Multifunctional oncolytic virus imitating nano-drug as well as preparation method and application thereof

Abstract

The invention provides a multifunctional mimic oncolytic virus nano-drug as well as a preparation method and application thereof. The multifunctional mimic oncolytic virus nano-drug comprises vesicles and a substance with a pyroptosis induction function, the vesicle is a shell of the multifunctional oncolytic virus imitating nano-drug; a substance with a pyroptosis induction function is loaded in the shell; the vesicles are formed by hybridizing an engineered cell membrane simulating a Newcastle disease virus envelope and a macrophage cell membrane; the multifunctional mimic oncolytic virus nano-drug realizes tumor killing, immune activation and tumor targeting through the characteristics of pyroptosis and oncolytic virus. The mimic oncolytic virus cell membrane and the macrophage cell membrane are hybridized to form the vesicles, and the vesicles are used as a shell of the drug to load the substance with the pyroptosis induction function in the vesicles, so that the in-vivo circulation time of the nanoparticles is prolonged, immune clearance caused by virus protein modification is avoided, and the drug delivery efficiency is improved. Therefore, the enrichment degree of the nanoparticles in tumors is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to a multifunctional oncolytic virus-mimicking nanomedicine and a preparation method and application thereof. Background Art

[0002] Melanoma is one of the most aggressive and lethal malignant tumors. In recent years, the annual incidence of melanoma in my country has increased rapidly at a rate of 3-6%. Because melanoma grows quickly and is difficult to contain, once the disease worsens, it is very easy to metastasize throughout the body, causing patients to miss the best time for surgery. Immune checkpoint inhibitor therapy can improve the survival prognosis of advanced melanoma to a certain extent, but its high non-response rate of 55% greatly limits the clinical benefit rate of immunotherapy. Therefore, finding new melanoma immunotherapy strategies has important scientific significance and clinical value.

[0003] Pyroptosis is a newly defined mode of programmed cell death, triggered by the Gasdermin family, characterized by cell bubbling and rupture. Compared with apoptosis, pyroptosis occurs faster, and its lytic properties make it more conducive to the release of tumor-associated antigens. In addition, the pro-inflammatory nature of pyroptosis has the potential to reshape the immune microenvironment. These characteristics suggest that pyroptosis is a desirable immunogenic cell death mode, and triggering cancer cell pyroptosis is expected to become a promising cancer treatment strategy. Studies in the prior art have shown that FeSO4 can enhance reactive oxygen species (ROS) signals initiated by CCCP and induce pyroptosis in melanoma cells. However, the drug accumulation of FeSO4 in tumors is unstable and uncontrollable. Since Gasdermin is widely expressed in various tissues, non-targeted induction of pyroptosis will inevitably lead to systemic toxicity. In addition, the heterogeneity of tumors, immunosuppressive tumor microenvironment and complex immune escape mechanisms make it more difficult to effectively induce tumor pyroptosis. Therefore, establishing an effective targeted pyroptosis induction strategy and combined treatment regimen is a key issue that needs to be addressed.

[0004] Virus-based immunotherapy is an emerging and promising strategy for tumor treatment. Newcastle disease virus (NDV) is an excellent oncolytic virus (OV) that has natural targeted infection properties for tumor cells. During the process of infection, replication, and release from cancer cells, oncolytic viruses interact with specific genes, proteins, or tissue environments of cancer, which may promote tumor growth and survival, making it possible for OVs to work synergistically with targeted therapies, which block tumor growth by interfering with specific molecules required for carcinogenesis and tumor growth. This process is due to the specific binding of the viral envelope protein hemagglutinin-neuraminidase (HN) to sialic acid highly expressed on the tumor surface, followed by fusion protein (F)-mediated fusion of the viral envelope with the tumor cell membrane, thereby delivering the viral genome into the host cell. At the same time, NDV envelope proteins have the properties of immune adjuvants, especially HN proteins, which have been shown to have good anti-tumor effects, mediating efficient lymphocyte proliferation responses, cytotoxic T lymphocyte (CTL) activity, and strong type I interferon responses. This will hopefully further amplify the therapeutic effects of pyroptosis, thereby overcoming the complex immune escape mechanisms of tumors.

[0005] In summary, since natural virus replication is uncontrollable and there is a risk of genetic material being inserted into the host genome, how to retain the targeted infection ability and adjuvant function of oncolytic viruses while avoiding the safety issues of natural viruses is the key to solving the problem. Summary of the invention

[0006] In view of the technical problems existing in the prior art, the purpose of the present invention is to provide a multifunctional oncolytic virus-mimicking nanomedicine and its preparation method and application, which can achieve efficient anti-tumor effects and reshape the immune microenvironment by synergizing the characteristics of pyroptosis and oncolytic viruses. The virus-mimicking vesicles provided by the present invention can retain the targeted infection ability and adjuvant function of oncolytic viruses while avoiding the safety issues of natural viruses.

[0007] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a multifunctional oncolytic virus-mimicking nanodrug, comprising a vesicle and a substance with pyroptosis inducing function; the vesicle is the shell of the multifunctional oncolytic virus-mimicking nanodrug; the substance with pyroptosis inducing function is loaded inside the shell; the vesicle is formed by hybridization of an engineered cell membrane that mimics the Newcastle disease virus envelope and a macrophage membrane; the multifunctional oncolytic virus-mimicking nanodrug achieves efficient anti-tumor effect by synergizing the characteristics of pyroptosis and oncolytic viruses.

[0008] The mechanisms of action include:

[0009] CCCP-mediated mitochondrial dysfunction prompts cancer cells to release ROS. ESIONPs-ADPS provides iron ions to amplify the CCCP-induced increase in ROS, ultimately inducing pyroptosis of melanoma cells through the ROS-Tom20-Caspase3-GSDME signaling pathway.

