Rubia cordifolia-derived exosome-like nanovesicles, artificial blood vessels, their preparation and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a madder-derived exosome-like nanovesicle, an artificial blood vessel, and their preparation and application. This invention utilizes differential centrifugation and sucrose density gradient centrifugation techniques to extract high-purity, highly active exosome-like nanovesicles from madder. Then, polydopamine is loaded onto the surface of a small-diameter polyurethane artificial blood vessel, which is then immersed in a suspension of madder-derived exosome-like nanovesicles to obtain a small-diameter artificial blood vessel loaded with madder-derived exosome-like nanovesicles. These madder-derived exosome-like nanovesicles can effectively promote endothelial cell proliferation and inhibit reactive oxygen species generation; animal experiments show unobstructed blood flow under ultrasound. The small-diameter artificial blood vessel loaded with madder-derived exosome-like nanovesicles can simultaneously achieve in vivo endothelialization and vascular patency, possessing significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a madder-derived exosome-like nanovesicle, artificial blood vessel, and their preparation and application. Background Technology

[0002] Small-diameter artificial blood vessels (typically ≤6 mm in diameter) are important implantable grafts in vascular surgery, primarily used for bypass grafting in peripheral vascular stenosis or occlusive lesions, and arteriovenous access in hemodialysis. However, existing small-diameter artificial blood vessels still face numerous technical challenges in long-term use. First, the surface of traditional small-diameter artificial blood vessel materials (such as polyurethane and polytetrafluoroethylene) lacks bioactivity, making it difficult to effectively promote endothelial cell adhesion and proliferation, leading to delayed endothelialization after implantation and increasing the risk of thrombosis and restenosis. Second, contact between the material surface and blood easily activates inflammatory responses, recruiting immune cells such as macrophages and releasing inflammatory factors, further exacerbating intimal hyperplasia and functional degeneration. Furthermore, existing methods for improving the performance of small-diameter artificial blood vessels through chemical modification or biomolecular coating (such as heparin and vascular endothelial growth factor) often suffer from problems such as cross-linking agent toxicity, low bioactivity retention, or poor load stability, making it difficult to achieve long-term functional maintenance.

[0003] Exosomes, due to their crucial role in intercellular communication and tissue repair involving the bioactive molecules they carry (such as proteins, lipids, and RNA), have been extensively studied for the functional modification of biomaterials. For example, patent CN119391645A discloses a genetically engineered exosome, its sustained-release method, and its applications. This genetically engineered exosome is derived from human adipose-derived stem cells and contains specific siRNA targeting the Tgfbr2 gene. By encapsulating the exosome in PHA biodegradable material and immersing it in artificial blood vessels, it improves the biocompatibility and functionality of the artificial blood vessels, preventing adverse remodeling and stenosis caused by implantation. However, current research largely focuses on the loading and application of animal-derived exosomes (such as those derived from stem cells), while the utilization of plant-derived exosomes remains in the exploratory stage. Therefore, a new method is needed to safely and efficiently bind plant exosomes with endothelialization and anti-inflammatory functions to the surface of materials, thereby solving technical challenges such as delayed endothelialization, inflammatory response, and insufficient long-term patency.

[0004] Rubia cordifolia, as a potential source of plant exosomes, holds promise for solving the aforementioned technical challenges. Rubia cordifolia is a traditional Chinese medicine with a long history of medicinal value, widely used for promoting blood circulation, reducing inflammation, and promoting tissue repair. Modern research shows that Rubia cordifolia is rich in various active ingredients (such as anthraquinones, flavonoids, and polysaccharides), and its extracts have significant antioxidant, anti-inflammatory, and angiogenic effects. This invention reveals that Rubia cordifolia-derived exosome-like nanovesicles (RELNs) are rich in active groups such as hydroxyl (-OH) and amino (-NH2) groups, which can specifically bind to the phenolic hydroxyl, quinone, and amino groups on the surface of polydopamine (PDA) molecules, achieving stable loading through hydrogen bonding, π-π conjugated stacking, and hydrophobic interactions. This characteristic provides a natural advantage for the efficient and controllable loading of exosomes onto the surface of small-diameter artificial blood vessels, potentially solving problems such as thrombosis and restenosis in existing small-diameter artificial blood vessels. Summary of the Invention

[0005] To address the clinical challenges of delayed endothelialization and excessive inflammatory response at the implantation site following implantation of existing small-diameter artificial blood vessels, one objective of this invention is to provide a method for preparing exosome-like nanovesicles derived from Rubia cordifolia. This invention employs differential centrifugation and a sucrose density gradient purification method. The resulting Rubia cordifolia-derived exosome-like nanovesicles exhibit endothelial cell migration-promoting and anti-inflammatory effects, providing technical support for subsequent functional modification of small-diameter artificial blood vessels.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The preparation method of exosome-like nanovesicles derived from madder root includes the following steps:

[0008] (1) After homogenizing the wall of madder, differential centrifugation was performed to obtain madder exosome suspension; the centrifugation parameters were 2000-4000g 15-30min, 4000-6000g 25-40min, and 9000-11000g 25-40min.

