Construction method and application of cell robot for enhancing cell therapy targeting and entity penetration

Abstract

The invention relates to the technical field of cell targeted therapy, in particular to a construction method and application of a cell robot for enhancing cell therapy targeting and entity penetration. The invention relates to a construction method of a cell robot for enhancing cell therapy targeting and entity penetration. The construction method comprises the following steps: coupling magnetic microfilaments on the surfaces of cells; the magnetic microfilament comprises a microfilament and magnetic particles attached to the microfilament; and the microfilaments are made of polylactic acid-glycolic acid copolymer. According to the technical scheme, the technical problem that in the prior art, chimeric antigen receptor modified macrophages with high targeting and penetrating capacity are lacked can be solved, and the technical problems that in the prior art, chimeric antigen receptor cells generally face infiltration of solid tissues and infiltration is not ideal are further solved. The engineering cell design combining the bionic sperm model and magnetic driving breaks through the bottleneck of the traditional cell therapy in solid tissue delivery, provides a brand new technical thought and method for wider cell therapy optimization, and has ideal application and popularization values.

Description

Technical Field

[0001] This invention relates to the field of cell-targeted therapy technology, specifically to a method for constructing a cell robot that enhances the targeting and solid penetration of cell therapy, and its application. Background Technology

[0002] Atherosclerosis (AS) is a vascular disease characterized by lipid deposition in the arterial intima, chronic inflammation, and fibrosis. Its pathological mechanisms include endothelial dysfunction, low-density lipoprotein (LDL) oxidation, macrophage infiltration, and foam cell formation. With the increasing aging of the global population, the incidence of atherosclerosis has risen significantly. According to the latest statistics, more than one billion people worldwide are affected by this disease, especially among those over 60 years of age, where the incidence rate has exceeded 45%, making it a leading cause of death from cardiovascular disease.

[0003] Atherosclerosis not only causes ischemic events (such as myocardial infarction and stroke) by leading to vascular stenosis and calcification, but also increases clinical harm due to arterial thrombosis triggered by the rupture of vulnerable plaques. Currently, the treatment of atherosclerosis mainly relies on lipid-lowering and anti-inflammatory drugs, aiming to reduce foam cell formation and inhibit excessive apoptosis. However, these treatments often struggle to reverse the pathological process, particularly when faced with the drug delivery challenges posed by the fibrous cap structure.

[0004] In recent years, chimeric antigen receptor-modified macrophage (CAR-M) therapy has emerged as a novel strategy, demonstrating unique advantages in the treatment of atherosclerosis. Compared with traditional CAR-T therapy, CAR-M therapy has the following characteristics: First, CAR-M can directly target and phagocytose target cells without relying on T cell-mediated cytotoxicity; second, it can secrete anti-inflammatory factors (such as IL-10 and TGF-β), helping to regulate the plaque microenvironment and reduce the risk of cytokine storms; finally, it penetrates deep into diseased tissue through chemotaxis, overcoming the challenges posed by the solid tumor-like barrier. However, the unique structure of atherosclerotic plaques (including an intact endothelial layer, a dense fibrous cap rich in collagen and smooth muscle cells, and a calcified core) greatly limits the targeting and penetration efficiency of CAR-M, hindering the effective delivery of cells, nanomaterials, and even drugs. Therefore, optimizing and modifying CAR-M to improve its targeting and penetration capabilities has become an important direction of current research. Summary of the Invention

[0005] The present invention aims to provide a method for constructing a cell robot that enhances the targeting and penetration of cell therapy into solid tissues, in order to solve the technical problem of the lack of chimeric antigen receptor-modified macrophages with strong targeting and penetration capabilities in the prior art, and further solve the technical problem that chimeric antigen receptor cells generally face unsatisfactory infiltration and penetration into solid tissues in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing a cell robot that enhances the targeting and solid penetration of cell therapy includes a process of coupling magnetic microfilaments to the cell surface; the magnetic microfilaments include microfilaments and magnetic particles attached to the microfilaments; the microfilaments are made of polylactic acid-glycolic acid copolymer.

[0007] Furthermore, the magnetic microwires are prepared by the following method: an aqueous solution containing ferrous ions and ferric ions is prepared, heated, and then an alkaline solution is added dropwise; ultrasonic treatment and cleaning are performed, followed by the addition of an acid solution; after ultrasonic treatment again, a suspension containing Fe3O4 nanoparticles is obtained. Polylactic acid-glycolic acid copolymer was shredded and dispersed in water, and magnetic beads were added; it was then ground using a cryogenic grinder and centrifuged to obtain a microfilament suspension; A suspension containing Fe3O4 nanoparticles was mixed with a microfilament suspension, and after incubation and magnetic separation, a magnetic microfilament suspension was obtained.

[0008] Further, 10.8 mg FeCl3·6H2O and 15.2 mg FeCl2·4H2O were dissolved in 5 mL of distilled water, heated to 50 °C, and 1 mL of 0.5 M NaOH solution was slowly added dropwise to the solution. After ultrasonic treatment and washing, the solution was resuspended in 1 mL of water, 10 μL of 5 M HCl was added, ultrasonic treatment was performed, and centrifugation was performed to obtain a suspension containing Fe3O4 nanoparticles.

[0009] Further, 500 mg of PLGA was cut into small pieces, 1 mL of distilled water and magnetic beads were added, and the mixture was ground for 10 min using a -20℃ refrigerated grinder. Then, it was centrifuged at 6000 rpm for 1 min and the supernatant was discarded. Finally, it was centrifuged at 1000 rpm for 10 s and the precipitate was discarded to obtain a microfilament suspension.

[0010] Furthermore, the microfilament suspension was mixed with a suspension containing Fe3O4 nanoparticles, placed in a shaker at 50 rpm overnight, and then separated magnetically to obtain a magnetic microfilament suspension.

[0011] Furthermore, the magnetic microfilaments form covalent connections with the cell surface through click chemical reactions.

[0012] Preferably, magnetic microfilaments are modified with polydopamine and then linked with azide tetraethylene glycol succinimide ester to obtain magnetic microfilaments modified with azide groups. Cells modified with alkyne groups were obtained by modifying the cell surface with dibenzocyclooctyne-tetraethylene glycol-succinimide ester; Azide-modified magnetic microfilaments and alkynyl-modified CAR-M cells were covalently linked via a click reaction, followed by magnetic separation to obtain a cell robot.