[0010] Preferably, the multifunctional oncolytic virus-mimicking nanodrug is prepared by hybridizing the oncolytic virus-mimicking cell membrane and the macrophage membrane, fusing the two cell membranes together to form a vesicle, and the substances (CCCP and ESIONPs) having pyroptosis-inducing function are loaded in the vesicle during membrane extrusion to obtain the multifunctional oncolytic virus-mimicking nanodrug.

[0011] Preferably, the hybridization comprises mixing the oncolytic virus-mimicking cell membrane and the macrophage membrane and then extruding the mixture through the membrane repeatedly.

[0012] Preferably, the substance having pyroptosis inducing function comprises extremely small iron oxide nanoparticles (ESIONPs-ADPS) and an oxidative phosphorylation uncoupler.

[0013] Preferably, the extremely small iron oxide nanoparticles are hydrophilic.

[0014] Preferably, the size of the extremely small iron oxide nanoparticles ranges from 3 to 5 nm;

[0015] Preferably, the oxidative phosphorylation uncoupler is carbonyl cyanide chlorophenylhydrazone (CCCP).

[0016] Preferably, the engineered cell membrane that mimics the Newcastle disease virus envelope comprises a 293T cell membrane that expresses the hemagglutinin-neuraminidase protein (HN) and fusion protein (F) of the Newcastle disease virus;

[0017] Preferably, the macrophage membrane comprises the cell membrane of RAW264.7 cells.

[0018] To achieve another object, the present invention also provides another technical solution, including a method for preparing the above-mentioned multifunctional oncolytic virus-mimicking nanodrug, which specifically includes the following steps:

[0019] S1. Construct an engineered cell line expressing oncolytic virus envelope protein, collect and lyse the cells, and obtain oncolytic virus-mimicking cell membranes;

[0020] S2. Cultivate macrophages, collect and lyse the cells, and obtain macrophage cell membranes;

[0021] S3. Synthesis of extremely small iron oxide nanoparticles (ESIONPs-ADPS);

[0022] S4. The oncolytic virus-like cell membrane and the macrophage membrane are mixed with a substance having a pyroptosis inducing function, and a multifunctional oncolytic virus-like nanomedicine is obtained by extruding the membrane. Through the hybridization of the oncolytic virus-like cell membrane and the macrophage membrane, that is, after the two cell membranes are mixed together, they are repeatedly extruded (more than 50 times of membrane extrusion), and the two cell membranes are fused together to form a vesicle, and the substance having a pyroptosis inducing function (CCCP and ESIONPs) is loaded in the vesicle during the membrane extrusion process.

[0023] The technical effects of the technical solution of the present invention are as follows:

[0024] 1. By adopting the technical solution of the present invention, the constructed hybrid oncolytic virus-mimicking cell membrane is used to simulate the oncolytic virus envelope, thereby improving the tumor targeting ability and cellular uptake efficiency of the loaded drug; in particular, the present invention hybridizes the oncolytic virus-mimicking cell membrane with the macrophage membrane to form a vesicle, and loads the substance with pyroptosis inducing function inside it as the shell of the drug, thereby prolonging the circulation time of the nanoparticles in the body, avoiding immune clearance caused by viral protein modification, and further improving the enrichment of the nanoparticles in the tumor.

[0025] 2. By adopting the technical solution of the present invention, a multifunctional oncolytic virus-mimicking nanomedicine is constructed, which is a nanomedicine that has both the tumor targeting characteristics and immune adjuvant function of oncolytic viruses, as well as the lytic and proinflammatory functions of pyroptosis. It reshapes the immune microenvironment by synergizing pyroptosis and oncolytic virus-mediated immune activation and proinflammatory response, thereby effectively inhibiting the growth of melanoma. The present invention provides a new combination strategy for targeted pyroptosis induction strategy and combined therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1a It is a schematic diagram of the preparation mechanism of the multifunctional oncolytic virus-mimicking nanomedicine prepared by the present invention.

[0027] Figure 1b It is a schematic diagram of the mechanism of inhibiting melanoma tumor growth by the multifunctional oncolytic virus-mimicking nanomedicine prepared by the present invention.

[0028] Figure 2 This is an immunofluorescence image verifying the expression of HN and F proteins on the cell membrane in the multifunctional oncolytic virus-mimicking nanomedicine obtained in Example 2 of the present invention.

[0029] Figure 3a and Figure 3b They are transmission electron micrographs of the extremely small iron oxide nanoparticles in the multifunctional oncolytic virus-mimicking nanomedicine and the multifunctional oncolytic virus-mimicking nanomedicine obtained in Example 2 of the present invention.

[0030] Figure 4This is the UV-visible absorption spectrum of the multifunctional oncolytic virus-mimicking nanodrug obtained in Example 2 of the present invention after being loaded with carbonyl cyanide chlorophenylhydrazone.

[0031] Figure 5 This is a fluorescence imaging image of the multifunctional oncolytic virus-mimicking nanomedicine obtained in Example 2 of the present invention in mice within 24 hours of injection.

[0032] Figure 6 It is a fluorescence image of the hybridized virus-simulated cell membrane and the unhybridized virus-simulated cell membrane obtained in Example 2 of the present invention in the blood of mice within 24 hours of injection.

[0033] Figure 7a-7c This is a result diagram of the multifunctional oncolytic virus-mimicking nanomedicine obtained in Example 2 of the present invention inducing pyroptosis of melanoma cells; wherein, Figure 7a It is the cell morphology after drug treatment; Figure 7b is the cell activity graph after drug treatment; Figure 7c This is the WB result of GSDME-N domain in melanoma cells after drug treatment.

