CN116004538A

CN116004538A - Nanometer vesicle carrying dsRNA generated in cells, preparation and application

Info

Publication number: CN116004538A
Application number: CN202211711080.2A
Authority: CN
Inventors: 张志平; 余育林; 孔丽
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2023-04-25

Abstract

The invention relates to a nano vesicle carrying dsRNA generated in cells, and preparation and application thereof, belonging to the technical field of antitumor pharmacy. The preparation method comprises the following steps: culturing the tumor cells in a medium comprising an epigenetic modulator that induces endogenous retroviral expression of the tumor cells, thereby producing dsRNA; collecting the cultured tumor cells, dispersing in buffer solution containing protease inhibitor, sequentially passing through membranes with large-to-small pore diameters by using a liposome extruder, collecting the extrusion liquid, and centrifuging to obtain the nano vesicles carrying cells for generating dsRNA. The invention uses the nanometer vesicle to carry dsRNA generated in cells, and the correspondingly obtained nanometer vesicle preparation can efficiently regulate tumor immunity microenvironment, promote dendritic cell maturation, polarize tumor-related macrophages, increase the number of killer T cells in tumor tissues, and enhance the activity of B cells in germinal centers, and can be particularly used for tumor immunity combined treatment.

Description

Nanometer vesicle carrying dsRNA generated in cells, preparation and application

Technical Field

The invention belongs to the technical field of antitumor pharmacy, and particularly relates to a nano vesicle carrying dsNRA (double-stranded ribonucleic acid) for generating dsRNA in cells, and preparation and application thereof.

Background

According to the recent daily release of the global latest cancer burden data of 2020 by the international cancer research Institute (IARC) of the world health organization, 457 thousands of new cancer cases are shown in 2020 of China, 300 thousands of new death cases are shown, the data is not slightly improved compared with the previous year, and the double-bit global first of the new disease cases and the new death cases shows that China is a large national cancer world. Cancer has become a major factor threatening the life and health of the national population, however, it is not currently available to cure cancer. Methods for cancer treatment are diverse, and traditional treatment methods include chemotherapy, radiation therapy, surgical excision, and the like; emerging therapies include immune checkpoint blocker therapy, cancer vaccines, proton therapy systems, and the like. However, for some malignant tumors, a good therapeutic effect is still not achieved. Therefore, the development of novel antitumor agents is of great importance for the treatment of malignant tumors.

In recent years, with intensive research on cancer, it was found that the balance of tumor immunity microenvironment seriously affects the anti-tumor therapeutic effect. Tumors can be classified into cold tumors and hot tumors according to their strong or weak immunity microenvironment. At present, most refractory tumors are cold tumors, and a few are hot tumors. The tumor can influence the expression of antigen molecules on the surface of tumor cell membrane and some co-stimulatory molecules by regulating the expression of self genes, thereby achieving the effect of evading immune cell monitoring. In addition, the tumor cells can secrete a large amount of immunosuppressive cytokines, so that the immunocyte activity in the tumor microenvironment is reduced, the number of immunosuppressive cells is increased, the immunosuppressive property of the tumor microenvironment is further enhanced, and the effect of cancer treatment is reduced. Therefore, the development of an immune preparation capable of regulating the tumor immune microenvironment is urgent.

At present, extracellular vesicles, particularly tumor cell-derived vesicles, are becoming a research hotspot for cancer treatment. Because of the advantages of excellent biocompatibility, natural targeting, low immunogenicity, carrying tumor cell antigen information and the like, tumor-derived cell vesicles are increasingly widely applied to anti-tumor immunotherapy. However, the information exposure of most tumor cell surface antigens is low, and the immune activation effect of single tumor cell derived vesicles is poor, so that the tumor cell derived vesicles are not satisfactory in the aspect of cancer treatment effect.

