A chemotactic extracellular vesicle and a preparation method and application thereof

Abstract

The application discloses chemotactic extracellular vesicles, and a preparation method and application thereof. The membrane of the chemotactic extracellular vesicles at least overexpresses one of the following chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 and CCR10. The chemotactic extracellular vesicles have the ability of directional migration to tumor tissues releasing chemokines in response to high release of chemokines by the tumor, thereby enhancing tumor enrichment of the encapsulated drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a chemotactic extracellular vesicle, its preparation method, and its application. Background Technology

[0002] Extracellular vesicles (EVs), naturally secreted by cells, are highly biocompatible natural delivery carriers with a diameter of less than 1000 nanometers. EVs with a diameter of approximately 200 nanometers, in particular, are considered more ideal drug carriers because they can more easily penetrate and passively accumulate in tumor tissue due to the larger gaps outside the tumor (high-permeability, long-retention effect). In the field of cancer treatment, EVs can ensure the stability of the encapsulated drug. However, due to insufficient targeting, EVs can non-specifically diffuse into multiple tissues, leading to drug dispersion. Therefore, currently used EVs for cancer treatment are primarily administered via intratumoral or intraperitoneal injection, and are not suitable for intravenous injection. Furthermore, the microenvironment and physical barriers of solid tumors limit the amount of EVs that can accumulate at the tumor site, significantly reducing the tumor-killing toxicity of the encapsulated drug. This necessitates increasing the injection dose and can easily cause drug toxicity in non-target organs. Summary of the Invention

[0003] To address the issue of weak targeting of natural extracellular vesicles to tumors or inflamed tissues, this invention proposes a chemotactic extracellular vesicle, its preparation method, and its applications. The chemokine receptors overexpressed on the membrane of this chemotactic extracellular vesicle respond to the high release of chemokines from tumors, endowing the extracellular vesicle with the ability to migrate directionally to tumor tissues releasing chemokines, thereby improving the enrichment of encapsulated drugs in tumor tissues.

[0004] The technical solution provided by this invention is as follows:

[0005] In a first aspect, the present invention provides a chemotactic extracellular vesicle whose membrane overexpresses at least one of the following chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR10.

[0006] In conjunction with the first aspect of the invention, in some embodiments, the chemotactic extracellular vesicles are derived from human immune cells and / or human tumor cells.

[0007] In conjunction with the first aspect of the invention, in some embodiments, the particle size of the chemotactic extracellular vesicle is ≤200 nm.

[0008] In conjunction with the first aspect of the invention, in some embodiments, chemotactic extracellular vesicles target tumors and / or inflammatory tissues; when tumors and / or inflammatory tissues highly express CXCL8, the surface of the chemotactic extracellular vesicles overexpresses CXCR1 and / or CXCR2 receptors; and / or, when tumors and / or inflammatory tissues highly express CXCL10 and / or CXCL11, the surface of the chemotactic extracellular vesicles overexpresses CXCR3 receptors; and / or, when tumors and / or inflammatory tissues highly express CXCL12, the surface of the chemotactic extracellular vesicles overexpresses CXCR4 receptors; and / or, when tumors and / or inflammatory tissues highly express CXCL13, the surface of the chemotactic extracellular vesicles overexpresses CXCR5 receptors; and / or, when tumors and / or inflammatory tissues highly express CCL5, the surface of the chemotactic extracellular vesicles overexpresses CCR1 receptors; and / or, when tumors and / or inflammatory tissues highly express CCL5, the surface of the chemotactic extracellular vesicles overexpresses CCR1 receptors; and / or, when tumors and / or inflammatory tissues highly express CXCL8, the surface of the chemotactic extracellular vesicles overexpresses CCR1 receptors; and / or, when tumors and / or inflammatory tissues highly express CXCL8, the surface of the chemotactic extracellular vesicles overexpresses CXCR1 receptors; and / or, when tumors and / or inflammatory tissues highly express CXCL8, the surface of the chemotactic extracellular vesicles overexpresses CXCR2 receptors; and / or, when tumors and / or inflammatory tissues highly express CXCL1, the surface of the chemotactic extracellular vesicles overexpresses CXCR3 receptors; and / or, when tumor In CCL2, CCR2 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, in tumor and / or inflammatory tissues, CCL11 is highly expressed, and CCR3 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, in tumor and / or inflammatory tissues, CCL22 is highly expressed, and CCR4 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, in tumor and / or inflammatory tissues, CCL3 is highly expressed, and CCR5 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, in tumor and / or inflammatory tissues, CCL20 is highly expressed, and CCR6 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, in tumor and / or inflammatory tissues, CCL19 is highly expressed, and CCR7 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, in tumor and / or inflammatory tissues, CCL27 and / or CCL28 are highly expressed, and CCR10 receptors are overexpressed on the surface of chemotactic extracellular vesicles.

