A membrane fusion type nanovesicle for constructing CAR-M in vivo and a preparation method and application thereof

Abstract

This invention discloses a membrane-fused nanovesicle for in vivo CAR-M construction, its preparation method, and its application, belonging to the fields of biomedicine and cell therapy. The membrane-fused nanovesicle comprises an extracellular vesicle mimic and a fusion factor DSPE-PEG anchored to its membrane surface. n - Fusion peptide. This invention obtains F-CAR EVMs with membrane fusion function by anchoring a fusion factor to the surface of CAR-expressing vesicles. After intravenous injection, F-CAR EVMs target the SR-BI receptor on the surface of monocytes via the fusion peptide, delivering CAR molecules to the monocyte membrane surface via membrane fusion to form CAR-Mo; CAR-Mo chemotacticly moves to the tumor site and differentiates into CAR-M, exerting an anti-tumor effect. Experiments show that the monocyte fusion rate of F-CAR EVMs reaches 9.18±1.39% at the optimal dose, and it has good biocompatibility.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cell therapy, specifically to a membrane-fused nanovesicle for in vivo construction of chimeric antigen receptor macrophages (CAR-M), its preparation method, and its application. Background Technology

[0002] Macrophages possess natural tumor infiltration and phagocytosis functions, and based on this, chimeric antigen receptor macrophage (CAR-M) technology has attracted widespread attention in recent years. CAR-M can not only directly kill tumor cells, but also activate adaptive immunity through antigen presentation and cytokine secretion, demonstrating its potential for solid tumor treatment.

[0003] Currently, CAR-M transfection mainly relies on in vitro viral transfection, which suffers from limited cell sources, weak amplification capacity, high production costs, and long preparation cycles. In vivo transfection using non-viral vectors such as lipid nanoparticles (LNPs) offers advantages such as eliminating the need for in vitro culture and amplification, significantly reducing costs, and shortening the preparation cycle. However, macrophages possess strong nucleic acid degradation capabilities, and existing non-viral vectors (such as LNPs) typically exhibit low in vivo gene transfection efficiency, with significant individual variability.

[0004] Therefore, constructing an efficient and targeted in vivo macrophage CAR transduction system is a key scientific problem that urgently needs to be solved in clinical practice. Summary of the Invention

[0005] This invention aims to solve the following technical problems in existing CAR-M preparation technologies: in vitro viral transduction strategies have drawbacks such as difficulty in transfecting mature macrophages, limited cell sources, weak in vitro expansion capacity, high production costs, and long preparation cycles; non-viral in vivo delivery technologies, represented by lipid nanoparticles, have problems such as low macrophage transfection efficiency, large individual differences, insufficient targeting, as well as liver and spleen accumulation and immunogenicity risks.

[0006] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a membrane-fused nanovesicle for constructing chimeric antigen receptor macrophages (CAR-M) in vivo, the membrane-fused nanovesicle comprising: Extracellular vesicle mimics, and fusion factors anchored to the membrane surface of the extracellular vesicle mimics; The fusion factor has the following structural formula: DSPE-PEG n - Fusion peptide; DSPE is distearate phosphatidylethanolamine, used for anchoring to the surface of extracellular vesicle mimic membranes; PEGn The linker is a polyethylene glycol linker, and n is the degree of polymerization; the fusion peptide is an amphiphilic peptide analog of apolipoprotein AI (apo AI).

[0007] Preferably, the extracellular vesicle mimic expresses a chimeric antigen receptor.

[0008] More preferably, the chimeric antigen receptor is NKG2D-CAR.

[0009] Preferably, the fusion peptide includes, but is not limited to, 18A, whose amino acid sequence is shown in SEQ ID NO: 1. In this invention, amphiphilic peptide analogs of apolipoprotein AI refer to a class of synthetically designed polypeptides capable of mimicking the function of natural apolipoprotein AI (apoA-I). The core structural feature of these polypeptides is their amphiphilic α-helix structure, and their design principles and representative sequences (such as 18A, 4F, etc.) are known in the art. Those skilled in the art will understand that the fusion peptide is not limited to a specific sequence, but rather encompasses all amphiphilic peptide analogs of apolipoprotein AI possessing an amphiphilic α-helix structure capable of mediating membrane fusion, as well as variants that retain the aforementioned structure and function.

