Erythrocyte vesicles coated with epo and methods of making and using the same

Abstract

The application discloses a kind of red blood cell vesicles coated EPO and its preparation method and application, belong to biomedical technology field.The technical problem to be solved is for the defects of low modification efficiency, many product impurities, wide particle size distribution and lack of lesion oxidation-reduction response release capacity of existing red blood cell vesicle process, provide a kind of preparation method of cholesterol-phenylboronic acid-disulfide bond modified red blood cell vesicle.The technical solution points are as follows: S1, take fresh red blood cells, wash by isotonic washing buffer, obtain purified intact red blood cells;S2, cholesterol-phenylboronic acid-disulfide bond modified EPO complex is incubated with intact red blood cells, to realize red blood cell in situ pre-modification;S3, temperature control induces red blood cell in situ budding, obtains red blood cell vesicle crude product;S4, gradient differential centrifugation purifies vesicle crude product, removes cell debris and unreacted raw materials, obtains red blood cell vesicle coated EPO.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an EPO-coated erythrocyte vesicle, its preparation method, and its application. Background Technology

[0002] Chronic liver fibrosis is a pathological process caused by excessive deposition of extracellular matrix following continuous liver damage. Without effective intervention, it can progress to cirrhosis and even liver cancer, seriously threatening patients' lives and health. Currently, there are no specific drugs that can directly reverse the progression of liver fibrosis. Existing treatments mainly focus on eliminating pathogenic factors and anti-inflammatory liver protection, but they are insufficient to inhibit excessive activation of hepatic stellate cells at the pathway level. Therefore, there remains a significant clinical need for anti-fibrotic therapy.

[0003] Erythrocyte vesicles (erythrocyte-derived extracellular vesicles) are nanoscale membrane vesicle structures produced from mature erythrocytes. Mature erythrocytes lack a nucleus, therefore erythrocyte vesicles do not carry genomic DNA, exhibiting advantages such as good biocompatibility, low immunogenicity, and long in vivo circulation time, making them a highly promising class of biopharmaceutical delivery carriers. Conventional erythrocyte vesicle preparation mainly falls into two mainstream approaches: one is to directly collect spontaneously secreted natural vesicles from erythrocytes; the other involves first hypotonic lysis of erythrocytes to obtain the erythrocyte chromophore membrane, then reconstructing the vesicle product through extrusion, ultrasound, etc., followed by post-incubation surface modification to endow the vesicles with targeting or responsive functions.

[0004] Existing preparation processes, which involve first lysing to prepare the erythrocyte membrane and then assembling vesicles, followed by post-modification after vesicle formation, have several inherent drawbacks. After erythrocyte lysis, repeated washing, sonication, and compression reconstruction, the natural protein structure of the cell membrane is damaged to some extent. In subsequent post-incubation modification, the modifying molecules can only non-specifically adsorb onto the vesicle surface, resulting in low modification binding efficiency. Furthermore, membrane fragments and impurities easily remain in the product system, leading to a wide vesicle size distribution, high polydispersity index, and significant batch-to-batch variations in properties, affecting the actual in vivo efficacy. Simultaneously, most conventional erythrocyte vesicles lack the ability to respond to the lesion microenvironment for release. Upon reaching liver fibrosis lesions, the controllability of active component release is insufficient, making it difficult to fully exert its regulatory effect on fibrosis signaling pathways.

[0005] Furthermore, unmodified erythrocyte vesicle carriers alone do not possess significant anti-fibrotic pharmacological activity; free active small molecules are easily metabolized and cleared from the body, resulting in low enrichment in liver lesions and making it difficult to achieve stable and effective intervention. Current technologies lack erythrocyte vesicle preparation methods that can simultaneously achieve high modification efficiency, high product purity, and lesion redox response release capabilities, thus limiting the practical application of erythrocyte vesicle carriers in the treatment of chronic liver fibrosis.

[0006] Therefore, developing a method for preparing erythrocyte vesicles that is simple to prepare, produces products with uniform particle size, has high modification efficiency, can achieve responsive release in the lesion microenvironment, and can effectively intervene in the progression of chronic liver fibrosis is of great practical significance.

[0007] Relevant patent documents retrieved: This document, published in China (CN116270532A) on June 23, 2023, discloses a vesicular drug delivery system targeting hepatic stellate cells, its preparation method, and its applications, belonging to the field of biomedical technology. It provides a vesicular drug delivery system targeting hepatic stellate cells, using erythrocytes for drug delivery, thus improving the local concentration and targeting of the drug. Using this vesicular drug delivery system loaded with BAPN can inhibit hepatic stellate cell activation and alleviate liver fibrosis, with the morphology and structure of RBCEVs remaining largely unaffected before and after modification. Compared with direct use of BAPN, BAPN delivered by cRGD-RBCEVs maintains a longer blood concentration, exhibits stronger liver targeting, and demonstrates more significant therapeutic effects, providing a new approach for treating fibrotic liver disease.

[0008] The publication, published in China (CN116059403A) on May 5, 2023, discloses a bile acid-modified erythrocyte vesicle, its preparation method, and its application, belonging to the biomedical field. The preparation method includes the following steps: (1) collecting blood and processing it to obtain erythrocyte vesicles; (2) electroporating the erythrocyte vesicles with LIMA1 siRNA to obtain erythrocyte vesicles carrying a small LIMA1 nucleic acid fragment; mixing the erythrocyte vesicles carrying the LIMA1 small nucleic acid fragment with a bile acid polymer solution to prepare a mixed suspension, reacting in a constant-temperature water bath, and washing afterward to obtain bile acid-modified erythrocyte vesicles. This paper describes how bile acid-modified erythrocyte vesicles encapsulate small nucleic acids. Animal experiments show that after oral administration, it has a good therapeutic effect on non-alcoholic fatty liver disease, reducing LIMA1 expression in liver tissue while effectively inhibiting lipid deposition in hepatocytes, alleviating the progression of non-alcoholic fatty liver disease, and causing no substantial damage to other organs.

[0009] This publication, published in China (CN119074770A) on December 6, 2024, discloses the application of apoptotic vesicles in the preparation of drugs for the prevention or treatment of heart failure. This paper is the first to discover the relationship between cardiac homeostasis and apoptosis, demonstrating this through myocardial injury and heart failure phenotypes in apoptosis-deficient mice. The paper finds that exogenous infusion of apoVs significantly improves cardiac function in apoptosis-deficient mice and Dox-induced mice, and significantly alleviates the pathological manifestations of Dox-induced heart failure in mice, suggesting that using apoVs to treat heart failure is a promising therapeutic strategy.

