Preparation and application of engineered cell membrane vesicles targeting tumor cells

Abstract

The invention discloses preparation and application of engineered cell membrane vesicles targeting tumor cells. According to the application, a drug-loading delivery system with a tumor targeting function is constructed through genetic engineering, CD6 molecules are highly expressed on the surface of membrane vesicles, and Il24 mRNA is encapsulated, so that an effective anti-tumor effect is achieved. The material design system is innovative, has very important significance for developing a novel tumor targeted drug delivery carrier, and provides important enlightenment for structural precision and functional diversification of the tumor targeted drug delivery carrier; meanwhile, the natural protection effect of cell membrane vesicles and a possible lysosome escape mechanism are combined, mRNA degradation is effectively avoided, and the intracellular delivery efficiency of mRNA is improved; through gene therapy and immunotherapy, the limitation of a single therapy is broken through, and a new design thought and theoretical basis are provided for tumor targeted therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the preparation and application of engineered cell membrane vesicles targeting tumor cells. Background Art

[0002] Tumor is an important disease affecting human health and has now become the second leading cause of death globally. Tumor treatment mainly includes traditional surgical treatment, radiotherapy, chemotherapy, and new treatment methods such as targeted therapy and immunotherapy that have developed rapidly in recent years. Targeted drugs have always been a hot topic in the research and development of anti-tumor drugs, and can generally be divided into two categories: monoclonal antibodies and small molecule compounds. In recent years, with the in-depth research, new targets have emerged continuously, and many breakthroughs have been made in the research and development of anti-tumor drugs. So far, the US Food and Drug Administration has approved tumor targeted therapy drugs for more than 30 targets, many of which have been approved for the treatment of multiple indications. The emergence of these drugs has brought new hope to tumor patients, but there are still problems such as large treatment doses and high dosing frequencies, which bring inconvenience to further transformation.

[0003] Existing drug delivery systems have technical bottlenecks in the comprehensive capabilities in five dimensions: biocompatibility, targeting specificity, treatment synergy, production and standardization, and the difficulty of drug loading capacity and release control. That is, improving biocompatibility often sacrifices the drug loading amount, enhancing targeting requires complex surface engineering, and multi-mechanism synergy leads to uncontrollable pharmacokinetic parameters.

[0004] Therefore, studying a drug delivery system that can target tumors is crucial for the effective treatment of tumors. Summary of the Invention

[0005] To make up for the deficiencies of the existing technology, the present invention provides the preparation and application of engineered cell membrane vesicles targeting tumor cells.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a genetically engineered membrane vesicle targeting tumors, which expresses a ligand of CD166 and encapsulates a drug.

[0008] Further, the ligand of CD166 is selected from CD6.

[0009] Further, the drug is selected from cytokines.

[0010] Further, the cytokine is selected from interleukins.

[0011] Further, the interleukin is selected from IL-24.

[0012] Furthermore, the administration form of the IL-24 is mRNA, namely Il24 mRNA.

[0013] Furthermore, the tumor is a tumor overexpressing CD166.

[0014] Furthermore, the tumor overexpressing CD166 is selected from colorectal cancer and oral squamous cell carcinoma.

[0015] Furthermore, the membrane vesicles are membrane vesicles of 3T3 cells.

[0016] The second aspect of the present invention provides a pharmaceutical composition for treating tumors, and the pharmaceutical composition comprises the genetically engineered membrane vesicles or CD6 and Il24 mRNA described in the first aspect of the present invention.

[0017] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0018] The third aspect of the present invention provides a preparation method of genetically engineered membrane vesicles targeting tumors, and the method comprises: constructing cell membrane vesicles overexpressing a ligand of CD166 and encapsulating a drug.

[0019] Furthermore, the step of constructing cell membrane vesicles overexpressing a ligand of CD166 comprises: constructing a cell line overexpressing a ligand of CD166 and separating the cells and the cell membrane vesicles.

[0020] Furthermore, a cell line overexpressing a ligand of CD166 is constructed by lentivirus infection.

[0021] Furthermore, the cells and the cell membrane vesicles are separated by chemical induction.

[0022] Furthermore, the reagent used in the chemical induction method is cytochalasin B.

[0023] Furthermore, the drug is encapsulated by a drug loading technology assisted by saponin.

[0024] Furthermore, the saponin is selected from digitonin.