Disclosure of Invention

In view of the above-identified deficiencies or improvements in the art, it is an object of the present invention to develop a nanovesicle preparation for producing dsRNA in a carrying cell for the treatment of a tumor. The novel nano vesicle preparation developed by the invention has more antigen information and immune cell stimulating molecules on one hand, and can enhance the recognition capability of antigen presenting cells (dendritic cells and macrophages) on tumor cells; on the other hand, the dsRNA generated in the nano vesicle carrying cells can effectively activate the corresponding intracellular interferon activating genes after being taken up by tumor cells and antigen presenting cells, and the interferon activating genes are stimulated to generate a type of interferon, so that immune cells are activated, the tumor immune microenvironment is regulated, the anti-tumor immune effect is enhanced, and the effect of achieving two purposes is achieved. The dsRNA-loaded nano vesicle developed by the invention is novel and effective, high in safety, convenient and quick in preparation method, low in cost and extremely high in clinical transformation potential and application prospect.

According to a first aspect of the present invention, there is provided a method for preparing a nanovesicle for intracellular dsRNA production, comprising the steps of:

(1) Culturing the tumor cells in a medium comprising an epigenetic modulator that induces endogenous retroviral expression of the tumor cells, thereby producing dsRNA;

(2) Collecting the tumor cells cultured in the step (1), dispersing in a buffer solution containing a protease inhibitor, sequentially passing through membranes with large pore diameters by using a liposome extruder, collecting the extruded liquid, and centrifuging to obtain the nano vesicles carrying cells for generating dsRNA.

Preferably, the epigenetic modulator is a DNA methyltransferase inhibitor.

Preferably, the DNA methyltransferase inhibitor is decitabine or zebralin, preferably decitabine.

Preferably, the tumor cell is a malignant tumor cell;

preferably, the malignant tumor cells are melanoma tumor cells, mouse triple negative breast cancer cells or mouse colorectal cancer cells.

According to another aspect of the present invention, there is provided a nanovesicle for producing dsRNA in a carried cell prepared by any one of the methods.

According to another aspect of the invention, there is provided the use of said nanovesicles carrying intracellular dsRNA for the preparation of a medicament for the treatment of tumors.

According to another aspect of the invention, there is provided the use of said nanovesicles carrying intracellular dsRNA for the preparation of an anti-tumour immune combination therapeutic agent.

Preferably, the nanovesicles are used to activate interferon genes within antigen presenting cells, thereby inducing interferon production.

Preferably, the nanovesicles are used to activate antitumor humoral immune response cells in draining lymph nodes.

Preferably, the nanovesicles are used to activate anti-tumor humoral and cellular immunity and inhibit tumor volume growth.

In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:

(1) The invention provides a novel preparation strategy of an anti-tumor pharmaceutical preparation, which is to induce tumor cells to generate dsRNA through epigenetic regulator, prepare nano vesicle preparation carrying the dsRNA generated by the cells through extrusion of the tumor cells, prepare vesicles by utilizing epigenetic regulated tumor cells and carry immune adjuvant dsRNA, and can realize stronger anti-tumor effect. Epigenetic modulators can enhance tumor cell immunogenicity, enhancing tumor cell responsiveness to immunotherapy. In addition, epigenetic modulators (e.g., DNMTi) are capable of inducing expression of retroviruses endogenous to certain tumor cells, thereby producing dsRNA. The dsRNA can be used as an immunoadjuvant for activating an intracellular interferon activating gene, inducing cells to generate a type of interferon, and assisting in activating immune cells such as dendritic cells, macrophages, natural killer cells, T cells and the like. Thus, epigenetic modulation of tumor cells can enhance tumor immunogenicity and stimulate production of dsRNA by tumor cells.

(2) The nano vesicle preparation has good uniformity; realizes the one-step preparation of the preparation carrier and the medicine carrying; solves the subsequent medicine carrying step of traditional vesicle medicine carrying, and improves the bioactivity of the vesicle; the preparation process is simple and convenient, and the large-scale production and clinical transformation are easy.