[0010] Preferably, the PEG n The molecular weight is 500~8000 Da.

[0011] More preferably, the PEG n The molecular weight is 1500 Da.

[0012] Secondly, the present invention provides a method for preparing membrane-fused nanovesicles for constructing chimeric antigen receptor macrophages in vivo, comprising the following steps: (1) Construct donor cells that stably express the target protein; (2) Preparation of extracellular vesicle mimics from the donor cells; (3) The fusion factor is co-incubated with the extracellular vesicle mimicry to anchor the fusion factor on the membrane surface of the extracellular vesicle mimicry, thus obtaining membrane fusion nanovesicles.

[0013] Preferably, in step (1), the target protein is a chimeric antigen receptor and the donor cell is a mammalian cell.

[0014] More preferably, the target protein is NKG2D-CAR, and the donor cell is HEK293T cell.

[0015] Preferably, in step (2), the method for preparing extracellular vesicle mimics includes: lysing donor cells with sodium deoxycholate, differential centrifugation, membrane extrusion, and ultracentrifugation.

[0016] More preferably, the membrane extrusion is performed sequentially through 0.45 μm and 0.22 μm filter membranes.

[0017] Preferably, in step (3), the concentration of the fusion factor is 5-80 μM.

[0018] Thirdly, the present invention provides the application of the membrane-fused nanovesicles in the preparation of drugs for constructing chimeric antigen receptor macrophages in vivo.

[0019] Preferably, the membrane-fused nanovesicles are administered via intravenous injection, targeting immune cells (e.g., monocytes) in the bloodstream, and delivering chimeric antigen receptor molecules to the surface of the immune cell membrane through membrane fusion, forming chimeric antigen receptor-modified immune cells; the chimeric antigen receptor-modified immune cells chemotactically move to the tumor site and differentiate into chimeric antigen receptor macrophages with anti-tumor activity.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) High efficiency: The membrane-fusion nanovesicles of this invention can efficiently deliver CAR molecules to the surface of monocyte membranes through a membrane fusion mechanism mediated by fusion factors. In vivo experiments showed that at the optimal dose of 4.50 mg / kg, the proportion of fusion-positive monocytes in the blood reached 9.18% ± 1.39% after 72 h of administration, and F-CAR Mo could effectively chemotactically attract to the tumor site and differentiate into F-CAR M.

[0021] (2) High specificity: In vivo distribution experiments showed that F-CAR EVMs exhibited significant specific fusion ability to monocytes. At the optimal dose, the fusion positivity rate of monocytes was much higher than that of T cells, B cells and NK cells (the positivity rates of T / B / NK cells were all less than 2.5%), indicating that F-CAR EVMs can accurately target monocytes.

[0022] (3) High safety: This invention uses a membrane fusion mechanism, which does not involve genome integration and avoids the risk of insertional mutations; the fusion factor is a non-viral synthetic polypeptide-phospholipid conjugate with low immunogenicity. In vitro cytotoxicity experiments show that the mononuclear cell viability is greater than 85% in the concentration range of 5-160 μg / mL, indicating good biocompatibility.

[0023] (4) Immediate availability: The nanovesicles of the present invention can be prepared in advance and stored stably. When used, only a single injection is required to initiate treatment in the body, which significantly simplifies the complex process of existing CAR-M cell therapy and is an "off-the-shelf" product.

[0024] (5) Simple operation and low cost: No in vitro cell culture and expansion are required, which significantly shortens the preparation cycle, reduces production costs, and is conducive to clinical translation and large-scale application.