[0009] (2) The obtained madder exosome suspension was centrifuged with a sucrose solution at a density gradient of 5-10%, 25-35%, 40-50%, and 55-65% to obtain madder exosome-like nanovesicles.

[0010] Preferably, in step (1), the fresh madder is washed with pure water and then homogenized by cell wall breaking.

[0011] Preferably, in step (1), madder root is added to pre-cooled PBS and homogenized at high speed using a high-speed blender to obtain coarse filtrate; after filtration through gauze, it is then centrifuged.

[0012] Preferably, in step (1), the mixture is homogenized at high speed using a blender for 8-10 minutes.

[0013] Preferably, in step (1), the centrifugation parameters are 3000g for 20min, 5000g for 30min, and 10000g for 30min.

[0014] Preferably, in step (2), the components are centrifuged at 120,000-180,000g at 2-8℃ for 1-3h, and the layered components are collected at the density interface of 25-35% and 40-50%. The components are then centrifuged at 100,000-150,000g at 2-8℃ for 60-120min to obtain madder exosome-like nanovesicles.

[0015] More preferably, in step (2), the first centrifugation condition is 150,000g at 4℃ for 2h; the second centrifugation condition after layering is 120,000g at 4℃ for 90min.

[0016] More preferably, in step (2), the density gradient of the sucrose solution is 8%, 30%, 45%, and 60%; the stratified components are collected at the 30% and 45% density interface.

[0017] As a preferred embodiment, the method for preparing the exosome-like nanovesicles derived from Rubia cordifolia includes the following steps:

[0018] Fresh madder was washed three times with pure water, then pre-cooled PBS was added and homogenized at high speed for 8-10 minutes using a high-speed blender to obtain a coarse filtrate. The coarse filtrate was then filtered twice with gauze to remove larger impurities. The resulting filtrate was then centrifuged sequentially at the following parameters: 3000g for 20 minutes, 5000g for 30 minutes, and 10000g for 30 minutes. The resulting precipitate was resuspended in an appropriate amount of PBS to obtain a madder exosome suspension.

[0019] Prepare sucrose solutions of different densities (8%, 30%, 45%, and 60%). Take 2 mL of each solution and slowly add it to a centrifuge tube from high to low concentration. Add 2 mL of the Rubia cordifolia exosome suspension obtained by resuspending in PBS to the top layer. Centrifuge at 150,000 g at 4°C for 2 h. Collect the stratified samples at densities of 30% and 45% into a new centrifuge tube and centrifuge at 120,000 g at 4°C for 90 min to obtain purified Rubia cordifolia exosome-like nanovesicles.

[0020] The exosome-like nanovesicles derived from Rubia cordifolia prepared in this invention can effectively promote the migration of vascular endothelial cells and accelerate the endothelialization of small-diameter artificial blood vessels by being stably loaded onto the surface of the artificial blood vessel. At the same time, they can exert significant anti-inflammatory effects by inhibiting the activation of inflammation-related signaling pathways and reducing the release of inflammatory factors, thereby improving the biocompatibility of small-diameter artificial blood vessels after implantation, reducing the occurrence of post-implantation complications, and achieving long-term vascular patency. This invention has important clinical application value.

[0021] The second objective of this invention is to provide a madder-derived exosome-like nanovesicle prepared by the aforementioned method.

[0022] To achieve the above objectives, the present invention adopts the following technical solution:

[0023] Rubia cordifolia-derived exosome-like nanovesicles were prepared using the aforementioned method.

[0024] Preferably, the exosome-like nanovesicles derived from Rubia cordifolia have a particle size of 80-120 nm and a zeta potential of -20 to -40 mV.

[0025] Preferably, the exosome-like nanovesicles derived from Rubia cordifolia have a particle size of 100-120 nm and a zeta potential of -20 to -25 mV.

[0026] More preferably, the exosome-like nanovesicles derived from Rubia cordifolia have a particle size of 110.4 nm and a Zeta potential of -22.78 mV.

[0027] The third objective of this invention is to provide a modified small-diameter artificial blood vessel.

[0028] To achieve the above objectives, the present invention adopts the following technical solution:

[0029] A modified small-diameter artificial blood vessel, wherein the surface of the modified small-diameter artificial blood vessel is loaded with the aforementioned madder-derived exosome-like nanovesicles.

[0030] Preferably, the inner diameter of the small-diameter artificial blood vessel is 1-3 mm, more preferably 1.4 mm.

[0031] Preferably, the length of the small-diameter artificial blood vessel is 1-5 cm, more preferably 1.2 cm.