[0034] Figure 8 This is a result diagram of the multifunctional oncolytic virus-mimicking nanomedicine obtained in Example 2 of the present invention inducing a type I interferon response; Figure 8 The horizontal axes 1-5 represent: 1-HVMNV@Fe-C; 2-VMNV@Fe-C; 3-ONV@Fe-C; 4-HVMNV5-ONV.

[0035] Figure 9a-9c This is a diagram showing the anti-tumor effect of the multifunctional oncolytic virus-mimicking nanomedicine obtained in Example 2 of the present invention. DETAILED DESCRIPTION

[0036] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0037] The present invention provides a multifunctional oncolytic virus-mimicking nanomedicine, in which an engineered cell membrane that imitates the Newcastle disease virus envelope is hybridized with a macrophage membrane to form a vesicle of the multifunctional oncolytic virus-mimicking nanomedicine as an outer shell; a substance with pyroptosis-inducing function is loaded inside the vesicle, and the obtained multifunctional oncolytic virus-mimicking nanomedicine achieves a highly efficient anti-tumor effect by synergizing the characteristics of pyroptosis and oncolytic viruses.

[0038] As a preferred embodiment, the engineered cell membrane that mimics the Newcastle disease virus envelope specifically refers to a 293T cell membrane that expresses the hemagglutinin-neuraminidase protein (HN) and fusion protein (F) of the Newcastle disease virus.

[0039] As a preferred embodiment, the macrophage membrane includes but is not limited to the cell membrane of RAW264.7 cells.

[0040] As a preferred embodiment, the engineered cell membrane that mimics the Newcastle disease virus envelope specifically refers to the 293T cell membrane that expresses the hemagglutinin-neuraminidase protein (HN) and fusion protein (F) of the Newcastle disease virus; the macrophage membrane includes but is not limited to the cell membrane of RAW264.7 cells.

[0041] As a preferred embodiment, the substance having pyroptosis inducing function specifically refers to hydrophilic extremely small iron oxide nanoparticles (ESIONPs-ADPS) and oxidative phosphorylation uncoupler.

[0042] As a preferred embodiment, the oxidative phosphorylation uncoupler includes carbonyl cyanide chlorophenylhydrazone (CCCP).

[0043] As a preferred embodiment, the size of the extremely small iron oxide nanoparticles is in the range of 3-5 nm.

[0044] As a preferred embodiment, see Figure 1a , provides a schematic diagram of the preparation mechanism of a multifunctional oncolytic virus-mimicking nanomedicine. As can be seen from the figure, the specific preparation method includes the following steps:

[0045] a. construct an engineered cell line expressing oncolytic virus envelope protein, collect and lyse the cells, and obtain oncolytic virus-mimicking cell membranes;

[0046] b. culturing macrophages, collecting and lysing the cells, and obtaining macrophage cell membranes;

[0047] c. Providing a substance having a pyroptosis-inducing function, wherein the substance having a pyroptosis-inducing function specifically refers to hydrophilic extremely small iron oxide nanoparticles (ESIONPs-ADPS) and oxidative phosphorylation uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP);

[0048] d. The above-mentioned oncolytic virus-mimicking cell membrane and macrophage membrane are mixed with a substance having pyroptosis inducing function, and a multifunctional oncolytic virus-mimicking nanodrug is obtained by extruding through the membrane.

[0049] As a preferred embodiment, the acquisition of the oncolytic virus-mimicking cell membrane in step (a) specifically includes the following steps:

[0050] (1) Insert the genes of HN and F proteins into the Plvx-puro vector;

[0051] (2) Obtaining a 293T engineered cell line that stably expresses HN and F proteins through lentiviral infection and drug resistance screening;

[0052] (3) collecting engineered 293T cells, washing them with phosphate buffer, and lysing the cells on ice using an ultrasonic cell disruptor;

[0053] (4) Collect cell membranes by centrifugation.

[0054] As a preferred embodiment, the power of cell lysis in step (3) is 10-30%, lysis is performed for 2-5 seconds, paused for 2-5 seconds, and lysis is continued for 5-10 minutes.

[0055] As a preferred embodiment, the solution for lysing cells in step (3) includes ddH2O containing 1× protease inhibitor and 0.5-2 mM EDTA.

[0056] As a preferred embodiment, in step (4), the centrifugation method is a double centrifugation method, including centrifugation at a speed of 1000-2000g for 5-10 minutes, collecting the supernatant, and centrifuging at a speed of 100,000g for 1-1.5 hours at 4°C to collect the precipitate to obtain white cell membrane fragments.

[0057] As a preferred embodiment, the acquisition of macrophage membrane in step (b) specifically comprises the following steps:

[0058] (1) culturing macrophages, collecting the cells, and washing them with phosphate buffered saline to remove residual culture medium;

[0059] (2) Lyse cells on ice using an ultrasonic cell disruptor.

[0060] (3) Collecting cell membranes by double centrifugation. The double centrifugation method refers to first centrifuging at a speed of 1000-2000 g for 5-10 minutes, collecting the supernatant, and then centrifuging at a speed of 100,000 g for 1-1.5 hours at 4°C to collect the precipitate, thereby obtaining milky white cell membrane fragments.

[0061] As a preferred embodiment, the power of cell lysis in step (2) and step (4) is 10-30%, lysis for 2-5 seconds, stop for 2-5 seconds, and continue lysis for 5-10 minutes.

[0062] As a preferred embodiment, the solution for lysing cells in step (2) and step (4) includes ddH2O containing 1× protease inhibitor and 0.5-2 mM EDTA.