[0025] Furthermore, the ligand of CD166 is selected from CD6.

[0026] Furthermore, the membrane vesicles are membrane vesicles of 3T3 cells.

[0027] Furthermore, the drug is selected from cytokines.

[0028] Furthermore, the cytokine is selected from interleukins.

[0029] Furthermore, the interleukin is selected from IL-24.

[0030] Furthermore, the administration form of the IL-24 is mRNA, that is, Il24 mRNA.

[0031] Furthermore, the mass ratio of Il24 mRNA to cell membrane vesicles is 2:5.

[0032] The fourth aspect of the present invention provides the use of the genetically engineered membrane vesicles or CD6 and Il24 mRNA described in the first aspect of the present invention in the preparation of a pharmaceutical composition for treating tumors.

[0033] Furthermore, the tumor is selected from tumors overexpressing CD166.

[0034] Furthermore, the tumors overexpressing CD166 are selected from colorectal cancer and oral squamous cell carcinoma.

[0035] Advantages and beneficial effects of the present invention:

[0036] In this application, a drug-loaded delivery system with tumor-targeting function is constructed by genetic engineering. By highly expressing CD6 molecules on the surface of membrane vesicles and encapsulating Il24 mRNA, an effective anti-tumor effect is achieved. The material design system of this application is innovative, which has great significance for the development of new tumor-targeted drug delivery carriers, and provides important inspiration for the precise structure and diverse functions. At the same time, it combines the natural protection of cell membrane vesicles and the possible lysosome escape mechanism, effectively avoiding mRNA degradation and improving the intracellular delivery efficiency of mRNA. Through gene therapy and immunotherapy, the limitations of single therapy are broken through, providing a new design idea and theoretical basis for tumor-targeted therapy. Description of the Drawings

[0037] Figure 1 It is the electron microscope image of CMVs (scale bar: 1 μm);

[0038] Figure 2 It is the preparation diagram of CD6-3T3 and CD6-CMVs. Among them, 2A is the statistical chart of the relative expression level of CD6, and 2B is the protein content diagram of CD6;

[0039] Figure 3 It is the loading diagram of CMVs on Il24 mRNA (confocal images of DiO and Cy5-labeled CMVs / Il24 mRNA (scale bar: 1μm), green represents the membrane of CMVs, and red represents Il24 mRNA);

[0040] Figure 4 It is the mRNA release curve diagram;

[0041] Figure 5It is the transport diagram of CMVs and CD6-CMVs to CT26 cells. Among them, 5A is the confocal photo of CT26 cells treated with DiO-labeled CMVs and CD6-CMVs (scale bar: 40μm). The blue represents the tumor cell nucleus, and the green represents the cell membrane vesicles with or without CD6 molecules. 5B is the comparison diagram of DiO fluorescence intensity under different treatments (****P<0.0001, n=3);

[0042] Figure 6 It is the expression diagram of Il24 mRNA in different treatment groups;

[0043] Figure 7 It is the comparison diagram of the killing ability of different treatment groups to CT26 cells (green represents live cells, and red represents dead cells);

[0044] Figure 8 It is the diagram of differentially expressed genes in the RNA sequencing analysis results. Among them, 8A is the volcano diagram of differentially expressed genes, and 8B is the heat map of differentially expressed genes;

[0045] Figure 9 It is the GSEA enrichment analysis diagram. Among them, 9A is the ES line chart of cytokine and chemokine-related gene sets, 9B is the ES line chart of immune response-related gene sets, and 9C is the ES line chart of DNA-related gene sets;

[0046] Figure 10 It is the research diagram of the targeting effect of CD6-CMVs. Confocal images of frozen sections (scale bar: 100μm) and local enlarged images (scale bar: 20μm) after tail vein injection of PBS, DiI / CMVs, and DiI / CD6-CMVs for 24h;

[0047] Figure 11 It is the in vivo distribution diagram of CMVs and CD6-CMVs;

[0048] Figure 12 It is the diagram of the effect of CD6-CMVs / Il24 mRNA on the growth of mouse colorectal cancer tumors. Among them, 12A is the change diagram of tumor volume over time after the first administration to mice (n=5), and 12B is the ordinary optical microscope images of tumor tissue sections under 20× (scale bar: 100μm) and 40× (scale bar: 50μm) after 2 weeks of administration;

[0049] Figure 13 It is the in vivo toxicity study of CMVs / Il24 mRNA and CD6-CMVs / Il24 mRNA (scale bar: 200μm). Specific implementation manners

[0050] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

[0051] The present invention provides a genetically engineered membrane vesicle targeting tumors, which expresses a ligand of CD166 and encapsulates a drug.