[0032] Preferably, the modified small-diameter artificial blood vessel is made of polyurethane substrate, and the madder-derived exosome-like nanovesicles form a stable coating on the polyurethane surface through in-situ self-polymerization of polydopamine.

[0033] Preferably, the binding mechanism between the madder-derived exosome-like nanovesicles and polydopamine includes hydrogen bonding, π-π conjugation stacking, and hydrophobic interaction.

[0034] The fourth objective of this invention is to provide a method for loading exosome-like nanovesicles derived from madder into small-diameter artificial blood vessels.

[0035] To achieve the above objectives, the present invention adopts the following technical solution:

[0036] A method for loading madder-derived exosome-like nanovesicles onto small-diameter artificial blood vessels includes the following steps:

[0037] 1) Immerse the polyurethane material in a weakly alkaline buffer solution containing dopamine hydrochloride to obtain a polyurethane material loaded with polydopamine.

[0038] 2) The polyurethane material loaded with polydopamine obtained in step 1) is immersed in the madder-derived exosome-like nanovesicle solution for incubation, so that a polydopamine coating loaded with exosomes is formed on the surface of the polyurethane material, and a small-diameter polyurethane artificial blood vessel loaded with madder-derived exosome-like nanovesicles is obtained.

[0039] This invention employs a combination of differential centrifugation and ultracentrifugation, along with sucrose density gradient centrifugation purification technology, to prepare high-purity, highly active exosome-like nanovesicles derived from Rubia cordifolia. The surface of these Rubia cordifolia-derived exosome-like nanovesicles is rich in active groups such as hydroxyl (-OH) and amino (-NH2) groups, which can specifically bind to the phenolic hydroxyl (-OH), quinone (C=O), and amino (-NH2) groups on the surface of polydopamine (PDA) molecules. Specifically, the hydroxyl groups on the surface of the Rubia cordifolia-derived exosome-like nanovesicles form stable hydrogen bonds with the phenolic hydroxyl and amino groups on the PDA surface. Furthermore, the groups containing aromatic ring structures on the RELNs surface and the benzene ring structure in the PDA molecule further enhance the binding stability through a π-π conjugated stacking effect, thereby achieving a stable binding between the two. Based on this characteristic, polydopamine was first loaded onto the surface of a small-diameter polyurethane artificial blood vessel. After constant temperature incubation until the polydopamine was firmly attached, the modified small-diameter artificial blood vessel was immersed in a suspension of exosome-like nanovesicles derived from Rubia cordifolia. Stable loading of nanovesicles on the surface of the small-diameter artificial blood vessel was achieved by incubation at 4 ℃ for 12-16 h.

[0040] Preferably, in step 1), the weakly alkaline buffer solution is a Tris-HCl buffer solution. Dopamine will undergo oxidation and self-polymerization in a weakly alkaline aqueous environment to form polydopamine.

[0041] Preferably, in step 1), the concentration of dopamine hydrochloride is 1-5 mg / mL, more preferably 2 mg / mL; and the reaction time is 40-60 h, more preferably 48 h.

[0042] Preferably, in step 2), the concentration of the madder-derived exosome-like nanovesicle solution is 1-5 mg / mL, more preferably 2 mg / mL.

[0043] The fifth objective of this invention is to provide an application of the aforementioned madder-derived exosome-like nanovesicles and / or the aforementioned small-diameter artificial blood vessels in the preparation of cardiovascular implantable medical devices.

[0044] To achieve the above objectives, the present invention adopts the following technical solution:

[0045] The aforementioned application of madder-derived exosome-like nanovesicles and / or the aforementioned small-diameter artificial blood vessels in the preparation of cardiovascular implantable medical devices.

[0046] Preferably, the application of the madder-derived exosome-like nanovesicles in the preparation of small-diameter artificial blood vessels.

[0047] Preferably, the madder-derived exosome-like nanovesicles are used in the preparation of products for promoting endothelial cell migration, anti-inflammation, improving the hydrophilicity of small-diameter artificial blood vessels, promoting endothelialization of small-diameter artificial blood vessels, and / or improving the patency of small-diameter artificial blood vessels.

[0048] Preferably, the product includes a drug and / or a medical device.

[0049] The beneficial effects of this invention are as follows:

[0050] 1. This invention employs a combination of differential centrifugation and ultracentrifugation, along with sucrose density gradient centrifugation purification technology, to prepare high-purity, highly active exosome-like nanovesicles derived from Rubia cordifolia. In vitro scratch assays showed that these Rubia cordifolia-derived exosome-like nanovesicles significantly promoted the migration ability of human umbilical vein endothelial cells, demonstrating that RELNs accelerate the endothelialization process of small-diameter artificial blood vessels and shorten the vascular function remodeling time. Simultaneously, reactive oxygen species (ROS) assays showed a significant decrease in ROS levels in macrophages after treatment with Rubia cordifolia-derived exosome-like nanovesicles, indicating their potential to inhibit inflammatory responses. This anti-inflammatory property helps alleviate local inflammation after implantation, reducing the risk of thrombosis and restenosis.