[0063] As a preferred embodiment, the obtaining of the iron oxide nanoparticles ESIONPs-ADPS in step (c) specifically comprises the following steps:

[0064] (1) Synthesis of oleic acid-capped extremely small iron oxide nanoparticles (ESIONPs-OA) by thermal decomposition;

[0065] (2) Pentafluorophenol-modified ESIONPs (ESIONPs-PFP) were obtained by exchanging the surface ligands of ESIONPs-OA with 3,4-dihydroxyphenylacetic acid (DOPA-PFP) activated by pentafluorophenol;

[0066] (3) The ESIONPs-PFP obtained in step (2) is reacted with 3-((3-aminopropyl)dimethylamino)propane-1-sulfonic acid (ADPS) to obtain hydrophilic iron oxide nanoparticles (ESIONPs-ADPS).

[0067] As a preferred embodiment, the preparation of the multifunctional oncolytic virus-mimicking nanodrug in step (d) specifically includes the following steps: mixing the virus-mimicking cell membrane and the macrophage membrane in a PBS solution; adding carbonyl cyanide chlorophenylhydrazone while sonicating, and removing the drug precipitate by low-speed centrifugation at 1000g; adding extremely small iron oxide nanoparticles to the supernatant after centrifugation, mixing evenly, and extruding through a porous membrane, and finally collecting the brown precipitate by centrifugation to obtain the multifunctional oncolytic virus-mimicking nanodrug.

[0068] As a preferred embodiment, in step (1), the virus-mimicking cell membrane and the macrophage cell membrane are mixed according to a protein mass ratio, and the mixing ratio is greater than or equal to 1:1.

[0069] As a preferred embodiment, in step (1), 0.5-1 mg of viral cell membrane and 0.5-1 mg of macrophage cell membrane are used.

[0070] As a preferred embodiment, the pore size of the polycarbonate porous membrane is 0.1-0.8 μm.

[0071] As a preferred embodiment, the porous membrane is a polycarbonate porous membrane.

[0072] As a preferred embodiment, the mixture is extruded through a polycarbonate porous membrane at least 50 times.

[0073] As a preferred embodiment, the centrifugation method includes collecting the brown precipitate through a centrifuge with a centrifugal force of 8000g.

[0074] As a preferred embodiment, the preparation of the multifunctional oncolytic virus-mimicking nanodrug in step (d) specifically includes the following steps: first dissolving carbonyl cyanide chlorophenylhydrazone into a solution, then adding the simulated virus cell membrane, macrophage membrane, carbonyl cyanide chlorophenylhydrazone solution and extremely small iron oxide nanoparticles into a PBS buffer solution and mixing them evenly, and then extruding through a polycarbonate porous membrane and collecting the brown precipitate by centrifugation to obtain the multifunctional oncolytic virus-mimicking nanodrug.

[0075] As a preferred embodiment, the dissolution of carbonyl cyanide chlorophenylhydrazone includes dissolving carbonyl cyanide chlorophenylhydrazone in DMSO-4PEG300-Tween-80-normal saline in sequence; preferably, the mass fractions of each solvent are 10% DMSO, 40% PEG300, 5% Tween-80, and 45% normal saline.

[0076] As a preferred embodiment, the addition ratio of each component is: 0.5-1 mg of virus-like cell membrane, 0.5-1 mg of macrophage membrane, 50-100 mM of carbonyl cyanide chlorophenylhydrazone and 50-100 μg of extremely small iron oxide nanoparticles.

[0077] As a preferred embodiment, the pore size of the polycarbonate porous membrane is 0.1-0.8 μm.

[0078] As a preferred embodiment, the mixture is extruded through a polycarbonate porous membrane at least 50 times.

[0079] As a preferred embodiment, the centrifugation in step (2) includes collecting the brown precipitate in a centrifuge with a centrifugal force of 8000 g.

[0080] Multifunctional oncolytic virus-mimicking nanomedicine prepared by the above technical solution

[0081] See also Figure 1b, which is a schematic diagram of the mechanism of the multifunctional oncolytic virus-mimicking nanomedicine provided by the present invention to inhibit the growth of melanoma tumors. Functional virus-mimicking nanomedicines can simulate the infection mode of Newcastle disease virus to deliver the loaded pyroptosis inducer to tumor cells in a targeted manner. Combined with the diagram, it is specifically manifested as follows: the virus-mimicking vesicles bind to the sialic acid receptors highly expressed on the surface of tumor cells through the surface-modified HN protein, and then deliver the loaded extremely small iron oxide nanoparticles (ESIONPs-ADPS) and carbonyl cyanide chlorophenylhydrazone (CCCP) into the tumor cells through membrane fusion. CCCP, as an oxidative phosphorylation uncoupler, can mediate mitochondrial dysfunction in tumor cells and release ROS signals. The iron ions provided by ESIONPs-ADPS can amplify the ROS signal induced by CCCP, thereby activating Caspase-3 protein, which can cleave GSDME, a key executor protein of pyroptosis, to expose the active GSDME-N domain, thereby inducing pyroptosis of tumor cells and releasing tumor-associated antigens and pro-inflammatory factors; HN protein exposed on the surface of tumor cells can mediate type I interferon response, thereby amplifying the anti-tumor effect caused by pyroptosis and reshaping the tumor immunosuppressive microenvironment.

[0082] The technical solution of the present invention is further described in detail below through specific embodiments.

[0083] Example 1

[0084] A method for obtaining a virus-like cell membrane based on an engineered cell line, specifically, the preparation comprises the following steps:

[0085] (1) Molecular cloning: The complete gene sequences including the transmembrane region, intracellular region, and extracellular region of the HN and F proteins were synthesized and ligated into the lentiviral vector pLVX-IRES-Puro; the obtained vectors containing the target sequences were named pLVX-HN and pLVX-F.

