Multiple cell membrane-mediated biomimetic nano / gene delivery platform, preparation method thereof, and pharmaceutical application targeting colon cancer

Abstract

The present invention discloses a multi-cell membrane-mediated biomimetic nano / gene delivery platform, its preparation method, and its pharmaceutical application for colon cancer targeting, belonging to the field of biomedicine. Zinc oxide nanoparticles and a miR21 antagonist are self-assembled in vitro to prepare a NPs nanocomplex. The NPs nanocomplex is then assembled with colon cancer cell membranes and macrophage membranes to construct a double-membrane-camouflaged biomimetic nano / gene delivery platform (M@NPs). This delivery platform can be used for in vivo delivery of gene drugs without the mediation of viral vectors. Based on the principles of homologous targeting mediated by tumor cell membranes and immune escape mediated by macrophage membranes, it achieves targeted delivery of nano / gene drugs for colon cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a multi-cell membrane-mediated biomimetic nano / gene delivery platform, a preparation method thereof, and pharmaceutical applications targeting colon cancer. Background Art

[0002] Gene therapy involves the use of genetic engineering methods to introduce exogenous genes, such as siRNA, shRNA, miRNA, or DNA vectors, into diseased cells in an effort to correct disease-causing genes through gene editing, gene interference, or gene modification, thereby achieving disease treatment. The prerequisite for achieving gene therapy is the delivery of gene therapy to cells. Different delivery vehicles exhibit varying efficiencies, which directly impact the effectiveness of gene therapy. Viruses naturally carry genetic material into cells. Modified viruses can be used as gene delivery vehicles to carry therapeutic genes (drugs) into cells. In addition to viruses, bacterial vectors are another commonly used gene delivery vehicle and are widely used for the in vivo delivery of small RNAs. These therapeutic genes (drugs) are encapsulated in modified bacteria in vitro, enabling in vivo delivery of gene therapy. Although viruses and bacteria have been engineered to possess excellent gene delivery capabilities, safety concerns associated with these viruses and bacteria remain to be addressed, significantly limiting their application in clinical research. In recent years, with the development of nanotechnology, an increasing number of nanobiomaterials, such as chitosan, PEI, PEG, and other polycationic materials, have been used for the delivery of gene drugs. Although these nanomaterials have higher gene delivery capabilities, due to the toxic side effects of polycationic complexes and the limitations of single-functional nanomaterials in tumor treatment applications, the development of multifunctional nanomaterials that not only have efficient gene delivery capabilities but can also effectively promote tumor cell apoptosis for tumor treatment is urgently needed.

[0003] Zinc oxide (ZnO) nanoparticles are widely used in tumor therapy research due to their small size and large surface area. Their zinc ions can induce ROS production, causing oxidative stress and promoting apoptosis in tumor cells, but they exhibit good biocompatibility with non-tumor cells. Therefore, by combining the inherent tumor-killing potential of ZnO nanoparticles with miRNAs that promote tumor cell apoptosis, a multifunctional anti-tumor nanoplatform has been constructed, which has important clinical applications for the treatment of colon cancer.

[0004] In vivo application of gene therapy must first overcome biological barriers. The clearance of exogenous substances by various degradative enzymes and immune cells in the body reduces the efficiency of nano-drug delivery systems. Researchers have attempted to improve their in vivo efficiency by physicochemically modifying nano-drug delivery systems through various methods. Inspired by the ability of native cells to avoid immune clearance, the development of pseudo-"cell-like" nano-platforms coated with cell membranes for in vivo delivery of gene-based drugs holds great promise. Cancer cells possess immune evasion capabilities due to the presence of antigens and immune adjuvants on their surface. Furthermore, leveraging the principles of homologous targeting of tumor cells and immune evasion mediated by macrophage membranes, the development of nano- / gene-based drug delivery systems camouflaged with both colon cancer cell and immune cell membranes has not only effectively avoided clearance but also achieved targeted drug delivery, improving the drug delivery efficiency of the delivery system and ultimately enabling its application in the treatment of colon cancer. Studies have shown that miR21 expression is upregulated in a variety of solid malignancies and hematological tumors. Numerous studies have confirmed that elevated miR21 expression is associated with poor prognosis in cancer patients. The mechanism of tumorigenesis by miR21 has been widely studied. It regulates various genes related to the pathogenesis of multiple tumors, including cell proliferation, invasion, migration, apoptosis, and resistance to chemotherapy.