[0013] Further, 5 mg of magnetic microfilaments were added to 10 ml of 0.5 mg / mL polydopamine solution, and the pH was adjusted to 8.5 using Tris solution. The solution was then placed in a shaker at 50 rpm for 2 h. After magnetic separation, a polydopamine-treated microfilament suspension was obtained. Azide tetraethylene glycol succinimide ester was added to the polydopamine-treated microfilament suspension, and the solution was placed in a shaker at 50 rpm for 40 min. After washing, azide-modified magnetic microfilaments were obtained. The azide-modified magnetic microfilaments were then sonicated until a click reaction was initiated. Add 1 mL containing 1×10 5 Dibenzocyclooctyne-tetraethylene glycol-succinimide ester was added to the DMEM medium for cells; after 40 min of culture, the medium was aspirated and the cells were washed to obtain CAR-M cells modified with alkyne groups. After mixing azide-modified magnetic microfilaments with alkynyl-modified CAR-M cells, the mixture was incubated at 50 rpm for 40 min and then magnetically separated to obtain Robotic CAR-M.

[0014] Furthermore, the cells used for coupling magnetic microfilaments are chimeric antigen receptor-modified macrophages.

[0015] This technical solution also provides a cell robot that enhances the targeting and solid penetration of cell therapy, which includes cells and magnetic microfilaments coupled to the cell surface; the magnetic microfilaments include microfilaments and magnetic particles attached to the microfilaments; the material of the microfilaments is polylactic acid-glycolic acid copolymer; Preferably, the magnetic particles are Fe3O4 nanoparticles; Preferably, the cells are chimeric antigen receptor-modified macrophages.

[0016] This technical solution also provides an application of a cell robot that enhances the targeting and solid penetration of cell therapy in the preparation of products for treating atherosclerosis.

[0017] Furthermore, a method for preparing chimeric antigen receptor mononuclear macrophages includes the following steps performed sequentially: S1: Integrate the gene fragment with the sequence shown in SEQ ID NO.7 into the multiple cloning site of the universal lentiviral expression vector GV492 to obtain the expression plasmid GV492-CAR-OPN; S2: HEK293T cells were transfected with the expression plasmid GV492-CAR-OPN, and the supernatant was collected after cell culture. The supernatant was centrifuged and the liquid was discarded to obtain lentivirus. S3: RAW264.7 macrophages were infected with lentiviruses and then sorted by flow cytometry to obtain chimeric antigen receptor mononuclear macrophages targeting OPN.

[0018] In S2, HEK293T cells in logarithmic growth phase were fed at a dose of 5 × 10⁻⁶. 6 Cells were seeded in a medium containing 10% serum. After 24 hours of culture, a transfection system containing expression plasmid GV492-CAR-OPN, pHelper 1.0 vector plasmid, and pHelper 2.0 vector plasmid was added. After culturing for another 6-8 hours, the medium was replaced with fresh medium containing 2% serum. After 40-42 hours of culture, the supernatant was collected from the culture dish.

[0019] In S3, RAW264.7 macrophages were infected with lentivirus for 24 hours at an MOI of 50, and then replaced with fresh DMEM high-glucose medium. After culturing for another 72 hours, chimeric antigen receptor mononuclear macrophages targeting OPN were obtained by sorting.

[0020] The technical principle of this technical solution is as follows: Based on the synergistic effect of biomimetic engineering and magnetic drive technology, this invention constructs a cell robot with active targeting and efficient solid penetration capabilities by precisely coupling magnetic microfilaments to the surface of chimeric antigen receptor-modified macrophages (CAR-M).

[0021] (1) Biomimetic construction of magnetic microfilaments The magnetic microfilaments utilize polylactic-glycolic acid copolymer (PLGA) as the substrate material, which possesses excellent biocompatibility and biodegradability, avoiding immune rejection or residual toxicity in vivo. Using PLGA microfilaments as a carrier, Fe3O4 nanoparticles are uniformly loaded onto the microfilament surface via liquid-phase deposition. First, a highly magnetically responsive Fe3O4 nanoparticle suspension is prepared through a co-precipitation reaction of ferrous and ferric ions, and ultrasonic dispersion technology is used to ensure the monodispersity of the nanoparticles. Subsequently, PLGA is sheared and dispersed, mixed with magnetic beads, and then cryo-milled to obtain a uniformly sized PLGA microfilament suspension. Finally, incubation allows the Fe3O4 nanoparticles to firmly attach to the PLGA microfilament surface through van der Waals forces and hydrogen bonds. Magnetic separation and purification yield magnetic microfilaments with both good flexibility and magnetic responsiveness. Their structure mimics the filamentous morphology of sperm tails, providing a highly efficient propulsion mechanism for cells.

[0022] (2) Coupling of magnetic microfilaments with CAR-M cells Click chemistry technology was employed to achieve specific covalent coupling between magnetic microfilaments and CAR-M cells. This reaction is characterized by high efficiency, high selectivity, and mild conditions, avoiding damage to the activity of CAR-M cells. Specifically, the surface of the magnetic microfilaments was first modified with polydopamine. The amino and carboxyl groups of polydopamine were then used to connect azido-tetraethylene glycol succinimidyl ester, functionalizing the surface of the magnetic microfilaments with azido groups. Simultaneously, the amino groups on the surface of CAR-M cells were modified with dibenzocyclooctyne-tetraethylene glycol-succinimidyl ester, introducing alkynyl groups onto the cell surface. Based on the click reaction between the azido and alkynyl groups, a stable triazole ring structure was rapidly formed without a catalyst, achieving stable covalent coupling between the magnetic microfilaments and CAR-M cells, ultimately constructing a magnetically driven cellular robot (Robotic CAR-M).

[0023] (3) Magnetic-driven targeting and solid penetration The targeting and penetration capabilities of cellular robots stem from the synergistic enhancement effect of magnetic drive and the chemotaxis of CAR-M cells themselves: On the one hand, CAR-M cells can recognize specific antigens in the microenvironment of atherosclerotic plaques through surface chimeric antigen receptors, generating a natural chemotactic migration ability; on the other hand, the external magnetic field can generate a directional driving force on the magnetic microfilaments on the cell surface through the magnetic field gradient, guiding the cellular robot to actively migrate towards the lesion site, overcoming the shear force of blood flow and the obstruction of vascular endothelium, and achieving precise targeting. When penetrating solid lesion tissue, the biomimetic filamentous structure of the magnetic microfilaments can reduce the frictional resistance of cells in dense tissue. At the same time, the external magnetic field can drive the magnetic microfilaments to produce a swinging motion similar to the tail of a sperm by adjusting the intensity and direction of the magnetic field, providing additional propulsion power for CAR-M cells, helping them penetrate the fibrous cap of atherosclerotic plaques (a dense structure rich in collagen and smooth muscle cells), break through the solid tissue barrier, and reach the core area of ​​the plaque to exert a therapeutic effect.