[0086] (2) Construction of stable cell lines: A three-plasmid system mediated by the lentiviral vector pLVX-IRES-Puro was used to construct a cell line with high expression of HN and F proteins. The system includes a transfer plasmid, a packaging plasmid, and an envelope plasmid.

[0087] When the degree of fusion of human renal epithelial cells (293T cells) reaches 70% to 80%, pLVX-HN (6 μg), pLVX-F (6 μg), Pspax2 (8 μg) and pMD2.G (2 μg) are respectively added to centrifuge tube A, and then 200 μL Opti-MEM is added and allowed to stand; at the same time, the transfection reagent Lipofectamine 2000 is placed in centrifuge tube B containing Opti-MEM and allowed to stand for 5 minutes. Centrifuge tubes AB are mixed and allowed to stand for 30 minutes to obtain a transfection complex.

[0088] Then, the culture medium of 293T cells was replaced with serum-free DMEM (Gibco), and the transfection complex was added for virus packaging. At 6 h of packaging, the complete culture medium (DMEM containing 10% fetal bovine serum) was replaced; at 8 h, the virus supernatant was collected, centrifuged at 2000 g for 10 min, the supernatant was taken, and the lentivirus was collected for subsequent lentiviral infection.

[0089] 293T cells were inoculated in a 60 mm culture dish until the cell confluence reached 70%. The pseudovirus collected in the above step was mixed with serum-free DMEM medium at a ratio of 1:1 (v / v), and then the infection-assisting agent polybrene was added to make the final concentration 10 μg / mL, and then added to the 293T cells after mixing, and replaced with complete medium (DMEM containing 10% fetal bovine serum) after 12 hours. After 48 hours, resistance screening was carried out for two consecutive weeks, and puromycin (final concentration of 6 μg / mL) was used to maintain resistance screening for two weeks. Among them, 293T cells successfully transfected with HN and F genes have puromycin resistance genes and can survive in the puromycin environment.

[0090] (3) Detection of HN and F protein expression in engineered cell lines: To determine whether HN and F proteins are expressed on the cell membrane, the transfected 293T cells were fixed with cold ethanol. After 10 min, the cells were blocked with 5% skim milk solution for 1 h. Mouse anti-HN protein monoclonal antibody and chicken anti-F protein polyclonal antibody were added respectively and incubated at 37°C for 1 h. After washing three times with PBST, Alexa Fluor 488-labeled goat anti-mouse IgG and Alexa Fluor Goat anti-chicken IgY labeled with 647 was stained for 30 min at 37° C. After washing three times with PBST, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature, and the cells were observed using a confocal microscope after 10 min.

[0091] Results Figure 2 ,The results of confocal microscopy showed that HN and F proteins were successfully expressed on the 293T cell membrane.

[0092] (4) Isolation of cell membrane: In order to obtain engineered virus-like cell membranes and macrophage cell membranes, HN-F engineered cells and RAW264.7 cells were washed three times with cold PBS. The cells were resuspended in ddH2O containing 1× protease inhibitors (including 20mM AEBSF, 3μM Aprotinin, 1.3mM Bestatin, 0.14mM E64 and 0.1mM Leupeptin) and 1mM EDTA. Then, the two types of cells were crushed on ice using an ultrasonic cell crusher with a crushing power of 20%, crushing for 5s and stopping for 5s, and continuing to crush for 5min. Centrifuge at 2000g for 5min and collect the supernatant. Subsequently, centrifuge at 100,000g for 1h at 4°C, collect the precipitate, and obtain white cell membrane fragments. Resuspend the precipitate in sterile PBS and then store at -80°C.

[0093] Example 2

[0094] This embodiment provides a preparation and characterization of a multifunctional oncolytic virus-mimicking nanomedicine, which specifically includes the following steps:

[0095] The virus-like cell membrane (1 mg) and macrophage membrane (1 mg) prepared in Example 1 were mixed in a PBS solution at a mass ratio of 1:1, and then 100 mM CCCP and 100 μg iron oxide nanoparticles (ESIONPs-ADPS) were added to the membrane. The mixture was repeatedly co-extruded more than 50 times using a small extruder and passed through 0.8, 0.4, and 0.2 μm polycarbonate porous membranes in sequence.

[0096] The mixture was repeatedly extruded through a membrane to hybridize the engineered cell membrane that mimics the Newcastle disease virus envelope with the macrophage membrane, and CCCP and iron oxide nanoparticles were loaded into the hybrid virus-mimicking vesicles.

[0097] The morphology of nanoparticles was observed by transmission electron microscopy, and the loading of nanodrugs on CCCP was detected by UV-visible spectrophotometer.

[0098] The preparation method of iron oxide nanoparticles (ESIONPs-ADPS) includes:

[0099] (1) Oleic acid-capped extremely small iron oxide nanoparticles (ESIONPs-OA) were synthesized by thermal decomposition; the preparation method can be referred to the method in the prior art, Y. Cao et al., Nanoscale, 12 (2020), 17502-17516.

[0100] (2) Pentafluorophenol-modified ESIONPs (ESIONPs-PFP) were obtained by exchanging the surface ligands of ESIONPs-OA with 3,4-dihydroxyphenylacetic acid (DOPA-PFP) activated by pentafluorophenol;

[0101] The specific steps include: dissolving ESIONPs-OA (30 mg) in 45 mL tetrahydrofuran (THF), adding DOPA-PFP (400 mg, 1.2 mmol), reacting at 50° C. overnight under nitrogen protection, and washing with excess dimethyl sulfoxide as a precipitant to obtain ESIONPs-PFP.