[0009] In conjunction with the first aspect of the invention, in some embodiments, the membrane of the chemotactic extracellular vesicle at least overexpresses the CXCR3 receptor and / or the CCR10 receptor.

[0010] In conjunction with the first aspect of the invention, in some embodiments, overexpression means that the expression level of the chemokine receptor on a single chemokine extracellular vesicle is ≥1 times that of a normal EV.

[0011] In a second aspect, the present invention provides a drug that targets tumors and / or inflamed tissues, the drug using the aforementioned chemotactic extracellular vesicles as a carrier.

[0012] Thirdly, the present invention provides a recombinant vector, which is a eukaryotic expression vector or a viral vector, and the vector contains recombinant genes of at least one of the following chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR10.

[0013] Fourthly, the present invention provides a recombinant cell comprising the above-described recombinant vector.

[0014] Fifthly, the present invention provides a method for preparing the above-mentioned chemotactic extracellular vesicles, comprising the following steps:

[0015] The gene of the chemokine receptor was constructed into a plasmid vector to obtain the target plasmid;

[0016] The target plasmid is packaged with lentivirus to obtain a recombinant vector;

[0017] Cells overexpressing chemokine receptors were screened by infecting cells with recombinant vectors.

[0018] Cells overexpressing chemokine receptors are cultured, and extracellular vesicles overexpressing chemokine receptors are collected and purified to obtain chemotactic extracellular vesicles.

[0019] In conjunction with the fifth aspect of the present invention, in some embodiments, the culture medium used to culture cells overexpressing chemokine receptors is α-MEM medium containing IL-15.

[0020] In conjunction with the fifth aspect of the present invention, in some embodiments, collecting and purifying extracellular vesicles overexpressing chemokine receptors includes: replacing the culture medium with a medium containing exosome-free serum at least 24 hours before collection; performing differential centrifugation on the culture medium after culture; and collecting the extracellular vesicles overexpressing chemokine receptors. Preferably, the differential centrifugation sequentially includes: centrifugation at 300g for 10 min, centrifugation at 2000g for 10 min, centrifugation at 10000g for 30 min, and centrifugation at 3000g for 30 min using an ultrafiltration tube with a cutoff of 100 kDa.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects:

[0022] 1. The extracellular vesicles provided by this invention possess the ability to migrate directionally to tumor tissues in response to the high release of chemokines from tumors, effectively overcoming the weakness of natural extracellular vesicles in targeting. Utilizing these extracellular vesicles for targeted drug delivery to inflamed tissues and tumors can significantly improve drug bioavailability. Furthermore, they are compatible with the delivery of various drugs, including small molecules, nucleic acids, and proteins, demonstrating high potential for clinical application.

[0023] 2. The extracellular vesicle preparation method provided by the present invention has high yield, which is up to 55 times higher than the prior art, and the purity of the obtained extracellular vesicles is close to 100%. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 : Schematic diagram of the differential centrifugation separation method of the present invention.

[0026] Figure 2 Map of CXCR3-CCR10 co-expression plasmids.