[0025] The key point of this invention lies in the innovative design and construction of a membrane-fusion-type F-CAR EVM, which has potential advantages such as simple operation, repeatable administration, and avoidance of risks associated with viral vectors, providing a "ready-to-use" carrier for in vivo CAR-M preparation. After intravenous injection, this carrier rapidly integrates CAR molecules into the surface of circulating monocytes via membrane fusion through the specific binding of the fusion peptide to the SR-BI receptor on the surface of monocytes, generating CAR-Mo rapidly without genetic modification. CAR-Mo can naturally chemotactically migrate to tumor sites and differentiate into CAR-Ms with anti-tumor activity, providing a safe and efficient new technical pathway for in vivo CAR-M engineering. Attached Figure Description

[0026] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 Schematic diagram of the preparation of F-CAR EVMs.

[0028] Figure 2 Microstructure of CAR EVMs (scale bar 200 nm, a), microstructure of F-CAR EVMs (scale bar 200 nm, b), particle size (c), zeta potential (d), protein composition characterization (e), and CAR protein quantification (f).

[0029] Figure 3 Stability of F-CAR EVMs under physiological conditions (a) and storage stability (b).

[0030] Figure 4 :Cytotoxicity of F-CAR EVMs.

[0031] Figure 5 : Fusion ratios of CAR EVMs and F-CAR EVMs with different immune cells in vivo at 24 h (a) and fusion ratios of F-CAR EVMs with different immune cells in vivo at 72 h (b).

[0032] Figure 6 Laser confocal image of tumor tissue sections 72 h after drug administration (scale bar 50 μm). Detailed Implementation

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0035] The present invention selects monocytes in the blood circulation rather than macrophages because: monocytes have low phagocytic activity, which helps to reduce the internalization and degradation of exogenous carriers, thereby improving the transduction efficiency of CAR molecules; at the same time, monocytes are widely distributed in the blood, with short delivery pathways and less influence from the internal environment.

[0036] The design concept of this invention is as follows: Figure 1 As shown, firstly, HEK293T cells expressing NKG2D-CAR stably and efficiently were obtained through lentiviral infection. Uniform CAR EVMs were prepared using a combination of membrane extrusion and differential centrifugation. Subsequently, after in vitro co-incubation, the fusion factor DSPE-PEG1500-18A was anchored to the surface of the CAR EVMs, successfully constructing F-CAR EVMs with membrane fusion function. The physicochemical properties of the F-CAR EVMs were then characterized, and the fusion efficiency between F-CAR EVMs and peripheral blood monocytes (Mo) was systematically studied. After intravenous injection, F-CAR EVMs specifically targeted Mo in the blood via the interaction of the fusion peptide 18A and the scavenger receptor class B type I (SR-BI) receptor. Simultaneously, they achieved membrane fusion with Mo via the SR-BI receptor-mediated lipid raft pathway, rapidly forming F-CAR Mo (membrane-fused CAR-modified monocytes). F-CAR Mo, leveraging the targeting effect of CAR and the inherent tumor chemotactic properties of Mo, actively chemotactically attracts tumor tissue. In the tumor microenvironment, F-CAR Mo differentiates into F-CAR M using its own differentiation potential, specifically recognizing and killing tumor cells with high NKG2D ligand expression, thereby inhibiting tumor growth.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Preparation of membrane-fused nanovesicles (F-CAR EVMs) 1.1 Construction of donor cells HEK293T cells stably expressing NKG2D-CAR (hereinafter referred to as NKG2D-CAR HEK293T cells) were constructed using conventional methods in the field (such as lentiviral infection) and used as donor cells.