[0052] In some embodiments, the ligand of CD166 includes but is not limited to CD6 (T cell surface glycoprotein CD6), CD318 (CDCP1, CUB domain-containing protein 1), CD44.

[0053] In a specific embodiment, the ligand of CD166 is selected from CD6.

[0054] In some embodiments, the drug is a drug for treating tumors, including but not limited to chemotherapeutic drugs (such as alkylating agents, antimetabolites, antitumor antibiotics, platinum drugs), targeted therapy drugs (such as tyrosine kinase inhibitors, antibodies, PARP inhibitors, kinase inhibitors), immunotherapy drugs (such as immune checkpoint inhibitors, cytokines, CAR-T cells).

[0055] In a preferred embodiment, the drug is selected from cytokines, and the cytokines include interferons (IFN) such as IFN-α, IFN-β, IFN-γ; interleukins such as IL-24, IL-2, IL-7, IL-12, IL-15, IL-21; colony-stimulating factors (CSF) such as GM-CSF (granulocyte-macrophage colony-stimulating factor), G-CSF (granulocyte colony-stimulating factor); tumor necrosis factors (TNF) such as TNF-α, tumor necrosis factor-induced protein.

[0056] Among them, interferons (IFN) include but are not limited to IFN-α, IFN-β, IFN-γ); interleukins include but are not limited to IL-24, IL-2, IL-7, IL-12, IL-15, IL-21; colony-stimulating factors (CSF) include but are not limited to GM-CSF (granulocyte-macrophage colony-stimulating factor), G-CSF (granulocyte colony-stimulating factor); tumor necrosis factors (TNF) include but are not limited to TNF-α, tumor necrosis factor-induced protein.

[0057] In a more preferred embodiment, the cytokine is selected from interleukins.

[0058] In a more preferred embodiment, the interleukin is selected from IL-24.

[0059] In a specific embodiment, the administration form of the IL-24 is mRNA, that is, Il24 mRNA.

[0060] In some embodiments, the tumor is a tumor overexpressing CD166, including but not limited to colorectal cancer, oral squamous cell carcinoma, liver cancer, breast cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, nasopharyngeal cancer, bladder cancer, melanoma.

[0061] In a specific embodiment, the tumor overexpressing CD166 is selected from colorectal cancer and oral squamous cell carcinoma.

[0062] The present invention provides a pharmaceutical composition for treating tumors, which comprises the above-mentioned genetically engineered membrane vesicles or CD6 and Il24 mRNA.

[0063] In some embodiments, for the regimen of CD6 and Il24 mRNA, the pharmaceutical composition comprises a single compound preparation or a combination of two separate single-agent preparations. Specifically, the compound preparation is a compound preparation containing CD6 and Il24 mRNA, and the combination of single-agent preparations is a combination of a single-agent preparation containing CD6 and a single-agent preparation containing Il24 mRNA. In a specific embodiment, the administration mode of the two single-agent preparations in the combination of single-agent preparations is simultaneous administration.

[0064] In some embodiments, CD6 and Il24 mRNA in the pharmaceutical composition can be administered simultaneously, separately or sequentially. Among them, simultaneous means that the two drugs are administered synchronously. If not administered simultaneously, they are administered sequentially within a time range such that both can be therapeutically effective within the same time range. Therefore, sequential administration allows the administration of the other drug within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours or several hours after the administration of one drug, provided that the circulating half-life of the first-administered drug allows for the simultaneous presence of a therapeutically effective amount of both. The time delay between the administrations of the components will vary depending on the exact nature of the components, the interactions between them, and their respective half-lives. Different from simultaneous or sequential, separate means that the interval between the administration of one drug and the other drug is significant, that is, when the second drug is administered, the first-administered drug may no longer be present in the bloodstream in a therapeutically effective amount.

[0065] The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0066] In some embodiments, pharmaceutically acceptable excipients include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. These media and solvents are well known in the art for use with pharmaceutically active substances. Its use in therapeutic compositions is contemplated unless any conventional medium or agent is incompatible with the active ingredient so far. In addition, various adjuvants commonly used in the art may be included.

