A functional artificial blood vessel and a preparation method and application thereof

Abstract

The application discloses a functional artificial blood vessel and a preparation method and application thereof, relates to the technical field of artificial blood vessels, and comprises a main support and functionalized red blood cell membrane vesicles which are covalently connected to the main support; the functionalized red blood cell membrane vesicles are covalently connected to functional groups; the covalent connection significantly improves the long-term stability of the membrane vesicles under blood flow flushing and complex physiological environments; in addition, the functionalized red blood cell membrane vesicles are covalently connected to functional groups, for example, the functional groups have immune, antibacterial and epidemic prevention functions, so that the functionalized red blood cell membrane vesicles are endowed with new functions.

Description

Technical Field

[0001] This invention relates to the field of artificial blood vessel technology, and in particular to a functional artificial blood vessel, its preparation method, and its application. Background Technology

[0002] Currently, small-diameter artificial blood vessels are widely used for vascular replacement and reconstruction. However, existing artificial blood vessels generally suffer from problems such as strong early inflammatory responses, high risk of implant-related infections, and insufficient long-term functional integration with host tissues in clinical applications. Existing technologies mainly address these issues by modifying the material surface, such as introducing anticoagulant coatings, modifying anti-inflammatory molecules, immobilizing endothelialization factors, and using antibiotics or antibacterial materials. Although these strategies have improved the performance of artificial blood vessels to some extent, they still suffer from problems such as highly homogenous functional design, non-physiological immune regulation mechanisms, poor sustainability of antibacterial strategies, insufficient interfacial stability, and a lack of personalized adaptation capabilities. Most existing functionalization strategies for artificial blood vessels rely on uniform materials and processing methods. Summary of the Invention

[0003] The main objective of this invention is to propose a functional artificial blood vessel, its preparation method, and its application, aiming to solve the problem of existing artificial blood vessels that lack personalized adaptability and have highly singular functional designs.

[0004] To achieve the above objectives, the present invention proposes a functional artificial blood vessel, comprising a main scaffold and functionalized erythrocyte membrane vesicles covalently connected to the main scaffold, wherein the functionalized erythrocyte membrane vesicles are covalently connected to functional groups.

[0005] In one embodiment, the main support structure comprises a porous tubular support; and / or, The functional groups include polypeptides or proteins; and / or, The main support structure is made of at least one of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, and polyurethane.

[0006] In one embodiment, the diameter of the artificial blood vessel is ≤6 mm.

[0007] This invention also provides a method for preparing a functional artificial blood vessel, comprising the following steps: S10. Obtain the main scaffold and erythrocyte membrane vesicles; S20. Functionalize the erythrocyte membrane vesicles to give the erythrocyte membrane vesicles a first connection site and a second connection site, thereby obtaining functionalized vesicles; S30. Activate the main scaffold to obtain a main scaffold with reaction sites, and incubate the main scaffold with reaction sites with the functionalized vesicles to covalently connect the erythrocyte membrane vesicles to the main scaffold through the first connection site and the reaction site. S40. Incubate the solution of the functional group with the functionalized vesicles, so that the functional group is attached to the second attachment site to obtain an artificial blood vessel.

[0008] In one embodiment, step S20 includes: S201. Separation of red blood cells from autologous peripheral blood; S202. The red blood cells are perturbed to cause them to undergo endogenous PS eversion, and during the process, they are mixed with a first lipid molecule and a second lipid molecule so that the functional groups in the first lipid molecule and the second lipid molecule are exposed on the vesicle surface, respectively, to obtain functionalized vesicles with corresponding first and second connection sites, wherein the first lipid molecule contains a first functional group and the second lipid molecule contains a second functional group.

[0009] In one embodiment, the first lipid molecule comprises DSPE-PEG-N3; and / or, The second lipid molecule includes DSPE-PEG-NHS; and / or, The first functional group includes an azide group; and / or, The second functional group includes active molecules having an amino group.

[0010] In one embodiment, in step S30, the host scaffold having reactive sites includes modifying groups of cyclooctyne and its derivatives.

[0011] In one embodiment, the mass ratio of the first lipid molecule to the second lipid molecule is (1:3) to (1:10). The present invention also provides an application of a functional artificial blood vessel, the application including a product made from an artificial blood vessel prepared by any of the artificial blood vessels described above or by any of the artificial blood vessel preparation methods described above.

[0012] The technical solution of the present invention includes a main scaffold and functionalized erythrocyte membrane vesicles covalently connected to the main scaffold. The main scaffold provides a carrier, and by covalently connecting the functionalized erythrocyte membrane vesicles, a stable functionalized interface of the membrane vesicles is formed. Compared with physical adsorption, covalent connection significantly improves the long-term stability of the membrane vesicles under blood flow and complex physiological environments. In addition, the functionalized erythrocyte membrane vesicles are covalently connected to functional groups, such as functional groups with immune, antibacterial and disease prevention functions, thus endowing the functionalized erythrocyte membrane vesicles with new efficacy. Attached Figure Description

[0013] 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 the structures shown in these drawings without creative effort.

