CN114908054B - Cell membrane vesicle and preparation method and application thereof - Google Patents

Abstract

The invention discloses a cell membrane vesicle and a preparation method and application thereof. The cell membrane vesicle of the invention can prepare the biological cell membrane surface over-expression IL-15/IL-15 Ralpha complex of the cell membrane vesicle. The invention establishes an IL-15/IL-15Rα stable cell line, prepares cell membrane vesicles which overexpress IL-15/IL-15Rα, is used for activating TRM cells of anti-tumor tissues, promoting the proliferation and survival of the TRM cells and enhancing the anti-tumor effect. In addition, the cell membrane vesicle can be used as a carrier for encapsulating immune checkpoint small molecule inhibitor PD-1/PD-L1inhibitor 1, blocking the inhibition effect of tumor cells on TRM cells, activating the TRM cells cooperatively, realizing the removal of solid tumor cells such as melanoma and the like and preventing tumor recurrence.

Description

Cell membrane vesicle and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a cell membrane vesicle and a preparation method and application thereof.

Background

In recent years, with the development of the disciplines of cell biology, molecular biology, tumor immunology, etc., tumor immunotherapy has become a revolutionary therapy for the fourth tumor therapy. Unlike traditional therapies, immunotherapy attacks and clears tumor cells by activating the human immune system itself. Tumor cells evade monitoring and clearance of the lymphatic system in the body through the modes of antigen loss, expression of immune checkpoint inhibitory ligands or utilization of inhibitory tumor microenvironment and the like, so that immune escape is realized. Tumor immunotherapy is the reactivation of the immune system by various means, which allows tumor cells to be recognized and cleared. Current tumor immunotherapy approaches include immune checkpoint inhibitors, small molecule immune drugs, and immune modulators.

Immunomodulators generally refer to a class of drugs that can modulate the immune system of the body for the treatment of disease, including immunostimulants, immunosuppressants, bi-directional immunomodulators, and the like. The immunomodulator comprises cytokines, chemokines, polysaccharide drugs and the like, has obvious regulation effect on the immune system, and comprises the functions of activating or inhibiting immune cells. In recent years, molecular biology and tumor immunology have been studied intensively, and the immune system has been known intensively, so that various immune mediators can be studied in various diseases such as tumors and autoimmune diseases. Immune checkpoint therapies targeting PD-1, PD-L1 and CTLA-4 have achieved tremendous success, providing a new approach to cancer treatment, inspiring tremendous motivation to find new potential immune targets. Although immune checkpoint therapy has achieved great success, it suffers from low clinical response rates and certain toxic and side effects. Thus, researchers are greatly developing new therapies for improving cancer treatment.

Because of its pleiotropic effects on immune cells and their activation in immunity, cytokines have become a research hotspot for cancer immunotherapy and are in important positions during cancer immunotherapy. Cytokines include a variety of immune or non-immune cell produced regulatory proteins that play a key role in regulating and modeling innate and adaptive immune responses. Cytokines are secreted and membrane-bound, acting locally in an autocrine, paracrine or remote manner. Membrane-bound cytokines act through cell-to-cell contact and communicate information with each other. The information transfer function of cytokines on target cells plays an important role in immune response, and the ability of cytokines to activate immune effector cells such as T cells and NK cells is critical for their immunotherapeutic potential. Currently, studies on cytokines including cytokine fusion receptor subunits, fc regions of cytokine fusion antibodies, or cytokine fusion tumor antigen specific antibodies, and the like, have been conducted in a deep effort. Compared with recombinant cytokines, genetically engineered cytokines have better activity, targeting and stability. Various cytokine-based combination therapeutic strategies, including cytokines or genetically engineered products with other anti-tumor drugs, such as chemotherapeutic drugs, other cytokines, agonists, tumor vaccines, tumor-specific antibodies, checkpoint blocking antibodies, adoptive cell therapies, and the like, can significantly improve anti-tumor effects.

Immune checkpoints are activity-regulating receptor molecules expressed on activated immune cells, particularly T cells, that prevent an excessively strong immune response from destroying normal cells themselves. Immune checkpoints are auxiliary molecules that can promote or inhibit T cell activation, maintaining the in vivo environment in a state of equilibrium. Immune checkpoints can be categorized into inhibitory immune checkpoints (co-inhibitory checkpoint) and stimulatory immune checkpoints (co-stimulatory checkpoint). Many inhibitory immune checkpoints currently under investigation include PD-1, CTLA-4, TIM-3, LAG-3, and the like. Inhibitory immune checkpoint receptors prevent autoimmune diseases caused by an excessively strong immune response that destroys its normal tissues. On the other hand, costimulatory immune checkpoint molecules such as CD137, OX40, CD27, CD28, etc. play a vital role in T cell activation, proliferation and memory T cell formation.

Activation of T cells is a tightly regulated process that first requires antigen presenting cells to recognize a first activation signal generated by binding to T cell antigen receptor (TCR) via major histocompatibility complex (major histocompatibility complex, MHC), while requiring the co-action of a second signal co-stimulatory factor and a third signal cytokine. Wherein the co-stimulatory factor as the second signal may provide a co-stimulatory signal not only by binding to a stimulatory immune checkpoint, but also by binding to an inhibitory immune checkpoint. Inhibitory immune checkpoints play an important role in autoimmune tolerance and tumor immune escape, and by "depleting" T cells, the immune system of the body is in an inhibited state and cannot be monitored and cleared. Among these immune checkpoints, PD-1, PD-L1 and CTLA-4 are the most studied. CTLA-4 is a homologous protein to CD28, both bind competitively to CD80 and CD86 proteins, whereas CTLA-4 has significantly higher affinity than CD28 protein. Regulatory T cells (regulatory T cell, tregs) use their surface CTLA-4 molecules to competitively bind with CD28 on the T cell surface to CD80 and CD86 on the DCs surface, thereby inhibiting T cell activation. PD-1 is highly expressed mainly in activated T cells and B cells, and is low-expressed in activated natural killer cells (natural killer cell, NK), monocytes and Dendritic Cells (DCs). PD-1 is combined with ligands PD-L1 and PD-L2 to phosphorylate intracellular end tyrosine residues to cause dephosphorylation of downstream Syk and PI3K protein kinase, so that ERK, AKT and other channels required by downstream promotion of T cell activation are inhibited, and finally T cell activation is inhibited, thereby exerting negative regulation and control effects. PD-L1 is the most important negative check point for regulating T cell activity, plays an immune tolerance role in peripheral tissues and tumor microenvironment, controls the response time and intensity of immune response, and reduces the damage of the immune response to healthy organs to the greatest extent.

Blocking the inhibitory immune checkpoints relieves the inhibition state of the tumor microenvironment on the T cells, so that antigen-specific T cells at the tumor infiltration position are activated again, proliferation of the T cells is promoted, and the tumor cells are continuously killed. Among them, the blocking therapeutic effect of PD-1/PD-L1 signal axis has strong response and superior durability, and is a research hot spot of tumor immunotherapy in recent years. Since 2010, the united states food and drug administration (Food and Drug Administration, FDA) approved a series of monoclonal drugs against immune checkpoints, opening a new era of tumor immunotherapy. Currently marketed PD-1 and PD-L1 monoclonal antibodies include nine types of monoclonal antibodies, such as Nivolumab, pembrolizumab, atezolizumab and Durvalumab, and are suitable for treating various cancers, such as metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, liver cancer and the like. In addition, combined immune checkpoint therapies are more effective than single therapies, such as nivolumab (PD-1 antibody) and Ipilimumab (CTLA-4) antibodies in combination for the treatment of advanced melanoma, advanced renal cell carcinoma and recurrent or advanced non-small cell lung carcinoma, and the like.

Small molecule compound drugs generally refer to chemically synthesized drugs having a molecular weight of less than 1000. Compared with protein medicines, the small molecules have the advantages of high efficiency and good tissue permeability, and can overcome the shortages of incapability of oral administration, difficult production, low response rate, difficult permeation and the like of monoclonal antibodies. The small molecular compound medicine can penetrate through cell membranes or other biological barriers, penetrate deeply into tumor microenvironment, and act on extracellular, cell membrane surfaces or intracellular specific targets. Meanwhile, the pharmacokinetics of the small molecule immunotherapy drug is superior to that of the macromolecular drug, including higher oral bioavailability and short blood half-life, so as to reduce biological side effects. With the development of tumor immunology, many cells, receptors and signaling pathways associated with activating or suppressing immune responses in the immune microenvironment are being extensively discovered and studied. Aiming at the potential anti-tumor targets, such as PD-1/PD-L1, toll-like receptor, indoleamine-2, 3-dioxygenase, chemokine receptor and interferon gene stimulating protein, relevant targeted small molecule drugs are continuously sent out at home and abroad. Among these potential targets, immune checkpoint receptors and ligand proteins, including PD-1, PD-L1, CTLA-4, and the like, are being studied in depth and have application prospects. Among them, a series of small molecule antagonists have been developed for the pair of signaling axes of PD-1 and PD-L1, such as BMS-1, BMS-142, BMS-200, BMS-202, BMS-242, BMS-1001, BMS-1166, and the like. Such small molecule drugs block tumor PD-L1-induced T cell failure by binding to PD-L1, blocking the binding of PD-L1 to PD-1.