[0005] However, currently, most reports focus on drug delivery platforms disguised as single cell membranes, such as red blood cell membranes for enhanced sustained drug release, tumor cell membranes for increased homologous targeting, and immune cell membranes for enhanced immune escape. However, these single drug delivery platforms often only meet one of these criteria, failing to address the dual or multifunctional effects of enhancing both homologous targeting and immune escape, significantly limiting the application of biomimetic nano-drug delivery platforms. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a multi-cell membrane-mediated biomimetic nano / gene delivery platform and its preparation method and pharmaceutical application targeting colon cancer, which can solve the technical difficulties that the prior art drug delivery platform cannot take into account the dual functions of both homologous targeting and improving immune escape.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses a multi-cell membrane-mediated biomimetic nano / gene delivery platform, which is assembled from nano zinc oxide particles, miRNA21 antagonists, colon cancer cell membranes and macrophage (Raw264.7) membranes.

[0009] Preferably, nano zinc oxide particles and miRNA21 antagonists are self-assembled in vitro to form NPs nanocomplexes, and then the NPs nanocomplexes are assembled with colon cancer cell membranes and macrophage membranes to construct a multiple cell membrane-mediated biomimetic nano / gene delivery platform M@NPs.

[0010] Preferably, the colon cancer cell membrane is MC38 tumor cell membrane.

[0011] Preferably, the miRNA21 antagonist is mmu-miR-21a-5pantagomir, which is synthesized by RiboBio, and its nucleotide sequence is shown in SEQ ID NO: 1. It is a miR21 inhibitor with strong stability.

[0012] Preferably, the volume ratio of colon cancer cell membrane to macrophage membrane is 2:1.

[0013] Preferably, the volume ratio of the nano zinc oxide particles to the miRNA21 antagonist is 4:1.

[0014] The specific preparation was as follows: 12 μL of ZnO (2 μg / μL) and 3 μL of miRNA21 (25 μM) were mixed in a volume ratio (v / v = 4:1) and sonicated for 30 seconds at t100 amplitude (Q700, Qsonica, USA). After standing at room temperature for 5 minutes, 15 μL of cell membrane (300 μg / mL) was added and allowed to stand at room temperature for 20 minutes.

[0015] The present invention also discloses a method for preparing the above-mentioned multiple cell membrane-mediated biomimetic nano / gene delivery platform, comprising:

[0016] 1) Synthesis of ZnO nanoparticles;

[0017] 2) ZnO nanoparticles and miRNA21 antagonists were self-assembled in vitro to form NPs nanocomplexes;

[0018] 3) The NPs nanocomplex was assembled with the colon cancer cell membrane and macrophage membrane to construct a multi-cell membrane-mediated biomimetic nano / gene delivery platform.

[0019] Preferably, in step 1), ZnONPs nanoparticles are synthesized by the following specific operations:

[0020] Dissolve Zn(OAc)2·2H2O and NaOH in deionized water to obtain Zn(OAc)2·2H2O solution and NaOH solution, respectively; fully mix the Zn(OAc)2·2H2O solution and the NaOH solution; collect the precipitate, wash, and dry it to obtain a ZnO precursor;

[0021] The ZnO precursor was continuously heated at 250°C for 3 hours to obtain ZnO nanoparticles with uniform particle size. The ZnO nanoparticles were modified with PEI to prepare ZnONPs nanoparticles.

[0022] Preferably, in step 2), ZnO nanoparticles and miRNA21 antagonists are mixed in a volume ratio of 4:1, and then ultrasonically treated at an amplitude of 100 for 90 seconds (Q700, Qsonica, USA) to assemble into ZnO / miR21 NPs. After standing at room temperature for 5 minutes, the cell membrane is added at a volume ratio of NPs to cell membrane of 1:1 and allowed to stand at room temperature for 20 minutes.

[0023] The present invention also discloses the application of the above-mentioned multiple cell membrane-mediated bionic nano / gene delivery platform in the preparation of anti-colon cancer drugs.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The multi-cell membrane-mediated biomimetic nano / gene delivery platform disclosed in this invention utilizes in vitro self-assembly of biomaterial zinc oxide nanoparticles with a miR21 antagonist to prepare a NPs nanocomplex. The NPs nanocomplex is then assembled with colon cancer cell membranes and macrophage membranes to create a dual-membrane-camouflaged biomimetic nano / gene delivery platform, M@NPs. This delivery platform, without the need for viral vectors, can be used for in vivo delivery of gene drugs. Based on the principles of homologous targeting mediated by tumor cell membranes and immune escape mediated by macrophage membranes, it achieves targeted delivery of nano / gene drugs for colon cancer treatment. This invention utilizes the dual membrane camouflage of tumor cells and macrophages (immune cells) to mitigate immune clearance caused by the nano-drug delivery platform. Furthermore, by leveraging the homologous targeting properties of tumor cells and immune escape properties of immune cells, it enhances drug utilization and reduces immune clearance.