(3) The nano vesicle preparation has good targeting of immune cells (dendritic cells and macrophages), has strong lysosome escape capability, can effectively activate interferon genes in antigen presenting cells, induces a large amount of interferon type I, and achieves the effect of effectively relieving tumor immunity microenvironment inhibition.

(4) The nano vesicle preparation can effectively activate anti-tumor humoral immune response cells-B lymphocytes in drainage lymph nodes at the same time, and is mainly characterized by increasing the number of the center of initiation and the number of the B cells.

(5) After the nano vesicle preparation is administrated in tumor, the anti-tumor humoral immunity and cellular immunity can be effectively activated, and the increase of the tumor volume can be effectively inhibited. The survival time of the position in the tumor-bearing of the mice can be prolonged by nearly one time after the cytokine interleukin-12 (IL-12) is combined.

(6) The invention firstly proposes that dsRNA generated in cells is taken as an immunoadjuvant and nano vesicles prepared by blast cells are carried for the immune combination treatment of malignant tumors.

(7) In summary, the dsRNA-loaded nanovesicle preparation of the present invention is used for malignant tumor immune combination therapy, for example, it can promote antigen presenting cells to take up and present tumor antigen information, and simultaneously stimulate tumor cells and immune cells to produce a large amount of one type of interferon, thereby improving tumor immunogenicity and immune cell activity, regulating tumor immunosuppressive microenvironment, and realizing strong anti-tumor effect.

Drawings

FIG. 1 shows the expression of dsRNA in B16F10-OVA cells at various times during decitabine treatment.

FIG. 2 is a graph showing particle size, potential and morphology characterization of nanovesicles according to the invention. Where (a) in fig. 2 is a DLS result, (b) in fig. 2 is a TEM result, and the scale shown in the lower left corner of (b) in fig. 2 represents 200nm.

FIG. 3 shows the results of flow analysis of tumor cells after decitabine treatment and vesicle surface signaling molecules according to the present invention. Wherein, (a), (b), (c) and (d) in FIG. 3 are the tumor cell surface CD80, CD86, MHC I and MHC II protein expression levels in sequence; in FIG. 3, (e) shows the MHC I protein expression level on the surface of nanovesicles.

FIG. 4 shows the result of immunoblotting analysis of Decitabine-treated tumor cells and vesicle surface proteins of the present invention.

FIG. 5 shows the results of induction of BMDC maturation by nanocapsules according to the invention.

FIG. 6 shows the results of the nanovesicle preparation of the invention inducing polarization of Raw264.7 cells.

Figure 7 shows the escape results of nanocapsules according to the invention in BMDC and raw264.7 cell lysosomes. FIG. 7 (a) shows NV _dsRNA Laser confocal co-localization results at BMDC cell lysosome escape (Red: NV) _dsRNA The method comprises the steps of carrying out a first treatment on the surface of the Green: lysosomes; blue: nucleus), FIG. 7 (b) is NV _dsRNA Laser confocal co-localization results at BMDC cell lysosome escape (Red: NV) _dsRNA The method comprises the steps of carrying out a first treatment on the surface of the Green: lysosomes; blue: a cell nucleus).

FIG. 8 shows qPCR analysis of the production of interferon-type (IFN. Beta.) by BMDC and Raw264.7 cells induced by nanovesicles according to the invention. Fig. 8 (a) shows BMDC qPCR results, and fig. 8 (b) shows raw264.7qPCR results.

FIGS. 9 and 10 show the results of inhibition of malignant melanoma B16F10-OVA subcutaneous tumor by nanovesicles according to the invention. Wherein fig. 9 is a tumor growth curve and fig. 10 is a life cycle curve.

FIG. 11 shows B cell immunofluorescence of B16F10-OVA tumor tissue (CD 20: red, CD3: green, DAPI: blue); FIG. 12 shows the results of immunofluorescence of CD4 and CD8T cells in B16F10-OVA tumor tissue (DAPI: blue, CD4: green, CD8: red); FIG. 13 shows the result of immunohistochemistry of M1 type macrophages (iNOS), M2 type macrophages (Arg 1), cytotoxic T cells (IFNγ) in B16F10-OVA tumor tissue; FIG. 14 shows the results of draining lymph node germinal center immunofluorescence of B16F10-OVA tumor bearing mice (DAPI: blue, ki67: green, CD20: red).