In 1994, two groups have commonly found interleukin-15 (IL-15) cytokines that promote T cell growth. IL-15 belongs to the family of IL-2 cytokines sharing a receptor yc chain, has a 4 alpha helix bundle structure and has a molecular weight of 14-15kDa. IL-15 is a multifunctional cytokine that acts on cells of the innate and adaptive immune systems. IL-15 regulates the functions of immune cell activation, proliferation, anti-apoptosis, steady state and the like, and plays a key role in resisting exogenous pathogens, resisting tumors and the like. IL-15 is widely available and is produced mainly by DCs, macrophages and monocytes, and is also expressed in mast cells, B cells, endothelial cells, bone marrow and lymph node stromal cells, and tissue cells. Although IL-15 has three forms of soluble IL-15, membrane-bound IL-15/IL-15Rα and secreted IL-15/IL-15Rα, under normal conditions of the body, IL-15 expression levels are picogram levels. Its expression level is tightly regulated during transcription, mRNA cleavage, translation, and intracellular trafficking to maintain homeostasis. Two typical isoforms of IL-15 cytokines are formed by selective cleavage, differing in that the signal peptides are of different lengths, 48 amino acids (long Signal Peptide, LSP) and 21 amino acids (Short Signal Peptide, SSP), respectively, but the mature peptides encoded by the two are identical in length. The signal peptide plays an important role in intracellular transport of IL-15 protein, and LSP-IL-15 can be secreted to the outside of the cell through the secretory pathway of the endoplasmic reticulum after translation, or can be combined with IL-15/IL-15Rα form to play a role on the cell membrane. SSP-IL-15 is not secreted after translation and is located in the cytoplasm and nucleus. IFN-gamma, double-stranded RNA, lipopolysaccharide and the like bind to Toll-like receptors when the body is infected by bacteria and viruses, or activate CD40 receptors to promote the production of IL-15 by DCs and monocytes, thereby improving the host's ability to resist and eliminate the invasion of exogenous microorganisms.

Immune checkpoint inhibitor therapy (PD-1/PD-L1 antibody), adoptive cell therapy, immunomodulator therapy and the like have significant therapeutic effects in clinical applications. However, these therapies have limitations such as clinical response rates of immune checkpoint antibodies below 30%, few types of immunomodulators used clinically, and depending on high doses, side effects are not negligible. At the same time, low dose of IL-2 promotes proliferation of Tregs and maintains immunosuppressive effect of Tregs in peripheral tissues. In tumor microenvironment, tregs inhibit the immune killing function of effector T cells, reducing the anti-tumor effect. Studies show that IL-15 does not activate Tregs and has no obvious effect on the biological functions. IL-15, in contrast to IL-2, does not participate in cell death induced by T cell activation, in contrast, exhibits anti-apoptotic effects and stimulates CD8 ⁺ Memory T cell production, proliferation and activation. At the same time, IL-15 does not lead to capillary leak syndrome, and hypotension, tachycardia and peripheral oedema caused by third interstitial fluid. In view of the above properties, IL-15 may be superior to IL-2 in the treatment of cancer, particularly as a component of a vaccine requiring a long-term immune response. However, because of the short blood half-life of IL-15, high doses of IL-15 are required to achieve therapeutic effects, which in turn results in toxicity, limiting its clinical use.

Based on the limitations of current immunotherapy, development of novel immunotherapy is urgently required. The cell membrane vesicle is composed of lipid, protein and a small amount of saccharides, and is used as a carrier for in vivo delivery of various medicaments, the protein on the surface of the vesicle is favorable for improving the targeting property of the vesicle, has good biocompatibility and low immunogenicity, is favorable for reducing the clearance of the liver and the kidney to the medicaments, has long half-life period in blood, and can improve the bioavailability of the medicaments. Therefore, the search for the use of cell membrane vesicles as drug carriers is of great value.

Disclosure of Invention

The primary object of the present invention is to overcome the disadvantages and shortcomings of the prior art and to provide a cell membrane vesicle.

It is another object of the present invention to provide a method for producing the above-mentioned cell membrane vesicle.

It is a further object of the present invention to provide the use of the above-described cell membrane vesicles.

The aim of the invention is achieved by the following technical scheme: a cell membrane vesicle, which is prepared by preparing a biological cell membrane surface over-expression IL-15/IL-15 Ralpha complex.

The biological cell membrane is derived from a NIH 3T3 cell line.

The preparation method of the cell membrane vesicle comprises the following steps:

(1) Respectively carrying out slow virus packaging on IL-15 and IL-15 Ralpha genes to obtain IL-15 virus and IL-15 Ralpha virus which are respectively fused to express fluorescent protein, purifying, respectively infecting cells by using the two viruses, screening drug resistance, and respectively obtaining an IL-15 cell line expressing fluorescence and an IL-15 Ralpha cell line expressing fluorescence;

(2) Infecting an IL-15Rα cell line expressing fluorescence by adopting the IL-15 virus fusion expressing fluorescent protein in the step (1), and screening drug resistance to obtain a cell line expressing an IL-15/IL-15Rα complex;

(3) Culturing the cell line expressing the IL-15/IL-15Rα complex in the step (2), collecting cells, inhibiting proteolysis, homogenizing, centrifuging to obtain supernatant, centrifuging, collecting precipitate, and filtering to obtain cell membrane vesicles.

Preferably, the IL-15 gene lentivirus packaged original vector in the step (1) is pLV-Hygro, the IL-15 gene and the mcherry gene are inserted into the XbaI-BamHI site, the target plasmid pLV-IL15-mcherry-Hygro is obtained through recombination, and the auxiliary plasmid is pH1 and pH2; more preferably, the mass ratio of the plasmids of interest pLV-IL15-mcherry-Hygro, helper plasmids pH1 and pH2 is 4:3:1.

Preferably, the original vector packaged by the IL-15 alpha gene lentivirus in the step (1) is pLV-EGFP-N, the IL-15 Ralpha gene is inserted into the XhoI-NotI site, the target plasmid pLV-IL15RA-EGFP-Puro is obtained by recombination, and the auxiliary plasmids are pH1 and pH2; more preferably, the mass ratio of the objective plasmid pLV-IL15RA-EGFP-Puro to the helper plasmid is 4:3:1 at pH1 and pH 2.

Preferably, the cells used in the step (1) for the infection are mouse fibroblast line NIH 3T3 cells.

Preferably, the infected cells of step (1) employ a cell density of 50-60%.

Preferably, the infected cells of step (1) are added Polybrene at a final concentration of 8 μg/mL.

Preferably, the drug resistance screening of virus-infected cells corresponding to the IL-15 gene in step (1) employs hygromycin B.

Preferably, the drug resistance screening of the virus-infected cells corresponding to the IL-15. Alpha. Gene in the step (1) employs puromycin.

Preferably, the drug resistance screening of step (2) employs puromycin and hygromycin B.

Preferably, step (3) is specifically as follows: culturing a large amount of cell lines which stably overexpress the IL-15/IL-15Rα complex, collecting cells, centrifuging at 4 ℃ at 800-1000rpm, collecting cell pellets, washing the cells with PBS containing a protease inhibitor, resuspending the cells with PBS containing a protease inhibitor, placing on ice, homogenizing 200 times, collecting homogenate, centrifuging at 4 ℃ for 5min, collecting supernatant again, centrifuging at 14800g for 40min at 4 ℃, collecting pellets, resuspending the pellets with PBS containing a protease inhibitor, and sequentially passing through a 0.8 μm filter head and a 0.22 μm filter head to obtain cell membrane vesicles.

Preferably, the PBS is 1 XPBS.

Preferably, the number of times of passing the 0.8 μm filter head and the 0.22 μm filter head is 8.

The application of the cell membrane vesicle in preparing tumor immunotherapy medicaments.

Preferably, the tumor immunotherapeutic agent is obtained by loading an immunosuppressant with a cell membrane vesicle.

Preferably, the immunosuppressant is PD-1/PD-L1 inhibitor 1.

Compared with the prior art, the invention has the following beneficial effects:

the invention establishes an IL-15/IL-15 Ralpha stable cell line and preparesThe cell membrane vesicle expressing IL-15/IL-15Rα is used for activating TRM cells of tumor tissues, promoting the proliferation and survival of the TRM cells and enhancing the anti-tumor effect. Furthermore, CD8 ⁺ TRM cells express high levels of checkpoint proteins, and PD-1/PD-L1 small molecule inhibitors can prevent PD-1 and PD-L1 from being in CD8 ⁺ Interactions between T cells and tumor cells. Therefore, the cell membrane vesicle can be used as a carrier to encapsulate the PD-1/PD-L1 immune checkpoint small molecule inhibitor PD-1/PD-L1 inhibitor 1, block the inhibition effect of tumor cells on TRM cells, cooperatively activate the TRM cells, realize the removal of solid tumor cells such as melanoma and the like and prevent tumor recurrence.