[0010] This document, published in China (CN120114617A) on June 10, 2025, discloses an extracellular vesicle-artesunate conjugate, its preparation method, and its application in dermatological drugs. The conjugate structure comprises an extracellular vesicle, a linker fragment, and artesunate. Taking into full account the active groups on the surface of extracellular vesicles and the unique chemical structure of artesunate, this document successfully designed and optimized a suitable linker fragment and selected appropriate linking strategies and biological sites. Utilizing the advantages of mild, non-toxic, efficient, and simple operation of bioorthogonal reactions, artesunate was linked to specific active functional groups on the lipid membrane surface of extracellular vesicles via a linker, successfully preparing the extracellular vesicle-artesunate conjugate. This biochemical conjugation process has advantages such as high loading efficiency, no impact on the morphology, membrane structure, and lipid membrane surface protein markers of extracellular vesicles, and preservation of the basic biological functions of extracellular vesicles.

[0011] This document, published in China (CN121943851A) on May 1, 2026, discloses a membrane-fused extracellular vesicle, its preparation method, and its application in targeting myocardial infarction. The document provides a membrane-fused extracellular vesicle comprising a mononuclear cell membrane and extracellular vesicles; the extracellular vesicles include circulating blood extracellular vesicles. The mononuclear cell membrane described in this document can target damaged cardiomyocytes and infarct sites through homing effects. Circulating extracellular vesicles are readily available, easily purified, and suitable for large-scale production, and can repair damaged hearts. The membrane-fused extracellular vesicles prepared by mixing and extruding the two components and filtering them through a membrane possess specific proteins of both mononuclear cell membranes and extracellular vesicles, enabling them to target damaged cardiac sites and providing a target for the development of novel cardiac targeted drugs.

[0012] Relevant non-patent literature retrieved: The journal *ORIGINAL ARTICLE*, with the article titled "Erythropoietin-Modified Mesenchymal Stem Cells Enhance Anti-fibrosis Efficacy in Mouse Liver Fibrosis Mode," Volume 17, published on July 3, 2020, discloses that mesenchymal stem cell (MSC)-based cell transplantation is an effective treatment for chronic liver injury, liver fibrosis, and end-stage liver disease. However, numerous studies have found that only a small number of transplanted cells can migrate to the site of injury or lesion, resulting in very limited repair effects. Methods: Bone marrow-derived mesenchymal stem cells (BM-MSCs) overexpressing the erythropoietin (EPO) gene were constructed using lentiviruses; cell viability of EPO-overexpressing BM-MSCs was detected using a CCK-8 assay; cell migration and apoptosis were detected using the Boyden chamber assay and flow cytometry, respectively; finally, the anti-fibrotic efficacy of EPO-MSCs was evaluated in vivo by immunohistochemical analysis. Results: EPO overexpression enhanced the cell viability and migration ability of BM-MSCs without inducing apoptosis; in a carbon tetrachloride (CCl4)-induced mouse liver fibrosis model, EPO-MSC intervention significantly alleviated liver fibrosis. Conclusion: Compared with BM-MSC treatment alone, EPO-MSCs exhibited higher cell viability and stronger migration ability, resulting in improved anti-fibrotic efficacy. These findings provide a potential therapeutic strategy for improving the efficiency of mesenchymal stem cell transplantation and its application in the treatment of liver fibrosis.

[0013] The journal *Bioengineering*, with the article titled "Low-Intensity Continuous Ultrasound Enhances the Therapeutic Efficacy of Curcumin-Encapsulated Exosomes Derived from Hypoxic Liver Cancer Cells via Homotropic Drug Delivery Systems," published on November 23, 2024, discloses that exosomes are extracellular nanovesicles secreted by cells, capable of efficiently delivering therapeutic payloads for cancer treatment. However, exosome production is low and targeting specificity is limited; therefore, previous studies have explored using intracellular and extracellular stress stimulation to enhance the efficiency of exosome action. Inspired by these studies, this research evaluated the uptake efficiency of exosomes secreted by cobalt chloride-induced hypoxic liver cancer cells. Western blotting and reverse transcription-PCR results showed that compared to normoxic exosomes (N-Exos), hypoxic exosomes (H-Exos) had increased secretion and altered protein composition. This study further stimulated H-Exos with low-intensity continuous ultrasound (LICUS) at 360 mW / cm² in vitro (3 MHz) and in vivo (1 MHz). The thermal effect and mechanical stress generated by ultrasound successfully enhanced cellular uptake of exosomes through the clathrin-mediated endocytosis pathway; laser confocal microscopy revealed that a large number of exosomes were located around the target cell nucleus. Finally, the hydrophobic drug curcumin was loaded into LICUS-treated H-Exos (H-Exo-Cur) to treat parental HepG2 liver cancer cells. UV-Vis spectrophotometry confirmed that the stability, solubility, and effective concentration of the drug were all improved after exosome encapsulation. MTT and flow cytometry results showed that, compared with the control group, LICUS-assisted H-Exo-Cur induced approximately 40-fold cell death; animal experiments showed that its inhibitory ability on tumor growth was approximately 10 times that of the control group.

[0014] The journal *Advanced NanoBioMed Research*, with the article titled "Surface Modification of Erythrocytes with Lipid Anchors: Structure-Activity Relationship for Optimal Membrane Incorporation, in vivo Retention, and Immunocompatibility," published in July 2022, discloses that PEGylated lipid-modified erythrocytes exhibit reduced macrophage uptake. Some derivatives promote antibody binding in human plasma and mouse serum, as well as a moderate increase in complement deposition and hemolysis, but these are unrelated to erythrocyte in vivo stability. Ultrastable anchors can functionalize erythrocytes for drug delivery, imaging, and sensing. Modification of erythrocytes and lipids is of great interest in biomedical applications. A fluorescent lipid library was tested for stable incorporation into human and mouse erythrocytes. The modified erythrocytes were then injected into mice, and lipid stability and circulation properties were tested. The structural requirements for reducing immune recognition were also investigated.