[0014] Figure 1 This is a schematic diagram of the process for preparing artificial blood vessels in Embodiment 1 of the present invention; Figure 2 A schematic diagram of the quantitative results of flow cytometry analysis of the vesicles prepared in Example 1 of the present invention; Figure 3 This is a schematic diagram illustrating the bacterial growth of vesicles prepared in Example 1 of the present invention; Figure 4 This is a schematic diagram illustrating the in situ implantation effect of the artificial blood vessel prepared in Example 1 of the present invention in a rabbit carotid artery model for evaluating the vascular graft effect. Figure 5 A schematic diagram showing the results of flow cytometry analysis of the functionalized vesicles prepared in Example 2 of the present invention. Figure 6 A schematic diagram showing the results of flow cytometry analysis of the functionalized vesicles prepared in Example 3 of this invention.

[0015] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0016] 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. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Existing materials for fabricating artificial blood vessels can be divided into two categories: synthetic polymers, such as polyester, expanded polytetrafluoroethylene, polyurethane, polyglycolic acid, and polyhydroxyalkanoates; and natural biomaterials, such as collagen, hyaluronic acid, silk fibroin, chitosan, and bacterial cellulose. Large-diameter artificial blood vessels (greater than 10 mm) synthesized from these materials have achieved significant clinical success. However, small-diameter artificial blood vessels (less than 6 mm), such as coronary arteries, synthesized from these materials suffer from problems like short-term easy clotting and long-term restenosis due to a lack of endothelialization. This means that existing materials still cannot meet clinical needs.

[0018] Currently, small-diameter artificial blood vessels are widely used for vascular replacement and reconstruction. However, existing artificial blood vessels generally suffer from problems such as strong early inflammatory responses, high risk of implant-related infections, and insufficient long-term functional integration with host tissues in clinical applications. Existing technologies mainly address these issues by modifying the material surface, such as introducing anticoagulant coatings, modifying anti-inflammatory molecules, immobilizing endothelialization factors, and using antibiotics or antibacterial materials. Although these strategies have improved the performance of artificial blood vessels to some extent, they still suffer from problems such as highly homogenous functional design, non-physiological immune regulation mechanisms, poor sustainability of antibacterial strategies, insufficient interfacial stability, and a lack of personalized adaptation capabilities. Most existing functionalization strategies for artificial blood vessels rely on uniform materials and processing methods.

[0019] In view of this, the present invention provides a functional artificial blood vessel, comprising a main scaffold and functionalized erythrocyte membrane vesicles covalently connected to the main scaffold, wherein the functionalized erythrocyte membrane vesicles are covalently connected to functional groups.

[0020] The technical solution of the present invention includes a main scaffold and functionalized erythrocyte membrane vesicles covalently connected to the main scaffold. The main scaffold provides a carrier, and by covalently connecting the functionalized erythrocyte membrane vesicles, a stable functionalized interface of the membrane vesicles is formed. Compared with physical adsorption, covalent connection significantly improves the long-term stability of the membrane vesicles under blood flow and complex physiological environments. In addition, the functionalized erythrocyte membrane vesicles are covalently connected to functional groups, such as functional groups with immune, antibacterial and disease prevention functions, thus endowing the functionalized erythrocyte membrane vesicles with new efficacy.

[0021] In some embodiments of the present invention, the main scaffold includes a porous tubular scaffold, and the material of the main scaffold includes at least one of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer and polyurethane. The material can be used to construct a porous fiber tubular scaffold with a natural blood vessel structure by electrospinning.

[0022] The diameter of the artificial blood vessel is ≤6 mm, and the artificial blood vessel is easy to obtain a small-diameter artificial blood vessel with a diameter of less than 6 mm.

[0023] The functional groups include polypeptides or proteins. Further, the functional groups include anticoagulant molecules, anti-inflammatory molecules, or antimicrobial peptides. For example, antimicrobial peptides are a broad category encompassing thousands of peptides with diverse structures and origins. Different antimicrobial peptides exhibit significant differences in their compatibility with antimicrobial spectrum, immunomodulatory properties, biocompatibility, and material surface presentation methods. Specifically, this invention preferably uses LL37 as a functionalized antimicrobial molecule, primarily based on the following considerations: First, LL37 is a human-derived cathelicidin antimicrobial peptide with broad-spectrum antimicrobial activity, effectively inhibiting Gram-positive bacteria, Gram-negative bacteria, and biofilm formation. Second, LL37 not only possesses direct bactericidal capabilities but also participates in regulating innate immune responses, promoting tissue repair and angiogenesis, and can avoid inducing excessive inflammatory responses under appropriate presentation methods. Third, LL37 has a moderate molecular weight and stable structure, containing multiple reactive primary amine sites, suitable for stable coupling through mild chemical methods, thus exhibiting good application compatibility on blood-contact biomaterial surfaces.