Drawings

FIG. 1 is a confocal microscopy image of NIH 3T3 cells overexpressing IL-15Rα, IL-15/IL-15Rα complexes; wherein a is an IL-15 expressing NIH 3T3 cell; b is an NIH 3T3 cell expressing IL-15 ra; c is NIH 3T3 cells expressing the IL-15/IL-15Rα complex; scale = 10 μm.

FIG. 2 is a graph showing the results of Western Blot verification of the expression of NIH 3T3 cell proteins overexpressing IL-15Rα, IL-15/IL-15Rα complexes; wherein, #1: NIH 3T3 cell lysate, #2: NIH 3T3 cell line lysate expressing IL-15 ra, #3: IL-15 expressing NIH 3T3 cell line lysates, #4: NIH 3T3 cell line lysate expressing IL-15/IL-15Rα complex, β -actin as an internal reference protein.

FIG. 3 is a graph showing the particle size distribution of NIH 3T3 cell membrane vesicles over-expressing IL-15Rα, IL-15/IL-15Rα complexes after passing through a 0.8 μm filter head; wherein, #1: NIH 3T3 cell membrane vesicles (free NVs), #2: IL-15 ra-NIH 3T3 cell membrane vesicles (IL-15 ra NVs), #3: IL-15-NIH 3T3 cell membrane vesicles (IL-15 NVs), #4: IL-15/IL-15Rα -NIH 3T3 cell membrane vesicles (IL-15/IL-15 Rα NVs).

FIG. 4 is a representation of detection of NIH 3T3 cell membrane vesicles overexpressing IL-15Rα, IL-15/IL-15Rα complexes; wherein; a is the cell membrane vesicle particle size distribution; b is the average cell membrane vesicle size; c is the average potential of cell membrane vesicles; d is IL-15/IL-15RαNVs transmission electron microscopy, scale = 100nm.

FIG. 5 is a graph showing the results of detection of the expression of cell membrane vesicle proteins; wherein a is a coomassie brilliant blue staining chart of cell lysate and cell membrane vesicle lysate; b is a structural schematic diagram of IL-15/IL-15RαNVs; c is an IL-15RαNVs laser confocal microscopic image; d is an IL-15 NVs laser confocal microscopic image; e is an IL-15/IL-15 ra NVs laser confocal microscopy, scale = 1 μm.

FIG. 6 is a graph showing the results of detection of expression of IL-2/IL-15Rβ - γc receptors on the surface of CTLL-2 cells.

FIG. 7 is a graph showing the results of detection of binding of cell membrane vesicles to CTLL-2 cells; wherein a is the combination condition of cell membrane vesicles and CTLL-2 cells observed by a laser confocal microscope, and the scale is 10 mu m; b is the binding of IL-15/IL-15RαNVs to CTLL-2 cells observed by laser confocal microscopy, scale = 20 μm.

FIG. 8 is a graph showing the results of a cell membrane vesicle hemolysis assay; wherein a is a cell membrane vesicle hemolysis experiment; b is a statistical graph of the absorbance of a hemolysis experiment of cell membrane vesicles, n=3; DI water: double distilled water.

FIG. 9 is a graph showing the results of toxicity test of cell membrane vesicles in vitro; wherein a is an in vitro toxicity experiment of cell membrane vesicles with different concentrations on B16-Luciferase cells; b is an in vitro toxicity experiment of cell membrane vesicles with different concentrations on 4T1-Luciferase cells; #1: free NVs, #2: IL-15RαNVs, #3: IL-15NVs, #4: IL-15/IL-15 ra NVs, n=4.

FIG. 10 is a graph showing the results of hemodynamic test detection of cell membrane vesicles in mouse blood; wherein a is a standard curve for measuring the vesicle protein of a cell membrane by a BCA method; bCy5.5 labeled cell membrane vesicle standard curve; c kinetics of cy5.5 labeled cell membrane vesicles in mouse blood; #1: free NVs, #2: IL-15RαNVs, #3: IL-15NVs, #4: IL-15/IL-15 ra NVs, n=3.

FIG. 11 is a graph showing experimental detection results of the distribution of cell membrane vesicles in mouse organs; wherein a is the distribution of cell membrane vesicles in the main organs and tumors of mice; b is a main organ and tumor fluorescence intensity statistical graph; #1: free NVs, #2: IL-15RαNVs, #3: IL-15 NVs, #4: IL-15/IL-15 ra NVs, n=3.

Fig. 12 is a diagram of an anti-melanoma model animal experimental protocol.

FIG. 13 is a statistical plot of tumor volume growth in anti-melanoma model treatment mice; wherein a is a mouse tumor growth curve; b individual growth curves of mice, n=5; g1 is PBS treatment, G2 is free NVs treatment, G3 is IL-15RαNVs treatment, G4 is IL-15 NVs treatment, G5 is PD-1/PD-L1 inhibitor1 treatment, G6 is IL-15/IL-15RαNVs treatment, and G7 is IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor1 treatment.

FIG. 14 is a statistical plot of tumor weight and mouse survival for anti-melanoma model treatment experiments; wherein a is the tumor weight of the mouse, n=4; b change in survival rate of mice after treatment with different drugs, n=10; g1 is PBS treatment, G2 is free NVs treatment, G3 is IL-15RαNVs treatment, G4 is IL-15 NVs treatment, G5 is PD-1/PD-L1 inhibitor1 treatment, G6 is IL-15/IL-15RαNVs treatment, and G7 is IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor1 treatment.

FIG. 15 is a graph showing the results of flow cytometry and immunofluorescence detection of mouse tumor T cell content; wherein a is CD8 in tumor microenvironment analyzed by flow cytometry after each group of mice is finished treatment ⁺ T cell content; b is CD8 in tumor microenvironment of mice in each group ⁺ T cell number analysis, n=4; c is immunofluorescence technique for analyzing CD4 at tumor ⁺ And CD8 ⁺ T cell content, scale = 100 μm; g1 is PBS treatment, G2 is free NVs treatment, G3 is IL-15RαNVs treatment, G4 is IL-15 NVs treatment, G5 is PD-1/PD-L1 inhibitor 1 treatment, G6 is IL-15/IL-15RαNVs treatment, and G7 is IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor 1 treatment.

FIG. 16 is a flow cytometry and immunofluorescence detection of T in tumor microenvironment _RM Cell content detection result diagram; wherein a is T at the tumor microenvironment analyzed by flow cytometry _RM Cell content; b is T in tumor microenvironment of mice in each group _RM Cell number analysis, n=4; c is the T at the tumor part analyzed by immunofluorescence technology _RM Cell content, scale = 100 μm.

FIG. 17 is a CD8 of each group ⁺ T cell activation level detection results; wherein a is flow cytometry analysis CD8 ⁺ T cell activation level; b is CD8 activated in tumor microenvironment of mice in each group ⁺ T cell number analysis, n=4.

FIG. 18 is a graph showing the results of perforin content detection in tumor microenvironment; wherein a is flow cytometry analysis CD8 ⁺ T cells secrete perforin levels; b is CD8 in tumor microenvironment of mice in each group ⁺ T cell secretion punch prime analysis, n=4.

FIG. 19 is a graph of the staining of the organ HE of mice and the dynamic change of body weight during different drug treatments during tumor-bearing period of mice; wherein a is HE staining pattern, scale = 100 μm; b is a dynamic change graph of body weight, n=5; g1 is PBS treatment, G2 is free NVs treatment, G3 is IL-15RαNVs treatment, G4 is IL-15 NVs treatment, G5 is PD-1/PD-L1 inhibitor 1 treatment, G6 is IL-15/IL-15RαNVs treatment, and G7 is IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor 1 treatment.

FIG. 20 is a map of the pLV-EGFP-N vector.

FIG. 21 is a map of the pLV-hygro vector.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the examples below, all data results are in mean.+ -. Standard deviation (mean.+ -. SD), unless otherwise indicated. All experiments were performed in triplicate or more, including biological replicates. The data differences for each group were calculated using statistical soft Spss16.0, and the differences for more than two groups were compared by one-or two-factor analysis of variance ANOVA) and verified using Tukey. The survival curves were compared using Log-Rank (Mantel-Cox) analysis. * P <0.001 is the most significant difference, P <0.01 is the significant difference, and P <0.05 is the statistical difference.

EXAMPLE 1IL-15/IL-15Rα cell membrane vesicle preparation and characterization

1.1 construction and amplification of IL-15Rα and IL-15 lentiviral vectors

The plasmids of interest, pLV-IL15RA-EGFP-Puro (IL-15 Rα (SEQ ID NO. 1) was inserted into the original vector pLV-EGFP-N (FIG. 20), cloning site XhoI-NotI) and pLV-IL15-mcherry-Hygro (IL-15 (SEQ ID NO. 2) and mcherry (SEQ ID NO. 3) were inserted into the original vector pLV-Hygro (FIG. 21), cloning site XbaI-BamHI, helper plasmids pH1 and pH2 (both purchased from Beijing England Biotech Co., ltd.) were amplified as follows:

(1) the TransStbl3 competent cells (full gold) were thawed on ice, 500ng of the 4 plasmids were added under the condition of an ultra clean bench, and the mixture was gently flicked and allowed to stand on ice for 30min.