[0015] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The intervention effect on chronic liver fibrosis in vivo is limited. The relevant evidence is as follows: Under the same animal model, dosage and dosing period conditions, the corresponding sample of publication number CN116270532A (i.e., Comparative Example 1 of this invention) has a certain liver-protective and anti-inflammatory effect, but it can only reduce the serum ALT and AST of model mice to 116.3±11.5U / L and 204.7±18.2U / L, respectively. The proportion of collagen area in the liver stained by Masson is still as high as 9.6±1.3%, and the relative expression level of TGF-β1 gene in the fibrosis pathway is 2.53±0.28, which is difficult to fully achieve efficient intervention on chronic liver fibrosis. Summary of the Invention

[0016] The purpose of this invention is to provide: A method for preparing EPO-coated erythrocyte vesicles, and related technologies thereof, to solve the technical problems of low modification efficiency, high impurity content in products, lack of redox response drug release capacity at lesions, and insufficient in vivo anti-liver fibrosis efficacy of existing erythrocyte vesicle preparation processes, or combinations thereof.

[0017] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0018] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0019] Definitions of standard terms can be found in the references *Encyclopedia of Chinese Medicine: Hematology* (China Union Medical University Press), *Internal Medicine* (10th Edition) (People's Medical Publishing House), and *Pathology* (9th Edition) (People's Medical Publishing House).

[0020] Unless otherwise stated, conventional methods within the scope of the art, such as transmission electron microscopy (TEM), zeta potential detection, immunofluorescence staining, etc., shall be used.

[0021] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0022] The “range” disclosed in this document takes the form of a lower limit and an upper limit. It can be one or more lower limits and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and composable; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if the minimum range values ​​are listed as 1 and 2, and if the maximum ranges are listed as 3, 4, and 5, then the following ranges are all expected: 1-2, 1-4, 1-5, 2-3, 2-4, and 2-5.

[0023] In this invention, unless otherwise specified, the numerical range "ab" represents a shortened representation of any combination of real numbers from a to b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed in this document, and "0-5" is simply a shortened representation of these numerical combinations.

[0024] The term "erythrocyte vesicle" used in this article refers to a vesicle of about 200 nanometers in size that is formed when erythrocytes bud and detach in situ due to the eversion of phosphatidylserine during apoptosis.

[0025] The term "liver fibrosis" used in this article refers to the pathological process in which the liver is continuously stimulated by various chronic damaging factors such as viruses, alcohol, and toxins, resulting in the activation of hepatic stellate cells and excessive and abnormal deposition of extracellular matrix (collagen, fibronectin, etc.). It is an intermediate pathological stage in the progression of chronic liver disease to cirrhosis.

[0026] The term "modification" used in this article refers to the artificial modification of membrane proteins and lipid components of vesicles (cells, cell membranes) through genetic engineering, chemical coupling, hydrophobic insertion, etc., to introduce specific functional groups, peptides or functional proteins into vesicles, thereby changing the biological properties of vesicles and achieving purposes such as targeting, stimulus response, and improved stability.

[0027] The term "anchoring" used in this article refers to a binding method in which functional molecules are stably bound and fixed to the lipid bilayer of vesicles / cell membranes using hydrophobic structures (such as cholesterol), transmembrane fragments, membrane affinity groups, etc., so that the functional molecules remain on the surface of the vesicle membrane and are not easily dissociated and detached.

[0028] The term "response" used in this article refers to the ability of a nanovesicle delivery system to sense changes in the lesion microenvironment (external / endogenous signals such as pH, redox, enzymes, and ultrasound), resulting in changes in molecular structure and triggering expected biological behaviors such as drug release and conformational changes.

[0029] The term “hematocrit” used in this article refers to the proportion of red blood cells that settle out of a certain amount of anticoagulated whole blood after centrifugation.

[0030] The term "coating" used in this article refers to the following: biomolecules such as functional proteins and peptides are positioned on the outer surface of the lipid bilayer of erythrocyte vesicles through anchoring molecular covalent binding or strong specific non-covalent binding, exposing the biomolecules to the external aqueous environment of the vesicle; this is different from the internal encapsulation method, which encapsulates the molecules inside the aqueous lumen of the vesicle.

[0031] The term "EPO" as used herein refers to erythropoietin, preferably recombinant human erythropoietin (rh-EPO), which is a glycoprotein cytokine. In this invention, after modification with cholesterol-PEG anchoring molecules, it binds to the outer surface of the erythrocyte vesicle membrane in a coated form, thus preserving its biological activity.

[0032] The term "NTA detection" used in this article refers to Nanoparticle-Tracking Analysis, a detection method used to characterize the particle size distribution and particle concentration of nanoparticles / vesicles. By tracking the Brownian motion of particles under laser irradiation, the particle size and particle number concentration of the sample are calculated, which is a routine physicochemical characterization method for extracellular vesicles in this field.

[0033] The term "SYBR Green method" as used in this article refers to a real-time quantitative PCR detection method in which SYBR-Green fluorescent dye is embedded in double-stranded DNA. After the dye binds to the amplification product, it generates a fluorescent signal. The relative expression level of target gene mRNA in the sample is quantitatively analyzed based on the changes in the fluorescent signal.

[0034] The terms “Col-I, α-SMA, TGF-β1” used in this article refer to: Col-I, type I collagen, which is a major component of the extracellular matrix and reflects the level of collagen deposition in tissues; α-SMA, α-smooth muscle actin, which is a marker protein for the activation of hepatic stellate cells; and TGF-β1, transforming growth factor-β1, which is a core signaling molecule driving the development of liver fibrosis. The three are classic detection targets for evaluating the degree of liver fibrosis.

[0035] The term "BCA method" used in this article refers to the BCA protein quantification method, which is based on the biuret reaction principle and uses colorimetry to determine the total protein concentration in a sample. It is a routine detection method for determining the protein content in biological samples.