[0024] The cell robot constructed in this invention has the following significant beneficial effects compared to existing CAR-M cell therapy and other cell therapy technologies: (1) Significantly improves targeting accuracy and reduces off-target toxicity Existing CAR-M cells mainly rely on their own chemotaxis to migrate to lesion sites, which is easily affected by blood flow and signals from normal tissues, resulting in poor targeting and high off-target rates, potentially causing damage to normal tissues. This invention utilizes a dual targeting mechanism of magnetic drive and CAR-M self-antigen recognition. An external magnetic field can precisely guide the cell robot to migrate to lesion sites such as atherosclerotic plaques, significantly improving targeting accuracy, reducing cell accumulation in normal tissues, decreasing off-target toxicity, and enhancing treatment safety.

[0025] (2) Effectively overcomes physical tissue barriers and solves the penetration problem The intact endothelial layer, dense fibrous cap, and calcified core of atherosclerotic plaques constitute a physiological barrier similar to that of solid tumors. Existing CAR-M cells and nanomedicines struggle to effectively penetrate this barrier to reach the core area of ​​the lesion, resulting in poor treatment outcomes. The magnetic microfilament biomimetic structure of this invention reduces frictional resistance during cell penetration. Simultaneously, the active propulsion provided by magnetic drive synergizes with the migration ability of CAR-M cells, effectively overcoming the aforementioned solid tissue barrier. This allows the cell robot to penetrate deep into the plaque core area, exerting its therapeutic effects by phagocytizing foam cells and secreting anti-inflammatory factors, significantly improving the effectiveness of cell therapy and solving the technical problem of unsatisfactory infiltration and penetration into solid tissues in existing cell therapies.

[0026] (3) Excellent biocompatibility and high safety The PLGA material used in this invention is biodegradable, gradually degrading into lactic acid and glycolic acid in vivo, and ultimately metabolized into carbon dioxide and water for excretion, leaving no residual toxicity. The Fe3O4 nanoparticles exhibit excellent biocompatibility, and the magnetic microfilament loading method avoids cytotoxicity caused by the free diffusion of nanoparticles. Furthermore, the click-coupling method is mild, does not damage the activity and function of CAR-M cells, and does not trigger a strong immune response. This design ensures the safety of the cell robot in vivo, laying the foundation for clinical translation.

[0027] (4) It has a wide range of applications and high promotion value. The technical solution of this invention is not only applicable to CAR-M cells, but can also be extended to other chimeric antigen receptor modified cells such as CAR-T and CAR-NK, as well as ordinary cells without chimeric antigen receptor modification. By coupling magnetic microfilaments to the surface of these cells, enhanced targeting and solid tissue penetration can be achieved. Therefore, this technology can be widely used in the treatment of various cell therapy-dependent diseases such as atherosclerosis and solid tumors, providing a universal technical solution for solving the cell therapy delivery challenges related to various solid tissues, and has extremely high promotional value and clinical application prospects.

[0028] In summary, this invention is the first to propose a robotic CAR-M engineered cell design concept that combines a biomimetic sperm model with magnetic drive, and experimentally verified its excellent targeting and penetration effects into solid tissues. This technology breaks through the key bottleneck of traditional cell therapy in solid tissue delivery, providing a new technical approach and method for optimizing a wider range of cell therapies. It is expected to drive technological innovation in the field of cell therapy and help more cell therapies move from the laboratory to clinical application. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the preparation process of the chimeric antigen receptor-modified macrophage robot of the present invention.

[0030] Figure 2 This is a plasmid map of the expression vector GV492-CAR-OPN from Example 1.

[0031] Figure 3 This is a flow cytometry sorting result of CAR-M cells that were successfully infected in Example 1.

[0032] Figure 4 The flow cytometry results for CAR-M targeted phagocytosis in Example 1 are shown in Figure 1 (A: Flow cytometry results; B: Statistical graph).

[0033] Figure 5 A schematic diagram of the Robotic CAR-M fabrication in Example 2 (A: DBCO-CAR-M: CAR-M cells modified with alkyne groups; N3-Micro-tail: magnetic microfilaments modified with azide groups) and a confocal fluorescence imaging image (B: DIO: green fluorescent dye used to label the cell membrane; Micro-Tail: magnetic microfilaments, stained with rhodamine; DAPI: blue fluorescent dye used to label the cell nucleus; Merge: overlay of the above three channels).

[0034] Figure 6 This is a schematic diagram of the detection device structure in Example 3 (hydrogel-filled channel 1, cell flow channel 2, magnet 3, cell flow direction 4, T-shaped channel 5).

[0035] Figure 7 The experimental results of Robotic CAR-M targeting and penetrating plaques in fluid in Example 3 are shown in Figure 3 (A: Schematic diagram of experimental principle; B: Fluorescence imaging; CAR-M cells are labeled with green fluorescent markers; foam cells are labeled with red fluorescent DIL markers; C: Statistical graph of experimental results).

[0036] Figure 8 The experimental results of Robotic CAR-M penetrating plaques in fluid in Example 3 are shown in Figure 3 (A: Schematic diagram of experimental principle; B: Fluorescence imaging; CAR-M cells are labeled with green fluorescence; magnetic microfilaments are labeled with Rhodamine 6G; C: Statistical graph of experimental results).

[0037] Figure 9 Typical microscopic images of failed robotic CAR-M preparations for comparison. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0039] The key technical terms used in this solution are explained as follows: The fibrous cap is a key structure in atherosclerotic plaques, located on the outermost layer of the plaque and in direct contact with the vascular lumen. It is mainly composed of smooth muscle cells (SMCs) and extracellular matrix (especially collagen), and plays a role in "covering" and "stabilizing" the lipid core inside the plaque.

[0040] Chimeric antigen receptor-modified macrophages (CAR-M) are an emerging cell immunotherapy strategy that uses genetic engineering to introduce chimeric antigen receptors (CARs) into macrophages, enabling them to specifically recognize and target the elimination of diseased cells (such as tumor cells or abnormal cells in atherosclerotic plaques) that express specific antigens.

[0041] Foam cells are hallmark cells in atherosclerosis (AS), formed by monocytes / macrophages or vascular smooth muscle cells after taking up large amounts of modified lipoproteins (especially oxidized low-density lipoprotein, ox-LDL). They are named for their "foamy" appearance under a microscope, due to the presence of lipid droplets in their cytoplasm. In this protocol, foam cells were obtained as follows: RAW264.7 cells were cultured to 80%-90% abundance, and 20 μL of 20 μg / μL ox-LDL was added to 1 mL of DMEM medium. The culture was then incubated at 37°C in a 5% CO2 incubator for 24 h. Successful foam cell model construction was confirmed by Oil Red O staining.