[0051] 2. The method for loading exosome-like nanovesicles derived from Rubia cordifolia onto small-diameter artificial blood vessels provided by this invention has multiple advantages: 1) A stable coating is formed on the polyurethane surface through in-situ self-polymerization of polydopamine. The operation is mild and the process is simple, requiring no complex equipment or toxic crosslinking agents, resulting in high biosafety. Furthermore, the polydopamine coating is firmly bonded to the substrate and is not easily detached, providing numerous active sites for exosome loading. 2) Highly efficient loading is achieved through the synergistic effects of hydrogen bonds, π-π stacking interactions, and hydrophobic interactions between polydopamine and exosomes. The loading process is mild and controllable, maximizing the preservation of the structural integrity and bioactivity of exosomes and avoiding damage to their function caused by chemical modifications. 3) Multiple non-covalent interactions effectively overcome interfacial electrostatic repulsion, significantly improving the loading capacity and binding stability of exosomes on the coating, which is beneficial for long-term sustained release. 4) The composite coating constructed in this invention can effectively improve the biocompatibility of the surface of small-diameter artificial blood vessels, provide a good microenvironment for the adhesion, growth and spread of vascular endothelial cells, accelerate the vascular endothelialization process, and reduce the risk of coagulation, inflammation and restenosis. It has good application prospects and clinical translational value in the fields of small-diameter artificial blood vessel modification and tissue-engineered blood vessels.

[0052] 3. After modification with polydopamine and madder-derived exosome-like nanovesicles, small-diameter artificial blood vessels can simultaneously achieve in vivo endothelialization and long-term vascular patency. One month post-operation ultrasound and scanning electron microscopy results showed continuous blood flow within the lumen of the RELNs@PDA / PU graft, with a complete monolayer of endothelial cells. In contrast, the unmodified PU material exhibited significant delayed endothelialization and poor tissue integration. This indicates that the technical solution of this invention can effectively solve the problem of stenosis or occlusion that easily occurs after implantation of traditional small-diameter artificial blood vessels, providing reliable technical support for the clinical application of small-diameter vascular grafts. Attached Figure Description

[0053] Figure 1 Figures show the experimental results related to the extraction and characterization of madder-derived exosome-like nanovesicles. In this figure, A shows the extraction process and transmission electron microscopy image of madder-derived exosome-like nanovesicles; B shows the CCK-8 detection results of different concentrations of madder exosomes; C shows the NTA detection results of the average particle size of madder exosome-like nanovesicles; and D shows the NTA detection results of the average potential of madder exosome-like nanovesicles.

[0054] Figure 2 The images show the hemolysis results of exosome-like nanovesicles derived from Rubia cordifolia. In Figure A, there is a photograph of exosome-like nanovesicles derived from Rubia cordifolia during the hemolysis experiment; and in Figure B, there are the results of the hemolysis rate detection of exosomes from Rubia cordifolia at different concentrations.

[0055] Figure 3 The results of the scratch healing assay are shown below. In Figure A, the cell migration in the scratched area of ​​each well is observed using an upright microscope at three time points: 0h, 12h, and 24h of culture. Figure B shows the results of the scratch width measurement and scratch closure rate calculation using ImageJ software.

[0056] Figure 4 The figures show the ROS experimental results of exosome-like nanovesicles derived from Rubia cordifolia. In the figure, A is the fluorescence micrograph of the RELNs group, the Control group, and the Blank group; B is the fluorescence intensity statistical graph of the RELNs group, the Control group, and the Blank group.

[0057] Figure 5 The images show the scanning electron microscope characterization results of the PU group, PDA / PU group, and RELNs@PDA / PU composite modified group.

[0058] Figure 6 The image shows the results of atomic force microscopy analysis of the average roughness of the PU group.

[0059] Figure 7The image shows the results of atomic force microscopy analysis of the average roughness of the PDA / PU group.

[0060] Figure 8 The figure shows the results of atomic force microscopy analysis of the average roughness of the RELNs@PDA / PU composite modified group.

[0061] Figure 9 The image shows the energy dispersive spectroscopy elemental analysis results for the PU group, PDA / PU group, and RELNs@PDA / PU composite modified group.

[0062] Figure 10 and Figure 11 The graph shows the water contact angle test results for the PU group, PDA / PU group, and RELNs@PDA / PU composite modified group.

[0063] Figure 12 Images show the appearance of a small-diameter artificial blood vessel and an image of the common carotid artery of a rat transplanted with a small-diameter artificial blood vessel. Image A shows the appearance of a small-diameter artificial blood vessel with an inner diameter of 1.4 m and a length of 1.2 cm; image B shows the common carotid artery of a rat transplanted with a small-diameter artificial blood vessel.