[0036] In a first aspect, the present invention provides: a method for preparing erythrocyte vesicles, comprising the steps of: S1: Separate and purify intact red blood cells to obtain purified red blood cells; S2: Resuspend the purified red blood cells in an isotonic buffer system to obtain a red blood cell suspension; S3: Add the cholesterol-phenylboronic acid-disulfide bond (Chol-SS-PBA) functional molecule and the Chol-PEG2000-EPO conjugate to the red blood cell suspension simultaneously and incubate them together. This allows the cholesterol hydrophobic ends of Chol-SS-PBA and Chol-PEG2000-EPO to insert into the lipid bilayer of the red blood cell membrane, completing the in situ co-pre-modification of intact red blood cells. This simultaneously anchors the dual-response groups and EPO on the outer surface of the red blood cell membrane, achieving EPO coating on the red blood cell membrane surface. S4: Incubate the pre-modified erythrocyte suspension to induce in situ budding and shedding of erythrocytes, releasing erythrocyte vesicles that are modified with Chol-SS-PBA and coated with EPO. S5: Gradient differential centrifugation to remove intact erythrocytes and membrane fragments, and collect the supernatant containing crude modified erythrocyte vesicles; S6: The crude vesicles are purified and eluted to remove uninserted free Chol-SS-PBA molecules, free Chol-PEG2000-EPO, and free impurities, resulting in purified erythrocyte vesicles modified with Chol-SS-PBA and coated with EPO.

[0037] The steps for separating and purifying intact red blood cells described in S1 are as follows: take fresh anticoagulated whole blood, centrifuge to remove plasma and white membrane layer; wash red blood cells with isotonic washing buffer to obtain purified intact red blood cells; Preferably, the isotonic washing buffer in S1 is selected from at least one of HEPES-buffered Hanks balanced salt solution (HEPES-HBSS washing buffer), HEPES-PBS buffer, and bicarbonate-buffered Hanks balanced salt solution (ordinary HBSS). More preferably, the isotonic washing buffer in S1 is HEPES-HBSS washing buffer.

[0038] Preferably, the number of times the red blood cells are washed in S1 is 1-5 times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, or any range derived therefrom; More preferably, the number of times the red blood cells are washed in S1 is 3.

[0039] Preferably, the centrifugation temperature in S1 is 2-8℃, for example, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, or any range derived therefrom; More preferably, the centrifugation temperature in S1 is 4°C.

[0040] Preferably, the centrifugal force in S1 is 400-600g, for example 400g, 430g, 460g, 480g, 500g, 520g, 550g, 580g, 600g, or any range derived therefrom; More preferably, the centrifugal force in S1 is 420g.

[0041] Preferably, the single centrifugation time in S1 is 6-12 min, for example 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, or any range derived therefrom; More preferably, the single centrifugation time in S1 is 8 minutes.

[0042] The isotonic buffer system described in S2 is selected from HEPES-HBSS buffer and PBS buffer. Preferably, the isotonic buffer system described in S2 is a HEPES-HBSS buffer solution; In some specific embodiments, a protease inhibitor may also be added to the isotonic buffer system described in S2; Specifically, the protease inhibitor may be selected from at least one of benzyl sulfonyl fluoride (PMSF), water-soluble serine protease inhibitor (AEBSF), leupeptin, aprotinin, and pepstatin A. More specifically, the protease inhibitor may be PMSF; More preferably, the isotonic buffer system described in S2 is a HEPES-HBSS buffer solution containing 0-0.1 mM PMSF; The aforementioned 0-0.1mM, such as 0mM, 0.02mM, 0.04mM, 0.05mM, 0.06mM, 0.08mM, 0.1mM, or any range derived therefrom; Most preferably, the isotonic buffer system described in S2 is a HEPES-HBSS buffer solution containing 0.05 mM PMSF.

[0043] Wherein, the osmotic pressure range of the isotonic buffer system described in S2 is 280-300 mOsm / kg, for example 280 mOsm / kg, 283 mOsm / kg, 286 mOsm / kg, 288 mOsm / kg, 290 mOsm / kg, 293 mOsm / kg, 296 mOsm / kg, 300 mOsm / kg, or any range derived therefrom; Preferably, the osmotic pressure range of the isotonic buffer system described in S2 is 292 mOsm / kg.

[0044] Wherein, the pH of the isotonic buffer system described in S2 is 7.20-7.50, for example 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, 7.50, or any range derived therefrom; Preferably, the pH of the isotonic buffer system described in S2 is 7.38.

[0045] In some specific embodiments, the hematocrit of the red blood cell suspension described in S2 is 2.0%-8.0% (v / v), for example 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, or any range derived therefrom; Preferably, the hematocrit of the red blood cell suspension described in S2 is 3.5% (v / v).

[0046] Wherein, the concentration of Chol-SS-PBA in the red blood cell suspension described in S3 is 3.0-12.0 μM, for example 3.0 μM, 4.5 μM, 6.0 μM, 7.5 μM, 9.0 μM, 10.5 μM, 12.0 μM, or any range derived thereof; Preferably, the concentration of Chol-SS-PBA in the red blood cell suspension described in S3 is 7.5 μM.

[0047] In S3, the final concentration of Chol-PEG2000-EPO conjugate in the erythrocyte suspension is 2-7 μM; Preferably, the final concentration of the Chol-PEG2000-EPO conjugate in the red blood cell suspension in S3 is 4.5 μM.

[0048] Wherein, the co-incubation temperature described in S3 is 25-34℃, for example 25℃, 27℃, 29℃, 30℃, 31℃, 32℃, 34℃, or any range derived therefrom; Preferably, the co-incubation temperature in S3 is 28°C.

[0049] The co-incubation time described in S3 is 15-40 min, for example, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, or any range derived therefrom; Preferably, the co-incubation period of S3 is 30 minutes.

[0050] Among them, the co-incubation method described in S3 is oscillation incubation; Preferably, the oscillation frequency of the co-incubation in S3 is 30-60 rpm, for example, 30 rpm, 35 rpm, 40 rpm, 45 rpm, 50 rpm, 55 rpm, 60 rpm, or any range derived therefrom; More preferably, the oscillation frequency of the co-incubation in S3 is 45 rpm.

[0051] In some specific embodiments, the Chol-SS-PBA described in S3 may be premixed with a solubilizer before being added to the red blood cell suspension; Preferably, the type of co-solvent is selected from anhydrous ethanol and dimethyl sulfoxide (DMSO); More preferably, the co-solvent is DMSO; More preferably, the volume fraction of DMSO in the red blood cell suspension is ≤1%; Most preferably, the volume fraction of DMSO in the red blood cell suspension is 0.4%.

[0052] The incubation temperature described in S4 is 32-37℃, for example, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, or any range derived therefrom; Preferably, the incubation temperature is 34-36°C; More preferably, the temperature for the constant temperature incubation is 35°C.

[0053] Wherein, the incubation time described in S4 is 20-60 min, for example 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, or any range derived therefrom; Preferably, the incubation time is 30-50 minutes; More preferably, the incubation time is 40 minutes.