[0024] It should be noted that this invention achieves stable anchoring and directional presentation of functional groups by covalently fixing functionalized erythrocyte membrane vesicles to the surface of the main scaffold and covalently introducing functional groups into the membrane vesicles. This maintains good biocompatibility while endowing the artificial blood vessel with long-lasting antibacterial properties. Compared with simple incubation methods such as introducing antimicrobial peptides (e.g., LL37), this method has strong binding ability, can achieve long-term stability, and the antimicrobial peptides are stable and have high bioactivity. This avoids the conformational changes that can easily occur during random adsorption of antimicrobial peptides, leading to a decrease in their bioactivity, or the possibility of non-specific adsorption causing hemolysis or inflammatory reactions, affecting blood compatibility and making it difficult to achieve stable and effective antimicrobial function.

[0025] This invention also provides a method for preparing a functional artificial blood vessel, comprising the following steps: S10. Obtain the main support frame; S20. Functionalize the erythrocyte membrane vesicles to give the erythrocyte membrane vesicles a first connection site and a second connection site, thereby obtaining functionalized vesicles; S30. Activate the main scaffold to obtain a main scaffold with reaction sites, and incubate the main scaffold with reaction sites with the functionalized vesicles to covalently connect the erythrocyte membrane vesicles to the main scaffold through the first connection site and the reaction site. S40. Incubate the solution of the functional group with the functionalized vesicles, so that the functional group is attached to the second attachment site to obtain an artificial blood vessel.

[0026] The method for preparing artificial blood vessels provided by this invention is easy to operate, the insertion process is gentle, it does not require organic solvents or strong reaction conditions, does not damage the natural protein composition of red blood cell membranes, and the density of functional groups can be controlled by the addition concentration, making it suitable for large-scale preparation.

[0027] In some embodiments of the present invention, step S20 includes: S201. Separation of red blood cells from autologous peripheral blood; S202. The red blood cells are perturbed to cause them to undergo endogenous PS eversion, and during the process, they are mixed with a first lipid molecule and a second lipid molecule so that the functional groups in the first lipid molecule and the second lipid molecule are exposed on the vesicle surface, respectively, to obtain functionalized vesicles with corresponding first and second connection sites, wherein the first lipid molecule contains a first functional group and the second lipid molecule contains a second functional group.

[0028] By adopting the above technical solution, the erythrocyte membrane vesicles are made to turn outward by external force, thus constructing an immune-friendly substrate. In this process, the substrate is modified by mixing with a solution of the first functional group and a solution of the second functional group, thereby obtaining functionalized vesicles with complementary functions.

[0029] It should be noted that in the everted state, the DSPE-PEG-N3 / DSPE-PEG-NHS subsequently inserted into the erythrocyte membrane vesicles (EMVs) and the covalently fixed LL37 are all modified on the surface of the everted PS vesicles, achieving a dual engineering of "immune silencing signal + antibacterial function". It should be noted that red blood cells are derived from the peripheral blood of donors or patients. The distribution of phospholipids in the natural red blood cell membrane is asymmetrical. Phosphatidylserine (PS) is mainly located in the inner lobules (cytoplasmic side). Therefore, PS is almost undetectable on the surface of healthy red blood cells.

[0030] Freeze-thaw and ultrasound are two types of intense physical stress. This process simulates the typical characteristics of apoptotic cells—PS eversion as a "eat me" signal. Physical perturbation leads to endogenous PS eversion, forming basic EMVs. Then, through lipid insertion and covalent coupling, antibacterial and programmable functions are superimposed on them to achieve the dual goals of "bionic immune camouflage and active antibacterial".

[0031] In some embodiments of the present invention, the first lipid molecule includes DSPE-PEG-N3, wherein N3 is an azide group for click chemistry, particularly for subsequent copper-free click reactions with DBCO (dibenzocyclooctylene) or alkynes.

[0032] In some embodiments of the present invention, the second lipid molecule includes DSPE-PEG-NHS (succinimide ester). It is understood that DSPE-PEG-NHS is a three-segment functionalized phospholipid molecule used to covalently couple amino-containing molecules (such as proteins, peptides, and antibodies) to the surface of a lipid membrane. DSPE is distearate phosphatidylethanolamine, a synthetic phospholipid commonly used in drug delivery, liposomes, and biomimetic membrane modification. It can connect other molecules (such as PEG) and can act as a membrane anchoring carrier, responsible for "pinning" functional molecules to the membrane. PEG (polyethylene glycol) can extend the terminal groups, reduce steric hindrance, and improve reaction efficiency. NHS is N-hydroxysuccinimide ester, which has an active ester group and can rapidly form a stable amide bond with primary amines (-NH2).