FIGS. 15 and 16 show the results of inhibition of malignant melanoma B16F10-OVA subcutaneous tumor by nanovesicles in combination with IL-12 according to the invention. It is shown in fig. 15 as a tumor growth curve, and fig. 16 as a survival curve.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

Decitabine (Decistabine) was used in the following examples from MCE and J2 mab from Youning vitamins company; the tumor model used can be established with reference to ACS Nano 2021,15,3123.

The invention relates to a nano vesicle preparation for carrying cell generated dsRNA for tumor treatment, which comprises dsRNA generated in cells and nano vesicles carrying the dsRNA, wherein the drug is an epigenetic regulator, and the nano vesicle preparation carrying cell generated dsRNA can regulate and control tumor immune microenvironment and/or polarize tumor-related macrophages and/or inhibit tumor growth and/or prolong the survival period.

The dsRNA is the dsRNA which is autonomously generated in cells after drug stimulation. The nanovesicles are derived from tumor cells.

In some embodiments, the epigenetic modulator is a DNA methyltransferase inhibitor (DNMTi).

In some embodiments, the tumor is a malignant tumor, preferably one of malignant melanoma tumor cells B16F10-OVA, mouse triple negative breast cancer cells 4T1, mouse colorectal cancer cells MC 38.

The invention relates to a preparation method of a nano vesicle preparation for carrying cells to generate dsRNA for malignant tumor treatment, which comprises the following steps:

(1) Dissolving epigenetic regulator (such as DNMTi) with DMSO to obtain medicinal mother solution with specific concentration;

(2) Inoculating tumor cells into a 100mm round cell culture dish, and culturing for 48 hours with a complete culture medium, wherein a certain volume of the drug mother liquor in the step (1) is added into the complete culture medium;

(3) Scraping and centrifugally collecting the cells cultured in the step (2), dispersing the cells in a certain volume of Phosphate Buffer Solution (PBS) containing protease inhibitors, sequentially passing through polycarbonate membranes with different pore diameters by using a liposome extruder, collecting the extruded liquid, and obtaining the dsRNA-carried nano vesicles by using a gradient centrifugation method.

In some embodiments, in step (1), the epigenetic modulator is decitabine or zebralin, preferably decitabine;

the concentration of the mother liquor of the medicine is 1-10 mu M, preferably 5 mu M.

In some embodiments, in step (2), the tumor cell is one of a mouse malignant melanoma tumor cell B16F10-OVA, a mouse triple negative breast cancer cell 4T1, and a mouse colorectal cell MC 38.

In some embodiments, in step (3), the polycarbonate membranes with different pore sizes are 10 μm, 5 μm, 1 μm and 0.4 μm, and the cell suspension is extruded sequentially from the pore size to the pore size during the preparation;

the gradient centrifugation method is to centrifuge 1500g for 30min, centrifuge 12000g for 2min, and centrifuge 20000g for 1h.

The following are specific examples

Example 1

Decitabine-induced tumor cells produced dsRNA validation: mouse melanoma cells B16F10-OVA were inoculated onto cell slide and cultured for 0h, 24h, 36h, 48h and 60h using complete medium containing 1. Mu.M decitabine. And collecting the cell climbing sheet, and sequentially fixing, rupture of membranes, sealing, J2 primary antibody staining, immunofluorescence secondary antibody staining, sealing and the like to prepare the dsRNA (double-strand ribonucleic acid) staining cell climbing sheet. Fluorescence intensity was observed using a laser confocal microscope. As a result, as shown in FIG. 1, a large amount of dsRNA was produced in the cells treated for 48h and 60h, respectively, compared to 0h, 24h and 36h, and 48h was preferable in view of the treatment time and the cell density.