[0054] S4 describes an incubation process that employs low-speed horizontal oscillation. Preferably, the oscillation rate is 25-55 rpm, and the system remains isotonic throughout the process without the application of ultrasound, high-pressure extrusion, or electric field perforation. Further preferably, the oscillation rate is 35-45 rpm; even more preferably, it is 40 rpm.

[0055] Wherein, the temperature of differential centrifugation described in S5 is 2-8℃, for example 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, or any range derived therefrom; Preferably, the temperature of the differential centrifugation in S5 is 4°C.

[0056] Among them, the differential centrifugation mentioned in S5 includes primary centrifugation and secondary centrifugation; Preferably, the centrifugal force of the single centrifugation is 300-380g, for example 300g, 320g, 340g, 360g, 380g, or any range derived therefrom; More preferably, the centrifugal force of the single centrifugation is 320g.

[0057] Preferably, the duration of one centrifugation is 8-15 min, for example 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, or any range derived therefrom; More preferably, the duration of one centrifugation is 12 minutes.

[0058] Preferably, the centrifugal force of the secondary centrifugation is 1600-2200g, for example 1600g, 1700g, 1800g, 1900g, 2000g, 2100g, 2200g, or any range derived therefrom; More preferably, the centrifugal force of the secondary centrifugation is 1800g.

[0059] Preferably, the duration of the secondary centrifugation is 30-60 minutes, for example, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or any range derived therefrom; More preferably, the duration of the secondary centrifugation is 45 minutes.

[0060] Wherein, the purification described in S6 is selected from at least one of size exclusion chromatography and ultrafiltration concentration-dialysis; Preferably, the purification described in S6 is performed using a qEV series size exclusion chromatography column.

[0061] Among them, the elution buffer used for elution in S6 is an isotonic PBS buffer or HEPES-HBSS buffer; Preferably, the elution buffer used for elution in S6 is HEPES-HBSS buffer.

[0062] Secondly, the present invention provides: a red blood cell vesicle obtained based on the above preparation method.

[0063] Thirdly, the present invention provides the application of the preparation method or the red blood cell vesicles in the preparation of drugs for treating liver fibrosis and in nanomedicine delivery.

[0064] In some specific embodiments, the application is preferably in the preparation of drugs for treating liver fibrosis.

[0065] In this invention, the Chol-SS-PBA molecule used in the preparation method inserts into the outer leaflet of the lipid bilayer of the intact erythrocyte cell membrane via the hydrophobic tail of cholesterol. This molecule is not merely a functional marker group on the membrane; its insertion behavior can alter the local physicochemical properties of the erythrocyte membrane, thereby actively regulating the budding behavior of the erythrocyte membrane.

[0066] The erythrocyte membrane naturally contains lipid raft microregions rich in cholesterol. These microregions have an ordered liquid-phase structure and are phase-separated from the surrounding disordered liquid-phase membrane regions. A strong interaction exists between the lipid raft regions and the membrane skeleton (spectrin-ankylin complex), restricting membrane bending and budding. The cholesterol skeleton of the Chol-SS-PBA molecule can embed into these lipid raft microregions. Simultaneously, the polar groups of the phenylboronic acid-disulfide bonds on the molecule are exposed to the outer aqueous phase, disturbing the molecular packing arrangement of the lipid raft microregions and altering the membrane thickness, membrane torsional modulus, and membrane-cytoskeleton binding force within these regions.

[0067] Under the constant temperature incubation conditions of this invention, the Chol-SS-PBA molecules inserted into the membrane synergistically promote transverse phase separation in the lipid raft microregions due to the temperature effect; the binding force between the membrane and the membrane skeleton in local areas weakens, and the membrane generates inward / outward bending stress, driving the cell membrane to bulge outward locally and sprout, eventually detaching to form modified erythrocyte vesicles.

[0068] The above process differs from the passive spontaneous release of vesicles by erythrocytes at 37°C: when the system does not contain Chol-SS-PBA and intact erythrocytes are treated under the same isothermal incubation conditions, only a very low yield of spontaneously occurring vesicles is obtained; after introducing Chol-SS-PBA pre-modification, the vesicle generation yield is significantly increased. This proves that the isothermal budding of the present invention is not a passive waiting for erythrocytes to release vesicles naturally, but rather that Chol-SS-PBA molecules modify the physicochemical properties of the membrane, and actively induce membrane budding and shedding in conjunction with temperature conditions.

[0069] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. This invention employs an in-situ pre-modification and in-situ budding process for intact red blood cells, which differs from the traditional post-modification method after lysing the blood shadow membrane. This significantly improves the surface modification efficiency of vesicles and solves the defects of low modification and binding efficiency and insufficient functional load in existing technologies.

[0070] 2. The present invention adopts a gradient differential centrifugation purification system, which effectively removes membrane fragments and residual impurities. The resulting vesicles have uniform particle size, good dispersibility, and stable physicochemical properties, significantly improving product purity and formulation uniformity.

[0071] 3. This invention introduces a Chol-SS-PBA disulfide bond redox-responsive structure, which enables the vesicles to have controlled drug release characteristics in the lesion microenvironment, and can release functional molecules in response to liver fibrosis sites, thus enhancing the targeted therapy capability.

[0072] 4. This invention effectively improves the problems of rapid metabolism, weak enrichment, and lack of efficacy of free drugs by leveraging the synergistic effect of erythrocyte vesicle carriers, EPO functional molecules, and responsive modifying groups. It can significantly inhibit liver inflammation, reduce collagen deposition, block the TGF-β / Smad fibrosis pathway, and efficiently reverse chronic liver fibrosis damage. The therapeutic effect is significantly better than that of existing technologies.

[0073] Furthermore, based on the present invention: 1. Based on the comparison of Example 1 and Comparative Examples 1-3, this invention employs a combination of in-situ pre-modification of intact red blood cells, temperature-controlled in-situ budding, gradient differential centrifugation purification, and modification with a cholesterol-phenylboronic acid-disulfide bond-EPO complex, achieving novel technical effects such as high modification efficiency, lesion redox-responsive drug release capability, and efficient in vivo intervention in chronic liver fibrosis. The combined technical effect is superior to the sum of the effects of each individual technique.