[0033] In some embodiments of the present invention, the first functional group includes an azide group and its derivatives. Compared with other functional groups, the azide group (-N3) has better reactivity, stability and biocompatibility. The azide group does not interfere with the normal function of the cell membrane, so the effect of modifying the cell membrane with the azide group is better.

[0034] In some embodiments of the present invention, the second functional group includes an active molecule with an amino group, that is, an active molecule with an amino group after functionalization of erythrocyte membrane vesicles. The active molecule with an amino group can be linked to other corresponding functional groups, such as anticoagulant molecules or antimicrobial peptides, which can be selected according to the actual situation.

[0035] In some embodiments of the present invention, in step S30, the host scaffold with reaction sites includes cyclooctylene and its derivatives. The cyclooctylene includes dibenzocyclooctylene (DBCO), and the derivatives of the cyclooctylene include DBCO-PEG (polyethylene glycol) derivatives. By modifying the surface of cell membrane vesicles with azide groups, the azide groups undergo a click chemical reaction with the cyclooctylene and its derivatives on the host scaffold, thereby covalently linking them to the host scaffold.

[0036] In some embodiments of the present invention, the mass ratio of the first lipid molecule to the second lipid molecule is (1:3) to (1:10).

[0037] Specifically, the first lipid molecule is DSPE-PEG-N3, and the second lipid molecule is DSPE-PEG-NHS. The ratio of DSPE-PEG-N3 to DSPE-PEG-NHS can be adjusted within a certain range to achieve synergistic optimization of anti-inflammatory and antibacterial properties while maintaining the stability of the artificial blood vessel interface. Preferably, the mass ratio of DSPE-PEG-N3 to DSPE-PEG-NHS can be controlled within the range of (1:3) to (1:10), and more preferably, it is 1:2 to 1:10. DSPE-PEG-N3 is mainly used for subsequent click reactions with the surface of alkyne-containing materials to ensure the stable fixation of erythrocyte membrane vesicles on the surface of the vascular stent; DSPE-PEG-NHS is used for covalent coupling with the amino group of LL37 peptide to introduce antibacterial function.

[0038] By adjusting the ratio of the two functionalized lipids, the density of antimicrobial peptides on the vesicle surface can be controlled without significantly affecting the innate immune regulatory properties of the erythrocyte membrane. When the DSPE-PEG-N3 content is dominant, the stability and anti-inflammatory immune regulation effects of the artificial blood vessel interface are more prominent; when the DSPE-PEG-NHS content is moderately increased, local antimicrobial capacity can be enhanced while maintaining immune homeostasis. This ratio range helps to avoid non-specific immune activation caused by excessive exposure of antimicrobial peptides, thereby achieving a balance and maximization of stability, anti-inflammatory and antimicrobial properties of the artificial blood vessel under long-term implantation conditions.

[0039] In some embodiments of the present invention, step S30 includes: activating the main scaffold to obtain an aminated scaffold; A DBCO solution with active groups is mixed with the aminated scaffold and incubated to undergo an amidation reaction, thereby obtaining a host scaffold with reaction sites.

[0040] Specifically, in this invention, the connection between LL37 and DSPE-PEG-NHS is achieved through a nucleophilic substitution reaction between the active ester group of N-hydroxysuccinimide (NHS) and the primary amine group (–NH2) and N-terminal amino group on the side chain of lysine residues in the LL37 molecule. Specifically, after DSPE-PEG-NHS is inserted into erythrocyte membrane vesicles, its terminal NHS ester remains reactive under aqueous conditions. It can undergo a covalent amidation reaction with the primary amine in the LL37 molecule under near-neutral to weakly alkaline conditions (preferably pH 7.4–8.5) to form a stable -CO-NH- bond, thereby anchoring LL37 to the vesicle membrane surface.

[0041] This connection method requires no catalyst, has mild reaction conditions, and does not damage the erythrocyte membrane structure or the secondary conformation of LL37. It is beneficial to achieve stable presentation and functional expression of antimicrobial peptides while maintaining the immunomodulatory function mediated by phosphatidylserine.

[0042] Specifically, the introduction of DSPE-PEG-N3 containing azide groups and DSPE-PEG-NHS containing active ester groups onto the surface of erythrocyte membrane vesicles is achieved through a lipid insertion strategy, rather than chemical covalent grafting. The principle is that the hydrophobic fatty acid chains in the DSPE molecule can spontaneously embed into the lipid bilayer of erythrocyte membrane vesicles during membrane structure rearrangement, thereby stably exposing the PEG terminal functional groups on the vesicle surface.