(2) Heat shock is carried out in a water bath at 42 ℃ for 50s, and ice bath is carried out for 3min.

(3) 200. Mu.L of LB medium was added to the super clean bench, and the mixture was shaken at 37℃and 220rpm for 1.5 hours to obtain a culture.

(4) mu.L of the culture was pipetted with 60. Mu.g/mL Amp plate medium and incubated in a bacterial incubator for 12h.

(5) Single colonies on the plate medium were picked up to LB shake tube containing 60. Mu.g/mL ampicillin, and shaken at 37℃and 220rpm for 12 hours to obtain a bacterial solution.

(6) 200 mu L of bacterial liquid is absorbed by each tube, sent to the field of the biotechnology of the department of Optimaceae the company limited performs base sequencing. The residual bacterial liquid is temporarily stored in a refrigerator at 4 ℃. Sequencing results are consistent with the IL-15Rα and IL-15 base sequences of the Gene database on NCBI, and the residual bacterial liquid is subjected to plasmid extraction by using a high-purity plasmid miniprep kit of the radix et rhizoma Tiandi, and the steps are carried out according to the specification. The extracted plasmid was assayed for concentration and purity, requiring OD ₂₆₀ /OD ₂₈₀ >1.8、OD ₂₆₀ /OD ₂₃₀ >2. After the detection, the plasmid was stored at-20 ℃.

1.2 packaging and purification of viruses

HEK 293T cells (Feng Hui organism, CRL-11268) were plated to 10cm dishes at 50% density. After overnight, 1mL of MEM basal medium without serum and antibiotics was added to each of the 4-tube 15mL centrifuge tubes in a biosafety cabinet. The two tubes were filled with 6. Mu.g of the plasmid of interest pLV-IL15RA-EGFP-Puro, 4.5. Mu.g of the helper plasmid pH1, 1.5. Mu.g of the plasmid of interest pLV-IL15-mcherry-Hygro, and 6. Mu.g of the helper plasmid pH1, 1.5. Mu.g of the plasmid of interest pLV-IL15-mcherry-Hygro, respectively. Another 2 tubes were each filled with 12. Mu.L Lipofectamine 2000 transfection reagent. After 5min of standing at room temperature, a tube of plasmid was mixed with a tube of Lipofectamine 2000, and the resulting plasmid and transfection reagent mixture was allowed to stand at room temperature for 30min. The HEK 293T cells plated in advance are taken out from a cell culture box, the cells are rinsed once by a basic culture medium MEM, then 4mL of MEM culture medium is added, the mixed solution of the plasmid and the transfection reagent is added dropwise, the mixed solution is put back into the culture box for culture for 8 hours, then 10% of south America foetus calf serum and 1% of penicillin/streptomycin DMEM culture medium are replaced for normal culture, cell supernatants are collected at 24 hours, 48 hours and 72 hours respectively, and fresh culture medium is added. The collected supernatant medium was centrifuged at 500g for 10min at 4℃and then the supernatant was filtered through a 0.45 μm filter in a biosafety cabinet, diluted to 1X with 5 Xlentiviral concentrate, gently swirled and refrigerated overnight at 4 ℃. The next day, the mixture was centrifuged at 3500g for 25min at 4 ℃. After removing the supernatant, the supernatant was centrifuged at 3500g for 5min at 4℃and the remaining liquid was removed by pipetting, and the pellet was resuspended in 1 XPBS, which had a volume of 1/100 of the original supernatant, and the IL-15 virus fusion-expressing the mCherry protein (hereinafter referred to as IL-15 virus) and the IL-15Rα virus fusion-expressing the EGFP protein (hereinafter referred to as IL-15Rα virus) were split into 6 portions and stored at-80 ℃.

1.3 construction of 3T3 cell lines stably overexpressing IL-15/IL-15Rα complexes

NIH 3T3 cells (Feng Hui organism, CL 0244) were plated in six well plates at 50% density. After overnight, cells were removed from the cell incubator, fresh medium was added in 500. Mu.L per well, polybrene (Biyun) was added at a final concentration of 8. Mu.g/mL, 20. Mu.L of IL-15Rα virus from step 1.2 was added to one well, and 20. Mu.L of IL-15 virus from step 1.2 was added to the other well. The cells were returned to the incubator and after 72 hours fluorescence was observed under a fluorescence microscope. Cells were digested with pancreatin and transferred to new six well plates, and NIH 3T3 cells expressing IL-15Rα and IL-15 alone were screened sequentially with puromycin and hygromycin B at a concentration gradient of 0-3 μg/mL. After 3-4 passages and screening, NIH 3T3 cell lines which independently and stably express IL-15Rα and IL-15 are screened, and a large number of NIH 3T3 cell lines are amplified and frozen. NIH 3T3 cell lines stably expressing IL-15 ra were plated in six well plates at 50% density. After overnight, 500. Mu.L of fresh medium containing Polybrene at a final concentration of 8. Mu.g/mL was changed, and one aliquot of IL-15 virus obtained in step 1.2 was added and incubated for 72 hours. After observing fluorescence, cells were passaged with pancreatin, and simultaneously screened with puromycin and hygromycin B (concentration gradient of 0-3 μg/mL), and after repeated passaging and screening, 3T3 cell lines stably overexpressing IL-15/IL-15 ra complex were selected, cells were expanded in large amounts and frozen.

1.4 laser confocal microscopy verification of IL-15/IL-15Rα Complex expression

The stable over-expressed IL-15/IL-15Rα complex 3T3 cells from step 1.3 were plated on confocal dishes at a density of about 60%. Overnight incubation, medium was aspirated, washed once with 1 XPBS, then fixed with 4% paraformaldehyde for 15min at room temperature, then washed once with 1 XPBS, 1mL of 1. Mu.g/mL DAPI dye was added, incubated at room temperature in the absence of light for 10min, and washed three times with 1 XPBS for 5min each. Finally, 20. Mu.L of anti-fluorescence quenching caplet was added and the resulting film was taken on a machine (FIG. 1).

1.5 Western Blot verification of IL-15/IL-15Rα Complex expression

Blank (NIH 3T3 cells), NIH 3T3 cells stably overexpressing IL-15Rα alone, IL-15 alone and IL-15/IL-15Rα complex stably overexpressing were plated in six well plates at densities up to 90% or more. After overnight, cells were collected on ice by cell scraping, centrifuged at 3500rpm for 10min at 4℃and then washed twice with 1 XPBS, resuspended in 150. Mu.L of RIPA lysate with protease inhibitors, disrupted by an ultrasonic cytobreaker, sonicated for 3s with 25% power on ice and then suspended for 3s, and the ultrasound repeated 4 times. The sonicated contents were centrifuged at 4℃and 12000g for 10min, and the supernatant was collected. The BCA protein concentration assay kit was then used to detect protein concentration in each supernatant set, and the procedure was followed according to the instructions. After the concentration was measured, 120. Mu.g of each group was taken, the volume was made up to 150. Mu.L with RIPA lysate, 37.5. Mu.L of 5 XSSPAGE protein loading buffer was added, and after mixing, heating was performed at 100℃for 10min, and then Western blot steps were performed as follows:

(1) SDS-PAGE gels were prepared at a concentration of 10% and run at a voltage of 70V at 20. Mu.L of sample per well.

(2) The PVDF membrane is activated by methanol in advance, and a transfer membrane sandwich is manufactured according to the blackboard-sponge-filter paper-glue-PVDF membrane-filter paper-sponge-whiteboard. The film transfer tank was placed in ice and 250mA was transferred constantly to the film for 2h.

(3) And 5% of skimmed milk powder is prepared, and the PVDF membrane is sealed at room temperature for 1h.

(4) Membrane cutting, incubating IL-15 antibody (abcam, ab 7213), IL-15Rα antibody (Santa Cruz Biotechnology, sc-374023) and β -actin antibody, respectively, at 4℃with a slow shaking table overnight.

(5) The membranes were washed three times with TBST on a shaker for 10min each. Secondary antibodies (Anti-mouse→ Thermo Fisher Scientific,31430; anti-Rabbit→ Thermo Fisher Scientific,31460; anti- β -actin→ Santa Cruz Biotechnology, sc-47778) were incubated at room temperature for 1h.

(6) The membranes were washed three times with TBST on a shaker for 10min each.

(7) BufferA and BufferB were set to 1: ECL luminescence solution (beijing enghen organism) was prepared in proportion of 1, and exposed to light.

Western blot results are shown in FIG. 2, in which IL-15 cell lines and IL-15/IL-15Rα cell lines significantly expressed IL-15 protein compared to the blank NIH 3T3 cells. Meanwhile, IL-15Rα cell lines and IL-15/IL-15Rα cell lines have distinct IL-15Rα protein bands. The above results indicate that the IL-15/IL-15Rα -NIH 3T3 stable cell line has been successfully constructed.