[0064] Figure 13 Image showing the results of color Doppler ultrasound evaluation of the in vivo performance of pure PU and RELNs@PDA / PU small-diameter artificial blood vessels.

[0065] Figure 14 Figure showing the results of scanning electron microscopy evaluation of the in vivo performance of pure PU and RELNs@PDA / PU small-diameter artificial blood vessels. Detailed Implementation

[0066] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0067] To enhance understanding of the present invention, certain key technical and scientific terms will be clearly defined below. Unless otherwise specified herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be noted that the terminology used herein is intended to describe specific embodiments and not to be construed as limiting.

[0068] Madder (Rubia cordifolia), also known as blood madder or blood-scaring madder, is a plant of the Rubia genus in the Rubiaceae family. It has the effects of cooling blood, removing blood stasis, stopping bleeding, and regulating menstruation. It is mainly used to treat hematemesis, epistaxis, metrorrhagia, traumatic bleeding, amenorrhea due to blood stasis, joint pain, and swelling and pain from falls.

[0069] Polydopamine (PDA) is formed by the oxidative self-polymerization of dopamine monomers under weakly alkaline conditions and has abundant amino and phenolic hydroxyl groups. PDA possesses high adhesion, biocompatibility, and photothermal conversion capabilities. It is not only simple to prepare but also adheres firmly to the surface of almost all materials, making it widely used in fields such as biomaterial modification, drug delivery, and biosensing.

[0070] Small-diameter artificial blood vessels: a type of synthetic vascular graft with an inner diameter of ≤6 mm, used to repair or replace damaged blood vessels. They are mainly made of materials such as polyurethane, polyester, and polytetrafluoroethylene, and are used for peripheral vascular bypass, bypass grafting, and vascular defect repair to reconstruct blood flow pathways and improve tissue blood supply.

[0071] Anti-inflammatory effects: By inhibiting the release of inflammatory factors (such as ROS, IL-6, TNF-α, etc.), it reduces vascular inflammatory response and lowers the risk of thrombosis.

[0072] Reactive oxygen species (ROS): A collective term for oxygen-containing free radicals and peroxides that readily form free radicals, which are involved in oxygen metabolism in living organisms. These include peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha-oxygen. Excessive intracellular oxygen free radicals can lead to oxidative stress and inflammation, and are an important indicator of vascular damage.

[0073] Example 1. Extraction and characterization of exosome-like nanovesicles derived from Rubia cordifolia

[0074] 1. Extraction of exosome-like nanovesicles from Rubia cordifolia

[0075] Purchase fresh madder root, wash it three times with pure water, add pre-cooled PBS, and homogenize it at high speed for 8-10 minutes using a high-speed blender to obtain a coarse filtrate. Filter the coarse filtrate twice with gauze to remove larger impurities. Centrifuge the resulting filtrate sequentially at the following parameters: 3000g for 20 minutes, 5000g for 30 minutes, and 10000g for 30 minutes. Resuspend the precipitate in an appropriate amount of PBS. Prepare sucrose solutions of different densities (8%, 30%, 45%, and 60%). Slowly add 2 mL of the solution from high to low concentration to centrifuge tubes. Add 2 mL of the madder exosome suspension resuspended in PBS to the top layer. Centrifuge at 150000g at 4℃ for 2 hours. Collect the stratified samples at 30% and 45% densities into new centrifuge tubes and centrifuge at 120000g at 4℃ for 90 minutes to obtain purified madder exosome-like nanovesicles.

[0076] 2. Characterization of Rubia cordifolia-derived exosome-like nanovesicles

[0077] This invention utilizes fresh madder root, purified by differential centrifugation and sucrose density gradient, to obtain purified plant exosome-like nanovesicles in layers at 30% and 45% concentrations. The prepared madder root exosome-like nanovesicles were characterized using transmission electron microscopy and NTA, with results as follows: Figure 1 As shown, a typical double-layer bright film structure was observed by transmission electron microscopy; the average particle size was measured to be 110.4 nm and the average potential was -22.78 mV by NTA.

[0078] Example 2. Bioactivity of Rubia cordifolia-derived exosome-like nanovesicles (RELNs)

[0079] 1. Hemolysis test

[0080] Exosome samples were placed in 1.5 mL centrifuge tubes, and 1 mL of physiological saline was added for pre-incubation at 37°C for 30 min. 20 μL of diluted blood was added (physiological saline:blood = 4:5), and the samples were incubated in a water bath at 37°C for 60 min. Physiological saline (negative control) and deionized water (positive control) groups were set up. After centrifugation at 2000 rpm for 10 min, images were taken, and 100 μL of the supernatant was aspirated to measure the absorbance at 545 nm. The hemolysis rate was calculated. The experimental results are as follows: Figure 2 As shown, the hemolysis rate of madder exosomes at different concentrations was less than 5%, which meets the national standard and does not cause a hemolytic reaction.