[0074] 2. Based on the comparison of Example 1 and Comparative Examples 1-3, this invention purposefully selects a narrow-range process path from the broad preparation ideas of red blood cell vesicles disclosed in the prior art, which is not mentioned in the prior art, to first pre-modify intact live red blood cells and then form them in situ under temperature control. This has achieved unexpected technical effects: it balances high surface modification efficiency with the integrity of vesicle membrane structure, realizes redox controlled release of drugs from liver fibrosis lesions, and achieves stronger in vivo anti-liver fibrosis effects by inhibiting liver damage, reducing collagen deposition, and downregulating the expression of fibrosis pathway molecules under the same administration conditions. Attached Figure Description

[0075] Figure 1 This is a graph showing the NTA detection in erythrocyte vesicles obtained in Example 1. Detailed Implementation

[0076] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0077] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0078] Example 1 A method for preparing erythrocyte vesicles includes the following specific steps: S1: Take fresh mouse whole blood anticoagulated with EDTA, centrifuge at 450g for 9min at 4℃, and discard the plasma and white membrane layer; wash the red blood cell pellet three times with pre-cooled HEPES-HBSS washing buffer, 420g for 8min each time, to remove platelets and white blood cells, and obtain purified intact red blood cells.

[0079] S2: Resuspend the erythrocyte pellet in pre-warmed 28°C isotonic HEPES-HBSS buffer (292 mOsm / kg, pH 7.38, containing 0.05 mM PMSF), adjust the hematocrit to 3.5% (v / v), and obtain the erythrocyte suspension.

[0080] S3: Add Chol-SS-PBA to a final concentration of 7.5 μM and pre-prepared Chol-PEG2000-EPO conjugate (final concentration of 4.5 μM); add a small amount of DMSO to aid dissolution, with a final volume fraction of 0.4%; incubate at 28°C and 45 rpm with low-speed horizontal shaking for 30 min to complete the in situ co-modification of intact red blood cells.

[0081] S4: Place the red blood cell suspension in a constant temperature incubator, oscillate horizontally at 35℃ and 40 rpm for 40 min, and incubate at a constant temperature to induce red blood cells to bud and detach in situ, releasing red blood cell vesicles that are modified with Chol-SS-PBA and coated with EPO.

[0082] S5: 4℃ gradient differential centrifugation: centrifuge at 320g for 12min to precipitate intact erythrocytes and aspirate the supernatant; then centrifuge at 1800g for 45min to remove large membrane fragments and collect the supernatant to obtain crude vesicles.

[0083] S6: Load the crude vesicle supernatant onto a qEV-35 size exclusion column, elute with HEPES-HBSS, collect the vesicle components; add 4% trehalose, aliquot and store at -80℃; thus obtaining cholesterol-phenylboronic acid-disulfide bond modified erythrocyte vesicles.

[0084] The NTA detection image of erythrocyte vesicles obtained in Example 1 is shown below. Figure 1 .

[0085] Comparative Example 1 Red blood cell vesicles prepared according to the technical solution described in Example 1 of the prior art patent document CN116270532A.

[0086] Comparative Example 2 Compared with Example 1, the only difference is that S5 uses constant-speed centrifugation, specifically 320g for 25min to collect the supernatant to obtain crude capsules, and the other conditions are basically the same as in Example 1.

[0087] Comparative Example 3 Compared with Example 1, the only difference is that all Chol-SS-PBA is replaced with hydrophobic curcumin, and the other conditions are basically the same as in Example 1.

[0088] Comparative Example 4 Compared with Example 1, the only difference is that Chol-SS-PBA is not added; the other conditions are basically the same as in Example 1.

[0089] Comparative Example 5 Compared with Example 1, the only difference is that the pre-prepared Chol-PEG2000-EPO conjugate is not added (i.e., the process without surface coating of EPO), and the other conditions are basically the same as those in Example 1.

[0090] Detection Example 1 The properties of the erythrocyte vesicles prepared in Example 1 and Comparative Examples 1-5 were characterized: (1) The test conditions for average particle size, polydispersity index (PDI), and zeta potential (mV) are as follows: Particle size, polydispersity index (PDI), and zeta potential were determined using a Malvern Zetasizer Nano ZS90 nanoparticle size and zeta potential analyzer. Red blood cell vesicle samples were diluted with PBS buffer at pH 7.4 and tested at 25°C. In dynamic light scattering mode, the scattering angle was 90°, and the equilibration period was 120 s. Each sample was measured in triplicate, and the Z-mean particle size and PDI were recorded. Zeta potential was measured using electrophoretic light scattering mode at a test temperature of 25℃, with an equilibration period of 120 s. Each sample was measured in triplicate, and the average value was taken. The obtained results are shown below: Table 1

[0091] As shown in Table 1, Example 1 exhibits the lowest PDI, the most uniform particle size distribution, a suitable Zeta potential, and good vesicle colloidal stability. Comparative Examples 1 and 2 show larger particle sizes, higher PDI, and decreased particle size uniformity; Comparative Examples 3 and 4 have inferior particle sizes and PDI compared to Example 1; Comparative Example 5, while having a better particle size distribution, lacks the EPO functional component. This demonstrates that the dual-modification scheme of the present invention can prepare erythrocyte vesicles with excellent particle size performance.

[0092] (2) Total protein content The BCA protein quantification kit was used for determination. Vesicle samples were taken, protein lysis buffer was added for lysis, and the operation was carried out according to the kit instructions. The absorbance at 562 nm was measured using an ELISA reader, and the total protein concentration (μg / mL) of the sample was calculated based on the standard curve.

[0093] (3) EPO coating efficiency The EPO content in vesicle samples was detected using an ELISA kit. EPO coating efficiency (%) = (mass of EPO bound to vesicle surface / total mass of EPO added) 100%; (4) Chol-SS-PBA modification efficiency The absorbance of the characteristic absorption peak of Chol-SS-PBA in vesicles was determined by ultraviolet spectrophotometry, and the content of Chol-SS-PBA bound on the membrane was calculated by referring to the standard curve. Chol-SS-PBA modification efficiency (%) = (mass of Chol-SS-PBA bound to vesicle membranes / total mass of Chol-SS-PBA added) 100%; (5) Disulfide bond content The Ellman reagent (DTNB) colorimetric method was used to determine the amount of disulfide bond material corresponding to each milligram of total vesicle protein, in nmol / mg protein.