[0067] Examples of pharmaceutically acceptable excipients include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as TWEENS; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.

[0068] The choice of pharmaceutically acceptable excipients depends essentially on the mode of administration of the active substance.

[0069] The pharmaceutical compositions described in the present application are preferably provided in unit dosage forms. As used herein, a unit dosage form is a composition containing a certain amount of the drug, suitable for administration to an animal, preferably a mammalian subject, in a single dose according to good pharmaceutical practice. However, the preparation of a single dose or unit dosage form does not mean that the dosage form is administered once a day or once per treatment course. Some dosage forms contemplate administration once, twice, three times or more per day, and may be administered as an infusion, or as a continuous infusion, over a period of time (e.g., from about 30 minutes to about 2 - 6 hours), and may be administered more than once during a treatment course, although single administration is not specifically excluded. Those skilled in the art will recognize that the formulation does not specifically contemplate the entire treatment course, and these decisions are left to those skilled in the art of the treatment area rather than the formulation area.

[0070] The useful drugs described above can be in any of a variety of suitable forms for a variety of routes of administration, e.g., for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular or other parenteral routes of administration. Those skilled in the art should understand that oral and nasal compositions include compositions administered by inhalation and are prepared using available methods. Depending on the particular mode of administration required, a variety of pharmaceutically acceptable excipients well known in the art can be used. Pharmaceutically acceptable excipients include, for example, solid or liquid fillers, diluents, solubilizers, surfactants and encapsulating substances. Optionally, pharmaceutically active materials can be included which essentially do not affect the inhibitory activity of the drug. The amount of excipient used with the drug is sufficient to provide the actual amount of material per unit dose of the drug for administration.

[0071] The present invention provides a method for preparing a genetically engineered membrane vesicle targeting a tumor, the method comprising: constructing a cell membrane vesicle overexpressing a ligand of CD166 and encapsulating a drug.

[0072] The steps for constructing cell membrane vesicles overexpressing the ligand of CD166 include: constructing a cell line overexpressing the ligand of CD166, and separating the cells from the cell membrane vesicles.

[0073] In some embodiments, the method for constructing a cell line overexpressing the ligand of CD166 can be any known method in the art, including but not limited to lentiviral infection method, plasmid transfection method, cytoplasmic microinjection method, electroporation method, CRISPR knock-in method, recombinant lentiviral plasmid method, adenovirus vector method.

[0074] In a specific embodiment, a cell line overexpressing the ligand of CD166 is constructed by the lentiviral infection method.

[0075] In some embodiments, the method for separating cells from cell membrane vesicles can be any known method in the art, including but not limited to chemical induction method, density gradient centrifugation method, size exclusion chromatography method, immunocapture method, immunoprecipitation method, ultracentrifugation method, buoyancy-activated cell sorting (BACS™), SPMNP technology.

[0076] In a preferred embodiment, the chemical induction method is used to separate cells from cell membrane vesicles. The reagents used in the chemical induction method include but are not limited to cytochalasin B, jasplakinolide, dithiothreitol (DTT), paraformaldehyde (PFA), calcium ion (Ca²⁺), ionophore (such as A23187), hydrogen peroxide (H2O2), ascorbic acid (vitamin C), methyl-β-cyclodextrin (MβCD), tannic acid, trypsin / collagenase.

[0077] In a specific embodiment, cytochalasin B is used to separate cells from cell membrane vesicles.

[0078] In some embodiments, the method for encapsulating drugs can be any known method in the art, including but not limited to saponin-assisted drug loading technology, incubation method, electroporation method, extrusion method, sonication method, freeze-thaw cycle method, genetic engineering method, low permeability dialysis method.

[0079] In a preferred embodiment, saponin-assisted drug loading technology is used to encapsulate drugs.

[0080] In some embodiments, the saponins include but are not limited to digitonin, notoginsenoside R1 (NGR1), ginsenoside Rg1, Re, Rb1, Rd, CK, QB-80 saponin, escin, Ziziphus spina-christi saponin (SRF), dioscin, α-hederin, QS-21.

[0081] In a specific embodiment, the saponin is selected from digitonin.