[0081] 2. Scratch test

[0082] The effect of RELNs on the migration ability of human umbilical vein endothelial cells (HUVECs) was detected using a scratch healing assay. Purified RELNs were processed at a final protein concentration of 2 μg / mL, and HUVECs at a concentration of 2 × 10⁻⁶. 5 Cells were seeded at a volume of 2 mL per well in six-well culture plates and incubated overnight at 37°C with 5% CO2 to allow for cell adhesion and growth. When cell confluence reached 90% or higher, uniformly wide scratches were made at the bottom of each well using a sterile pipette tip. The plates were then gently rinsed three times with sterile phosphate-buffered saline (PBS) to remove cell debris. After replacing the culture medium with fresh PBS, the plates were incubated at 37°C with 5% CO2. Cell migration in the scratched areas was observed at 0h, 12h, and 24h using an upright microscope. Three replicates were used for each group. The scratch width was measured and the scratch closure rate was calculated using ImageJ software. The experimental results are as follows: Figure 3 As shown, compared with the control group, the migration ability of HUVECs in the RELNs-treated group was significantly improved, and the scratch closure rate was significantly higher than that in the control group, confirming that RELNs can effectively promote endothelial cell migration and provide direct cellular experimental evidence for the endothelialization process of small-diameter artificial blood vessels.

[0083] 3. ROS Experiment

[0084] RAW264.7 macrophages at a density of 1×10 6 Cells were seeded in six-well plates and incubated overnight at 37°C with 5% CO2. Lipopolysaccharide (LPS, 100 ng / mL) was added for inflammatory stimulation for 12 h. After stimulation, the supernatant was replaced, and 2 mg / mL RELNs were added for further incubation for 24 h. After incubation, the medium was discarded, and serum-free medium containing 10 μm DCFH-DA was added. The plates were incubated in the dark for 30 min, followed by washing three times with pre-cooled PBS to remove uninternalized probes. Results were captured using a fluorescence microscope. Figure 4 As shown, the green fluorescence signal in the RELNs group was significantly weaker than that in the Control group, indicating that LPS-induced ROS generation was significantly inhibited after RELNs treatment. RELNs alleviated the LPS-induced inflammatory response by reducing ROS levels and inhibiting the activation of ROS-mediated inflammatory signaling pathways, thus exerting a good anti-inflammatory effect.

[0085] Example 3. Preparation of small-diameter artificial blood vessels (RELNs@PDA / PU) loaded with madder-derived exosome-like nanovesicles

[0086] Dopamine hydrochloride was dissolved in Tris-HCl buffer at a concentration of 2 mg / mL. Polyurethane tubular materials were then immersed in the dopamine reaction solution for 48 h to obtain PDA-loaded polyurethane material (PDA / PU). Dopamine oxidizes and self-polymerizes in a weakly alkaline aqueous environment to form polydopamine, resulting in a brownish-red surface on the polyurethane. Subsequently, the PDA / PU was immersed in a 2 mg / mL solution of madder-derived exosome nanovesicles at 4°C for 16 h to obtain RELNs@PDA / PU. RELNs are loaded onto the PDA surface through hydrogen bonding and π-π conjugated stacking, forming an exosome and polydopamine coating on the polyurethane material. This bonding process requires no exogenous additives and relies solely on the inherent interactions between the two components. The PDA cross-linked network is rich in hydroxyl, amino, benzene rings, and hydrophobic regions, while the exosome membrane surface contains hydroxyl, amide groups, and aromatic amino acids from membrane proteins, and the membrane interior contains hydrophobic tails of phospholipids. When these two come into contact, hydrogen bonding, π-π stacking interactions, and hydrophobic interactions work synergistically. Specifically, the hydroxyl and imino groups in PDA can form hydrogen bonds with the hydroxyl and amide groups on the exosome membrane surface; the benzene rings of polydopamine can generate π-π stacking interactions with the aromatic amino acids on the exosome membrane proteins; and the hydrophobic regions of polydopamine can interact hydrophobically with the hydrophobic tails of the exosome membrane phospholipids. These forces effectively achieve a stable bond between plant exosomes and the polydopamine coating.

[0087] Example 4. Characterization of small-diameter artificial blood vessels loaded with madder-derived exosome-like nanovesicles

[0088] This embodiment utilizes scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) elemental analysis, atomic force microscopy (AFM), and water contact angle measurement to characterize the loading of madder-derived exosome-like nanovesicles onto small-diameter artificial blood vessels. The results are as follows: Figures 5-11 As shown. Specifically, the scanning electron microscopy characterization results are as follows: Figure 5 As shown, obvious particulate deposits are visible on the surface of the small-diameter artificial blood vessel in the PDA / PU modified group, indicating that polydopamine has been successfully loaded onto the surface of the polyurethane (PU) small-diameter artificial blood vessel matrix. In the SEM image of the RELNs@PDA / PU composite modified group, more small-sized particulate materials can be observed on the surface, which directly confirms that the exosome-like nanovesicles (RELNs) derived from Rubia cordifolia have been successfully loaded onto the surface of the PU small-diameter artificial blood vessel after PDA modification.