[0094] The obtained results are shown below: Table 2

[0095] As shown in the table above, Example 1 simultaneously achieves high total protein content, optimal EPO coating efficiency, and high Chol-SS-PBA modification efficiency and disulfide bond retention. Comparative Examples 3 and 4 lack reactive groups and cannot achieve responsive release; Comparative Example 5, although possessing excellent reactive group performance, lacks EPO drug loading. This demonstrates that the dual modification scheme of this invention can synergistically achieve vesicle production, drug loading, and responsive release. Example 2: Chronic liver fibrosis model induced by intraperitoneal injection of carbon tetrachloride (CCl4) 1. Laboratory animals C57BL / 6J mice, 7-8 weeks old, male or female, weighing 18-22g; n=6-8 mice per group.

[0096] Feeding conditions: Temperature 22±2℃, 12h light-dark cycle, free access to food and water.

[0097] 2 Experimental Groups Sham control group (olive oil control group): Intraperitoneal injection of the same volume of olive oil; tail vein injection of the same volume of PBS.

[0098] CCl4 model group: CCl4 modeling; tail vein injection of an equal volume of PBS.

[0099] CCl4+RBC-EVs group (unmodified extraerythrocyte vesicles): CCl4 modeling; empty extraerythrocyte vesicles were administered via tail vein.

[0100] CCl4+ Example 1: CCl4 modeling; functionalized extracellular vesicles of erythrocytes were administered to the tail vein.

[0101] CCl4+ Comparative Group 1: CCl4 modeling; functionalized extracellular vesicles of erythrocytes were administered to the tail vein.

[0102] CCl4+ Comparative Group 2: CCl4 modeling; tail vein was given functionalized erythrocyte extracellular vesicles.

[0103] CCl4+ Comparative Group 3: CCl4 modeling; tail vein was given functionalized erythrocyte extracellular vesicles.

[0104] CCl4+ Comparative Group 4: CCl4 modeling; tail vein was given functionalized erythrocyte extracellular vesicles.

[0105] CCl4+ Comparative Group 5: CCl4 modeling; tail vein administration of functionalized extraerythrocyte vesicles CCl4+ free drug (Chol-SS-PBA) control group.

[0106] CCl4+ positive drug control group (metformin): CCl4 modeling; positive drug administration.

[0107] 3. Preparation of modeling reagents CCl4-olive oil solution, 20% (v / v) (1 part CCl4 + 4 parts olive oil), prepared fresh and used immediately, and mixed thoroughly.

[0108] 4. Modeling process and drug administration regimen (1) Modeling process The CCl4 oil solution was injected intraperitoneally at a rate of 5 mL / kg body weight twice a week (Monday and Thursday); the Sham group received an equal volume of pure olive oil intraperitoneally at the same frequency.

[0109] Vesicle intervention drug administration (starting at the end of the 4th week of modeling, i.e., after early fibrosis has formed at 4 weeks of modeling; CCl4 stimulation was continued throughout the modeling process to simulate continuous liver injury).

[0110] (2) Extraerythrocyte vesicle delivery method: The RBC-EVs were administered via tail vein injection at a dose of 150-250 μg total protein per animal, resuspended in 100-150 μL sterile PBS; administered once every 3 days for 4 weeks of PMC.

[0111] The Sham control group and the CCl4 model group were given the same volume of sterile PBS via tail vein injection, with the same frequency of administration.

[0112] Experimental endpoint: At the end of week 8, mice were anesthetized and euthanized, and samples were collected.

[0113] Alternative short-cycle protocol: 6 weeks to establish model (initiating moderate liver fibrosis), vesicle intervention starting from week 3, and termination of the experiment in week 6.

[0114] 5 Sample Collection Blood was drawn from the orbital vein / heart after anesthesia; the serum was separated by centrifugation after standing at room temperature and stored at -80℃ for liver function biochemical testing.

[0115] Complete liver removal: A portion of liver tissue: fixed with 4% paraformaldehyde for histopathological staining of paraffin-embedded sections.

[0116] Remaining liver tissue: rapidly frozen in liquid nitrogen and stored at -80°C for extraction of tissue proteins and RNA.

[0117] Record the mouse's body weight and liver weight, and calculate the liver index (liver weight / body weight × 100%).

[0118] 6. Testing methods and results All measurement data are expressed as mean ± standard deviation. Statistical analysis was performed using data analysis software. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 indicates a statistically significant difference compared to the CCl4 model group; P < 0.01 indicates a highly statistically significant difference compared to the CCl4 model group; #P < 0.05 indicates a statistically significant difference compared to the Example 1 group; ##P < 0.01 indicates a highly statistically significant difference compared to the Example 1 group.

[0119] 6.1 General physical signs, weight, and liver index testing During the experiment, the fasting weight of mice was recorded every 7 days; after dissection, the wet weight of the liver was measured and the liver index was calculated. At the same time, the mental state, feeding status, fur condition, activity level and survival status of each group of mice were observed and recorded.

[0120] The test results are shown below: Table 3

[0121] As can be seen from the above, the CCl4 model group showed a decrease in body weight and a significant increase in liver index; Example 1 significantly improved body weight and liver index, which was superior to the comparative examples, free drugs and positive drugs; Comparative example 2 had limited overall improvement due to the large amount of vesicle impurities.

[0122] 6.2 Serum liver function biochemical tests The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total albumin (ALB) were measured using a fully automated biochemical analyzer according to the kit instructions. Each sample group was tested in triplicate.

[0123] The specific test results are shown below: Table 4

[0124] As can be seen from the above, Example 1 significantly reduced ALT / AST / ALP and increased albumin; Comparative Examples 1 and 3 had a certain liver-protective effect, but were significantly worse than Example 1; Comparative Example 2 and the free drug group showed only weak improvement in liver damage.