[0082] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0083] Example 1 Preparation of CD6-CMVs / Il24 mRNA (CD6-enriched cell membrane vesicles carrying Il24 mRNA)

[0084] 1. Experimental materials

[0085] MC 3T3 cells, CD6-eGFP lentivirus, infection enhancer, αMEM medium, 0.25% trypsin solution, PBS buffer, cytochalasin B, dimethyl sulfoxide, digitonin.

[0086] 2. Experimental methods

[0087] First, confirm the optimal multiplicity of infection (MOI) of CD6-eGFP lentivirus for infecting 3T3 cells. Prepare a 2 mL suspension of 3T3 cells at a density of 4×10 4 cells / mL in complete medium. Take 100 μL and add it to a 96-well plate, with a total of 12 wells. Culture at 37 °C for 24 h until the cell confluence reaches 20-30%. Discard the medium, inoculate the virus according to MOI = 0 / 10 / 50 / 100, and add the infection enhancer respectively. After 16 h of infection, change the medium with complete medium. After 72 h of infection, observe using a fluorescence microscope. Select the infection conditions and MOI corresponding to the group with an infection efficiency of about 80% and good cell growth as the subsequent infection experimental conditions. After determining the lentivirus infection conditions, transfect 3T3 cells. After 48-72 h of lentivirus infection, continue to culture the cells in a medium containing 5 μg / mL puromycin. After screening for 48 h, obtain a stable strain of CD6-3T3 (3T3 cells enriched with CD6).

[0088] When the density of CD6-3T3 cells cultured in a 10 cm culture dish reaches 90%-100%, CMVs are prepared. Discard the culture medium, wash the cells 3 times with PBS buffer, add an appropriate amount of serum-free αMEM medium containing 10 μg / mL cytochalasin B, and incubate at 37 °C for 30 minutes. Wash the cells 3 times with PBS buffer to remove the residual drug. Add an appropriate amount of trypsin solution to cover the cell surface, incubate at 37 °C until the cells completely detach, and add an equal volume of complete medium to terminate digestion. Transfer the cell suspension to a 15 mL centrifuge tube, vortex for 30 s to separate CMVs from the cells, and then centrifuge (300 g, 6 min) to remove the cells. After centrifugation, transfer the supernatant to a 1.5 mL EP tube and continue centrifugation (5000 g, 30 min). Discard the supernatant and resuspend the precipitate with serum-free αMEM medium.

[0089] Using serum-free αMEM as the loading buffer, add 5 µg CMVs, 2 µg mRNA, and 0.1 µg digitonin to each 100 μL loading system. After mixing, oscillate in a metal bath at 37 °C and 300 rpm for 20 minutes, add 0.1 μg CaCl2, and continue to oscillate in the metal bath under the above conditions for 10 minutes. Centrifuge at 7500 rpm for 30 minutes to collect the precipitate, and resuspend with PBS buffer for the next experiment.

[0090] 3. Experimental Results

[0091] For CMVs observed by transmission electron microscopy, CMVs generally appear spherical or ellipsoidal and are composed of a complete membrane structure, as Figure 1 shown.

[0092] The results of qRT-PCR and Western Blot experiments show that 3T3 cells infected with CD6-eGFP lentivirus highly express CD6 mRNA and protein, as Figure 2 shown. CMVs were prepared using 3T3 cells with high CD6 expression, and the CD6 protein content was detected. The results of the Western Blot experiment show that CMVs prepared from 3T3 cells with high CD6 expression also have a high CD6 level.

[0093] Using digitonin to permeabilize DiO-labeled CMVs and loading Cy5-fluorescently labeled Il24 mRNA, it can be seen under a confocal microscope that vesicles with green fluorescent labels co-localize with mRNA with red fluorescent tags, as Figure 3 shown.

[0094] Subsequently, the release of CD6-CMVs / Il24 mRNA at 4 °C was studied. It was found that the mRNA content remained basically stable within 6 hours of vesicle loading, with no significant difference statistically; after 12 hours of loading, the mRNA content began to decrease to 65.9% of the initial level and was completely released from 24 hours to 48 hours, as Figure 4 shown.

[0095] Example 2 Verification of the in vitro and in vivo targeting and anti-tumor effects of CD6-CMVs / Il24 mRNA (CD6-enriched cell membrane vesicles loaded with Il24 mRNA)

[0096] 1. Experimental methods

[0097] To verify the difference in the targeting effects of CMVs and CD6-CMVs, CMVs and CD6-CMVs labeled with DiO fluorescence were co-cultured with CT26 cells for 6 h and then fixed, observed under a confocal microscope, and the fluorescence intensity was analyzed by Image J software.