[0027] Figure 3 : Separation results of EV; where, A: EV yield of different separation methods; B: EV purity analysis obtained by differential centrifugation, determined by nanoparticle tracking analysis and counting; C: EV appearance and diameter determination under transmission electron microscopy.

[0028] Figure 4 : Expression levels of chemokine receptors on the membrane of unmodified PBMC-NK-EV (measured by flow cytometry); where A: expression level of CCR10; B: expression level of CXCR3; C: expression level of CXCR4.

[0029] Figure 5 PBMC-NK-EV's ability to migrate in a directional manner in response to chemokines.

[0030] Figure 6 : Chemotactic trajectory diagram of chemokines in response to PBMC-NK-EV; the horizontal direction from left to right is the direction of the chemokine concentration gradient. Detailed Implementation

[0031] The present invention will be further explained below with reference to the embodiments and accompanying drawings. The embodiments and accompanying drawings are for illustrative purposes only and are not intended to limit the scope of the invention. It will be understood by those skilled in the art that certain well-known structures and their descriptions in the embodiments and accompanying drawings may be omitted.

[0032] The term "transfer plasmid" refers to a plasmid carrying a target gene. The target gene is the gene that this application intends to express in the host cell, such as a protein-coding gene. The main function of a transfer plasmid is to transfer this target gene into the host cell, enabling it to undergo transcription and translation processes within the host cell, thereby producing the corresponding protein product.

[0033] The term "packaging plasmid" refers to a viral vector system in which the packaging plasmid contains viral packaging signals and some essential viral protein genes. Its function is to help package the target gene (present in the transfer plasmid) into viral particles. For example, in lentiviral vector systems, the packaging plasmid can provide components such as the viral core proteins, allowing RNA containing the target gene to be packaged into the viral capsid, forming infectious viral particles that then infect host cells.

[0034] The term "enveloped plasmid" primarily refers to the envelope proteins of viral particles. Envelope proteins play a crucial role in viral entry into host cells; they bind to receptors on the surface of host cells, facilitating viral entry. For example, in some retroviral vectors, proteins expressed by envelope protein genes in the envelope plasmid can alter the virus's host range, enabling it to infect specific cell types.

[0035] Currently, the main source of EVs used in tumor therapy is mesenchymal stem cells (MSCs), which produce a high percentage of EVs. Clinical data show that these MSC-derived EVs are non-specifically enriched in many organs, thus exhibiting weak tumor enrichment. Existing technology reports that NK cell-derived EVs (hereinafter referred to as NK-EVs) possess certain tumor recognition capabilities. The inventors discovered that NK-EVs highly express chemokine receptors on their membranes, while tumor tissues release high levels of chemokines, suggesting that the reason NK-EVs possess certain tumor recognition capabilities is due to the high expression of chemokine receptors on their membranes. This invention experimentally overexpressed chemokine receptors on the membranes of human NK-EVs, finding that the tumor-tending ability of human NK-EVs was enhanced, demonstrating that overexpressing chemokine receptors on the EV membrane can enhance its tumor tropism. Therefore, this invention proposes that overexpressing one or more chemokine receptors on EVs from various immune cell sources can make them suitable for targeted delivery of tumor drugs. Table 1 shows different chemokine receptors and their corresponding chemokines. Those skilled in the art can adjust the expression of EVs to high levels of the corresponding chemokine receptors based on the chemokines released by different tumors.

[0036] Table 1. Different chemokine receptors and their corresponding chemokines

[0037]

[0038]

[0039] The chemokine extracellular vesicles provided by this invention have membranes that overexpress at least one of the following chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR10. For example, chemokine receptors such as CXCR3 and CCR10 can recognize and bind to tumor tissues containing highly expressed chemokines CXCL10 and CXCL11, as well as CCL27 and CCL28. Given the high expression of CXCL10 and CXCL11 in various tumor tissues, EVs overexpressing CXCR3 and CCR10 have broad application potential in the field of multi-cancer tumor drug delivery.