[0125] 6.3 Liver histopathological examination Liver tissue was fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin to prepare 5μm serial sections, which were then stained with hematoxylin and eosin (HE) and Masson's trichrome staining, respectively. The morphology of the liver tissue was observed under a microscope, and the proportion of collagen fibers in the Masson-stained sections was quantitatively analyzed using ImageJ software. HE slides were used to perform semi-quantitative scoring of inflammatory infiltration, with a score range of 0-4 points. The higher the score, the more severe the inflammation and hepatocellular necrosis. Table 5. Semi-quantitative scoring criteria for inflammatory infiltration-hepatocyte necrosis in HE sections

[0126] Five slices were randomly selected from each group, and five fields of view were selected from each slice for statistical analysis. The experimental results are shown below: Table 6

[0127] 6.4 RT-qPCR gene expression detection Frozen liver tissue was harvested, and total RNA was extracted using the Trizol method, followed by reverse transcription to obtain cDNA. Real-time quantitative PCR using the SYBR Green method was employed to detect the mRNA expression levels of fibrosis-related genes Col-I, α-SMA, TGF-β1, and the inflammatory gene TNF-α, with GAPDH used as an internal reference gene. -^ΔΔCt The relative expression levels of each gene were calculated using a method that normalized the gene expression of the Sham control group to 1.0; each group had 3 biological replicates and 3 technical replicates.

[0128] The specific test results are shown below: Table 7

[0129] As can be seen from the above, Example 1 can significantly downregulate collagen, stellate cell activation, TGF-β pathway and inflammatory factors; the downregulation magnitude of each pair is weaker than that of Example 1; the free drug has almost no gene regulation effect.

[0130] 6.5 Western blot protein expression detection Total protein was extracted from liver tissue and quantified using the BCA method. After SDS-PAGE electrophoresis, membrane transfer, blocking, incubation with primary and secondary antibodies, and ECL chemiluminescence imaging, the protein band grayscale was quantitatively analyzed using ImageJ software to detect the relative expression levels of Col-I, α-SMA, and p-Smad2 / 3 proteins. The protein expression of the Sham control group was normalized to 1.0.

[0131] The specific test results are shown below: Table 8

[0132] As can be seen from the above, the protein results are consistent with the trend of qPCR: Example 1 significantly inhibited the activation of the TGF-β / Smad fibrosis signaling pathway.

[0133] In summary, compared with the CCl4 model group, the cholesterol-phenylboronic acid-disulfide bond modified erythrocyte vesicles prepared in Example 1 can significantly improve the survival status of mice, reduce liver index, reduce serum ALT, AST and ALP levels, increase serum albumin, reduce liver collagen deposition and inflammatory cell infiltration, significantly downregulate the expression of α-Col-I, α-SMA, TGF-β1, and TNF-α genes and proteins, and inhibit the activation of TGF-β / Smad2 / 3 fibrosis signaling pathways, thus exhibiting excellent anti-chronic liver fibrosis therapeutic effects.

[0134] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing EPO-coated erythrocyte vesicles, characterized in that, Including the following steps: S1: Separate and purify intact red blood cells to obtain purified red blood cells; S2: Resuspend the purified red blood cells in an isotonic buffer system to obtain a red blood cell suspension; S3: Add Chol-SS-PBA functional molecules and Chol-PEG2000-EPO conjugate to the red blood cell suspension simultaneously and co-incubate to allow the cholesterol hydrophobic ends of Chol-SS-PBA and Chol-PEG2000-EPO to insert into the lipid bilayer of the red blood cell membrane, thereby completing the in situ co-pre-modification of intact red blood cells and achieving EPO coating on the surface of the red blood cell membrane. S4: Incubate the pre-modified erythrocyte suspension to induce in situ budding and shedding of erythrocytes, releasing erythrocyte vesicles that are modified with Chol-SS-PBA and coated with EPO. S5: Gradient differential centrifugation to remove intact erythrocytes and membrane fragments, and collect the supernatant containing crude modified erythrocyte vesicles; S6: The crude vesicles are purified and eluted to remove uninserted free Chol-SS-PBA molecules, free Chol-PEG2000-EPO, and free impurities, resulting in purified erythrocyte vesicles modified with Chol-SS-PBA and coated with EPO.

2. The preparation method according to claim 1, characterized in that, The steps for separating and purifying intact red blood cells described in S1 are as follows: take fresh anticoagulated whole blood, centrifuge to remove plasma and white membrane layer; wash red blood cells with isotonic washing buffer to obtain purified intact red blood cells.

3. The preparation method according to claim 2, characterized in that, The isotonic washing buffer described in S1 is selected from at least one of HEPES-HBSS washing buffer, HEPES-PBS buffer, and bicarbonate-buffered Hanks balanced salt solution. S1 describes washing the red blood cells 1-5 times; The centrifugation temperature described in S1 is 2-8℃, the centrifugal force is 400-600g, and the centrifugation time for a single cycle is 6-12min.

4. The preparation method according to claim 1, characterized in that, The isotonic buffer system described in S2 is selected from either HEPES-HBSS buffer or PBS buffer. The isotonic buffer system described in S2 has an osmotic pressure range of 280-300 mOsm / kg and a pH of 7.20-7.

50. The hematocrit of the red blood cell suspension described in S2 is 2.0 v / v%-8.0 v / v.

5. The preparation method according to claim 1, characterized in that, The concentration of Chol-SS-PBA in the red blood cell suspension described in S3 is 3.0-12.0 μM, and the final concentration of Chol-PEG2000-EPO conjugate is 2-7 μM. The co-incubation temperature described in S3 is 25-34℃, the duration is 15-40 min, and the method is shaking incubation.

6. The preparation method according to claim 1, characterized in that, S3 describes that the Chol-SS-PBA is premixed with a solubilizer before being added to the red blood cell suspension; The type of co-solvent is selected from anhydrous ethanol and DMSO; The volume fraction of the solubilizer in the red blood cell suspension is ≤1%.

7. The preparation method according to claim 1, characterized in that, The incubation temperature described in S4 is 32-37℃, and the incubation time is 20-60 min; The incubation process described in S4 employs low-speed horizontal oscillation; the oscillation rate is 25-55 rpm.

8. The preparation method according to claim 1, characterized in that, The temperature for differential centrifugation described in S5 is 2-8℃; The differential centrifugation described in S5 includes primary centrifugation and secondary centrifugation; The centrifugal force for each centrifugation is 300-380g, and the duration is 8-15min. The centrifugal force for the second centrifugation is 1600-2200g, and the duration is 30-60min; The purification described in S6 is selected from at least one of size exclusion chromatography and ultrafiltration concentration-dialysis. The elution buffer used for elution in S6 is either isotonic PBS buffer or HEPES-HBSS buffer.

9. A red blood cell vesicle obtained by the preparation method according to any one of claims 1-8.

10. The preparation method according to any one of claims 1-8 or the red blood cell vesicles according to claim 9 in the preparation of drugs for treating liver fibrosis and in nanomedicine delivery.

Images