1.6 cell membrane vesicle fabrication and characterization detection

NIH 3T3 cells stably overexpressing IL-15Rα, stably overexpressing IL-15/IL-15Rα complex were cultured in large quantities in 15cm cell culture dishes, and then harvested by cell scraping, centrifugation at 800rpm at 4℃and cell pellet was collected. Cells were washed twice with 1 XPBS containing protease inhibitor cocktail (Sieimer's fly) and resuspended with 4mL of 1 XPBS containing protease inhibitor cocktail. After the glass homogenate was sterilized with alcohol and ultraviolet light, it was placed on ice, 4mL of the cell resuspension was added, and the mixture was homogenized manually 200 times. Collecting homogenate, centrifuging at 4deg.C for 5min at 1000g, collecting supernatant, and removing precipitate. The supernatant was centrifuged at 3000g for 5min at 4 ℃. The supernatant was again removed, precipitated and centrifuged at 14800g at 4℃for 40min. Finally removing the supernatant, taking the precipitate, re-suspending the precipitate by using 1 XPBS containing protease inhibitor mixture, and sequentially passing through a 0.8 μm filter head eight times and a 0.22 μm filter head eight times to obtain the cell membrane vesicle.

The particle size of the cell membrane vesicles, the particle size after passing through a 0.8 μm filter head eight times and the Zeta potential were measured with a nanoparticle size and Zeta analyzer (FIGS. 3 and 4). The average particle size of NIH 3T3 cell membrane vesicles of the over-expressed IL-15/IL-15Rα complex is 162.9nm, the zeta potential is-9.4 mV, the cell membrane vesicles can circulate in the body, and the therapeutic effect of the loaded drugs is improved.

After the copper net is clamped by tweezers, the dovetail clamp is added for fixation, so that the copper net is prevented from falling off. The forceps were fixed on ice with an adhesive tape, 10. Mu.L of a cell membrane vesicle solution was dropped, left stand for 5min, the liquid was sucked from the edge of the copper mesh with filter paper, the dropping was repeated six times, then 10. Mu.L of 3% uranium acetate was dropped for 5min, and the liquid was sucked from the edge with filter paper. Finally, airing the copper net at room temperature, observing the shape of the cell membrane vesicle by using a 120kV transmission electron microscope and photographing, and displaying the result that the cell membrane vesicle is of a double-layer membrane closed ring structure. In the experimental process, gun heads, filter papers and the like related to uranium acetate are treated according to safety standards.

1.7 determination of cell membrane vesicle concentration

And (3) measuring the concentration of the cell membrane vesicles obtained in the step (1.6) by using a BCA protein concentration measuring kit of Biyun days. 0.5mg/mL BSA protein standard was diluted in a gradient to give 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL standard solutions, and cell membrane vesicles were incubated with 1 XPBS according to 2:18, namely diluting by 10 times to obtain a sample to be detected. Taking 20 mu L of standard substance solution with each concentration and a sample to be detected, respectively mixing with 200 mu L of BCA working solution by vortex, then sucking 200 mu L of mixture to an ELISA plate, sleeving PE film gloves, and incubating for 30min in an incubator at 37 ℃. Standard and sample were read using a562 absorbance of the microplate reader. And (3) according to the detection result, a concentration-reading standard curve is manufactured by using the reading of the standard substance, a formula and an R square value are calculated, and the formula and the R square value are substituted into the reading of the sample to be detected, so that the diluted sample concentration is calculated.

1.8 Coomassie brilliant blue staining to verify cell membrane vesicle protein expression

5 Xloading buffer was added to each of the cell lysate and cell membrane vesicles, diluted to 1X, and boiled at 100℃for 10min, followed by electrophoresis with 20. Mu.L of 10% SDS-PAGE gel at 250V per well. After electrophoresis, adding coomassie brilliant blue dye solution, boiling for 1min, boiling for 15min by changing distilled water, and then photographing. The cell membrane vesicle sample had a reduced protein level compared to the cell lysate sample, but still contained protein, indicating that there was no significant loss of protein from the cell membrane surface during the cell membrane vesicle preparation (fig. 5 a).

1.9 laser confocal microscopy of cell membrane vesicles

Cell membrane vesicles made of NIH 3T3 cells which stably overexpress IL-15Rα, stably overexpress IL-15 and stably overexpress IL-15/IL-15Rα complex are dripped on a glass slide with 10 mu L, covered with a cover glass, and nail oil sealing sheets are added around, and the glass slide is aired at room temperature in a dark place. The fluorescence of the cell membrane vesicles was then observed under a confocal laser microscope and the results are shown in FIGS. 5c, d and e. IL-15Rα is a membrane protein, and shows obvious green fluorescence under the mirror, which indicates that IL-15Rα is still expressed on the surface of cell membrane vesicles. IL-15 protein alone is overexpressed in cells and is present in the cytoplasm in free form, so IL-15 protein is not present on the membrane. However, in the preparation of cell membrane vesicles, small amounts of IL-15 protein may be entrapped inside the cell membrane vesicles, and thus small amounts of red fluorescence may also be observed. Simultaneously, when IL-15Rα and IL-15 are overexpressed in cells, both are assembled into IL-15/IL-15Rα complex in the endoplasmic reticulum, and then expressed on the surface of cell membrane. Under confocal laser microscopy, green fluorescence and red fluorescence were clearly observed and co-localized together, indicating that the surface of the cell membrane vesicles contained the IL-15/IL-15 ra complex.

EXAMPLE 2 in vitro biological Activity of IL-15/IL-15Rα cell membrane vesicles

1.1 cell membrane vesicle-bound cell experiments

The IL-15/IL-15Rα compound on the cell surface acts on the IL-2/IL-15Rβ -yc receptor of the target cell mainly by a trans-presentation method, activates the internal passage of the receptor cell, and regulates the physiological activity. Thus, we examined whether IL-15/IL-15Rα cell membrane vesicles could bind to cells expressing the relevant receptor. We performed experiments with the mouse IL-2 dependent cytotoxic T cell line CTLL-2 as target cell. The cell membrane vesicles prepared in step 1.6 of example 1 were subjected to a 0.22 μm filter in a biosafety cabinet to remove the foreign bacteria. CTLL-2 cells (Feng Hui organisms, CL 0078) were resuspended in 1640 medium in a special dish for confocal microscopy, at a density of approximately 50%, and 80. Mu.g of cell membrane vesicles were added and mixed horizontally. Culturing for 6h in a cell culture box, taking out the culture dish, and adding Hoechst dye solution to dye the cell nuclei. Co-localization of cell membrane vesicles and cells was observed under a confocal laser microscope. Western blot was used to detect expression of IL-2/IL-15Rβ - γc receptor on CTLL-2 cell surface (detection procedure was the same as in Western blot detection in example 1). Cell membrane vesicles that do not overexpress IL-15/IL-15Rα are added to CTLL-2 cells as a blank.

The results show that compared to blank CTLL-2 cells (no IL-15/IL-15Rα overexpressing cell membrane vesicles added to CTLL-2 cells), the IL-15/IL-15Rα overexpressing cell membrane vesicles have a distinct IL-15/IL-15Rα protein band (FIG. 6), indicating that the cell membrane vesicles contain the IL-15/IL-15Rα complex and bind to target cells.

The results show that the green fluorescent EGFP protein expressed by the fusion of IL-15Rα and the red fluorescent mCherry protein expressed by the fusion of IL-15 are co-localized on the surface of CTLL-2 cell membrane, which indicates that the IL-15/IL-15Rα cell membrane vesicles can bind with receptors on the cell surface, and the biological activity of the IL-15/IL-15Rα over-expressed on the cell membrane vesicles is not obviously affected. Meanwhile, since the cells endocytose the nanoparticles, the blank cell membrane vesicles do not express the related ligand and cannot bind to CTLL-2 cells, so they are endocytosed into the cells (fig. 7). The above results indicate that the cell membrane vesicles contain IL-15/IL-15Rα complexes and are able to bind to target cells.

1.2 cell membrane vesicle hemolysis experiments

Materials entering the organism need to have good biocompatibility, avoid causing the change of physicochemical properties in the organism, and maintain the stability of the internal environment. Among them, blood compatibility is an important study of biocompatibility. The blood compatibility of the material mainly comprises a hemolysis experiment and a coagulation experiment. The hemolysis experiment aims at exploring whether the material damages erythrocyte membrane and increases the free hemoglobin content in plasma so as to judge whether biological toxic effect exists or not. 6w age C57BL/6 mice were bled through the orbit, allowed to stand in an anticoagulant tube for 20min, centrifuged at 2000g for 5min at 4deg.C, the supernatant removed and resuspended in 1 XPBS to 2.5% erythrocyte solution. mu.L of the erythrocyte solution was placed in a common EP tube, 150. Mu.g of cell membrane vesicles was added, the volume was made up to 1mL with 1 XPBS, and the solution was diluted to a concentration of 2%. The negative control group was 1×pbs; the positive control group was a 2% strength red blood cell solution diluted with double distilled water. The experimental and control tubes were placed in a shaker at 37℃at 100rpm for 1h. Then, 2000g of the mixture was centrifuged at 4℃for 5 minutes, 200. Mu.L of the supernatant was taken up to the ELISA plate, the absorption wavelength was set to 540nm, and the absorbance OD was measured. Three complex wells were repeated for each group of material and the calculation formula for the hemolysis rate was as follows:

Hemolysis(％)＝(ODS-ODN)/(ODP-ODN)×100％；

Wherein ODS refers to the OD value of the sample, ODN refers to the OD value of the negative control PBS group, ODP refers to the OD value of the positive control double distilled water group.