[0040] In some embodiments, when tumor and / or inflammatory tissue highly expresses CXCL8, the surface of chemokinetic extracellular vesicles overexpresses CXCR1 and / or CXCR2 receptors; and / or, when the tumor and / or inflammatory tissue highly expresses CXCL10 and / or CXCL11, the surface of chemokinetic extracellular vesicles overexpresses CXCR3 receptors; and / or, when the tumor and / or inflammatory tissue highly expresses CXCL12, the surface of chemokinetic extracellular vesicles overexpresses CXCR4 receptors; and / or, when the tumor and / or inflammatory tissue highly expresses CXCL13, the surface of chemokinetic extracellular vesicles overexpresses CXCR5 receptors; and / or, when the tumor and / or inflammatory tissue highly expresses CCL5, the surface of chemokinetic extracellular vesicles overexpresses CCR1 receptors; and / or, when the tumor and / or inflammatory tissue highly expresses CCL2, the surface of chemokinetic extracellular vesicles overexpresses CXCR1 and / or CXCR2 receptors. Overexpression of CCR2 receptor; and / or, when the tumor and / or inflammatory tissue highly expresses CCL11, overexpression of CCR3 receptor on the surface of chemokinetic extracellular vesicles; and / or, when the tumor and / or inflammatory tissue highly expresses CCL22, overexpression of CCR4 receptor on the surface of chemokinetic extracellular vesicles; and / or, when the tumor and / or inflammatory tissue highly expresses CCL3, overexpression of CCR5 receptor on the surface of chemokinetic extracellular vesicles; and / or, when the tumor and / or inflammatory tissue highly expresses CCL20, overexpression of CCR6 receptor on the surface of chemokinetic extracellular vesicles; and / or, when the tumor and / or inflammatory tissue highly expresses CCL19, overexpression of CCR7 receptor on the surface of chemokinetic extracellular vesicles; and / or, when the tumor and / or inflammatory tissue highly expresses CCL27 and / or CCL28, overexpression of CCR10 receptor on the surface of chemokinetic extracellular vesicles.

[0041] In some embodiments, the membrane of the chemotactic extracellular vesicle is at least overexpressing CXCR3 receptors and / or CCR10 receptors.

[0042] In some implementations, overexpression means that the expression level of the chemokine receptor on a single chemokine extracellular vesicle is ≥1 times that of the normal EV.

[0043] In some embodiments, chemotactic extracellular vesicles are derived from human immune cells and / or human tumor cells. Tumor cells exhibit homing behavior, and by collecting and modifying extracellular vesicles derived from tumor cell lines, it is hoped that the passive enrichment effect of extracellular vesicles on tumor cells can be improved. However, some studies have shown that tumor-derived chemotactic extracellular vesicles may still have pro-tumorigenic effects; therefore, tumor-derived chemotactic extracellular vesicles are not suitable as drug carriers. Chemotactic extracellular vesicles derived from human immune cells, after overexpressing chemokine receptors, can recognize and autonomously target tumor tissues that release chemokines, which may be suitable for tumor drug delivery in various cancers and has high application potential. In addition, chemotactic extracellular vesicles derived from human immune cells have low immunogenicity and are not easily recognized and cleared by the immune system in vivo, enabling more effective drug delivery to the target site and improving drug efficacy. The aforementioned human immune cells include NK cells and human T cells, preferably derived from T lymphocytes of peripheral blood mononuclear cells from healthy individuals. Chemokine receptors expressed on extracellular vesicle membranes can respond to in vivo and in vitro microenvironments containing chemokines, exhibiting innate chemotaxis. For example, CXCL10 and CXCL11, as chemokines of CXCR3, are highly expressed in breast cancer, prostate cancer, and ovarian cancer. Therefore, NK-EVs overexpressing CXCR3 show significantly improved chemotactic sensitivity and response rate in tumor or inflammatory tissues compared to unmodified NK-EVs, and the enrichment level of EVs is also effectively increased.