[0098] To detect the expression of IL-24 after adding CD6-CMVs / Il24 mRNA, PBS, CMVs, CD6-CMVs, CMVs / Il24 mRNA, and CD6-CMVs / Il24 mRNA were co-cultured with CT26 cells for 48 h, then the samples were collected, total proteins were collected, and Western Blot experiments were performed to examine the protein content.

[0099] A subcutaneous tumor model was established in BALB / c mice using CT26 cells. First, CT26 cells were cultured in 1640 medium containing 10% FBS. When the cells grew to the logarithmic growth phase, the cells were collected to prepare a cell suspension, and the concentration was adjusted to 1×10 6 cells / 0.1 mL. Healthy 5-6-week-old BALB / c mice were selected, and the cell suspension was inoculated subcutaneously into the right axilla of the mice.

[0100] The in vivo distributions of CMVs and CD6-CMVs were studied by small animal in vivo imaging. After 2 h of tail vein injection of CMVs and CD6-CMVs labeled with DiI fluorescence into tumor model mice, the in vivo fluorescence distribution was observed using a small animal in vivo imager. The mice were sacrificed, and tumor tissues and visceral organs were taken, and the fluorescence distribution was observed using a small animal in vivo imager. The mice were sacrificed 24 h after injection to obtain tumors. The removed tissues were gently rinsed and quickly placed in a pre-cooled freezing rack containing OCT to ensure that the tissues were completely wrapped. The wrapped tissues were quickly placed in liquid nitrogen for freezing. The frozen tissues were sectioned using a cryostat, the cell nuclei were labeled with DAPI, and the fluorescence intensity was observed under a confocal microscope.

[0101] To study the tumor-suppressive effect of CD6-CMVs / Il24 mRNA, when the tumor cells were inoculated for 7 days and the subcutaneous tumor volume was approximately 100 mm 3 the mice were randomly divided into 3 groups. (a) Control group: injected with PBS only; (b) injected with CMVs / Il24 mRNA; (c) injected with CD6-CMVs / Il24 mRNA. The drugs were administered 3 times a week via the tail vein injection. Meanwhile, the body weight and tumor volume of the mice were measured 3 times a week, and the tumor volume was calculated according to the formula 0.52×length×width 2 . During the experiment, the health status of the mice was observed regularly to ensure no obvious discomfort until the tumor volume of the control group reached 1500 mm 3 and then the mice were sacrificed to collect tumor tissues and organs for detection.

[0102] The morphological characteristics of the tissues were studied and analyzed by sectioning and staining. First, the sections were placed in xylene and alcohol for dewaxing and gradient hydration treatment respectively. Next, the sections were placed in hematoxylin solution for staining for 5 min, rinsed with double-distilled water for 10 minutes, stained with eosin solution for 1 minute, and then rinsed with double-distilled water. Gradient dehydration was carried out with alcohol, placed in xylene, and sealed with neutral balsam. The tissue sections were observed and photographed under an optical microscope.

[0103] 2. Experimental results

[0104] CMVs labeled with DiO fluorescence and CD6-CMVs were co-cultured with CT26 cells for 6 hours and then fixed. The nuclei of CT26 cells were labeled with DAPI, and the entry of vesicles into cells was observed under a confocal microscope. It was found that after 6 h, more CD6-CMVs were transported into cells compared with CMVs, indicating that CMVs with the targeting molecule CD6 could be transported to tumor cells faster, as Figure 5 shown.

[0105] CD6-CMVs / Il24 mRNA was added to CT26 cells and cultured for 24 hours, then the cells were collected for Western Blot experiment to detect the expression of IL-24 protein in the cells. The results of the Western Blot experiment showed that the CD6-CMVs / Il24mRNA group highly expressed IL-24, and its expression level was higher than that of the CMVs / Il24 mRNA group without targeting molecules, while only the groups adding CMVs, CD6-CMVs and the blank control group had low expression of IL-24, as Figure 6 shown. These results indicated that CMVs could enter CT26 cells, and the loaded Il24 mRNA could be transported into cells and expressed normally. Meanwhile, the transfection ability of CMVs with the CD6 targeting molecule was stronger than that of CMVs without targeting molecules.