For ease of comparison, the positive group hemolysis rate was defined as 100%. The hemolysis rate of the cell membrane vesicle is 0.95 percent, which is less than 5 percent of the accepted standard, thus showing that the cell membrane vesicle has good blood compatibility and does not destroy the morphology and the function of red blood cells in the circulatory system.

As a result, as shown in FIG. 8, the hemolysis rates of the PBS group, the free NVs group, the IL-15Rα NVs group, the IL-15NVs group and the IL-15/IL-15Rα NVs group were 0.95%, 1.07%, 0.61% and 0.95%, respectively, which were less than 5% of the accepted standard, indicating that the cell membrane vesicles were excellent in blood compatibility, and the morphology and function of erythrocytes were not destroyed in the circulatory system.

1.3 in vitro toxicity experiments of cell membrane vesicles

The contamination path of the nano material has in vitro toxicity, and the cell strain can be used for researching in vitro toxicity. Thus, applicants have further investigated the toxic effects of cell membrane vesicles on cells under in vitro conditions. B16-F10-Luciferase cells (Feng Hui organisms, FH 0518) and 4T1-Luciferase (Feng Hui organisms, WC 0004) were seeded into 96-well plates, adjusted to a density of 104/well and incubated overnight. The DMEM medium containing 10% FBS was aspirated, 100. Mu.L of fresh medium containing cell membrane vesicles was added to each well, the cell membrane vesicles concentrations were 5, 10, 25, 50, 75 and 125. Mu.g/mL, respectively, and incubation was continued for 24h in the cell incubator. The medium was then aspirated, washed 3 times with 1 XPBS, fresh medium containing 10% CCK-8 solution was added and incubated for 2h at 37 ℃. Then using a multifunctional enzyme labeling instrument to measure absorbance OD at the wavelength of 450nm, repeating four compound holes of each group of materials, and calculating the cell survival rate according to the following formula:

Cell viability(％)＝(ODS-ODb)/(ODc-ODb)×100％；

Wherein ODS refers to the OD of the sample, ODb refers to the OD of the fresh medium to which only 10% CCK-8 solution was added, ODC refers to the OD of the fresh medium to which 10% CCK-8 solution was added to the blank.

As shown in FIG. 9, the cell viability of the PBS group was defined as 100%, and the cell viability of B16-Luciferase cells incubated with different concentrations of cell membrane vesicles at cell membrane vesicle concentrations of 5, 10, 15, 25, 50 and 75. Mu.g/mL was 90% or more, indicating low toxicity; when the cell membrane vesicle concentration is 125. Mu.g/mL, the survival rate of the cells is less than 85%. After 4T1-Luciferase cells are incubated with cell membrane vesicles at different concentrations, the cell viability is over 85% under the conditions that the concentrations are 5, 10, 15, 25, 50 and 75 mug/mL, and the cell viability is obviously reduced when the cell membrane vesicle concentration is 125 mug/mL.

1.4 hemodynamic experiments of cell membrane vesicles in mouse blood

The concentration of the medicine in the blood of the organism changes with time, and the acting strength is in direct proportion to the blood medicine concentration. Thus, the applicant has further explored the hemodynamic behaviour of cell membrane vesicles in vivo. The length of the kinetic elimination time of the cell membrane vesicles in the blood reflects the blood half-life, clearance rate and dosing interval of the cell membrane vesicles. The protein concentration of the cell membrane vesicle is measured by using a BCA kit, cy5.5 solution is added to the cell membrane vesicle for marking, the cell membrane vesicle is incubated on a shaker at room temperature for 40min, and the cell membrane vesicle is centrifuged at 14800g for 40min at 4 ℃ to remove the supernatant. Cell membrane vesicles were washed twice with 1 XPBS, and finally resuspended in 1 XPBS, 300. Mu.g/150. Mu.L of each rat tail was intravenously injected, and the supernatants were collected by anticoagulation tube orbit extraction for 2min, 3h, 5h, 14h, 24h, 42h, respectively, then allowed to stand at room temperature for 2h, centrifuged at 3000rpm for 10 min. The supernatant was aspirated to an ELISA plate and absorbance was measured at 670nm using a multifunctional microplate reader.

As a result, as shown in FIG. 10, the blood concentration gradually decreased with time in the blood circulation, and after 5 hours and 42 hours of blood circulation, the blood concentration was 30% and 4.1%, indicating that the cell membrane vesicles had high retention ability in the blood, and the drug action time was prolonged.

1.5 experiments on the distribution of cell membrane vesicles in mouse organs

The medicine menstrual blood enters the body, most of the medicine menstrual blood can be cleared by the liver and the kidney, and part of the medicine reaches the treatment part. Thus, the application investigated the amount of cell membrane vesicles that reached major organs and tumor sites in the body after administration of menstrual blood. Mice were dehaired from the left abdomen and each mouse was subcutaneously injected with 106B 16-F10-Luciferase cells. Five days later, the cell membrane vesicles labeled with cy5.5 dye were intravenously injected into the tail of the tumor-forming mice, the mice were then sacrificed at 8h, 24h and 32h cervical dislocation, the mice were dissected, the mice were heart, liver, spleen, lung, kidney and tumor were dissected, and the cell membrane vesicle content levels of each organ were detected using a small animal in vivo imager, selecting a cy5.5 imaging system.

As a result, as shown in FIG. 11, cell membrane vesicles mainly accumulated in the liver, kidney and tumor sites. At 8h, a large number of cell membrane vesicles reached the liver and kidneys and a small number reached the tumor site. At 24h, the liver and kidneys still contained a large number of cell membrane vesicles, and the cell membrane vesicle content reaching the tumor site was further increased. At 32h, the level of cell membrane vesicles in the liver and kidney was still high, and the level of cell membrane vesicles decreased at the tumor site.

Example 3 IL-15/IL-15Rα cell membrane vesicle treatment of mouse tumor model

1.1 murine melanoma model treatment experiments

To investigate the therapeutic effect of co-expressed IL-15/IL-15Rα -NVs on tumors, applicant established a mouse melanoma model. Abdomen of 8 w-age C57BL/6 mice was dehaired one day in advance, and subcutaneously injected 10 the next day ⁶ Individual melanoma cells of murine origin B16-F10-luciferases. After tumorigenesis, mice are randomly divided into 7 groups and receive different treatments: (1) PBS group (1 XPBS 150. Mu.L), (2) free NVs group (150. Mu.L, vesicle 300. Mu.g), (3) IL-15Rα NVs group (150. Mu.L, vesicle 300. Mu.g), (4) IL-15 NVs group (150. Mu.L, vesicle 300. Mu.g), (5) PD-1/PD-L1 inhibitor 1 group (150. Mu.L, PD-1/PD-L1 inhibitor 1 50. Mu.g), (6) IL-15-IL-15Rα NVs group (150. Mu.L, vesicle 300. Mu.g), (7) IL-15-IL-15Rα NVs-PD-1/PD-L1 inhibitor 1 group (150. Mu.L, vesicle 300. Mu.g, PD-1/PD-L1 inhibitor 1 50. Mu.g); each group of vesicles and/or PD-1/PD-L1 inhibitor 1 was injected into 150. Mu.L of 1 XPBS. The administration frequency is once every three days, and the administration is four times. Body weight and tumor length and width of the mice were measured every three days, and tumor volume (volume=major diameter x minor diameter x 1/2) was calculated and weighed. When the volume of the mice exceeds 1500mm ³ At that time, animals were sacrificed according to ethical requirements and survival was recorded (fig. 12).

As a result, as shown in FIG. 13, the volume of the mouse melanoma increased rapidly in PBS group, while the volume of the mouse melanoma increased relatively slowly in IL-15/IL-15RαNVs group and IL-15/IL-15RαNVs-PD-1/PD-L1inhibitor 1 group. The therapeutic effect of IL-15/IL-15RαNVs group was more pronounced than that of free NVs group (P < 0.01), and the tumor growth rate was reduced to a certain level compared with that of IL-15 NVs group and PD-1/PD-L1inhibitor 1 group. The volume of the IL-15/IL-15RαNVs-PD-1/PD-L1inhibitor 1 group grows slowest (P < 0.001), and the combination therapy is obvious compared with the therapy of the PD-1/PD-L1inhibitor 1 drug alone, which shows that the combined therapy has obvious anti-tumor effect.