[0102] (3) Dissolve ESIONPs-PFP (7.5 mg) in 5 mL DMSO, weigh the amino-containing zwitterionic small molecule ADPS (32.4 mg, 0.2 mmol) and dissolve it in 5 mL DMSO, mix the two solutions, add DIPEA (300 μL), and stir at room temperature for 48 h. After the reaction, add 10 mL of deionized water and continue stirring for 24 h. After 3 days of dialysis, filter through a 0.45 μm filter membrane to obtain hydrophilic ESIONPs-ADPS.

[0103] See also Figure 3a , which is the result of observing the morphology of ESIONPs-ADPS by transmission electron microscopy. As can be seen from the figure, ESIONPs-ADPS is uniform, monodisperse spherical, and the average diameter is about 3nm.

[0104] See also Figure 3b , Figure 3b The morphology of the multifunctional oncolytic virus-mimicking nanomedicine HVMNV@Fe-C is shown; as can be seen from the figure, the average diameter of HVMNV@Fe-C formed by co-extrusion with the engineered cell membrane of the Newcastle disease virus envelope and the macrophage membrane increases to 246nm. As can be seen from the figure, ESIONPs-OA and CCCP are dispersed in the vesicles formed by the hybridization of the engineered cell membrane of the Newcastle disease virus envelope and the macrophage membrane.

[0105] See also Figure 4 , is the UV-visible absorption spectrum of the multifunctional oncolytic virus-mimicking nanodrug loaded with CCCP. Compared with HVMNV, HVMNV@Fe-C shows an absorption peak similar to CCCP at 380nm, proving that CCCP is successfully loaded into the nanodrug.

[0106] Example 3

[0107] Using the nanomedicine prepared from untransfected 293T cell membrane as a control, the targeting of the oncolytic virus-mimicking nanomedicine to melanoma in vivo was investigated as follows:

[0108] The 293T cell membrane-coated nanodrug (ONV@Fe-C) and the oncolytic virus-mimicking membrane-coated nanodrug (HVMNV@Fe-C) were labeled with Did fluorescent probes and injected into tumor-bearing mice through the tail vein. In vivo imaging of the mice was performed at 2h, 4h, 6h, 8h, 10h, and 24h after injection.

[0109] See also Figure 5 , showing the enrichment of the two nanodrugs in the tumor site of mice. Compared with ONV@Fe-C, HVMNV@Fe-C was effectively enriched in the tumor site.

[0110] Example 4

[0111] The effect of macrophage hybridization on the in vivo circulation time of nanoparticles was investigated, including the following steps:

[0112] Preparation of nanovesicles (VMNV): The viral cell membrane (1 mg) prepared in Example 1 was repeatedly co-extruded more than 50 times using a small extruder, and passed through 0.8, 0.4, and 0.2 μm polycarbonate porous membranes in turn to obtain nanovesicles (VMNV) prepared by simulating viral cell membranes.

[0113] Preparation of hybrid nanovesicles (HVMNV): The virus-mimicking cell membrane (1 mg) and macrophage membrane (1 mg) prepared in Example 1 were mixed in a PBS solution at a mass ratio of 1:1. The mixture was repeatedly co-extruded more than 50 times using a small extruder, and passed through 0.8, 0.4, and 0.2 μm polycarbonate porous membranes in turn to obtain hybrid nanovesicles (HVMNV) prepared by mixing the virus-mimicking cell membrane and the macrophage membrane.

[0114] The nanovesicles prepared by mimicking viral cell membrane (VMNV) and hybrid nanovesicles prepared by mixing mimicking viral cell membrane and macrophage membrane (HVMNV) were labeled with Did fluorescent probe (DiD cell membrane red fluorescent probe, brand: Thermo Fisher), and injected into mice through the tail vein, and then the tail vein blood was drawn at 5min, 2h, 4h, 6h, 8h, 10h, 12h, and 24h after the injection to measure the fluorescence intensity in the blood.

[0115] Results Figure 6 , 6h to 24h after injection, the fluorescence signal of HVMNV / Did was significantly higher than that of VMNV / Did. This proves that the hybridization of the virus-mimicking cell membrane and the macrophage membrane can avoid immune clearance caused by viral protein modification, thereby prolonging the circulation time of nanoparticles in the blood.

[0116] Example 5

[0117] This example uses human melanoma cells A375 as a model to investigate the ability of the oncolytic virus-mimicking nanomedicine obtained in Example 2 to induce tumor cell pyroptosis. The specific rubbing method includes the following steps:

[0118] The specific steps are as follows: A375 cells were inoculated into a 96-well plate and cultured in a 37°C CO2 incubator overnight. 100 ng (based on the mass of iron nanoparticles) of the virus-mimicking nanodrug HVMNV@Fe-C was added to each well, and the same volume of PBS was used as a negative control. After culturing in a 37°C CO2 incubator for 8 hours, the cell state was observed under a microscope. The cell activity after treatment was detected by CCK-8, and the cleavage of the pyroptosis execution protein GSDME was detected by WB. The test results are shown in Figure 7a-7c .

[0119] Figure 7a The cell morphology after the nano drug treatment is shown, and the cells show typical pyroptosis characteristics such as bubbling and rupture. The results show that the virus-mimicking nano drug prepared by the present invention can effectively induce pyroptosis of melanoma cells.

[0120] Figure 7b The cell survival rate after nanodrug treatment is shown, and the results indicate that the viral-mimicking nanodrug co-loaded with ESIONPs and CCCP can effectively kill melanoma cells.

[0121] Figure 7c The cleavage of GSDME after nanodrug treatment is shown, proving that the viral-mimicking nanodrug can cause the cleavage of GSDME and expose the active N-terminal domain.