[0042] CAR-M cells, short for chimeric antigen receptor-macrophages, are precisely targeted immune cells obtained by genetically modifying macrophages. The CAR (chimeric antigen receptor) on the surface of CAR-M cells consists of four key domains that determine its targeting and function: Antigen-binding domain (e.g., single-chain antibody scFv): responsible for recognizing specific antigens on the surface of target cells; Hinge region: A flexible structure that increases the flexibility of CAR binding to target antigens; Transmembrane region: anchors the CAR to the macrophage membrane; Intracellular signaling domains (such as CD3ζ, FcRγ, etc.): When CAR recognizes the target antigen, this region will activate the phagocytic signaling pathway of macrophages, triggering the function of phagocytosis and degradation of target cells.

[0043] The mechanism of action of CAR-M is a combination of "specific recognition + macrophage innate function". Through the antigen-binding domain of CAR, it precisely binds to antigens on the surface of target cells; after CAR binds to the antigen, the intracellular signaling domain initiates the phagocytic program of macrophages, which engulf, phagocytose and degrade the target cell; after macrophages engulf the target cell, they can also present the target cell's antigen to other immune cells (such as T cells), further amplifying the immune response.

[0044] The overall technical flowchart for preparing chimeric antigen receptor-modified macrophage robots (Robotic CAR-M cells) and performing solid targeting and penetration can be found in [link to technical documentation]. Figure 1 Examples 1 and 2 detail the construction process of Robotic CAR-M cells. Example 3 examines the therapeutic effect of Robotic CAR-M cells on plaques using a mouse model of atherosclerosis. In this technical solution, modifying the CAR-M surface with magnetic microfilaments significantly enhances the targeting, penetration, and therapeutic effects of CAR-M on atherosclerotic plaques.

[0045] Example 1: Preparation of CAR-M This embodiment uses chimeric antigen receptor-modified macrophages targeting OPN (osteopontin) as an example to illustrate the preparation process of CAR-M. This technical solution involves modifying the cell surface with magnetic microfilaments. The modified cells are not limited to CAR-M cells or macrophages, but can also include other cell types. In this solution, DBCO-PEG4-NHS ester (introducing an alkyne group) is modified on the cell surface, i.e., a click chemical reaction site is introduced to connect with the magnetic microfilaments. The NHS ester (N-hydroxysuccinimide ester) in DBCO-PEG4-NHS ester is an active group that couples with the cell surface. Under mild conditions, it can undergo a specific amidation reaction with the primary amino groups (-NH2) on cell surface proteins and glycoproteins to form stable amide bonds, achieving covalent anchoring of the reagent on the cell surface. Since cell surface proteins and glycoproteins exist on any cell surface, this method can achieve covalent connection between magnetic microfilaments and various cell types.

[0046] First, a chimeric antigen receptor plasmid targeting OPN was constructed and packaged into a lentivirus.

[0047] The chimeric antigen receptor of this scheme comprises, sequentially, CD8α, an anti-OPN single-chain variable region, a CD8 hinge region, a CD28 transmembrane region, and a CD3ζ cytoplasmic domain, wherein the amino acid sequences of CD8α and the single-chain variable region of OPN are shown in SEQ ID NO. 1. MASPLTRFLSLNLLLLGESIILGSGEAYIQMTQSPASLSVSVGETVTITCRASENIYSFLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQFSLKINSLQSEDFGTYYCQHFWGTPFTFGSGTKLEIKR GGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKISCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSQSNNYTTYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQMGDYWGQGTTLTVSS (SEQ ID NO.1).

[0048] The single-stranded variable regions of CD8α and OPN, with nucleotide sequences shown in SEQ ID NO.2: ATGGCCTCACCGTTGACCCGCTTTCTGTCGCTGAACCTGCTGCTGCTGGGTGAGTCGATTATCCTGGGGAGTGGAGAAGCTTACATCCAGATGACTCAGAGCCCCGCCAGCCTGTCCGTGAGCGTGGGCGAGACTGTGACCATCACATGCCGGGCCAGCGAAAACATTTACTCATTTCTGGCCTGGTACCAGCAGAAACAGGGAAAGTCCCCACAGCTGCTGGTGTACGCCGCCACCAACCTGGCCGACGGCGTGCCTTCTAGATTCTCTGGCTCTGGCTCCGGAACCCAGTTCAGCCTCAAGATTAACTCCCTGCAAAGCGAGGACTTTGGCACATACTATTGTCAGCACTTCTGGGGCACCCCCTTTACTTTCGGCAGCGGCACCAAGCTGGAAATTAAAAGGGGCGGCGGAGGCTCTGGCGGCGGAGGCTCCGGGGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTGGTGCAGCCTAAAGGCTCCCTGAAGATCAGCTGTGCTGCCAGCGGGTTTACCTTTAACATCTACGCTATGAACTGGGTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTGGCCCGCATCCGCTCCCAGTCTAATAACTACACAACCTACTACGCCGATTCTGTGAAGGACAGGTTTACTATCAGCAGAGATGACTCCCAGTCTATGCTGTACCTGCAGATGAATAACCTGAAGACAGAGGACACAGCCATGTACTACTGTGTGAGACAGATGGGAGACTACTGGGGCCAGGGAACCACCCTCACTGTGAGCTCC (SEQ ID NO.2).

[0049] The transmembrane domain (hinge region + transmembrane region) includes CD8 and CD28, and the amino acid sequence is shown in SEQ ID NO.3: TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLS (SEQ ID NO.3).

[0050] The transmembrane domains include CD8 and CD28, and their nucleotide sequences are shown in SEQ ID NO.4: ACTACTACCAAGCCAGTGCTGCGAACTCCCTCACCTGTGCACCCTACCGGGACATCTCAGCCCCAGAGACCAGAAGATTGTCGGCCCCGTGGCTCAGTGAAGGGGACCGGATTGGACTTCGCCTGTGATATTTACATCTGGGCACCCTTGGCCGGAATCTGCGTGGCCCTTCTGCTGTCCTTGATCATCACTCTCATCTGC (SEQ ID NO. 4).

[0051] The intracellular domain includes CD3ζ, and its amino acid sequence is shown in SEQ ID NO.5: RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR (SEQ ID NO. 5).

[0052] The intracellular domain includes CD3ζ, and its nucleotide sequence is shown in SEQ ID NO. 6: AGAGCAAAATTCAGCAGGAGTGCAGAGACTGCTGCCAACCTGCAGGACCCCAACCAGCTCTACAATGAGCTCAATCTAGGGCGAAGAGAGGAATATGACGTCTTGGAGAAGAAGCGGGCTCGGGATCCAGAGATGGGAGGCAAACAGCAGAGGAGGAGGAACCCCCAGGAA GGCGTATACAATGCACTGCAGAAAGACAAGATGGCAGAAGCCTACAGTGAGATCGGCACAAAAGGCGAGAGGCGGAGAGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGCACTGCCACCAAGGACACCTATGATGCCCTGCATATGCAGACCCTGGCCCCTCGC (SEQ ID NO.6).