[0043] (1) Introduction method of DSPE-PEG-N3 During the process of forming membrane vesicles by ultrasonic recombination after the red blood cell membrane is subjected to freeze-thaw treatment, DSPE-PEG-N3 is added to the system simultaneously.

[0044] Under sonication conditions (42 kHz, 20 W, 20% power, 5 min), the erythrocyte membrane undergoes transient disruption and rearrangement. The hydrophobic chains of DSPE in the DSPE-PEG-N3 molecule can interact hydrophobically with membrane lipids and insert into the lipid bilayer structure. After sonication, the uninserted free DSPE-PEG-N3 is removed by centrifugation at 20,000 g for 30 min to obtain EMVs-PS / N3 with azide groups on the surface.

[0045] This insertion method does not involve covalent modification of membrane proteins, which is beneficial for maintaining the natural protein composition and biological activity of erythrocyte membranes.

[0046] (2) Introduction methods of DSPE-PEG-NHS and LL37 The introduction of DSPE-PEG-NHS also employs a lipid insertion strategy. DSPE-PEG-NHS is added during the sonication and recombination stage of membrane vesicles. The hydrophobic chain of DSPE is embedded in the membrane lipid bilayer, while the active ester group of NHS at the PEG terminus is exposed on the vesicle surface. After sonication and centrifugation purification, the resulting vesicles are co-incubated with LL37 peptide in PBS at 4°C or room temperature. The NHS ester can undergo nucleophilic substitution reactions with lysine residues or N-terminal amino groups in the LL37 molecule, thereby achieving covalent fixation of LL37 on the vesicle surface.

[0047] (3) Construction of multifunctional engineered erythrocyte membrane vesicles When DSPE-PEG-N3 and DSPE-PEG-NHS are added simultaneously during the sonication phase, the two functional lipids can be synchronously inserted into the lipid bilayer of erythrocyte membrane vesicles. After centrifugation and purification, and then co-incubated with LL37 for 12 h, engineered erythrocyte membrane vesicles (EMVs-PS / N3 / LL37) with both azide group and antimicrobial peptide functions can be obtained.

[0048] It should be noted that both DSPE-PEG-N3 and DSPE-PEG-NHS are amphiphilic lipid molecules with hydrophobic lipid tails and hydrophilic PEG segments. The DSPE portion can insert into the lipid bilayer structure of the cell membrane through hydrophobic interactions, thereby achieving stable anchoring of the lipid molecules on the membrane surface. In this invention, ultrasonic treatment promotes sufficient contact between the lipid molecules and membrane vesicles, causing the hydrophobic fatty chain of DSPE to spontaneously embed into the lipid bilayer structure of the membrane vesicles, while the PEG chain and its terminal azide group (–N3) or NHS active ester group are exposed on the membrane surface. This introduces functional groups that can further undergo chemical reactions on the surface of the membrane vesicles. Therefore, both DSPE-PEG-N3 and DSPE-PEG-NHS are stably introduced into the surface of erythrocyte membrane vesicles through an ultrasound-induced lipid insertion mechanism.

[0049] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0050] Experimental materials DSPE-PEG-N3 and DSPE-PEG-NHS were purchased from Aladdin Reagents Ltd. (Aladdin, China), with DSPE-PEG-N3 catalog number D163600 and DSPE-PEG-NHS catalog number D163641; DBCO-Cy5 and NH2-FITC were purchased from Aladdin Reagents Ltd. (Aladdin, China), with DBCO-Cy5 catalog number D485480 and NH2-FITC catalog number A110141.

[0051] Example 1 A functional artificial blood vessel, the preparation steps are as follows: 1) Peripheral blood was collected and centrifuged at 2500 rpm for 15 min at room temperature. The obtained red blood cells were resuspended in diluted 0.25×PBS and incubated at 4℃ for 90 min, followed by centrifugation at 8000 rpm at 4℃ for 10 min. The supernatant was discarded. After washing / centrifuging with PBS for 3 cycles, the red blood cell membrane was obtained. The red blood cell membrane suspension was obtained by hypotonic treatment and stored at -80℃ for 24 h.