[0044] In some implementations, the extracellular vesicles have a particle size ≤200 nm. In the field of tumor therapy, extracellular vesicles serve as drug carriers, ensuring the effective stability of the encapsulated drug. Furthermore, due to their small size, these extracellular vesicles can penetrate gaps outside the tumor, enter and accumulate within the tumor tissue, thereby significantly enhancing the drug's tumor-killing efficacy.

[0045] The drug for targeting tumors and / or inflamed tissues provided by this invention uses the aforementioned chemotactic extracellular vesicles as carriers. Utilizing the chemotactic extracellular vesicles' attraction to tumors and / or inflamed tissues, the drug can be specifically enriched at the tumor and / or inflamed tissue sites, thereby effectively reducing toxicity to non-target organs. After intravenous injection, the drug actively accumulates at the tumor tissue, significantly enhancing the tumor-killing effect of the delivered drug. This drug successfully solves the problem that naturally delivered extracellular vesicle drugs suffer from insufficient tumor targeting and weak tumor-killing ability, leading to the need for increased drug dosages and consequently, excessive drug toxicity in non-target organs.

[0046] The recombinant vector provided by this invention is a eukaryotic expression vector or a viral vector, on which at least one gene of the following chemokine receptors is recombined: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR10. This recombinant vector can be introduced into source cells, causing the source cells to overexpress the corresponding chemokine receptors, thereby leading to overexpression of the chemokine receptors on the extracellular vesicles secreted by the source cells.

[0047] This invention relates to a genetically engineered recombinant cell in which a specific recombinant vector is embedded. In this way, the recombinant cell can overexpress genes contained in the recombinant vector within the cell. Specifically, these genes encode proteins that are chemokine receptors, which are highly expressed on the cell membrane. Therefore, the extracellular vesicles secreted by these recombinant cells also correspondingly overexpress these chemokine receptors on their surface.

[0048] The method for preparing the above-mentioned chemotactic extracellular vesicles provided by the present invention includes the following steps:

[0049] The gene of the chemokine receptor was constructed into a plasmid vector to obtain the target plasmid;

[0050] The target plasmid is packaged with lentivirus to obtain a recombinant vector;

[0051] Cells overexpressing chemokine receptors were screened by infecting cells with recombinant vectors.

[0052] Cells overexpressing chemokine receptors are cultured, and extracellular vesicles overexpressing chemokine receptors are collected and purified to obtain chemotactic extracellular vesicles.

[0053] This method utilizes modified source cells to produce natural chemotactic extracellular vesicles, involving fewer chemical modification steps and requiring no special culture components, thus ensuring the safety of the chemotactic extracellular vesicles and their good stability in vivo. The yield of chemotactic extracellular vesicles obtained in this invention is over 85%, with a purity approaching 100%.

[0054] In some implementations, the culture medium used to culture cells overexpressing chemokine receptors is α-MEM medium containing IL-15. IL-15 is an important cytokine that promotes cell proliferation and differentiation while enhancing cellular immune activity. Using α-MEM medium containing IL-15 not only provides the nutrients required for cell growth but also further optimizes the growth environment of cells overexpressing chemokine receptors through the action of IL-15, thereby increasing the yield and quality of extracellular vesicles.