[0039] 1.2 Preparation of CAR EVMs NKG2D-CAR HEK293T cells were cultured to 80% confluence under serum-free conditions. Cells were collected by scraping, centrifuged at 100 g for 5 min at 4°C, and the supernatant was discarded. Cells were resuspended in pre-chilled PBS, counted, and centrifuged again at 100 g for 5 min at 4°C. The cells were then resuspended in 0.045% (w / v, g / 100 mL) sodium deoxycholate (containing 1 mM benzyl sulfonyl fluoride (PMSF)) and vigorously shaken at 4°C for 30 min. After centrifugation at 2500 g for 15 min at 4°C, the supernatant was collected and squeezed through a membrane (0.45 μm 10 times, 0.22 μm 10 times). The cells were then ultracentrifuged at 100,000 g for 4 h, and the pellet was collected. The pellet was resuspended in PBS to obtain CAR EVMs, which were stored at -80°C. All steps were performed on ice to avoid cell aggregation and maintain the viability of the CAR EVMs.

[0040] 1.3 Synthesis and Anchoring of Fusion Factors (1) Synthesis of fusion factors Precisely weigh DSPE-PEG 1500 -Mal and HS-18A were dissolved in 4 mL and 2 mL of PBS (pH 7.4, containing 4 mM EDTA), respectively, and sonicated until dissolved to obtain DSPE-PEG with a concentration of 1.5 mM. 1500 -Mal reaction solution and 6 mM HS-18A reaction solution. Under nitrogen protection, the HS-18A reaction solution was added dropwise to DSPE-PEG. 1500 In the -Mal reaction solution, the mixture was stirred at room temperature for 12 h. After the reaction, the reaction solution was removed and unreacted raw materials were removed using a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da. The water was changed every 4 h, and the mixture was dialyzed with ultrapure water for 48 h. After dialysis, the mixture was freeze-dried to obtain DSPE-PEG. 1500 -18A. Spectrophotometer (H NMR) 1 The product structure was verified by H NMR and found to be correct.

[0041] (2) Anchoring of fusion factors fusion factor DSPE-PEG 1500 -18A is added to freshly prepared CAR EVMs to make DSPE-PEG 1500The -18A concentration was 10 μM, and the mixture was incubated at 37°C with 5% CO2 for 20 min. The resulting solution was then transferred to an ultrafiltration tube (MWCO: 100 kDa), centrifuged at 3000 g for 5 min, and washed three times with PBS buffer to remove any residual unanchored DSPE-PEG. 1500 -18A yields CAR EVMs with membrane fusion capability, namely F-CAR EVMs.

[0042] Example 2: Characterization of F-CAR EVMs This embodiment characterizes the physicochemical properties and protein composition of CAR EVMs and F-CAR EVMs.

[0043] 2.1 Particle size, potential and protein concentration analysis To detect the amount of CAR protein on CAR EVMs and F-CAR EVMs, samples were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer on ice for 30 min, with shaking every 10 min to ensure complete lysis. Total protein was quantified using the BCA method; simultaneously, CAR protein was quantified according to the NKG2D ELISA kit instructions.

[0044] CAR EVMs and F-CAR EVMs resuspended in PBS (protein concentration 1 μg / mL) were analyzed for particle size, polydispersity index (PDI) and zeta potential using a dynamic light scattering analyzer.

[0045] 2.2 Microscopic Morphological Characterization Morphological observation of CAR EVMs and F-CAR EVMs was performed using transmission electron microscopy. 20 μL of sample suspension was dropped onto a copper mesh grid for the electron microscope, allowed to stand for 1 min, and then fixed with 1% phosphotungstic acid solution for 1 min. Residual liquid was blotted off with filter paper, and the samples were allowed to air dry at room temperature before being observed and imaged under a transmission electron microscope.