[0052] 2) Preparation of 15% (w / v) polycaprolactone (PCL, average molecular weight 45,000, Macklin, China) electrospinning solution: Dissolve PCL in hexafluoroisopropanol (Aladdin, USA) and stir at room temperature for 10 h. Load the electrospinning solution into a 5 mL syringe equipped with a No. 21 needle and apply a voltage of 6.5 kV at an injection rate of 0.3 mm / min using an injection pump (ET-1334H, Beijing Ucalery, China) for electrospinning. The collection device is a stainless steel rod with a diameter of 2 mm. After spinning at 30 r / min for 70 min, the PCL vessel is removed from the stainless steel rod and placed in 10 mL of a solution containing 10% (w / v) 1,6-hexanediamine. It is then incubated at 37°C for 1 h for amination modification and rinsed three times with ultrapure water. Subsequently, the amination-modified PCL vessel is immersed in 10 mL of PBS solution containing 0.1 mMDBCO-PEG4-NHS and incubated for 1 h. It is then rinsed three times with ultrapure water to obtain DBCO-modified vessels, which are the host scaffolds with reactive sites.

[0053] 3) Place the thawed red blood cell membrane suspension in an ultrasonic disruptor (42 kHz, 20 W; JY92-IIN, XINZHI, CN) and sonicate at 20% power for 5 min to obtain EMVs-PS, which are red blood cell vesicles.

[0054] 4) During the functional modification process, the prepared EMVs-PS suspension was divided into three groups for modification: Group 1: 1 mg / mL DSPE-PEG-N3 was added during the sonication phase, and the precipitate was collected after centrifugation at 20,000 g for 30 min to obtain EMVs-PS / N3.

[0055] Group 2: 1 mg / mL DSPE-PEG-NHS was added during sonication, and after centrifugation, it was co-incubated with 1 mg / mL LL37 peptide for 12 h to prepare EMVs-PS / LL37.

[0056] Group 3: DSPE-PEG-N3 (0.1 mg / mL) and DSPE-PEG-NHS (1 mg / mL) were added simultaneously, with a mass ratio of DSPE-PEG-N3 to DSPE-PEG-NHS of 1:10. After centrifugation, the mixture was incubated with 1 mg / mL LL37 for 12 h to obtain EMVs-PS / N3 / LL37. These vesicles were named engineered erythrocyte membrane vesicles, and all the obtained vesicles were stored in PBS at 4°C.

[0057] 5) The main scaffold with reactive sites is incubated with the obtained vesicles. The main scaffold is 1.5 cm long and 2 mm in diameter. The vesicle concentration is 20 μg / mL and the volume is 1 mL. Incubation is carried out at room temperature for 1 h to obtain the corresponding artificial blood vessel. The specific flowchart for preparing the artificial blood vessel is as follows: Figure 1 As shown.

[0058] Example 2 Unlike Example 1, in step 4), the red blood cell membrane vesicles were functionalized by lipid insertion. DSPE-PEG-N3 and DSPE-PEG-NHS were added to the red blood cell membrane suspension at a mass ratio of 1:1, with a final concentration of 0.5 mg / mL for both. The functionalized lipid molecules were inserted into the vesicle membrane structure by ultrasonic treatment to obtain functionalized vesicles.

[0059] Example 3 Unlike Example 1, in step 4), the red blood cell membrane vesicles were functionalized by lipid insertion. DSPE-PEG-N3 (0.1 mg / mL) and DSPE-PEG-NHS (0.5 mg / mL) were added to the red blood cell membrane suspension at a mass ratio of 1:5. The functionalized lipid molecules were inserted into the vesicle membrane structure by sonication to obtain the functionalized vesicles of Example 3.

[0060] Performance testing 1) The vesicles prepared in Example 1 were tested. In this example, "control" refers to untreated ordinary red blood cell membranes, and "EMVs-PS / N3-LL37" is the vesicle obtained in Example 1. The x-axis represents fluorescence intensity, and the y-axis represents particle number. Vesicles were labeled using three commercially available, qualified probes: DBCO-Cy5, Annexin V-FITC, and LL37-FITC, using conventional fluorescent labeling methods. DBCO-Cy5 (recognizing N3), Annexin V-FITC (recognizing PS), and LL37-FITC (replacing LL37) were used to label the engineered vesicles. Quantitative analysis was performed using flow cytometry (CytoFLEX, Beckman Coulter, USA). The flow cytometry quantitative results are shown below. Figure 2 As shown, the outward conversion rate of phosphatidylserine (PS) reached 77.4%, which is significantly higher than the outward conversion level of erythrocytes under normal physiological conditions; the grafting efficiency of N3 group reached 93.4%, indicating that it has achieved high-density and stable covalent grafting on the surface of membrane vesicles; the grafting rate of LL37 peptide was 91.1%, indicating that the antimicrobial peptide has achieved efficient and stable immobilization on the surface of vesicles.