[0055] In some embodiments, collecting and purifying extracellular vesicles overexpressing chemokine receptors includes: replacing the culture medium with one containing exosome-free serum at least 24 hours before collection; centrifuging the culture medium after culture; and collecting the extracellular vesicles overexpressing chemokine receptors. The centrifugation process includes: centrifugation at 300g for 10 min, centrifugation at 2000g for 10 min, centrifugation at 10000g for 30 min, and centrifugation at 3000g for 30 min using an ultrafiltration tube with a cutoff of 100 kDa. This centrifugation process is designed to progressively separate and purify different components in the sample, with each step having a specific purpose and function. The 300g centrifugation for 10 minutes removes large particulate impurities such as cell debris and unbroken cells. These large particles settle to the bottom at lower centrifugal forces, resulting in a clearer supernatant and providing a purer sample for further separation. The 2000g centrifugation for 10 minutes further removes smaller particles from the supernatant, such as mitochondria and incompletely broken cell debris. These particles are smaller than cell debris, but they can settle under centrifugation at 2000g, further purifying the supernatant. Centrifugation at 10000g for 30 minutes is used to separate even smaller particles in the supernatant, such as microparticles and protein complexes. Centrifugation at 10000g causes these lighter particles to settle to the bottom, resulting in a purer supernatant. This step is very effective in removing tiny particles and impurities from the sample, ensuring that the substances obtained in subsequent steps are purer. Centrifugation at 3000g for 30 minutes using an ultrafiltration tube with a cutoff of 100kDa is used to further concentrate and purify the target substance in the supernatant. The 100kDa cutoff of the ultrafiltration tube means that substances with a molecular weight greater than 100kDa will be retained in the ultrafiltration tube, while substances with a molecular weight less than 100kDa will pass through the filter membrane into the collection tube. Centrifugation at 3000g for 30 minutes concentrates the target substance in the ultrafiltration tube, increasing its concentration for easier subsequent analysis and application. The centrifugation steps described above are designed to progressively remove particles and impurities of different sizes from the sample, ultimately yielding the target substance in high purity. The centrifugal force and time for each step are carefully selected based on the physical properties of the target substance (such as sedimentation coefficient and molecular weight) to ensure optimal separation results.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Example 1

[0058] (1) Obtain EVs from peripheral blood mononuclear cells (PBMCs)

[0059] Cell acquisition followed the manufacturer's instructions for Ficoll Plaque gradient centrifugation buffer (17144002, Cytivia) to centrifuge and purify peripheral blood. For example, after 14 days of cell culture using the Daco NK cell expansion system (681353, Daco), CD3- cells were sorted using the EasySep Negative selection kit. Flow cytometry analysis showed that >85% of the cells contained CD3-CD56+ viable cells, ready for NK cell EV culture. Before collecting EVs, the cell culture medium was replaced with medium containing 10% exosome-free serum, and cultured at 37°C for 48 hours. After culture, the cells were centrifuged in a 5000g ultrafiltration tube (100kD, PALL) for 5 minutes, and 95% of the supernatant was concentrated to more than 50 times, yielding cells with a density higher than 1.0 × 10⁻⁶. 10 EVs were analyzed. EV components were identified by flow cytometry and transmission electron microscopy. Purification methods are described in [link to purification method]. Figure 1 .

[0060] This embodiment evaluated the responsiveness of extracellular vesicles derived from human peripheral blood NK cells (PBMC-NK-EV) and extracellular vesicles derived from NK-92 cells (NK-92EV) to several NK cell-sensitive chemokines. Figure 4 The results showed that the chemokine receptors CCR10, CXCR3, and CXCR4 in NK-92EV and PBMC-NK-EV were expressed at intermediate to high levels. Figure 4 A-4C), which also explains the varying degrees of chemotactic response of these extracellular vesicles to the chemokines CCL27, CCL5, and CXCL10. Figure 5 The above results show that PBMC-NK-EVs possess chemokine properties, responding to CCL27 and CXCL10. The applicant hypothesizes that the expression level of chemokine receptors on the NK-EV surface is correlated with its responsiveness to chemokines CCR10 and CXCR3; it is highly likely that high expression levels of chemokine receptors enhance the EV's sensitivity to chemokines, thereby increasing its chemokine response. The chemokine trajectory diagram of PBMC-NK-EVs in response to chemokines is shown below. Figure 6 This demonstrates the ability of PBMC-NK-EV to migrate directionally to regions with high chemokine concentrations.

[0061] This invention lists PBMC-NK-EV as a candidate source for subsequent modification of NK-EV.