Example 2

dsRNA Nanovesicles (NV) _dsRNA ) Preparation: decitabine was first prepared as a 5mM stock solution in DMSO. An appropriate amount of mouse melanoma cells B16F10-OVA were inoculated into 100mm round cell culture dishes and cultured in complete medium containing 1. Mu.M decitabine for 48h. Cells were collected and washed once with PBS. Cells were re-dispersed with an appropriate amount of protease inhibitor-containing PBS and passed sequentially through 10 μm, 5 μm, 1 μm and 0.4 μm polycarbonate membranes using a liposome extruder. Purifying the obtained vesicle crude product solution by a gradient centrifugation method, wherein the specific method comprises the following steps: firstly, 1500g is centrifuged for 30min, then 12000g is centrifuged for 2min, finally 20000g is centrifuged for 1h, the sediment is collected, and is dispersed by filling with a proper amount of PBS and is preserved at 4 ℃.

And further carrying out physical and chemical property evaluation on the dsRNA-carried nanovesicles. Observing the morphological characteristics of the nano vesicle preparation by adopting a Transmission Electron Microscope (TEM); the particle size distribution and potential information of the nanovesicle preparations were determined using a dynamic light scattering particle size analyzer (DLS). As shown in FIG. 2, the nano vesicles have a particle size of about 200nm, uniform particle size dispersion, and have a complete vesicle structure from the TEM result, and have a particle size of about 200nm, and good dispersibility.

Example 3

Tumor cells after decitabine treatment and expression conditions of vesicle surface related proteins prepared by the tumor cells: inoculating proper amount of B16F10-OVA cells into a 6-well plate, culturing for 48 hours by using a complete culture medium containing 1 mu M decitabine, collecting tumor cells and nano vesicles prepared by using the tumor cells, and performing flow cytometry analysis; tumor cells immunoblotted after 24h and 48h of collection treatment. The results are shown in fig. 3, 4, and the results in fig. 3 show that the tumor cells treated with decitabine and some proteins on the surface of nanovesicles prepared from the treated cells were significantly increased compared to the control (DMSO treatment). FIG. 4 shows that there is a significant increase in both tumor associated antigen (TRP 2) and some immunostimulatory molecules (HSP 70, gp 100) following surface decitabine treatment.

Example 4

NV _dsRNA Curing effect on cells derived from mouse bone marrow: extraction of mouse bone marrow-derived dendritic cells (BMDC), culture until day seven, saline, and empty vesicles (NV) _blank ,50μg/mL)、NV _dsRNA After BMDC (50. Mu.g/mL) and lipopolysaccharide (LPS, 1. Mu.g/mL) were treated for 24h, cells were collected, stained with CD80, CD86 and CD11c flow antibody at 4℃for 30min, and then analyzed by flow cytometry. Results are shown in FIG. 5, NV _dsRNA After treatment of immature BMDC, the cell surface co-stimulatory molecules CD80, CD86 were significantly upregulated, and the proportion of mature DC (CD 80CD86 double positive cells) increased from 20% to 40% in the untreated group, indicating NV _dsRNA Has DC maturation promoting effect, and can be used for resisting antigen presenting process in tumor cell immune response.

Example 5

NV _dsRNA Effect on macrophage polarization: inoculating appropriate amount of Raw254.7 cells into 24-well plate, and administering physiological saline, 50 μg/mL NV _blank ，50μg/mL NV _dsRNA After 24h of treatment, the cells were collected, stained with F4/80, CD86 flow antibody at 4℃for 30min, and then examined by flow cytometry. The results are shown in FIG. 6, NV _dsRNA The treated Raw264.7 cell surface CD86 was highly expressed, rising from 5% to 60% of untreated, indicating NV _dsRNA Has strong capability of promoting macrophage to change into M1 type phenotype, and is helpful for resisting tumor humoral immune responseB cells of the medium.