[0106] The anti-tumor effect of CD6-CMVs / Il24 mRNA in vitro was further investigated. First, the killing ability of CD6-CMVs / Il24 mRNA on CT26 cells was examined by a live-dead assay, as Figure 7 shown. The results showed that the number of dead cells in the treatment group with CD6-CMVs / Il24mRNA was significantly higher than that in the CD6-CMVs treatment group without Il24 mRNA, the CMVs group without targeting molecules, and the CMVs / Il24 mRNA group.

[0107] CT26 cells untreated and treated with CD6-CMVs / Il24 mRNA were further subjected to RNA sequencing to further explore the effect of CD6-CMVs / Il24 mRNA. Differentially expressed genes were screened with the absolute value of fold change (FC) greater than 1.5 and P<0.05, Figure 8 A volcano plot shows the gene distribution. Genes downregulated in the treatment group relative to the untreated group are on the left, and genes upregulated in the treatment group relative to the untreated group are on the right. 186 genes were upregulated and 175 genes were downregulated in the CD6-CMVs / Il24 mRNA group compared with the Blank group. Further clustering analysis of these differentially expressed genes was performed to draw a heat map, and the results are as Figure 8 shown in B.

[0108] Gene Set Enrichment Analysis (GSEA) was further performed. Enriched gene sets were screened with a false discovery rate (FDR) <0.25 and p<0.05. The results showed that multiple cytokine and related receptor-related pathway sets were significantly enriched, such as cytokine activity, cytokine receptor activity, cytokine binding, chemokine activity, etc. In addition, the TNF-related pathway was upregulated, as Figure 9 shown in A, further confirming the expression of Il24 mRNA and the immune regulation mediated by cytokines. GSEA analysis showed that immune response-related pathways were upregulated, including leukocyte, neutrophil, monocyte, macrophage chemotaxis, regulation of monocyte and myeloid dendritic cell differentiation, upregulation of cellular MHC class I molecules, as Figure 9 shown in B. At the same time, the phenomenon of inhibition of DNA-related pathways was observed. The downregulation of genes related to DNA replication, DNA double-strand depolymerization, and DNA unwinding suggested cell cycle arrest or proliferation inhibition, while the downregulation of genes related to DNA recombination and DNA repair suggested impaired DNA damage repair ability, making tumors more prone to DNA damage-related apoptosis and having the ability to increase sensitivity to PARP inhibitors or DNA-damaging drugs (such as cisplatin and paclitaxel), as Figure 9 shown in C.

[0109] Furthermore, mouse models of colorectal cancer and oral squamous cell carcinoma were further established to verify the in vivo targeting and anti-tumor effects of CD6-CMVs / Il24 mRNA. DiI-labeled CMVs and CD6-CMVs were injected via the tail vein, and mice injected with PBS were set as the control group. After 24 h of injection, tumor tissues were taken for frozen section to observe the transfection of DiI / CMVs and DiI / CD6-CMVs. Under a confocal microscope, a large area of sheet-like red fluorescence regions could be seen in the CD6-CMVs group, with scattered dot-like fluorescence appearing locally; the fluorescence area of CMVs was relatively small, with only a small part of the region showing sheet-like fluorescence distribution, and the rest showed scattered dot-like red fluorescence, as Figure 10 shown.

[0110] The targeting effect of CD6-CMVs was further verified in the mouse colorectal cancer model. The in vivo imaging results showed that 2 h after injection, the results showed that after injection of CMVs, the fluorescence signal was mainly enriched in the liver, and weak fluorescence signals were visible in the tumor, heart, lung, and spleen; while after injection of CD6-CMVs, the fluorescence signal in the liver region was significantly reduced, and at the same time, the fluorescence signal in the tumor region increased, as Figure 11 shown. The results indicated that the introduction of the targeting molecule CD6 could enhance the tumor targeting of CMVs, and CD6-CMVs could be effectively enriched in the tumor region.