[0061] 2) The effect of engineered erythrocyte membrane vesicles (50 μg / mL) on bacterial growth was investigated. Four types of erythrocyte membrane vesicles were used, where control was untreated ordinary erythrocyte membrane vesicles, and EMVs-PS, EMVs-PS / N3, EMVs-PS / LL37, and EMVs-PS / N3 / LL37 were obtained from EMVs-PS, EMVs-PS / N3, EMVs-PS / LL37, and EMVs-PS / N3 / LL37 prepared in Example 1, respectively. These were then compared with 1×10⁻⁶ ions / mL. 6 A mixture of CFU / mL Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) was thoroughly prepared, and 100 μL of the mixture was spread onto a plate. After incubation at 37°C for 24 h, colony counting was performed. The results are as follows: Figure 3 The image shows the antibacterial effects against *Escherichia coli* and *Staphylococcus aureus* under different treatment conditions. From left to right, treatment intensity increases (e.g., higher concentration or longer treatment time). The results show that with increasing treatment intensity, the colony-forming units (CFU) of both bacteria significantly decreased, indicating that this treatment method has broad-spectrum and dose-dependent antibacterial activity.

[0062] 3) Testing Methods: The orthotopic implantation effect of vascular grafts was evaluated using a rabbit carotid artery model. Eighteen New Zealand white rabbits (2.2-2.5 kg) were randomly divided into three groups, with different types of vascular grafts implanted in each group. The vascular grafts included: unmodified PCL-based artificial blood vessels (PCL group), PCL-based artificial blood vessels modified with EMVs-PS / N3 and EMVs-PS / N3 / LL37 (EPNP group and EPNLP group), respectively. Rabbits were anesthetized by injecting 5 mL of 3% sodium pentobarbital solution into the ear vein, and heparin (100 units / kg) was injected simultaneously to prevent thrombosis. After shaving the neck hair and disinfecting the area, the epidermis was incised to expose and separate the left carotid artery. A segment of the artery was clamped with a vascular clamp, and a vascular graft of approximately 1.2 cm in length was implanted into the defect site and sutured. The vascular clips were removed to restore blood flow, and the incision was sutured with 4-0 nylon sutures. Three months post-implantation, all rabbits were euthanized, and the vascular grafts were removed, fixed in 4% paraformaldehyde, and subjected to histological analysis and immunofluorescence staining. In short, paraffin sections were dewaxed, then sequentially immersed in a series of ethanol solutions (containing 75% alcohol) and rinsed with reverse osmosis water. Frozen sections were thawed, fixed, and stained, including eosin staining, differentiation, and bluing. After hematoxylin staining, the sections were dehydrated and sequentially immersed in a clearing agent. Masson's trichrome staining and alizarin red staining were used to evaluate collagen fiber regeneration and calcification after vascular graft implantation. For immunofluorescence assessment, fresh tissue was fixed in 4% PFA and embedded in paraffin; sections were dewaxed and hydrated. After antigen retrieval, samples were blocked with 3% BSA at room temperature for 30 min. Sections were stained with primary antibodies against CD31, α-SMA, TNF-α, and TGF-1β, followed by nuclear staining with corresponding fluorescently labeled secondary antibodies and DAPI (4′,6-diamidindole). The staining results are shown below. Figure 4 Histological staining results (H&E, M&T) showed that Figure 4H&E staining was used to assess tissue regeneration; Masson's trichrome staining was used to assess collagen fiber regeneration; Alizarin Red staining was used to assess calcification; CD31 staining was used to observe endothelialization; α-SMA staining was used to assess smooth muscle cell regeneration; TNF-α staining was used to assess the distribution of M1 macrophages; and TGF-β1 staining was used to assess the distribution of M2 macrophages. The EPNLP group formed continuous and uniform vascular tissue with regularly arranged collagen fibers, showing significantly better regeneration performance than the PCL group (tissue loss) and the EPNP group (intima hyperplasia). Engineered erythrocyte membrane vesicles significantly reduced calcification in PCL vascular grafts, an improvement that may be related to their ability to promote a repair-promoting immune microenvironment. Three months after implantation, the EPNLP lumen surface was covered with a complete and continuous endothelial cell layer. Furthermore, engineered erythrocyte membrane vesicles promoted angiogenesis while inhibiting excessive proliferation of vascular smooth muscle cells. Immunofluorescence results using M1 macrophage marker TNF-α and M2 macrophage marker TGF-β1 showed that the PCL group exhibited significant M1 macrophage recruitment accompanied by a strong inflammatory response; in contrast, the EPNP and EPNLP groups showed significantly reduced inflammation and both showed stronger M2 macrophage recruitment.

