood flow diversion device; S2: Prepare the first raw material solution of the base layer, and place the treated blood flow guiding device into the first raw material solution for room temperature deposition to obtain a blood flow guiding device with a base layer; S3: Prepare the second raw material solution for the outer layer, and place the blood flow guiding device with the base layer into the second raw material solution for room temperature deposition and drying to obtain the functional integrated blood flow guiding device of the endothelial-derived exosome.

[0029] In one specific embodiment, in step S2, the time for room temperature deposition is 6-24 hours; in step S3, the time for room temperature deposition is 12-24 hours.

[0030] Example 1 Taking a nickel-titanium substrate and a nickel-titanium alloy blood flow guiding device as an example, the functionalized coating is prepared on it using the preparation method described in this invention, such as... Figure 2 As shown, the specific steps include: (1) First, adjust the pH of the deionized water with Tris buffer to 8.0-8.5 with dilute hydrochloric acid, then add 0.25 mg / ml dopamine hydrochloride and 0.05 mg / ml copper chloride hexahydrate and stir evenly to obtain the raw material solution of the base layer. (2) The cleaned and dried nickel-titanium substrate and blood flow guiding device were placed in the raw material solution one and deposited at room temperature for 12 hours to obtain a base sample with polydopamine chelated copper ions; (3) Add 4 mg / ml of phosphoric acid choline (PC), 0.2 mg / ml of Chemerin 15 peptide, 0.2 mg / ml of REDV peptide, and 0.2 ug / ml of VEGF recombinant protein to ultrapure water (UP), stir well, and obtain the second raw material solution of lipid bilayer; (4) The sample with the base layer in step (2) is immersed in the second raw material solution and deposited at room temperature for 12 hours. After drying, a nickel-titanium substrate and a nickel-titanium alloy blood flow guiding device with the functionalized coating are obtained.

[0031] Example 2 Unlike Example 1, in step (3) of this example, the concentration of Chemerin 15 peptide is 0.05 mg / ml, the concentration of REDV peptide is 0.05 mg / ml, the concentration of VEGF recombinant protein is 0.1 ug / ml, and the concentration of phosphocholine is 3 mg / ml.

[0032] Example 3 Unlike Example 1, in step (3) of this example, the concentration of Chemerin 15 peptide is 1 mg / ml, the concentration of REDV peptide is 1 mg / ml, the concentration of VEGF recombinant protein is 2 ug / ml, and the concentration of phosphocholine is 7 mg / ml.

[0033] Comparative Example 1 Unlike Example 1, the concentration of phosphocholine in step (3) of this comparative example is 1 mg / ml.

[0034] Comparative Example 2 Unlike Example 1, the concentration of phosphocholine in step (3) of this comparative example is 2 mg / ml.

[0035] Comparative Example 3 Unlike Example 1, the concentration of phosphocholine in step (3) of this comparative example is 8 mg / ml.

[0036] Test Example 1 The surface morphology of the deposited coatings was observed using field emission scanning electron microscopy when the concentrations of polydopamine-chelated copper ions in the substrate and phosphocholine (PC) varied at 1 mg / ml, 2 mg / ml, 4 mg / ml, and 8 mg / ml. The results are as follows: Figure 3 As shown. From Figure 3 It can be seen that when the concentration of phosphocholine (PC) is 1 mg / ml or 2 mg / ml, the substrate cannot be completely covered; when the concentration of phosphocholine (PC) is 4 mg / ml, the substrate is completely and uniformly covered; when the concentration of phosphocholine (PC) is 8 mg / ml, the coating becomes thicker on the basis of completely covering the substrate.

[0037] Test Example 2 The surface morphology of the nickel-titanium alloy sheet (plate) and the nickel-titanium alloy blood flow guide device before and after modification in Example 1 was observed using field emission scanning electron microscopy. The results are as follows: Figure 4 As shown. From Figure 4 It can be seen that the endothelial-derived exosome-modified coating was successfully and uniformly constructed on the substrate.