[0046] 2.3 Protein component characterization Membrane proteins were extracted using non-denaturing cell lysis buffer. The working lysis buffer was prepared by adding PMSF to RIPA lysis buffer to a final PMSF concentration of 1 mM. After centrifugation to precipitate the samples, 250 μL of lysis buffer was added to each tube, gently pipetted to mix, and lysed on ice for 10 min. The lysis buffer was then centrifuged at 4°C and 14,000 rpm for 5 min, and the supernatant was collected into pre-chilled 1.5 mL centrifuge tubes. The supernatant was then added to 5× protein loading buffer at a 1:4 (v / v) ratio, mixed, and heated at 100°C for 4 min to denature the proteins. The samples were stored at -20°C for later use. For protein electrophoresis, 20 μg of protein sample was loaded into 4-12% FuturePAGE. TM In the gel wells, electrophoresis was performed at 120 V for 1 h in a BIO-RAD electrophoresis system until the protein bands migrated to the bottom of the gel. After electrophoresis, the gel was placed in Coomassie Brilliant Blue staining solution and stained with shaking at room temperature for 1 h; then destained with destaining solution with shaking at room temperature for at least 5 h, changing the destaining solution 4 times during this period, until the gel background was transparent. Finally, the gel was placed in a protein imaging system to complete image acquisition.

[0047] 2.4 Stability Test To evaluate the stability of F-CAR EVMs under physiological conditions, F-CAR EVMs were resuspended in PBS buffer, RPMI 1640 medium containing 10% fetal bovine serum (FBS), PBS containing 10% FBS, or physiological saline, and incubated on a shaker at 37°C. The particle size and zeta potential of the F-CAR EVMs were measured at predetermined time points. Furthermore, to assess their storage stability, F-CAR EVMs were stored at 4°C, and their particle size and zeta potential were measured for 7 consecutive days.

[0048] 2.5 Characterization Results The particle size and zeta potential of CAR EVMs and F-CAR EVMs were determined using dynamic light scattering, and their morphology was characterized using transmission electron microscopy. See Table 1 and... Figure 2 Images (a), (b), and (c) show that the average particle size of CAR EVMs is 149.47 ± 2.43 nm, and the average particle size of F-CAR EVMs is 152.73 ± 4.00 nm. Transmission electron microscopy images show that both exhibit typical spherical particles with membrane structures.

[0049] The results of the zeta potential measurement are as follows: Figure 2 As shown in (d), since the main structure of the cell membrane is a negatively charged phospholipid bilayer, both CAR EVMs and F-CAR EVMs derived from the cell membrane showed negative Zeta potentials; however, the negative potential of F-CAR EVMs was slightly lower than that of CAR EVMs, which may be related to DSPE-PEG.1500 The shielding effect of the PEG chain on surface charge after -18A insertion into CAR EVMs membranes is related.

[0050] SDS-PAGE protein electrophoresis results are as follows: Figure 2 As shown in (e), the membrane protein components of NKG2D-CAR HEK293T cells are almost completely preserved in F-CAR EVMs, indicating that F-CAR EVMs possess biological characteristics related to NKG2D-CAR HEK293T cells.

[0051] CAR protein quantification results as follows Figure 2 As shown in (f), the CAR protein content in each microgram of F-CAR EVMs total protein was 0.973 ± 0.029 ng, indicating that DSPE-PEG 1500 Insertion of -18A into the CAR EVM membrane did not affect the CAR protein content.

[0052] The stability test results are as follows Figure 3 As shown, the particle size and zeta potential of F-CAR EVMs remained stable for more than 14 days in a simulated physiological environment and for more than 48 hours under storage conditions at 4℃, indicating that the system has good physical stability.

[0053] Table 1. Reproducibility study of particle size, PDI, and potential of CAR EVMs and F-CAR EVMs (n=3) CAR EVMs 149.47 ± 2.43 0.42 ± 0.04 -7.52 ± 0.07 F-CAR EVMs 152.73 ± 4.00 0.22 ± 0.02 -6.07 ± 0.13 Example 3: Effects of F-CAR EVMs on Monocyte Viability This embodiment examines the cytotoxicity of F-CAR EVMs to mononuclear cells (Mo) to evaluate their biosafety.

[0054] 3.1 Experimental Methods Mo is applied at a rate of 1 × 10⁻⁶ per well. 4 F-CAR EVMs were seeded at a density of 100 μL in each well of a 96-well plate. Then, different volumes of F-CAR EVMs were added to each well, and the volume was brought up to the same final volume with blank medium, so that the final concentrations of F-CAR EVMs were 0, 5, 10, 20, 40, 80, 160, 320, 640, and 1280 μg / mL, respectively, and the plates were cultured for another 24 h.