Example 6

NV _dsRNA At BMDC and raw264.7 cell lysosomal escape: inoculating appropriate amount of cells on cell slide, and using NV containing DiD label _dsRNA The cells were treated for 4h. The cell climbing slices are collected and then are processed,

fixing cells, labeling lysosomes with lysosomes green fluorescent dye, sealing, observing NV under a confocal laser microscope _dsRNA Lysosomal escape. The results are shown in FIG. 7, which shows NV _dsRNA Can escape from BMDC and Raw264.7 cell lysosomes, which indicates that dsRNA carried by the dsRNA can enter cytoplasm and further activate interferon activating genes.

Example 7

NV _dsRNA Induction of BMDCs and raw264.7 cells to produce interferon-type (ifnβ) qPCR analysis: collecting NV _dsRNA BMDC and Raw264.7 cells after 24h treatment are washed once by PBS, cells are collected, a proper amount of Trizol is used for extracting total RNA, the total RNA is reversely transcribed into cDNA, an IFN beta primer sequence is added, qPCR operation is carried out, and the expression quantity of the corresponding genes is analyzed. The results are shown in FIG. 8, which shows NV _dsRNA The treated BMDC and Raw264.7 cells all had higher IFN beta expression.

Example 8

NV _dsRNA Inhibiting malignant melanoma growth: NV was prepared as in example 1 _dsRNA Nano vesicles. Subsequently, inoculate 2X10 ⁵ B16F10-OVA cells A mouse melanoma subcutaneous model was established in the left armpit of C56BL/6 female mice when tumor volume was about 50mm ³ At the time, tumor-bearing mice were randomly divided into 3 groups of 7 mice each, and physiological saline and NV were injected intratumorally _blank ，NV _dsRNA (25. Mu.L in volume, 100. Mu.g/dose protein) was administered once every three days for a total of 3 doses. In the treatment process, a vernier caliper is used for monitoring the growth condition of the tumor and calculating the size of the tumor volume, wherein the formula is as follows: tumor volume (mm) ³ ) =length (mm) ×width (mm)/2. The tumor growth and weight change of the mice are monitored for a long time, when the mice appear to have weight reduction of 20%, the tumor volume exceeds 1500mm ³ The length of the unilateral tumor exceeds 20mm or is smallerWhen the state of the mice is obviously poor, the observation of the mice is finished, and after the experiment is finished, a tumor growth curve and a survival period are drawn. The results are shown in FIG. 9 and FIG. 10, and the average volume of tumor in saline-treated mice was increased to 1500mm at 15 th balance ³ Above, and NV _blank Tumor volume of mice in treatment group is only 500-600mm at 15 th balance ³ Corresponding NV _dsRNA Mice on day 15 had an average tumor volume of only 100mm in the treatment group ³ Left and right. Indicating NV _dsRNA Has strong tumor growth inhibiting effect, and can be used for treating NV due to its lifetime _dsRNA Median survival in treated mice provided about 50%.

Example 9

NV _dsRNA Analysis of the immune mechanism of inhibition of malignant melanoma: mouse tumor and draining lymph node tissue after treatment was obtained as in example 8, paraffin fixed, immunohistochemistry was performed and immunofluorescence analysis of CD3, CD4, CD8, CD20, iNOS, arg1, ifnγ in tumor tissue was performed; ki67, CD20 in lymph nodes were drained. The results are shown in FIG. 11, FIG. 12, FIG. 13, FIG. 14, FIG. 11, compared to normal saline and NV _blank Treatment group, NV _dsRNA Treatment group tumor tissue group had more B cells (CD 20) indicating NV _dsRNA Can activate stronger anti-tumor humoral immune response in tumor tissues; FIG. 12 shows NV _dsRNA The CD4 and CD8T cell density in tumor tissues can be effectively increased, which indicates that the tumor cells can better activate the anti-tumor cell immune response; FIG. 13 shows NV _dsRNA The number of M1 type macrophages (iNOS) in tumor tissues of mice in the treatment group is obviously increased, the number of M2 type macrophages (Arg 1) is reduced, and the number of cytotoxic T cells (IFNgamma) is obviously increased, which indicates NV _dsRNA Can effectively induce the immunity level of anti-tumor cells in tumor tissues; FIG. 14 shows NV _dsRNA More germinal centers (Ki 67/CD 20) appeared in draining lymph nodes of mice in the treatment group, indicating NV _dsRNA Can well activate B cells in lymph nodes and enhance anti-tumor humoral immune response.