[0038] Test Example 3 The modified sample from Example 1 was characterized using fluorescence staining, X-ray photoelectron spectroscopy, and atomic force microscopy (AFM). The results are as follows: Figure 5 As shown. From Figure 5 It can be seen that the modified sample successfully assembled Chmerin 15 peptide, VEGF recombinant protein, REDV peptide and divalent copper ions, and the modified coating is an ultrathin nanoscale structure.

[0039] Test Example 4 The nickel-titanium alloy substrate and blood flow diverting device used in Example 1, without the addition of nitric oxide donors, and the modified nickel-titanium alloy substrate and blood flow diverting device, with the addition of nitric oxide donors, were incubated in whole blood and platelet-rich plasma (PRP) at 37°C for 1 hour under static conditions. The results are as follows: Figure 6 As shown. From Figure 6 It can be seen that the number of red blood cells and platelets on the surface of the modified sample was significantly reduced compared with that of the unmodified sample, indicating that the modified sample has good antithrombotic and anticoagulant effects.

[0040] Test Example 5 The nickel-titanium alloy substrate used in Example 1, without the addition of nitric oxide donors, and the modified nickel-titanium alloy substrate with the addition of nitric oxide donors, were directly cultured with endothelial cells and smooth muscle cells for 2 h, 24 h, or 72 h, respectively; and directly cultured with macrophages for 24 h. The fluorescence patterns after culture were then observed, and the results are as follows: Figures 7-9 As shown. From Figures 7-9 It can be seen that the number of endothelial cells on the modified coating is significantly increased, while the number of smooth muscle cells and macrophages is significantly reduced, indicating that the modified sample has excellent potential to promote endothelialization, inhibit intimal hyperplasia, and resist inflammation.

[0041] The above description is merely a representative embodiment of the present invention and is not intended to limit the present invention in any way. Any embodiment made by those skilled in the art without departing from the scope of the present invention and utilizing the disclosed technical content is an equivalent embodiment of the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An endothelium-mimicking, functionally integrated blood flow diverter device comprising a blood flow diverter device and a functionalized coating integrated on the blood flow diverter device, characterized in that, The functionalized coating includes a base layer capable of catalytically releasing endogenous nitric oxide and an outer layer loaded on the surface of the base layer, wherein the outer layer and the base layer can together form an exosome-like biofunctional integrated surface. The outer layer is a phosphoric acid choline lipid bilayer containing bioactive factors / molecules, including Chemerin 15 peptide, REDV peptide, and VEGF recombinant protein. The outer layer is loaded onto the surface of the base layer through electrostatic self-assembly. During electrostatic self-assembly, the concentration of phosphoric acid choline is 3-7 mg / ml.

2. The functionally integrated blood flow guiding device mimicking endothelial-derived exosomes according to claim 1, characterized in that, The base layer is polydopamine that chelates copper ions.

3. The functionally integrated blood flow guiding device mimicking endothelial-derived exosomes according to claim 1, characterized in that, During electrostatic self-assembly, the concentration of the Chemerin 15 peptide is 0.05-1 mg / ml, the concentration of the REDV peptide is 0.05-1 mg / ml, and the concentration of the VEGF recombinant protein is 0.1-2 ug / ml.

4. The functionally integrated blood flow guiding device based on endothelial-derived exosomes according to any one of claims 1-3, characterized in that, The thickness of the outer layer is 5-500 nm.

5. The method for preparing the functionally integrated blood flow guiding device based on endothelial-derived exosomes as described in any one of claims 1-4, characterized in that, Includes the following steps: S1: Prepare the blood flow diversion device, and clean and dry it to obtain the processed blood flow diversion device; S2: Prepare the first raw material solution of the base layer, and place the treated blood flow guiding device into the first raw material solution for room temperature deposition to obtain a blood flow guiding device with a base layer; S3: Prepare the second raw material solution for the outer layer, and place the blood flow guiding device with the base layer into the second raw material solution for room temperature deposition and drying to obtain the functional integrated blood flow guiding device of the endothelial-derived exosome.

6. The preparation method according to claim 5, characterized in that, In step S2, the time for room temperature deposition is 6-24 hours.

7. The preparation method according to claim 5, characterized in that, In step S3, the time for room temperature deposition is 12-24 hours.

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