[0055] After incubation, 10 μL of Cell Counting Kit-8 (CCK8) solution was added to each well. A blank control well containing only culture medium and CCK8 solution but no cells was also included. The 96-well plate was incubated at 37°C with 5% CO2 for 2 hours. During this time, the absorbance of each well was measured at 450 nm every 30 minutes using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (OD) 加药组 - OD 空白组 ) / (OD 未加药组 - OD 空白组 ) × 100% In the formula, OD 加药组 Wells containing cells, CCK8, and F-CAR EVMs; OD 空白组 These are cell-free wells containing only culture medium and CCK8; OD 未加药组 These are wells containing cells and CCK8 but not F-CAR EVMs. All experiments were performed in duplicate, with three independent replicates.

[0056] 3.2 Experimental Results The results are as follows Figure 4 The results showed that the Mo activity was greater than 85.00% in the concentration range of 5-160 μg / mL, indicating that F-CAR-EVMs have good biocompatibility.

[0057] Example 4: Evaluation of F-CAR EVMs for In Vivo Targeted Fusion with Mononuclear Cells This embodiment examines the ability of F-CAR EVMs to target and fuse with monocytes in vivo and determines the optimal dosage.

[0058] 4.1 Experimental Methods CAR EVMs and F-CAR EVMs were labeled with DiO fluorescent dye, respectively. DiO-labeled CAR EVMs (4.50 mg / kg) and different doses (2.25, 4.50, 6.75 mg / kg) of F-CAR EVMs were injected into BALB / c mice via the tail vein.

[0059] Animals were sacrificed 24 h and 72 h after injection, respectively. Blood was collected via the orbital cavity into heparin-anticoagulated tubes and diluted 1:1 (v / v) with pre-chilled Duchenne phosphate-buffered saline (D-PBS) and stored on ice. PBMCs were then extracted according to the instructions of the mouse peripheral blood mononuclear cell (PBMC) extraction kit. The specific steps were as follows: Blood samples were transferred to centrifuge tubes containing 5 mL of erythrocyte lysis buffer, mixed thoroughly by pipetting, and incubated on ice for 10 min; lysis was terminated by adding PBS, and the samples were centrifuged at 450 g for 5 min at 4°C, and the supernatant was discarded. The precipitate was resuspended in 1 mL of PBS, filtered through a 200-mesh nylon screen into EP tubes, and stored on ice for later use.

[0060] Take 1 × 10 6 Cells were blocked with 5% rat serum on ice for 30 min in the dark. After separation, flow cytometry antibodies (CD3, CD14, CD19, CD49b) targeting different immune cell markers were added to separate tubes, and staining was performed on ice for 1 h in the dark. After washing with PBS, CD3+ cells in peripheral blood were analyzed by flow cytometry. 3+ DiO + T cells, CD14⁺DiO⁺Mo, CD19 + DiO + B cells and CD49b + DiO + NK was used for quantitative analysis.

[0061] 4.2 Experimental Results The results are as follows Figure 5 As shown, consistent with in vitro membrane fusion experiments, Mo's fusion ability with F-CAR EVMs was significantly higher than that with T cells, B cells, and NK cells.

[0062] As the injection dose increases, DiO + The Mo ratio increased accordingly; 72 hours after injection, the optimal injection dose was determined to be 4.50 mg / kg, at which point DiO2... + The Mo ratio was 9.18 ± 1.39%. Furthermore, the fusion efficiency of T cells, B cells, and NK cells increased only slightly with increasing dose, indicating that although F-CAR EVMs can effectively fuse Mo, they have no significant effect on the fusion of other cells, highlighting the specific fusion effect of F-CAR EVMs.

[0063] Example 5: Investigation of F-CAR Mo forming F-CAR M at the tumor site This embodiment examines whether F-CAR Mo can chemotactically reach the tumor site and differentiate into macrophages.