As a result, as shown in FIG. 14, the average tumor weight of the IL-15/IL-15RαNVs-PD-1/PD-L1inhibitor 1 group was the lightest compared to PBS (G1), and there was a significant difference (P)<0.001). IL-15/IL-15RαNVs group inhibited tumor growth more effectively than the free NVs group (P<0.01 The therapeutic effect of the IL-15/IL-15RαNVs-PD-1/PD-L1inhibitor 1 group is obvious compared with that of the PD-1/PD-L1inhibitor 1 alone (P)<0.001). At the same time, the survival rate of different drugs to mice is detectedIs a function of (a) and (b). Tumor volume was up to 1500mm ³ And drawing a survival curve graph of the mice for ethical death points of the mice, and analyzing the influence of the drug on the survival period of the tumor-bearing mice. Compared with a control group, the survival period of two groups of mice of IL-15/IL-15RαNVs (G6) and IL-15/IL-15RαNVs-PD-1/PD-L1inhibitor is obviously prolonged, and the survival rate is improved. In particular to IL-15/IL-15RαNVs-PD-1/PD-L1inhibitor 1 group which greatly prolongs the average survival time of tumor-bearing mice.

1.2 flow detection of mouse tumor immune cells and cytokines

To further compare the effect of different treatments on tumors 3 days after the end of the mice dosing, flow cytometry was used to detect immune-related cell content and cytokine levels at the tumor. After killing the mice with cervical dislocation, the subcutaneous tumors in the abdomen of the mice were peeled off, washed twice with 1×pbs, and the blood filaments were removed. After blotted dry with filter paper, the tumors were weighed and recorded. After weighing, the tumors were placed on a 70 μm cell sieve, under the sieve followed by a 50mL centrifuge tube, and handled on ice throughout. After wetting with 1 XPBS containing 2% FBS, the procedure was followed by gentle grinding and rinsing with 1 XPBS containing 2% FBS to give a single cell suspension. The single cell suspension was centrifuged at 350g for 5min at 4℃and the supernatant removed, then washed twice with 1 XPBS plus 2% FBS, resuspended in 500. Mu.L of 2% FBS, and split into 1.5mL EP tubes for flow experiments as follows:

1. cell surface molecular detection:

(1) the cell volume per tube was resuspended at 50. Mu.L with 1 XPBS of 2% FBS, and the flow-through antibody was added and incubated at 4℃for 30min. (extracellular antibody: anti-CD8 alpha antibody abcam ab22378; recombinant Anti-CD4 antibody abcam ab183685; PE/Cyanine7-Anti-CD8a BioLegend 100721;Pacific Blue-Anti-CD69 BioLegend 104524; APC/Cy7-Anti-CD3 ε BioLegend 100330; BV 510) ^TM -anti-CD8a BioLegend 100752; FITC-anti-CD4BioLegend 100406; APC-anti-CD103 BioLegend 121413; APC-anti-CD3 BioLegend 100236; PE-anti-CD 4BioLegend 100408; intracellular antibodies: APC-anti-Perforin BioLegend 154304)

(2) After staining was completed, cells were resuspended by washing twice with 1 XPBS of 2% FBS, and 500. Mu.L of 1 XPBS of 2% FBS was used for detection on-machine as soon as possible.

2. Cell endocrine cytokine detection:

(1) the tumor suspension was inoculated into a 48-well plate containing 500. Mu.L 1640 culture solution, PMA at a final concentration of 50ng/mL and ionomycin at 1. Mu.g/mL were added, and the culture was continued in a cell incubator for 1 hour, and brefeldin A at a final concentration of 25. Mu.M was added, followed by further culture for 5 hours.

(2) After removal of the medium, the medium was washed twice with 1 XPBS. The cell volume per tube was resuspended at 50. Mu.L with 1 XPBS of 2% FBS, surface molecular flow antibody was added and incubated at 4℃for 30min.

(3) After the staining was completed, the cells were washed twice with 1×pbs containing 2% fbs, and incubated with 4% paraformaldehyde at room temperature for 20min under dark conditions.

(4) Centrifugation was performed at 350g for 5min at 4℃and the supernatant was removed and washed twice with 1 XPBS in 2% FBS.

(5) The fixed cells were broken by adding 0.2% Trixton-x100, incubated at room temperature for 20min, and centrifuged at 350g for 5min.

(6) Each tube of cells was resuspended with 50. Mu.L of 0.01% Trixton-x100, intracellular flow antibody was added and incubated for 30min at 4 ℃.

(7) Centrifugation was performed at 350g for 5min at 4℃and the supernatant was removed and washed twice with 1 XPBS in 2% FBS.

(8) The stained cells were resuspended in 1 XPBS of 500. Mu.L 2% FBS and detected on-line as soon as possible using a flow cytometer.

As shown in FIGS. 15a and b, CD8 of the IL-15/IL-15Rα NVs group and IL-15/IL-15Rα NVs-PD-1/PD-L1 inhibitor 1 group compared to the PBS group, free NVs group, IL-15Rα NVs group, IL-15 NVs group and PD-1/PD-L1 inhibitor 1 group ⁺ Levels of T cell content rise to varying degrees. Wherein, in the tumor microenvironment treated with IL-15/IL-15RαNVs group, CD8 compared to PBS group and free NVs group ⁺ The infiltration level of T cells was increased to 42.7% (P)<0.05). CD8 of IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor 1 ⁺ The highest T cell content is higher than that of PD-1/PD-L1 inhibitor 1 group (P<0.05 Indicating that the CD8 in the tumor microenvironment can be further increased after the combined administration ⁺ The infiltration level of T cells enhances the anti-tumor effect.

1.3 immunofluorescence detection of mouse tumor T cell content

After the tumor of the mice is stripped, the mice are washed twice by 1X PBS, the filter paper absorbs the water, the mice are embedded by OCT embedding agent, and the mice are frozen in a refrigerator at the temperature of minus 20 ℃ until the OCT embedding agent is solidified. The frozen microtome was precooled, the slice thickness was adjusted to 7. Mu.m, and the slice was taken. And (3) placing the cut slices for 1-2 hours at room temperature, and then performing downstream operation to prevent slicing. The sections returned to room temperature were immersed in 1×pbs for 15min, and OCT embedding medium was removed. Most of the water is sucked by filter paper, and an immunohistochemical pen is used for drawing circles far away from tumor tissues, so that the edge effect is avoided. mu.L of 0.2% Trixton-x100 in 3% BSA was added, incubated at room temperature for 30min, and after blotting the liquid, a mixed primary antibody of CD4 and CD8 (primary antibody was extracellular antibody for cell surface molecular detection) was added and placed in a wet box at 4℃overnight. The next day the primary antibody was recovered, soaked in 1 XPBS and washed three times for 10min each on a shaker. After the liquid is sucked by filter paper, adding fluorescent secondary antibody 488 abcam ab150113；/>647 abcam ab 150159) and incubated at room temperature in the dark for 1h. After the secondary antibody was blotted off, it was soaked in 1 XPBS and washed three times for 10min on a shaker. After the major part of the water was removed by filtration, DAPI dye solution was added to a final concentration of 1. Mu.g/mL, and incubated at room temperature for 15min in the dark. After the dye solution was sucked away, it was soaked in 1 XPBS and washed three times for 10min each on a shaker. After the water is absorbed, an anti-fluorescence quenching agent sealing piece is dripped, a cover glass is covered, and the periphery of the cover glass is fixed by nail polish. As soon as possible, observation and shooting with a laser confocal microscope.

The tumor microenvironment is a complex environment containing a large number of tumor cells, lymphocytes and non-lymphoid parenchymal cells, which interact and interact. Among many lymphocytes, CD8 ⁺ T cells are the primary effector cells that directly kill tumor cells, and their levels of activation and proliferation. Representing the intensity of the anti-tumor effect. Thus, applicants examined tumor micro-organisms after completion of various drug treatmentsContent of T cells in the environment. Immunofluorescence results are shown in FIG. 15c, CD8 infiltrated in tumor tissue of IL-15/IL-15Rα NVs group and IL-15/IL-15Rα NVs-PD-1/PD-L1 inhibitor 1 group ⁺ T cells were significantly increased, consistent with flow cytometry results. By combining flow cytometry and immunofluorescence experimental results, the IL-15/IL-15Rα NVs group and the IL-15/IL-15Rα NVs-PD-1/PD-L1 inhibitor 1 group can improve CD8 in tumor tissues ⁺ T cell content (fig. 15 c).

Among cd8+ T cells, TRM cells are more sensitive to IL-15 cytokines and have lower activation thresholds, reduced dependence on co-stimulatory factors, demonstrate a greater ability to lyse tumor cells, providing a powerful anti-tumor effect. In tumor treatment, the presence and abundance of TRM cells is positively correlated with the cure rate and overall survival cycle of the patient. Thus, applicants examined the level of TRM cells in tumor tissue. The results are shown in FIG. 16, which shows that IL-15/IL-15RαNVs and IL-15/IL-15RαNVs-PD-1/PD-L1inhibitor1 effectively increase the TRM cells infiltrated in the tumor microenvironment, exert stronger anti-tumor effect, inhibit the growth of tumor and prolong the survival period of tumor-bearing mice.