[0062] (2) Obtaining EVs overexpressing chemokine receptors through genetic modification

[0063] (2.1) Construction of the target plasmid

[0064] The target plasmid was constructed using pCDH-CMV-MCS-EF1-Puro as the plasmid vector, and multi-fragment PCR amplification and splicing techniques were employed. The PCR reaction volume was 50 μL, with 0.5 μL each of template A and template B. After PCR amplification, all products were recovered by electrophoresis for subsequent seamless cloning experiments. Furthermore, the primers used for PCR amplification were sequenced to ensure fragment accuracy. A detailed construction diagram of the CXCR3-CCR10 co-expression plasmid can be found in [link to diagram]. Figure 2 .

[0065] (2.2) Constructing recombinant lentiviruses:

[0066] 2 μg of CXCR3-CCR10 co-expression plasmid was added to 100 μL of DH5-α competent cell suspension to prepare recombinant lentivirus. Subsequently, the yield and concentration of recombinant lentivirus were accurately determined using NanoDrop, and subsequent sequencing analysis was performed.

[0067] In containing 1×10 7 HEK 293T cells were cultured in T75 culture flasks with 1 μg of transfer plasmid (i.e., CXCR3-CCR10 co-expression plasmid), 0.5 μg of packaging plasmid, and 0.5 μg of envelope plasmid, and co-cultured with a liposome mixture at 37°C for 48 hours to concentrate lentivirus. Finally, the expression of the target gene was detected by flow cytometry, and the lentivirus titer was calculated using the formula: Lentiviral titer = Cell number × Cell positivity rate / Lentiviral volume.

[0068] Take 5.0 × 10 4 Mix with 8 μg / mL polybrene and lentiviral concentrate, dilute to 1 mL with culture medium, centrifuge at 550 g for 1 h at 37 °C. After centrifugation, discard 90% of the supernatant, resuspend the cell pellet in 10% supernatant, add 0.9 mL of fresh NK-92 medium, mix well, and incubate in an incubator; replace with fresh NK-92 medium after 24 h; determine the positive rate by flow cytometry after 72 h.

[0069] (3) Preparation of EVs with high expression of chemokine receptors

[0070] Flow cytometry was used to screen immune cells that overexpress chemokine receptors. For example, the NK cell line NK-92 was infected with recombinant lentivirus, and flow cytometry was used to harvest NK cells that overexpressed chemokine receptors.

[0071] NK cells overexpressing chemokine receptors were cultured in α-MEM medium containing IL-15 (20 ng / mL) prepared according to the American Tissue Culture Collection (ATCC) protocol. Twenty-four hours before collecting extracellular vesicles overexpressing chemokine receptors, the serum component in the α-MEM medium was replaced with exosome-free serum. After 48 hours of culture, differential centrifugation was performed to collect the extracellular vesicles overexpressing chemokine receptors, i.e., chemotactic extracellular vesicles. The differential centrifugation program was: 300 g, 10 min; 2000 g, 10 min; 10000 g, 30 min. The collected supernatant was obtained by centrifugation at 3000 g for 30 min using an ultrafiltration tube with a cutoff of 100 kDa.

[0072] To compare several traditional EV separation methods, the inventors tested various methods, including ultrafiltration, reagent extraction (SEC), and size exclusion chromatography, to separate EVs. Figure 3 A) Based on yield and purity, the differential centrifugation method described above was ultimately selected. For example... Figure 3 As shown in A, although all four methods above can be used to separate EVs, when the differential centrifugation separation method of the present invention is used, the yield and purity of EVs are both higher, and the recovery rate of EVs is significantly improved compared with traditional EV separation methods.

[0073] like Figure 3 As shown in Figure B, the components obtained by the gold standard separation method are basically similar to those obtained by the differential centrifugation method of this invention. The EVs (<200 nm) highly expressing chemokine receptors account for more than 80% of the total extracellular vesicles. The total yield of extracellular vesicles is increased from 1.1 × 10⁻⁶ in the prior art. 9 10 8 The number of cells per 100 mL increased to 5.5 × 10⁻⁶. 10 10 8 (100 mL per cell), equivalent to an average of 550 EV particles collected per NK-92 cell, which is higher than other separation methods. Figure 3 As shown in C, the isolated product was confirmed by transmission electron microscopy, and the diameter of the main components was within the range of extracellular vesicle diameter.