[0053] The complete coding gene for the chimeric antigen receptor is shown in SEQ ID NO.7:

[0054] The gene sequence fragment shown in SEQ ID NO.7 was integrated into the multiple cloning site of the universal lentiviral expression vector GV492 (purchased from Shanghai Jikai Gene Biotechnology Co., Ltd.) using conventional methods, resulting in the plasmid GV492-CAR-OPN expressing a CAR targeting the OPN target. The plasmid map is shown below. Figure 2 As shown, the vector GV492 is a lentiviral gene overexpression vector based on HIV1, which can be used for CAR sequence expression. The CAR sequence is obtained by sequentially linking CD8α, anti-OPN-scFv (anti-OPN single-stranded variable region), CD8 hinge region, CD28 transmembrane region, and CD3ζ sequence fragment (i.e., the sequence shown in SEQ ID NO.7). The vector also carries selection sites such as gcGFP and puromycin.

[0055] Next, lentivirus packaging was performed. 48 hours before transfection, HEK293T cells in logarithmic growth phase were digested with trypsin and adjusted to approximately 5 × 10⁶ cells / day in DMEM medium containing 10% serum. 6 Cells were re-seeded in 10 cm cell culture dishes and cultured at 37°C in a 5% CO2 incubator. The prepared DNA solutions were added to a sterile centrifuge tube, including 20 μg of GV492-CAR-OPN plasmid, 15 μg of pHelper 1.0 vector plasmid, and 10 μg of pHelper 2.0 vector plasmid. Both pHelper 1.0 and pHelper 2.0 vector plasmids were purchased from the lentiviral packaging kit of GKG Gene. Following the instructions, the mixed plasmids were thoroughly mixed with the corresponding volume of GKG transfection reagent, adjusting the total volume to 1 mL. The mixture was incubated at room temperature for 15 min. The transfection mixture was slowly added dropwise to the culture medium of HEK293T cells, mixed well, and cultured at 37°C in a 5% CO2 incubator. After 6-8 h of culture, the culture medium containing the transfection mixture was discarded, and the cells were washed once with 10 mL of PBS. The culture dish was gently shaken to wash away any remaining transfection mixture before being discarded. Slowly add 12 mL of cell culture medium containing 2% serum and incubate at 37°C in a 5% CO2 incubator for 48 h.

[0056] Then, lentivirus concentration and purification were performed. HEK293T cell supernatant was collected 48 h post-transfection (0 h is considered the start time after transfection) based on cell state. The supernatant was centrifuged at 4000 g for 10 min at 4 °C to remove cell debris and impurities. The supernatant was filtered through a 0.45 μm filter into 40 mL ultracentrifuge tubes. Samples were balanced, and each ultracentrifuge tube containing the viral supernatant was placed into a Beckman ultracentrifuge. Centrifugation was performed at 25000 rpm for 2 h at 4 °C. After centrifugation, the supernatant was discarded, and as much liquid as possible remained on the tube walls was removed. A volume of standard preservative solution corresponding to the viral volume was added, and the precipitate was resuspended by gentle pipetting. After thorough dissolution, the precipitate was centrifuged at 10000 rpm for 5 min. The supernatant was then aliquoted as required to obtain the concentrated viral solution (lentivirus), and the viral titer was determined.

[0057] After obtaining the viral concentrate, CAR-M was constructed. CAR plasmids and lentiviruses targeting the OPN target were constructed using the aforementioned method to prepare CAR-M. RAW264.7 macrophages were infected with the lentivirus. Viral concentrate was added to RAW264.7 macrophages (purchased from Guangdong Huiyu Technology Co., Ltd.) at an MOI of 50, and the cells were incubated at 37°C in a 5% CO2 incubator. After 24 hours, the medium was replaced with fresh DMEM high-glucose medium (without virus), and the cells were amplified. Infection status was observed after 96 hours. Figure 3 To use flow cytometry to sort successfully infected positive CAR-M cells.

[0058] Next, flow cytometry analysis was performed to detect the phagocytic capacity of CAR-M cells. CAR-M cells (green fluorescently labeled) were prepared and sorted according to the above steps, or unmodified macrophages (macrophages transfected with an empty vector) were used. These were then co-cultured with foam cells (red fluorescently labeled DILs) for 24 hours (effect cell to target ratio 2:1), and phagocytic efficiency was detected by flow cytometry. HUVEC cells (human umbilical vein endothelial cells, which do not produce OPN) were used as a control. The FITC channel detected GFP fluorescence in CAR-M cells or macrophages, and the PE channel detected red fluorescence of DILs in foam cells or HUVEC cells. "Double-positive cells" expressing both green and red fluorescent signals represent effector cells that phagocytosed target cells; the proportion of this cell population represents the phagocytic efficiency. Figure 4 The flow cytometry results for CAR-M-targeted phagocytosis show that the CAR-M phagocytic efficiency reached 8.37%, significantly higher than the control group. Macrophages themselves have low phagocytic efficiency against foam cells, and both CAR-M and macrophages have low phagocytic efficiency because they do not target HUVEC cells. The CAR modification using this method significantly enhances the targeted phagocytic ability of macrophages against foam cells, and this phagocytic activity is highly specific.

[0059] Example 2: Preparation of Robotic CAR-M 10.8 mg FeCl3·6H2O and 15.2 mg FeCl2·4H2O were dissolved in 5 mL of distilled water. After heating to 50 °C, 1 mL of 0.5 M NaOH solution was slowly added dropwise to the solution, resulting in a black solution with a pH of approximately 11. The solution was sonicated (20 kHz, 0.35 W) for 60 min and then centrifuged three times at 10000 rpm for 15 min. The solution was then resuspended in 1 mL of water, and 10 μL of 5 M HCl was added. After sonication (20 kHz, 0.35 W) for 60 min, a positively charged Fe3O4 nanoparticle solution was obtained. Take a small piece of PLGA (500mg, polylactic acid-glycolic acid copolymer; Qiyue Biotechnology, CAS No.: 34346-01-5), cut it into small pieces, and put it into a 1.5mL EP tube containing 1mL distilled water and magnetic beads (TDK, catalog No. 907743). Grind it for 10min using a cryogenic grinder (-20℃; 10s grinding per cycle, 20s interval; 20 cycles). Then centrifuge at 6000rpm for 1min and discard the supernatant, followed by centrifugation at 1000rpm for 10s and discard the precipitate to obtain a microfilament suspension. Mix all of the obtained microfilament suspension with all of the obtained Fe3O4 nanosolution and incubate overnight (approximately 8h) on a shaker at 50rpm. Then perform magnetic separation 2-3 times to obtain a magnetic microfilament suspension.