In tumor microenvironment, CD8 ⁺ T cell anti-tumor effects are not only closely related to their number, but also to their level of activation. Thus, we used flow cytometry to detect CD8 in tumors ⁺ Activation level of T cells. CD69 is an activation-inducing molecule involved in intracellular signaling of T cells. Thus, applicants detected CD8 by flow cytometry ⁺ The expression level of CD69 molecules in T cells can be presumed to be CD8 in different treatment groups ⁺ Activation level of T cells. The results are shown in FIG. 17, which shows that PD-1/PD-L1 inhibitor 1 and IL-15/IL-15RαNVs both promote activation of T cells in tumor microenvironment, and that IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor 1 further promotes activation of CD8+ T cells in tumor after combined use.

After antigen-specific T cells recognize tumor cells, perforin, granzyme B, IFN-gamma, TNF-alpha and other effector molecules are secreted to play an immune role. Perforin breaks membranes and holes on the surface of tumor cells, changes osmotic pressure to dissolve cells, or cooperates with granzyme B to induce apoptosis of tumor cells. These effector molecules play an important role in the immunotherapy of tumors. Thus, applicants examined the perforin content of the tumor microenvironment. As shown in FIG. 18, the IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor 1 increased most significantly, and the expression levels were higher than those of the single treatment PD-1/PD-L1 inhibitor 1 and IL-15/IL-15RαNVs, indicating that the combination therapy of IL-15/IL-15RαNVs-PD-1/PD-L1 inhibitor 1 was superior to the single treatment, and simultaneously activated T cells and blocked the inhibition of T cells by tumor cells, inducing a strong immune effect.

1.4 mouse organ HE staining

After killing the mice by dislocation of cervical vertebrae, the heart, liver, spleen, lung and kidney of the mice were taken, washed twice with 1×pbs, then the water was sucked off with filter paper, fixed with 4% paraformaldehyde at 4 ℃ for 48 hours, then the tissues were dehydrated with 75% ethanol, and paraffin-embedded. The paraffin microtome was adjusted to a slice thickness of 5 μm and sectioned. The slices are soaked in xylene for dewaxing for 5min, repeated for 3 times, soaked in absolute ethyl alcohol for 5min,95% ethyl alcohol for 5min,80% ethyl alcohol for 5min and 70% ethyl alcohol for 5min in sequence, distilled water for 2min, and gradient hydration is carried out. Then placing the slices into hematoxylin working solution for dyeing for 5min, washing with tap water, differentiating for several seconds with 1% hydrochloric acid alcohol, and stopping differentiation with tap water. The slices were soaked in eosin dye solution for 1min and washed with tap water. Dehydrating with 70% ethanol, 80% ethanol, 95% ethanol, and twice absolute ethanol for 5min each time, permeabilizing with xylene for 5min, repeating twice, and sealing with neutral resin. Microscope imaging.

As a result, as shown in FIG. 19a, the main organs of each group of mice were not significantly damaged and were morphologically normal after the treatment with the different drugs. Meanwhile, the dynamic change of body weight during the administration period was counted, and as a result, as shown in fig. 19b, the body weight was not significantly changed. The results show that the drug delivery system has no toxic or side effect on the tissues of mice and has high safety.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.

Sequence listing

<110> university of Zhongshan same form

<120> a cell membrane vesicle, and preparation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 375

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> IL-15Rα

<400> 1

gccaccatga ccacagagac agctattatg cctggctcca ggctgacacc atcccaaaca 60

acttctgcag gaactacagg gacaggcagt cacaagtcct cccgagcccc atctcttgca 120

gcaacaatga ccttggagcc tacagcctcc acctccctca ggataacaga gatttctccc 180

cacagttcca aaatgacgaa agtggccatc tctacatcgg tcctcttggt tggtgcaggg 240

gttgtgatgg ctttcctggc ctggtacatc aaatcaaggc agccttctca gccgtgccgt 300

gttgaggtgg aaaccatgga aacagtacca atgactgtga gggccagcag caaggaggat 360

gaagacacag gagcc 375

<210> 2

<211> 492

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> IL-15

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gccaccatga aaattttgaa accatatatg aggaatacat ccatctcgtg ctacttgtgt 60

ttccttctaa acagtcactt tttaactgag gctggcattc atgtcttcat tttgggctgt 120

gtcagtgtag gtctccctaa aacagaggcc aactggatag atgtaagata tgacctggag 180

aaaattgaaa gccttattca atctattcat attgacacca ctttatacac tgacagtgac 240

tttcatccca gttgcaaagt tactgcaatg aactgctttc tcctggaatt gcaggttatt 300

ttacatgagt acagtaacat gactcttaat gaaacagtaa gaaacgtgct ctaccttgca 360

aacagcactc tgtcttctaa caagaatgta gcagaatctg gctgcaagga atgtgaggag 420

ctggaggaga aaaccttcac agagtttttg caaagcttta tacgcattgt ccaaatgttc 480

atcaacacgt cc 492

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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<223> mcherry

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atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60

gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120

cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180

ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240

cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300

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ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420

atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480

gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540

gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600

aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660

cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtg a 711

Claims (6)

1. A method for preparing a cell membrane vesicle, comprising the steps of:

(1) Respectively carrying out slow virus packaging on IL-15 and IL-15 Ralpha genes to obtain IL-15 virus and IL-15 Ralpha virus which are respectively fused to express fluorescent protein, purifying, respectively infecting cells by using the two viruses, screening drug resistance, and respectively obtaining an IL-15 cell line expressing fluorescence and an IL-15 Ralpha cell line expressing fluorescence;

(2) Infecting an IL-15Rα cell line expressing fluorescence by adopting the IL-15 virus fusion expressing fluorescent protein in the step (1), and screening drug resistance to obtain a cell line expressing an IL-15/IL-15Rα complex;

(3) Culturing the cell line expressing the IL-15/IL-15Rα complex in the step (2), collecting cells, inhibiting proteolysis, homogenizing, centrifuging to obtain supernatant, centrifuging, collecting precipitate, and filtering to obtain cell membrane vesicles;

the IL-15 gene lentivirus packaged original vector is pLV-Hygro, an IL-15 gene and an mcherry gene are inserted into an XbaI-BamHI site, and a target plasmid pLV-IL15-mcherry-Hygro is obtained through recombination, and the auxiliary plasmid is pH1 and pH2;

the IL-15 alpha gene slow virus packaged original vector is pLV-EGFP-N, the IL-15 Ralpha gene is inserted into XhoI-NotI site, the target plasmid pLV-IL15RA-EGFP-Puro is obtained by recombination, and the auxiliary plasmid is pH1 and pH2;

the surface of the biological cell membrane for preparing the cell membrane vesicle is over-expressed with IL-15/IL-15 Ralpha complex.

2. The method of claim 1, wherein the biological cell membrane is derived from NIH 3T3 cell line.

3. The method for producing a cell membrane vesicle according to claim 1, wherein,

The mass ratio of the objective plasmid pLV-IL15RA-EGFP-Puro to the auxiliary plasmid pH1 to pH2 is 4:3:1;

the mass ratio of the objective plasmid pLV-IL15-mcherry-Hygro to the helper plasmid pH1 to the helper plasmid pH2 is 4:3:1.

4. The method for producing a cell membrane vesicle according to claim 1, wherein,

the drug resistance screening of virus infected cells corresponding to the IL-15 gene adopts hygromycin B;

the drug resistance screening of virus infected cells corresponding to the IL-15 alpha gene adopts puromycin;

and (3) screening the drug resistance by using puromycin and hygromycin B.

5. The method for producing a cell membrane vesicle according to claim 1, wherein,

the cells adopted by the infected cells in the step (1) are mouse fibroblast line NIH 3T3 cells;

the infected cells of step (1) adopt a cell density of 50-60%;

the infected cells of step (1) were added with Polybrene at a final concentration of 8. Mu.g/mL.

6. The method of claim 1, wherein step (3) is specifically as follows: culturing a large amount of cell lines which stably overexpress the IL-15/IL-15Rα complex, collecting cells, centrifuging at 4 ℃ and 800-1000 rpm, collecting cell pellets, washing the cells with PBS containing a protease inhibitor, resuspending the cells with PBS containing a protease inhibitor, placing on ice, homogenizing 200 times, collecting homogenate, centrifuging at 4 ℃ and 1000 g for 5 min, collecting supernatant, centrifuging at 4 ℃ for 5 min at 3000 g, collecting supernatant again, centrifuging at 14800 g for 40 min at 4 ℃, collecting pellets, resuspending the pellets with PBS containing a protease inhibitor, and sequentially passing through a 0.8 mu m filter head and a 0.22 mu m filter head to obtain cell membrane vesicles.