[0064] 5.1 Experimental Methods A NOG mouse model bearing HCT116 tumor was established. Mice were randomly divided into three groups and administered saline, DiR-labeled CAR EVMs, and DiR-labeled F-CAR EVMs via tail vein injection, respectively, at a dose of 4.50 mg / kg.

[0065] 72 h after drug administration, mice were euthanized and tumor tissue was removed. The tumor tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with 4',6-diamidinyl-2-phenylindole (DAPI), and then imaged using a pathological scanner.

[0066] F-CAR EVMs or CAR EVMs were labeled with DiR dye (shown in red), while macrophages were labeled with CD11b-FITC and the nuclei were stained with DAPI (shown in blue). Fluorescence imaging was performed on HCT116 tumor tissue sections.

[0067] 5.2 Experimental Results The results are as follows Figure 6 As shown, the red fluorescence signal in the DiR-CAR EVMs group was weak and did not overlap with the green fluorescence signal, indicating that the CAR EVMs failed to fuse with Mo, and their tumor accumulation mainly depended on the targeting properties of the formulation itself. In contrast, in the DiR-F-CAR EVMs group, obvious co-localization signals of red and green fluorescence (appearing yellow) were observed, indicating that after the F-CAR EVMs fused with Mo, they migrated together to the tumor tissue and further differentiated into macrophages, confirming that F-CARMo differentiated into F-CAR M at the tumor site.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A membrane-fused nanovesicle for constructing chimeric antigen receptor macrophages in vivo, characterized in that, The membrane-fused nanovesicles comprise: an extracellular vesicle mimic, and a fusion factor anchored to the membrane surface of the extracellular vesicle mimic; the fusion factor has the following structural formula: DSPE-PEG n - Fusion peptide; DSPE is distearate phosphatidylethanolamine, used for anchoring to the surface of extracellular vesicle mimic membranes; PEG n The linker is a polyethylene glycol linker, and n is the degree of polymerization; the fusion peptide is an amphiphilic peptide analog of apolipoprotein AI (apo AI).

2. The membrane-fused nanovesicle according to claim 1, characterized in that, The extracellular vesicle mimic expresses a chimeric antigen receptor.

3. The membrane-fused nanovesicles according to claim 2, characterized in that, The chimeric antigen receptor is NKG2D-CAR.

4. The method for preparing membrane-fused nanovesicles for in vivo construction of chimeric antigen receptor macrophages (CAR-M) according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Construct donor cells that stably express the target protein; (2) Preparation of extracellular vesicle mimics from the donor cells; (3) The fusion factor is co-incubated with the extracellular vesicle mimicry to anchor the fusion factor on the membrane surface of the extracellular vesicle mimicry, thus obtaining membrane fusion nanovesicles.

5. The preparation method according to claim 4, characterized in that, In step (1), the target protein is a chimeric antigen receptor, and the donor cell is a mammalian cell.

6. The preparation method according to claim 5, characterized in that, The target protein is NKG2D-CAR, and the donor cells are HEK293T cells.

7. The preparation method according to claim 4, characterized in that, In step (2), the method for preparing extracellular vesicle mimics includes: lysing donor cells with sodium deoxycholate, differential centrifugation, membrane extrusion, and ultracentrifugation.

8. The preparation method according to claim 4, characterized in that, In step (3), the concentration of the fusion factor is 5-80 μM.

9. The use of the membrane-fused nanovesicles according to any one of claims 1-3 in the preparation of a medicament for constructing chimeric antigen receptor macrophages in vivo.

10. The application according to claim 9, characterized in that, The membrane-fused nanovesicles are administered via intravenous injection, targeting immune cells in the bloodstream. Through membrane fusion, chimeric antigen receptor molecules are delivered to the surface of the immune cell membrane, forming chimeric antigen receptor-modified immune cells. These chimeric antigen receptor-modified immune cells chemotactically move to the tumor site and differentiate into chimeric antigen receptor macrophages with anti-tumor activity.

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