[0060] Place the centrifuge tube containing the mixture (microfilament suspension and Fe3O4 nanoparticle solution) on a magnetic separator, ensuring the magnet is firmly attached to the outer wall of the tube. Let it stand at room temperature for 5-10 minutes. Under the influence of the magnetic field, the microfilaments bound to the Fe3O4 nanoparticles are adsorbed to the side of the centrifuge tube closest to the magnet, forming a distinct black aggregate. Discard the supernatant, add an appropriate amount of PBS buffer to the precipitate, and gently pipette or vortex to redisperse the magnetic microfilaments evenly, forming a suspension. Place the resuspended suspension back on the magnetic separator and repeat the above steps until the supernatant becomes clear. Finally, a pure magnetic microfilament suspension is obtained, which can be used for subsequent experiments.

[0061] Cellular robots were synthesized using click chemistry. On one hand, 5 mg of magnetic microfilaments were added to 10 ml of 0.5 mg / mL polydopamine (C8H2O). 11In the NO2)4) solution, add 220 μl of 10 mg / mL Tris solution to adjust the pH of the polydopamine solution to approximately 8.5. Incubate at 50 rpm for 2 h, then perform conventional magnetic separation 2-3 times to obtain polydopamine-treated magnetic microfilaments. Finally, resuspend the polydopamine-treated magnetic microfilaments in 1 mL of PBS to obtain a polydopamine-treated microfilament suspension. Add N3-PEG4-NHS ester solution (40 mM solute, dissolved in DMSO) to all the polydopamine-treated microfilament suspension obtained in the previous steps at a volume ratio of 1:1000, and incubate at 50 rpm for 40 min. Then wash 2-3 times with PBS to obtain azide-modified magnetic microfilaments. N3-PEG4-NHS ester is azide-modified polyethylene glycol succinimide ester (CAS No.: 944251-24-5), composed of: an azide group (N3) located at one end of the molecule, capable of click chemistry; a polyethylene glycol (PEG4) chain serving as a flexible linker, enhancing water solubility and biocompatibility; and an NHS ester group at the other end, an active group that can react with amino groups to form stable amide bonds. It is important to note that after obtaining the azide-modified magnetic microfilaments, continuous sonication (20 kHz, 0.35 W) is necessary until the next reaction (click chemistry to connect the magnetic microfilaments and cells) can proceed. Failure to continuously sonicate will lead to microfilament aggregation, resulting in the failure of cell robot preparation. Extensive research has shown that continuous sonication yields the best results, superior to magnetic stirring, mechanical vibration, and 50 rpm shaking. The ultrasonic treatment method described in this scheme can achieve a long-term stable monodisperse state of microfilaments without damaging the integrity of the microfilament structure or the living cells subsequently connected to it.

[0062] On the other hand, at a volume ratio of 1:1000, 1×10 5 DBCO-PEG4-NHS ester solution (40 mM solute, dissolved in DMSO) was added to the CAR-M cell culture dish; 1 μL of DBCO-PEG4-NHS ester solution was added to 1 mL of solution containing 1×10⁻⁶ ppm of HCl. 5The cells were cultured in DMEM medium. Specifically, DBCO-PEG4-NHS ester refers to dibenzocyclooctylene-tetraethylene glycol-succinimide ester (CAS No.: 1427004-19-0), composed of: a DBCO group (dibenzocyclooctylene): located at one end of the molecule, capable of copper-free click chemistry; a tetraethylene glycol (PEG4) chain: serving as a flexible linker, enhancing water solubility and biocompatibility; and an NHS ester group (N-hydroxysuccinimide ester): an active group at the other end, capable of reacting specifically with amino groups. Cells were placed in an incubator, and after 40 minutes, the culture medium was aspirated. The cells were washed 2-3 times with PBS to obtain CAR-M cells modified with alkyne groups, and fresh culture medium was added to the cells. All the aforementioned magnetic microfilaments modified with azide groups were added to a culture dish and mixed with all the aforementioned CAR-M cells modified with alkyne groups. That is, using equal concentrations of N3-PEG4-NHS ester and DBCO-PEG4-NHS ester to treat magnetic microfilaments (initial dose 5 mg) and CAR-M cells (initial dose 1 × 10⁻⁶ mg), respectively. 5 (Each CAR-M cell) was modified, and then all the modified magnetic microfilaments were mixed with CAR-M cells. The culture dish was then placed in a shaker at 50 rpm for 40 min. The azide-modified magnetic microfilaments and alkynyl-modified CAR-M cells were covalently linked via a click reaction. Magnetic separation was performed 1-2 times to obtain Robotic CAR-M cells. The morphology of the Robotic CAR-M cells was observed using a laser confocal microscope (LSM900, Zeiss, Germany). Figure 5 A schematic diagram and confocal fluorescence imaging were created for the Robotic CAR-M, demonstrating its successful construction. Cells with varying numbers of microfilaments exhibit different degrees of magnetic response; cells with multiple connected microfilaments show stronger magnetism. By conventionally controlling the conditions for magnetic separation, [further details can be obtained]. Figure 5 The cell shown is connected by a single microfilament.

[0063] Example 3: Experimental results of in vitro simulation of the enhanced targeting and penetration effects of robotic CAR-M (1) Targeting capability of Robotic CAR-M in fluid A 5% (w / v) GelMA solution was prepared using intact macrophage culture medium as the solvent. The intact macrophage culture medium was DMEM (Qiyun Biotechnology, ZQ-120); the GelMA was methacrylated gelatin (EFL, EFL-GM-30). 1×10⁻⁶ GelMA solution was added to each mL of GelMA solution. 6Foam cells and 1 μL of 20 ng / μL LPS (Sigma-Aldrich; L2880-25MG) were added, along with the photoinitiator Irgacure 2959 at a final concentration of 0.25% (w / v). After these steps, a hydrogel solution was obtained. The hydrogel was infused into the shorter side of a "T"-shaped microfluidic chip up to the intersection. Crosslinking was performed under UV light (405 nm, 3 cm distance) to obtain a Gel MA hydrogel. Robotic CAR-M (1×10⁻⁶) was continuously infused into the longer channel at a rate of 1 cm / s. 4 / mL (prepared using DMEM medium), a magnet with a magnetic force of 20 mT was placed on the other side of the GelMA hydrogel. After a period of time (30 min), the magnet was removed, and the aggregation of Robotic CAR-M on one side of the hydrogel was observed and photographed using a Zeiss LSM800 confocal microscope.