[0122] Example 6

[0123] This example is an example of investigating the ability of the oncolytic virus-mimicking cell membrane obtained in Example 2 to induce type I IFN response. The specific investigation method includes the following steps:

[0124] PMBCs were isolated from the serum of C57BL / 7 mice (6 weeks), and A375 cells were treated with hybrid virus-mimicking cell membrane-coated nanodrugs HVMNV@Fe-C, virus-mimicking cell membrane-coated nanodrugs VMNV@Fe-C, unmodified 293T cell membrane-coated nanodrugs ONV@Fe-C, single hybrid virus-mimicking cell membrane HVMNV, and PBS for 12 h. Subsequently, tumor cells pretreated with different nanodrugs were incubated with fresh PMBCs for another 24 h. The supernatants of the five groups of cells were collected, and the IFN-α levels in the supernatants were measured using a commercial mouse IFN-α ELISA kit.

[0125] Results Figure 8, a single hybrid virus-like cell membrane HVMNV can effectively stimulate PMBCs to secrete IFN-α, proving the adjuvant function of the virus-like vesicles. When HVMNV@Fe-C was used, the IFN-α level was further improved, proving that pyroptosis and the adjuvant function of the virus-like vesicles have a synergistic amplification effect.

[0126] Example 7

[0127] This example is an evaluation of the anti-tumor effectiveness of the virus-mimicking nanomedicine obtained in Example 2. The specific evaluation method is as follows:

[0128] A375 cells were inoculated subcutaneously on the back of mice to establish a melanoma model. The mice were divided into 5 groups as follows:

[0129] a. Normal saline control group (PBS);

[0130] b. Very small iron oxide nanoparticles and CCCP control group (Free Fe-C);

[0131] c. The control group of nanodrugs coated with unmodified 293T cell membranes (ONV@Fe-C);

[0132] d. Nanodrug group coated with virus-like cell membrane (VMNV@Fe-C);

[0133] e. Nanodrug group coated with macrophage hybrid virus-mimicking cell membrane (HVMNV@Fe-C).

[0134] The above five groups of models were injected with the corresponding drugs through the tail vein, once every three days, for a total of three treatments. The tumor size was measured every two days. The tumor volume was calculated according to the formula L×W2 / 2, where L and W represent the length and width of the tumor, respectively. Tumor tissue was collected on the 35th day after treatment.

[0135] See also Figure 9a , the figure shows the tumor anatomy after 35 days of treatment,

[0136] See also Figure 9b , The figure shows the tumor growth of different treatment groups.

[0137] See also Fig.9c , The figure shows the curves of tumor growth volume changes over time in different control groups.

[0138] Depend on Figure 9a-9c The results shown clearly confirm that the multifunctional oncolytic virus-mimicking nanodrug HVMNV@Fe-C can effectively inhibit the growth of melanoma.

[0139] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or by being able to achieve the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A multifunctional oncolytic virus-mimicking nanomedicine, characterized in that: Including HVMNV@Fe-C morphologies formed by vesicles and substances with pyroptosis-inducing function; The vesicle is the shell of the multifunctional oncolytic virus-mimicking nanomedicine; the substance with pyroptosis inducing function is loaded inside the shell; The vesicle is formed by hybridization of an engineered cell membrane that mimics the Newcastle disease virus envelope and a macrophage membrane; The multifunctional oncolytic virus-mimicking nanomedicine achieves tumor killing, immune activation and tumor targeting by synergizing the characteristics of pyroptosis and oncolytic viruses.

2. The multifunctional oncolytic virus-mimicking nanomedicine according to claim 1, characterized in that: The multifunctional oncolytic virus-mimicking nanomedicine is prepared by hybridizing the oncolytic virus-mimicking cell membrane and the macrophage cell membrane, and fusing the two cell membranes together through extrusion through the membrane to form a vesicle; the substances (CCCP and ESIONPs) having pyroptosis inducing function are loaded in the vesicle during the extrusion through the membrane to obtain the multifunctional oncolytic virus-mimicking nanomedicine; And / or, the hybridization includes mixing the oncolytic virus-mimicking cell membrane and the macrophage membrane and then extruding the mixture through the membrane repeatedly to form the vesicle.

3. The multifunctional oncolytic virus-mimicking nanomedicine according to claim 2, characterized in that: The substance having pyroptosis inducing function includes extremely small iron oxide nanoparticles (ESIONPs-ADPS) and an oxidative phosphorylation uncoupler; and / or, the extremely small iron oxide nanoparticles are hydrophilic; and / or, the size of the extremely small iron oxide nanoparticles ranges from 3 to 5 nm; And / or, the oxidative phosphorylation uncoupler is carbonyl cyanide chlorophenylhydrazone (CCCP); And / or, the engineered cell membrane that mimics the Newcastle disease virus envelope comprises a 293T cell membrane that expresses the hemagglutinin-neuraminidase protein (HN) and fusion protein (F) of the Newcastle disease virus; And / or, the macrophage cell membrane comprises a cell membrane of a RAW264.7 cell.

4. A method for preparing a multifunctional oncolytic virus-mimicking nanodrug according to any one of claims 1 to 3, characterized in that: The specific steps include: S1. Construct an engineered cell line expressing oncolytic virus envelope protein, collect and lyse the cells, and obtain oncolytic virus-mimicking cell membranes; S2. Cultivate macrophages, collect and lyse the cells, and obtain macrophage cell membranes; S3. Synthesis of extremely small iron oxide nanoparticles (ESIONPs-ADPS); S4. The oncolytic virus-mimicking cell membrane and the macrophage cell membrane are mixed with a substance having a pyroptosis-inducing function, and a multifunctional oncolytic virus-mimicking nanomedicine is obtained by extruding the mixture through a membrane.