Example 10

NV _dsRNA Combination of IL-12 inhibits malignant melanoma growth: NV was prepared as in example 1 _dsRNA Nano vesicles. Subsequently, inoculate 2X10 ⁵ B16F10-OVA cells A mouse melanoma subcutaneous model was established in the left armpit of C56BL/6 female mice when tumor volume was about 50mm ³ At the time, tumor-bearing mice were randomly divided into 4 groups of 7 mice each, and physiological saline and NV were injected intratumorally _blank ，NV _dsRNA ，NV _dsRNA +IL-12 (25. Mu.L volume, protein concentration 100. Mu.g/dose, IL-12 100 ng/dose) was administered once every three days for a total of 3 times. In the treatment process, a vernier caliper is used for monitoring the growth condition of the tumor and calculating the size of the tumor volume, wherein the formula is as follows: tumor volume (mm) ³ ) =length (mm) ×width (mm)/2. The tumor growth and weight change of the mice are monitored for a long time, when the mice appear to have weight reduction of 20%, the tumor volume exceeds 1500mm ³ When the length of the unilateral tumor exceeds 20mm or the state of the mice is obviously bad, the observation of the mice is finished, and after the experiment is finished, a tumor growth curve and a survival period are drawn. The results are shown in FIG. 15 and FIG. 16, in which the average volume of the tumors in the saline-treated mice was increased to 1500mm at 15 th balance ³ Above, and NV _blank Tumor volume of mice in treatment group is only 500-600mm at 15 th balance ³ Corresponding NV _dsRNA Mice on day 15 had an average tumor volume of only 100mm in the treatment group ³ Left and right, combined treatment group (NV) _dsRNA +il-12) mice had minimal tumor volume. Indicating NV _dsRNA And the combination treatment group has a strong tumor growth inhibition effect, and the median survival time of the mice in the combination treatment group is about 100 percent from the survival time, which indicates that the combination treatment greatly prolongs the survival time of the mice. Therefore, the dsRNA-carried nano vesicle can effectively inhibit the malignant melanoma growth of mice and obviously prolong the survival period of the mice, and has extremely high clinical transformation potential.

It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A method for preparing a nanovesicle carrying dsRNA produced in a cell, comprising the steps of:

2. The method for producing a nanovesicle carrying an intracellular dsRNA according to claim 1, wherein the epigenetic regulator is a DNA methyltransferase inhibitor.

3. The method for producing a nanovesicle for intracellular production of dsRNA according to claim 2, wherein the DNA methyltransferase inhibitor is decitabine or zebralin, preferably decitabine.

4. The method for producing a nanovesicle for intracellular production of dsRNA according to claim 1, wherein the tumor cell is a malignant tumor cell;

5. The nanovesicles prepared by the method according to any one of claims 1 to 4, which carry intracellular dsRNA-producing nanovesicles.

6. The use of a nanovesicle harboring an intracellular dsRNA of claim 5 for the preparation of a medicament for treating tumor.

7. The use of a nanovesicle harboring an intracellular dsRNA of claim 5 for the preparation of an anti-tumor immune combination therapeutic agent.

8. The use according to claim 6 or 7, wherein the nanovesicles are used to activate interferon genes in antigen presenting cells, thereby inducing interferon production.

9. The use according to claim 6 or 7, wherein the nanovesicles are used to activate antitumor humoral immune response cells in draining lymph nodes.

10. The use according to claim 6 or 7, wherein the nanovesicles are used to activate anti-tumor humoral and cellular immunity and to inhibit tumor volume growth.