[0064] See the schematic diagram of the testing equipment. Figure 6 (Hydrogel-filled channel 1, cell flow channel 2, magnet 3, cell flow direction 4, T-channel 5). T-channel 5 includes hydrogel-filled channel 1 and cell flow channel 2. Hydrogel containing foam cells fills hydrogel-filled channel 1, and Robotic CAR-M cells flow along cell flow direction 4 in cell flow channel 2. Under the influence of magnet 3, Robotic CAR-M cells aggregate and penetrate to one side of hydrogel-filled channel 1.

[0065] (2) Penetration capability of Robotic CAR-M in fluid The experimental setup and methods were the same as in (1). After a period of time (2h), the magnet was removed, and the Robotic CAR-M or magnetic microfilaments in the hydrogel were observed and photographed using a Zeiss LSM800 confocal microscope. The penetration depth was quantified using ImageJ.

[0066] For detailed experimental results, please refer to Figure 7 and Figure 8 The experimental results are fluorescence imaging images of Robotic CAR-M targeting and penetrating plaques in fluid, indicating that Robotic CAR-M significantly enhances the targeting and penetration effects.

[0067] The above model simulates atherosclerosis in vitro. This embodiment 3's in vitro experimental design aligns with the pathological characteristics of atherosclerosis, and the experimental model simulates the pathological microenvironment of atherosclerosis in vitro. The in vitro model constructed in this embodiment highly replicates the key features of atherosclerotic plaques, providing pathologically relevant support for the functional validation of Robotic CAR-M. The foam cells added to the GelMA hydrogel in the model are hallmark cells of atherosclerotic plaque formation. After mononuclear cells infiltrate the vascular endothelium, they differentiate into macrophages. Macrophages, after engulfing excess lipids, transform into foam cells, and the accumulation of foam cells is a core step in plaque formation. A fluid microfluidic system simulates the intravascular blood flow environment. The experiment uses a "T"-shaped microfluidic chip to perfuse Robotic CAR-M at a rate of 1 cm / s, simulating the hemodynamic environment within the artery. Blood flow disturbances exist in the vessels at the atherosclerotic lesion site; this model can verify the targeting ability of Robotic CAR-M under blood flow impact. In addition, LPS simulates the local inflammatory microenvironment of the plaque. The lipopolysaccharide (LPS) added to the experimental model is a typical inflammatory inducing factor. Atherosclerosis is essentially a chronic inflammatory disease with a large number of inflammatory factors present in the plaque. The addition of LPS can simulate the inflammatory background at the plaque site and verify the targeting specificity of RoboticCAR-M in the inflammatory microenvironment.

[0068] against Figure 7 The experiment included a "Without Magnet" group and a "Mag" group (guided by a 20 mT magnet), simulating both a fluid environment (continuous perfusion of Robotic CAR-M) and a plaque model (GelMA hydrogel + foam cells). In the "Without Magnet" group (Control), at 0 min and 30 min, the green-labeled Robotic CAR-M were dispersed and did not aggregate towards the red foam cells (plaque area). This indicates that under fluid flushing and without an applied magnetic field, Robotic CAR-M has difficulty autonomously targeting the plaque area. In the "Mag" group (Magnetic Feld): at 30 min, the green-labeled Robotic CAR-M were significantly concentrated near the GelMA hydrogel area where the red foam cells were located. This demonstrates that under magnetic guidance, Robotic CAR-M can overcome fluid effects and target and aggregate at the plaque model.

[0069] For the non-magnetic groups (0 min, 30 min), the aggregation level of Robotic CAR-M was low and did not change significantly, indicating that Robotic CAR-M is difficult to target plaques in a fluid environment without magnet guidance.

[0070] In the group with magnets (30 min), the aggregation level of Robotic CAR-M was significantly higher than that in the group without magnets, with a statistically significant difference. This quantitatively validated the targeting ability of Robotic CAR-M under magnetic influence in fluid conditions.

[0071] This demonstrates that, in a fluid environment (simulating blood flow in vivo), under magnetic guidance, robotic CAR-M can achieve targeted aggregation of foam cells and hydrogel plaque models.

[0072] against Figure 8 The experiment included a Control group (non-magnetic guidance) and an MF group (20 mT magnet guidance), and simulated the in vivo fluid environment (1 cm / s perfusion of Robotic CAR-M) and the plaque model (GelMA hydrogel simulated plaque).

[0073] Figure 8 In Figure B, the green (Robotic CAR-M) and red (magnetic microfilaments within the plaque) signals are co-localized. Guided by a 20mT magnet, the Robotic CAR-M can overcome fluid erosion and penetrate into the internal region of the plaque simulated by GelMA.

[0074] For the Control group (without magnetic guidance), the penetration distance was almost zero. This indicates that without magnetic guidance, Robotic CAR-M is almost unable to penetrate the GelMA simulated plaque under fluid scouring. For the MF group, the penetration distance increased significantly, with a statistically significant difference. This indicates that magnetic guidance effectively enhances the penetration ability of Robotic CAR-M into plaques in a fluid environment.

[0075] Two experiments in this embodiment directly verified the targeting and enrichment capabilities and penetrating infiltration capabilities of Robotic CAR-M on atherosclerotic plaques. These two capabilities are key to overcoming current bottlenecks in atherosclerosis treatment. Through its targeting capability, it overcomes blood flow interference to achieve plaque-specific aggregation. In the experiment, the magnetic response of a magnet was used to guide Robotic CAR-M to aggregate towards the hydrogel side loaded with foam cells under fluid conditions, simulating the process by which Robotic CAR-M targets and anchors to atherosclerotic plaques in vivo. Combining the characteristics of CAR-M, its surface CAR (chimeric antigen receptor) can specifically recognize foam cell targets (OPNs), while magnetic responsiveness further enhances targeting efficiency, solving the problem that traditional drugs or cells are difficult to accumulate at plaque sites due to blood flow. Through its penetrating capability, it overcomes the physical barrier of plaques to achieve deep infiltration. Atherosclerotic plaques (especially late-stage plaques) have a dense fibrous cap and extracellular matrix barrier, making it difficult for traditional drugs or cells to penetrate the barrier and reach the core region of the plaque. In this embodiment, the penetration depth of Robotic CAR-M in the hydrogel was quantified by Image J, directly verifying its ability to overcome the matrix barrier; magnetic response drive provides the power for active penetration of Robotic CAR-M, which, combined with the migration characteristics of macrophages themselves, enables them to penetrate the plaque fibrous cap and go deep into the plaque to exert their effects (such as phagocytizing lipids, secreting anti-inflammatory factors, and killing abnormal foam cells).

[0076] Based on the in vitro experimental results of this embodiment, Robotic CAR-M shows promise as a novel strategy for the treatment of atherosclerosis, and its potential value lies in: (1) Precision targeted therapy: Through the magnetic response + CAR dual-targeting mechanism, the plaque-specific delivery of drugs or therapeutic factors is achieved, reducing the toxic side effects on normal tissues throughout the body.