[0089] Energy dispersive spectroscopy elemental analysis results further validated the successful loading of RELNs. (See Tables 1-3 and...) Figure 9 As shown, phosphorus (P) was detected in the RELNs@PDA / PU group, with a mass fraction of 2.41%, while the P content in the pure PU group and the PDA / PU modified group was 0%. Since the RELNs cell membrane surface is rich in specific P (mainly derived from membrane phospholipids), the above results clearly confirm at the elemental level that RELNs have been stably bound to the surface of small-diameter artificial blood vessels.

[0090] Atomic force microscopy test results as follows Figures 6-8 As shown, compared with the pure PU group, the average roughness of the small-diameter artificial blood vessel surface of the PDA / PU group and the RELNs@PDA / PU group was significantly reduced, indicating that PDA particles and RELNs can fill the micro-pores on the PU matrix surface to a certain extent and optimize the micro-morphology of the material surface.

[0091] Water contact angle test results are as follows Figure 10 and Figure 11 As shown, after PDA and RELNs composite modification, the hydrophilicity of the surface of small-diameter artificial blood vessels is significantly improved. Good surface hydrophilicity can provide a suitable microenvironment for the adhesion and proliferation of vascular endothelial cells, further supporting the endothelialization process of small-diameter artificial blood vessels.

[0092] Table 1. Energy dispersive spectroscopy elemental analysis results of the PU group

[0093]

[0094] Table 2. Energy dispersive spectroscopy elemental analysis results of PDA / PU groups

[0095]

[0096] Table 3. Energy dispersive spectroscopy elemental analysis results of the RELNs@PDA / PU group

[0097]

[0098] Example 5. In vivo experiment

[0099] 1. Materials and Methods

[0100] (1) Animal models and implantation surgery

[0101] Healthy male SD rats aged 8-10 weeks and weighing 220-300 g were selected and housed in an SPF-grade animal facility with free access to food and water. All animal experimental protocols were approved by the institution's animal ethics committee. After one week of acclimatization, the rats were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg body weight). Following neck preparation and disinfection with iodine, a midline incision was made, and the left common carotid artery (LCCA) was dissected. Both ends of the vessel were clamped with microarterial clips to block blood flow. After severing the artery, a 1.4 mm inner diameter and 1.2 cm length polyurethane (PU) or RELNs@PDA / PU small-diameter artificial blood vessel graft was connected to the autologous blood vessel using a cannula method. Figure 12 As shown. After confirming that the blood vessels are patent and there is no leakage, the incision is sutured layer by layer.

[0102] (2) Postoperative ultrasound hemodynamic assessment

[0103] One month after implantation of a small-diameter artificial blood vessel, rats underwent color Doppler ultrasound examination of the common carotid artery to assess vascular patency and hemodynamic status. Ultrasound coupling agent was applied to the rat's neck, the inner diameter of the small-diameter artificial blood vessel was measured, and the blood flow filling within the vessel lumen was observed to confirm vascular patency. Relevant hemodynamic parameters were collected, and image acquisition and data recording were completed.

[0104] (3) Sample collection and scanning electron microscopy (SEM) sample preparation

[0105] After ultrasound evaluation, rats were euthanized by anesthesia with an overdose of sodium pentobarbital. The implanted small-diameter artificial blood vessel and the autologous blood vessel segments connected at both ends were quickly removed. The samples were gently rinsed with pre-cooled phosphate-buffered saline (PBS, pH 7.4) to remove residual blood and tissue debris. The samples were immediately fixed in electron microscopy fixative for 24 h, followed by stepwise dehydration. The dried samples were then fixed on the sample stage with conductive adhesive and observed under a scanning electron microscope at an accelerating voltage of 3.0 kV. Images were acquired at magnifications of ×500 and ×2000 to assess the endothelialization and tissue integration of the vascular graft.

[0106] 2. Comprehensive performance evaluation results of small-diameter artificial blood vessels implanted in rats one month after implantation.