5. The method for preparing the multifunctional oncolytic virus-mimicking nanomedicine according to claim 4, characterized in that: In S1, the method for preparing the oncolytic virus-mimicking cell membrane comprises the following steps: (1) Insert the genes of HN and F proteins into the Plvx-puro vector; (2) Obtaining a 293T engineered cell line that stably expresses HN and F proteins through lentiviral infection and drug resistance screening; (3) The engineered 293T cells were collected, washed with phosphate buffer, and lysed on ice using an ultrasonic cell disruptor; (4) collecting cell membrane fragments by centrifugation, namely the oncolytic virus-mimicking cell membrane; Wherein, in step (3), the power of cell lysis is 10-30%, lysis is continued for 2-5 seconds after lysis, and lysis is continued for 5-10 minutes. The lysis solution is ddH2O containing 1× protease inhibitor and 0.5-2mM EDTA; And / or, in step (4), the centrifugation is a double centrifugation method, including centrifugation at a speed of 1000-2000g for 5-10 minutes, collecting the supernatant, and centrifuging at a speed of 100,000g for 1-1.5 hours at 4°C to collect the precipitate to obtain white cell membrane fragments.

6. The method for preparing the multifunctional oncolytic virus-mimicking nanomedicine according to claim 4, characterized in that: In S2, the method for preparing the macrophage membrane specifically comprises the following steps: (1) culturing macrophages, collecting the cells, and washing them with phosphate buffered saline to remove residual culture medium; (2) Lyse cells on ice using an ultrasonic cell disruptor; (3) Collecting cell membranes by centrifugation; Wherein, in step (2), the lysis efficiency of the cells is 10-30%, the lysis is performed for 2-5 seconds, the lysis is stopped for 2-5 seconds, and the lysis is continued for 5-10 minutes. The lysis solution is ddH2O containing 1× protease inhibitor and 0.5-2 mM EDTA; And / or, the protease inhibitor specifically comprises 20 mM AEBSF, 3 μM Aprotinin, 1.3 mM Bestatin, 0.14 mM E64 and 0.1 mM Leupeptin; And / or, in step (3), the centrifugation method is a double centrifugation method, including centrifugation at a speed of 1000-2000g for 5-10 minutes, collecting the supernatant, and centrifuging at a speed of 100,000g for 1-1.5 hours at 4°C to collect the precipitate to obtain milky white cell membrane fragments.

7. The method for preparing the multifunctional oncolytic virus-mimicking nanomedicine according to claim 4, characterized in that: In S3, the method for preparing the extremely small iron oxide nanoparticles specifically comprises: (1) Synthesis of oleic acid-capped extremely small iron oxide nanoparticles (ESIONPs-OA) by thermal decomposition; (2) Pentafluorophenol-modified ESIONPs (ESIONPs-PFP) were obtained by exchanging the surface ligands of ESIONPs-OA with 3,4-dihydroxyphenylacetic acid (DOPA-PFP) activated by pentafluorophenol; (3) ESIONPs-PFP reacts with 3-((3-aminopropyl)dimethylamino)propane-1-sulfonic acid (ADPS) to obtain hydrophilic ESIONPs-ADPS.

8. The method for preparing the multifunctional oncolytic virus-mimicking nanomedicine according to any one of claims 4 to 7, characterized in that: In S4, the preparation method of the multifunctional oncolytic virus-mimicking nanomedicine specifically comprises: mixing the virus-mimicking cell membrane and the macrophage membrane in a PBS solution; adding carbonyl cyanide chlorophenylhydrazone while sonicating, and removing the drug precipitate by centrifugation; finally, adding extremely small iron oxide nanoparticles to the supernatant after centrifugation, mixing evenly, and extruding through a porous membrane, and finally collecting the brown precipitate by centrifugation, which is the multifunctional oncolytic virus-mimicking nanomedicine; Or, in S4, the preparation method of the multifunctional oncolytic virus-mimicking nanodrug specifically includes the following steps: first dissolving carbonyl cyanide chlorophenylhydrazone to prepare a carbonyl cyanide chlorophenylhydrazone solution, then adding the simulated virus cell membrane, macrophage membrane, carbonyl cyanide chlorophenylhydrazone solution and hydrophilic extremely small iron oxide nanoparticles into a PBS buffer solution and mixing them evenly, and extruding them through a porous membrane, and finally collecting the brown precipitate by centrifugation to obtain the multifunctional oncolytic virus-mimicking nanodrug; wherein the preparation of the carbonyl cyanide chlorophenylhydrazone solution includes dissolving carbonyl cyanide chlorophenylhydrazone in DMSO-PEG300-Tween-80-physiological saline in sequence.

9. The method for preparing the multifunctional oncolytic virus-mimicking nanomedicine according to claim 8, characterized in that: The virus-mimicking cell membrane and the macrophage cell membrane are mixed according to a mass ratio of protein, and the mixing ratio is greater than or equal to 1:1; and / or, the porous membrane comprises a polycarbonate porous membrane; And / or, the pore size of the polycarbonate porous membrane is 0.1 to 0.8 μm; and / or, the mixture is extruded through a polycarbonate porous membrane for ≥50 times; And / or, the centrifugation method includes collecting the brown precipitate through a centrifuge with a centrifugal force of 8000g; And / or, the amount of the virus-mimicking cell membrane added is 0.5-1 mg; and / or, the amount of the macrophage membrane added is 0.5-1 mg; And / or, the amount of carbonyl cyanide chlorophenylhydrazone added is 50-100 mM; And / or, the added amount of the extremely small iron oxide nanoparticles is 50-100 μg.

10. Use of the multifunctional oncolytic virus-mimicking nanomedicine as described in any one of claims 1 to 3 in inhibiting the growth of melanoma; improving the targeting of nanomedicine to tumors through virus-mimicking modification, and synergizing pyroptosis and oncolytic virus-mediated immune activation and pro-inflammatory response to reshape the immune microenvironment.

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