[0077] (2) Plaque repair and reversal: By utilizing the natural function of macrophages and combining genetic engineering, RoboticCAR-M can play a role in phagocytizing lipids and inhibiting inflammatory responses within plaques, thereby stabilizing or even reversing plaques.

[0078] (3) Overcoming the limitations of clinical treatment: Current drug treatment for atherosclerosis has systemic side effects, and interventional treatment is prone to restenosis; the targeting and penetration of Robotic CAR-M provides a new technical approach to address these clinical pain points.

[0079] Comparative Example Robotic CAR-M was prepared according to the method in Example 2. However, after obtaining the magnetic microfilaments modified with azide groups, they were placed at 50 rpm for about 2 hours before being click-reacted with CAR-M cells modified with alkyne groups. After completing all the preparation steps corresponding to Example 2, the morphology of the Robotic CAR-M was observed under a microscope. Figure 9 It was found that the cells were not effectively connected to the magnetic microfilaments, and that the magnetic microfilaments aggregated.

[0080] Initially, the research did not anticipate that continuous, low-intensity sonication (20 kHz frequency, 0.35 W power) would become a crucial technical step in the successful fabrication of the cell robot. Conventionally, sonication is typically used only for the initial dispersion of materials. However, this study unexpectedly discovered that after the azide-modified magnetic microfilaments were prepared, continuous application of sonication at these specific parameters was necessary to effectively suppress microfilament aggregation, thereby ensuring efficient and uniform coupling with cells. This seemingly simple operation actually played a decisive role in maintaining system stability, ensuring reaction uniformity, and the functional integrity of the final product, representing a significant technological breakthrough for the project.

[0081] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a cell robot that enhances the targeting and solid penetration of cell therapy, characterized in that: The process includes coupling magnetic microfilaments to the cell surface; the magnetic microfilaments include microfilaments and magnetic particles attached to the microfilaments; the material of the microfilaments is polylactic acid-glycolic acid copolymer.

2. The method for constructing a cell robot that enhances targeting and solid penetration in cell therapy according to claim 1, characterized in that: The magnetic microwires are prepared by the following method: an aqueous solution containing ferrous ions and ferric ions is prepared, heated and then an alkaline solution is added dropwise; ultrasonic treatment and cleaning are performed, followed by the addition of an acid solution; after ultrasonic treatment again, a suspension containing Fe3O4 nanoparticles is obtained. Polylactic acid-glycolic acid copolymer was shredded and dispersed in water, and magnetic beads were added; it was then ground using a cryogenic grinder and centrifuged to obtain a microfilament suspension; A suspension containing Fe3O4 nanoparticles was mixed with a microfilament suspension, and after incubation and magnetic separation, a magnetic microfilament suspension was obtained.

3. The method for constructing a cell robot that enhances cell therapy targeting and solid penetration according to claim 2, characterized in that: 10.8 mg FeCl3·6H2O and 15.2 mg FeCl2·4H2O were dissolved in 5 mL of distilled water. After heating to 50 °C, 1 mL of 0.5 M NaOH solution was slowly added dropwise. After sonication and washing, the solution was resuspended in 1 mL of water, 10 μL of 5 M HCl was added, and after sonication and centrifugation, a suspension containing Fe3O4 nanoparticles was obtained.

4. A method for constructing a cell robot that enhances cell therapy targeting and solid penetration according to claim 2, characterized in that: Take 500mg of PLGA, cut it into small pieces, add 1mL of distilled water and magnetic beads, grind it for 10min using a -20℃ refrigerated grinder, then centrifuge at 6000 rpm for 1min and discard the supernatant, then centrifuge at 1000 rpm for 10s and discard the precipitate to obtain a microfilament suspension.

5. The method for constructing a cell robot that enhances cell therapy targeting and solid penetration according to claim 2, characterized in that: The microfilament suspension was mixed with a suspension containing Fe3O4 nanoparticles and placed in a shaker at 50 rpm overnight. Then, magnetic microfilament suspension was obtained by magnetic separation.

6. The method for constructing a cell robot that enhances cell therapy targeting and solid penetration according to claim 1, characterized in that: Magnetic microfilaments form covalent connections with the cell surface through click chemical reactions. Preferably, magnetic microfilaments are modified with polydopamine and then linked with azide tetraethylene glycol succinimide ester to obtain magnetic microfilaments modified with azide groups. Cells modified with alkyne groups were obtained by modifying the cell surface with dibenzocyclooctyne-tetraethylene glycol-succinimide ester; Azide-modified magnetic microfilaments and alkynyl-modified CAR-M cells were covalently linked via a click reaction, followed by magnetic separation to obtain a cell robot.

7. A method for constructing a cell robot that enhances targeting and solid penetration in cell therapy according to claim 6, characterized in that: 5 mg of magnetic microfilaments were added to 10 mL of 0.5 mg / mL polydopamine solution, and the pH was adjusted to 8.5 using Tris solution. The mixture was then treated on a shaker at 50 rpm for 2 h. After magnetic separation, a polydopamine-treated microfilament suspension was obtained. Azide tetraethylene glycol succinimide ester was added to the polydopamine-treated microfilament suspension, and the mixture was treated on a shaker at 50 rpm for 40 min. After washing, azide-modified magnetic microfilaments were obtained. The azide-modified magnetic microfilaments were then sonicated until a click reaction was initiated. Add 1 mL containing 1×10 5 Dibenzocyclooctyne-tetraethylene glycol-succinimide ester was added to the DMEM medium for cells; after culturing the cells for 40 min, the medium was aspirated and the cells were washed to obtain CAR-M cells modified with alkyne groups. After mixing azide-modified magnetic microfilaments with alkynyl-modified CAR-M cells, the mixture was incubated at 50 rpm for 40 min and then magnetically separated to obtain Robotic CAR-M.

8. A method for constructing a cell robot that enhances targeting and solid penetration in cell therapy according to any one of claims 1-7, characterized in that: The cells used for coupling magnetic microfilaments are chimeric antigen receptor-modified macrophages.

9. A cell robot that enhances the targeting and solid penetration of cell therapy, characterized in that: It includes cells and magnetic microfilaments coupled to the cell surface; the magnetic microfilaments include microfilaments and magnetic particles attached to the microfilaments; the material of the microfilaments is polylactic acid-glycolic acid copolymer; Preferably, the magnetic particles are Fe3O4 nanoparticles; Preferably, the cells are chimeric antigen receptor-modified macrophages.

10. The application of the cell robot according to claim 9, which enhances cell therapy targeting and solid penetration, in the preparation of products for treating atherosclerosis.

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