[0107] One month post-surgery, the in vivo performance of pure PU and RELNs@PDA / PU small-diameter artificial blood vessels was systematically evaluated from both in vivo functional and ultrastructural dimensions using color Doppler ultrasound and scanning electron microscopy (SEM). The ultrasound evaluation results are as follows: Figure 13 As shown, the RELNs@PDA / PU group exhibited excellent patency of small-diameter artificial blood vessels. Ultrasound revealed continuous blood flow in the left common carotid artery (LCCA), with no obvious stenosis or occlusion in the lumen of the small-diameter artificial blood vessel. The vessel wall thickness was uniform (approximately 0.12 cm), and there were no signs of abnormal intimal hyperplasia. Under color Doppler ultrasound, the blood flow signal within the vessel lumen was well-filled, with no filling defects, turbulence, or other abnormalities, confirming that the vessel remained patent throughout.

[0108] SEM ultrastructural observation further confirmed the histological differences between the two groups of blood vessels. For example... Figure 14 As shown, in the pure PU group, poor tissue integration was observed on the graft surface under low magnification (×500), with numerous exposed porous structures, a rough surface, and no continuous cell cover. Under high magnification (×2000), numerous collapsed fibrous strands and irregular pores were further observed, with no typical cobblestone-like endothelial cell monolayer, suggesting limited biocompatibility of the unmodified PU material and a slow endothelialization process in vivo. In contrast, in the RELNs@PDA / PU group, under ×500, the lumen surface was completely covered by dense, continuous new tissue, completely isolating the underlying PU substrate; ×2000 high magnification revealed morphologically intact and tightly arranged endothelial cells.

[0109] The above results corroborate each other, jointly confirming that RELNs@PDA / PU modification can simultaneously achieve in vivo endothelialization and vascular patency of small-diameter artificial blood vessels, and its overall performance is significantly better than that of the pure PU control group.

Claims

1. A method for preparing madder-derived exosome-like nanovesicles, characterized in that, Includes the following steps: (1) After homogenizing the wall of madder, differential centrifugation was performed to obtain madder exosome suspension; the centrifugation parameters were 2000-4000g 15-30min, 4000-6000g 25-40min, and 9000-11000g 25-40min. (2) The obtained madder exosome suspension was centrifuged with a sucrose solution at a density gradient of 5-10%, 25-35%, 40-50%, and 55-65% to obtain madder exosome-like nanovesicles.

2. The production method according to claim 1, characterized by, In step (2), centrifugation at 120,000-180,000g at 2-8℃ for 1-3 h was performed, and the layered components were collected at the density interface of 25-35% and 40-50%. Centrifugation at 100,000-150,000g at 2-8℃ for 60-120 min was performed to obtain madder exosome-like nanovesicles.

3. Rubia cordifolia-derived exosome-like nanovesicles prepared by the preparation method according to any one of claims 1 to 2.

4. The Rubia sourced exosome-like nanovesicle of claim 3, wherein, The exosome-like nanovesicles derived from Rubia cordifolia have a particle size of 80-120 nm and a zeta potential of -20 to -40 mV.

5. A modified small diameter vascular graft, characterized by, The modified small-diameter artificial blood vessel is loaded with the exosome-like nanovesicles derived from Rubia cordifolia as described in any one of claims 3 to 4.

6. The modified small-diameter artificial blood vessel according to claim 5, characterized in that, The inner diameter of the small-diameter artificial blood vessel is 1-3 mm; the modified small-diameter artificial blood vessel uses a polyurethane substrate, and the exosome-like nanovesicles derived from Rubia cordifolia form a stable coating on the polyurethane surface through in-situ self-polymerization of polydopamine.

7. A method for loading exosome-like nanovesicles derived from madder root onto small-diameter artificial blood vessels, characterized in that, Includes the following steps: 1) Immerse the polyurethane material in a weakly alkaline buffer solution containing dopamine hydrochloride to obtain a polyurethane material loaded with polydopamine. 2) The polyurethane material loaded with polydopamine obtained in step 1) is immersed in the madder-derived exosome-like nanovesicle solution for incubation, so that a polydopamine coating loaded with exosomes is formed on the surface of the polyurethane material, and a small-diameter polyurethane artificial blood vessel loaded with madder-derived exosome-like nanovesicles is obtained.

8. The method according to claim 7, characterized in that, In step 1), the weakly alkaline buffer is Tris-HCl buffer; the concentration of dopamine hydrochloride is 1-5 mg / mL, and the reaction time is 40-60 h; In step 2), the concentration of the exosome-like nanovesicle solution derived from Rubia cordifolia is 1-5 mg / mL; the incubation conditions are: incubation at 4℃ for 12-16 h.

9. The use of the madder-derived exosome-like nanovesicles according to any one of claims 3 to 4 and / or the small-diameter artificial blood vessels according to any one of claims 5 to 6 in the preparation of cardiovascular implantable medical devices.

10. The use of the madder-derived exosome-like nanovesicles according to any one of claims 3 to 4 in the preparation of products for promoting endothelial cell migration, anti-inflammation, improving the hydrophilicity of small-diameter artificial blood vessels, promoting endothelialization of small-diameter artificial blood vessels, and / or improving the patency of small-diameter artificial blood vessels.

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