[0074] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A chemotactic extracellular vesicle, characterized in that: The membrane of the chemokine extracellular vesicles overexpresses at least one of the following chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR10.

2. The chemotactic extracellular vesicle according to claim 1, characterized in that: The chemotactic extracellular vesicles are derived from human immune cells and / or human tumor cells.

3. The chemotactic extracellular vesicle according to claim 1, characterized in that: The particle size of the chemotactic extracellular vesicles is ≤200nm.

4. The chemotactic extracellular vesicle according to claim 1, characterized in that: The chemotactic extracellular vesicles target tumor and / or inflamed tissues; When the tumor and / or inflammatory tissue highly expresses CXCL8, CXCR1 and / or CXCR2 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, When the tumor and / or inflammatory tissue highly expresses CXCL10 and / or CXCL11, CXCR3 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, When the tumor and / or inflammatory tissue highly expresses CXCL12, CXCR4 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, When the tumor and / or inflammatory tissue highly expresses CXCL13, CXCR5 receptors are overexpressed on the surface of chemotactic extracellular vesicles; and / or, When the tumor and / or inflammatory tissue highly expresses CCL5, the surface of chemotactic extracellular vesicles overexpresses CCR1 receptors; and / or, When the tumor and / or inflammatory tissue highly expresses CCL2, the surface of chemotactic extracellular vesicles overexpresses CCR2 receptors; and / or, When the tumor and / or inflammatory tissue highly expresses CCL11, the surface of chemotactic extracellular vesicles overexpresses CCR3 receptors; and / or, When the tumor and / or inflamed tissue highly expresses CCL22, the surface of chemotactic extracellular vesicles overexpresses CCR4 receptors; and / or, When the tumor and / or inflamed tissue highly expresses CCL3, the surface of chemotactic extracellular vesicles overexpresses CCR5 receptors; and / or, When the tumor and / or inflammatory tissue highly expresses CCL20, the surface of chemotactic extracellular vesicles overexpresses CCR6 receptors; and / or, When the tumor and / or inflammatory tissue highly expresses CCL19, the surface of chemotactic extracellular vesicles overexpresses CCR7 receptors; and / or, When the tumor and / or inflammatory tissue highly expresses CCL27 and / or CCL28, the CCR10 receptor is overexpressed on the surface of chemotactic extracellular vesicles.

5. A drug targeting tumors and / or inflamed tissues, characterized in that: The drug uses the chemotactic extracellular vesicles as described in any one of claims 1 to 4 as a carrier.

6. A recombinant vector, characterized in that: The vector is a eukaryotic expression vector or a viral vector, and the vector contains at least one of the following chemokine receptor genes: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR10.

7. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 6.

8. A method for preparing the chemotactic extracellular vesicles of claim 1, characterized in that, Includes the following steps: The gene of the chemokine receptor was constructed into a plasmid vector to obtain the target plasmid; The target plasmid is packaged with lentivirus to obtain a recombinant vector; Cells overexpressing chemokine receptors were screened by infecting cells with recombinant vectors. Cells overexpressing chemokine receptors are cultured, and extracellular vesicles overexpressing chemokine receptors are collected and purified to obtain chemotactic extracellular vesicles.

9. The method according to claim 8, characterized in that: The culture medium used to culture cells overexpressing chemokine receptors was α-MEM medium containing IL-15.

10. The method according to claim 8, characterized in that: The process of collecting and purifying extracellular vesicles overexpressing chemokine receptors includes: replacing the culture medium with a medium containing exosome-free serum at least 24 hours before collection; performing differential centrifugation on the culture medium after culture; and collecting the extracellular vesicles overexpressing chemokine receptors.

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