[0063] 5) The functionalized vesicles obtained in Example 2 were labeled using two commercially available qualified probes, DBCO-Cy5 and NH2-FITC, via conventional fluorescent labeling methods. DBCO-Cy5 was used to initiate a copper-free click reaction with the azide group; NH2-FITC was used to initiate an amidation reaction with the NHS ester group. After the reaction, unreacted free molecules were removed by centrifugation and washing. Finally, flow cytometry was used to detect the fluorescence intensity of the Cy5 and FITC channels, and the grafting efficiency of the functional groups on the vesicle surface was quantitatively analyzed. The test results are as follows: Figure 5 The results indicate that when DSPE-PEG-N3 and DSPE-PEG-NHS are co-inserted into erythrocyte membrane vesicles at a 1:1 mass ratio, the co-grafting efficiency of bifunctional groups on the vesicle surface is low, only about 3%. This result suggests that the two types of functionalized lipids compete with each other during membrane insertion. Due to the large steric hindrance of the azide group (N3) and the conformational difference of the PEG chain, its insertion and orientation in the membrane may have a certain impact on the integration of the other type of functionalized lipid (DSPE-PEG-NHS), thereby limiting the synergistic modification efficiency of the bifunctional groups.

[0064] 6) Two probes were simultaneously added to the functionalized vesicles obtained in Example 3 for labeling the reaction. One probe, DBCO-Cy5, was used to conduct a copper-free click reaction with the azide group; the other probe, NH2-FITC, was used to conduct an amidation reaction with the NHS ester group. After the reaction, unreacted free molecules were removed by centrifugation and washing. Finally, flow cytometry was used to detect the fluorescence intensity of the Cy5 and FITC channels to quantitatively analyze the grafting efficiency of functional groups on the vesicle surface. The analytical results are as follows: Figure 6 As shown, when DSPE-PEG-N3 and DSPE-PEG-NHS were co-inserted into erythrocyte membrane vesicles at a mass ratio of 1:5, the proportion of the upper right quadrant (double-positive region) in the flow cytometry plot, i.e., the co-grafting efficiency of the bifunctional groups on the vesicle surface, was relatively high, approximately 64%. This indicates that both groups were successfully inserted into the erythrocyte membrane vesicles.

[0065] In summary, the cell membrane proteins on the artificial blood vessels provided by this invention have strong activity and good stability. The cell membrane can also promote the rapid adhesion of cells to the artificial blood vessels, enabling them to perform their functions.

[0066] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A functional artificial blood vessel, characterized in that, It includes a main scaffold and functionalized erythrocyte membrane vesicles covalently connected to the main scaffold, wherein the functionalized erythrocyte membrane vesicles are covalently connected to functional groups.

2. The functional artificial blood vessel as described in claim 1, characterized in that, The main support structure includes a porous tubular support; and / or, The functional groups include polypeptides or proteins; and / or, The main support structure is made of at least one of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, and polyurethane.

3. The functional artificial blood vessel as described in claim 1, characterized in that, The diameter of the artificial blood vessel is ≤6 mm.

4. A method for preparing a functional artificial blood vessel as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S10. Obtain the main scaffold and erythrocyte membrane vesicles; S20. Functionalize the erythrocyte membrane vesicles to give the erythrocyte membrane vesicles a first connection site and a second connection site, thereby obtaining functionalized vesicles; S30. Activate the main scaffold to obtain a main scaffold with reaction sites, and incubate the main scaffold with reaction sites with the functionalized vesicles to covalently connect the erythrocyte membrane vesicles to the main scaffold through the first connection site and the reaction site. S40. Incubate the solution of the functional group with the functionalized vesicles, so that the functional group is attached to the second attachment site to obtain an artificial blood vessel.

5. The method for preparing a functional artificial blood vessel as described in claim 4, characterized in that, Step S20 includes: S201. Separation of red blood cells from autologous peripheral blood; S202. The red blood cells are perturbed to cause them to undergo endogenous PS eversion, and during the process, they are mixed with a first lipid molecule and a second lipid molecule so that the functional groups in the first lipid molecule and the second lipid molecule are exposed on the vesicle surface, respectively, to obtain functionalized vesicles with corresponding first and second connection sites, wherein the first lipid molecule contains a first functional group and the second lipid molecule contains a second functional group.

6. The method for preparing a functional artificial blood vessel as described in claim 4, characterized in that, The first lipid molecule includes DSPE-PEG-N3; and / or, The second lipid molecule includes DSPE-PEG-NHS; and / or, The first functional group includes an azide group; and / or, The second functional group includes active molecules having an amino group.

7. The method for preparing a functional artificial blood vessel as described in claim 4, characterized in that, In step S30, the host scaffold with reaction sites includes modifying groups of cyclooctyne and its derivatives.

8. The method for preparing an artificial blood vessel as described in claim 4, characterized in that, The mass ratio of the first lipid molecule to the second lipid molecule is (1:3) to (1:10).

9. An application of a functional artificial blood vessel, characterized in that, The application uses a product made from an artificial blood vessel prepared by a method comprising any one of the artificial blood vessels as described in claims 1 to 3 or any one of the artificial blood vessels as described in claims 4 to 8.

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