[0111] To verify the in vivo anti-tumor effect of CD6-CMVs / Il24 mRNA, on the seventh day after subcutaneous injection of CT26 cells, when a tumor of approximately 100 mm 3 could be palpated, the mice were randomly divided into 3 groups, and CMVs / Il24 mRNA or CD6-CMVs / Il24 mRNA were injected via the tail vein respectively, and mice injected with PBS were set as the control group; injected 3 times a week, and the tumor volume was measured 3 times a week to observe the change of tumor volume. After 2 weeks of drug administration, the mice were sacrificed to take tissues. The results showed that both CMVs / Il24 mRNA and CD6-CMVs / Il24 mRNA had obvious inhibitory effects on tumor growth, and the inhibitory effect of CD6-CMVs / Il24 mRNA was stronger, showing a synergistic effect, as Figure 12As shown in Figure A. The results of H&E staining showed that in the control group, the tumor cells were arranged closely, with obvious nuclear atypia, increased mitotic figures, less stroma, and no obvious necrosis area or immune cell infiltration. In the CMVs / Il24 mRNA and CD6-CMVs / Il24 mRNA groups, the cell density decreased, and the cell spaces increased in some areas. The nuclear atypia was still obvious, but the nuclei in some areas were darker stained than those in the control group, which might indicate chromatin condensation, an early manifestation of apoptosis or necrosis. Some vacuolated cells were visible locally, suggesting cell damage or necrosis. In addition, the necrosis area in the CD6-CMVs / Il24 mRNA group increased significantly, and nuclear pyknosis, karyorrhexis, and karyolysis were observed in some areas, which are typical features of necrosis or apoptosis. At the same time, increased local inflammatory cell infiltration was visible, indicating enhanced immune response of the body, such as Figure 12 as shown in Figure B.

[0112] Finally, the in vivo toxicity of CMVs / Il24 mRNA and CD6-CMVs / Il24 mRNA was studied. Histological observations were made on the main organs, and the results showed that there were no obvious differences in the heart, liver, spleen, lung, and kidney tissues of different injection groups compared with the PBS group, as Figure 13 shown, indicating that CMVs / Il24 mRNA and CD6-CMVs / Il24 mRNA have no significant in vivo toxicity.

[0113] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A gene - engineered membrane vesicle targeting tumors, characterized in that, The genetically engineered membrane vesicles express ligands of CD166 and encapsulate drugs.

2. The genetically engineered membrane vesicle according to claim 1, wherein The ligands of CD166 are selected from CD6.

3. The genetically engineered membrane vesicle according to claim 1, wherein The drugs are selected from cytokines; Preferably, the cytokines are selected from interleukins; Preferably, the interleukins are selected from IL-24; Preferably, the administration form of IL-24 is mRNA.

4. The genetically engineered membrane vesicle according to claim 1, wherein The tumor is a tumor overexpressing CD166; Preferably, the tumors overexpressing CD166 are selected from colorectal cancer and oral squamous cell carcinoma; Preferably, the membrane vesicles are membrane vesicles of 3T3 cells.

5. A pharmaceutical composition for treating tumors, characterized in that, The pharmaceutical composition comprises the genetically engineered membrane vesicles according to any one of claims 1-4 or CD6 and Il24 mRNA; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

6. A method for preparing a tumor-targeting genetically engineered membrane vesicle, characterized in that, The method comprises: constructing cell membrane vesicles overexpressing ligands of CD166 and encapsulating drugs.

7. The method according to claim 6, wherein The step of constructing cell membrane vesicles overexpressing ligands of CD166 comprises: constructing a cell line overexpressing ligands of CD166 and separating the cells from the cell membrane vesicles; Preferably, the cell line overexpressing ligands of CD166 is constructed by lentiviral infection; Preferably, the cells are separated from the cell membrane vesicles by chemical induction; Preferably, the reagent used in the chemical induction method is cytochalasin B.

8. The method according to claim 6, wherein The drugs are encapsulated using a saponin-assisted drug loading technique; Preferably, the saponin is selected from digitonin.

9. The method according to claim 6, characterized in that, The ligands of CD166 are selected from CD6; Preferably, the membrane vesicles are membrane vesicles of 3T3 cells; Preferably, the drugs are selected from cytokines; Preferably, the cytokines are selected from interleukins; Preferably, the interleukins are selected from IL-24; Preferably, the administration form of IL-24 is mRNA; Preferably, the mass ratio of Il24 mRNA to cell membrane vesicles is 2:

5.

10. Use of the genetically engineered membrane vesicles according to any one of claims 1-4 or CD6 and Il24 mRNA in the preparation of a pharmaceutical composition for treating tumors; Preferably, the tumors are selected from tumors overexpressing CD166; Preferably, the tumors overexpressing CD166 are selected from colorectal cancer and oral squamous cell carcinoma.

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