[0026] Through experimental verification, the bionic nano / gene delivery platform M@NPs of the present invention can effectively promote the targeting of gene drugs to tumor tissues, and can also effectively inhibit the growth of tumor cells and promote tumor cell apoptosis. In addition, the "pseudo-tumor drug" based on double membrane camouflage can effectively escape the body's immune clearance and increase the drug's circulation cycle in the body, which can not only reduce the frequency of administration, but also increase the bioavailability of the drug. By selecting two animal models of subcutaneous colon cancer and lung metastasis to detect the effect of the drug delivery system, the results showed that the double membrane camouflaged bionic nano / gene delivery platform M@NPs can effectively inhibit the development of tumors. Therefore, the double membrane camouflaged bionic nano / gene delivery platform M@NPs is expected to be developed as an effective means to treat cancers such as colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 3 represents the expression levels of miR21 in colorectal cancer tissue samples and corresponding adjacent normal tissues and colon cancer cell lines; A represents the RT-qPCR detection of miR21 in tumor tissues and adjacent normal tissues of colon cancer patients; B represents the in situ hybridization detection of miR21 in tumor tissues and adjacent normal tissues of colon cancer patients; C represents the expression detection of miR21 in human colon cancer cells; and D represents the expression detection of miR21 in mouse cell lines (**P<0.01, ***P<0.001).

[0028] Figure 2 Figure 2 is the result of ZnO nanomaterial cytotoxicity test, ns, no significant difference, **P<0.01, ***P<0.001; A is L929 cell; B is MC38 cell;

[0029] Figure 3 Live-Dead staining experiment to detect the apoptosis of MC38 and L929 cells by ZnO nanomaterials;

[0030] Figure 4 The binding and release of nano-ZnO materials and miRNAs were detected. A: agarose gel electrophoresis detection of the binding of ZnO and miRNA at different volume ratios; B: semi-quantitative analysis of RNA in A; C: agarose gel electrophoresis detection of miRNA release by nano-complexes after treatment of ZnO / miRNA gene complexes with different concentrations of heparin; D: semi-quantitative analysis of C.

[0031] Figure 5 This is the result of testing the ability of ZnO to deliver miRNA genes in mammalian cells;

[0032] Figure 6 Live / Dead staining and CCK-8 assay results; A shows the live / dead staining of MC38 cells apoptosis by NPs / miR21; B shows the CCK-8 assay of MC38 cells by NPs / miR21 (**P<0.01, ***P<0.001);

[0033] Figure 7 The results show that NPs / miR21 inhibits the expression of Ki67 and Bcl2 in mouse colorectal cancer cells MC38;

[0034] Figure 8 Transmission electron microscopy images of mixed cell membrane fragments and M@NPs;

[0035] Figure 9Fluorescence imaging was used to detect the accumulation of M@NPs in tumor tissues and their clearance from the body. A shows the fluorescence imaging results of near-infrared labeled cell membranes assembled with cy5 fluorescently labeled cy5NPs 10 hours after intravenous injection into tumor-bearing mice. B shows the comparison of the clearance of M@NPs by the body at different time points.

[0036] Figure 10 Treatment of subcutaneous colon cancer tumor model mice; A is the flow chart of the subcutaneous injection experiment; B is a photo of colon cancer tumor tissue; C is body weight; D is tumor volume; E is tumor weight;

[0037] Figure 11 HE staining and immunofluorescence staining of tumor tissue; A is the HE staining result of tumor tissue; B is the immunofluorescence staining result of Bcl2 and Ki67 in tumor tissue;

[0038] Figure 12 The figure shows the accumulation of M@NPs in lung tumor tissue and the inhibition of lung tumor growth. A is the fluorescence imaging result of near-infrared labeling; B is a photo of lung tumor tissue growth.

[0039] Figure 13 HE staining of the lungs of the mouse model of lung metastasis of M@NPs;

[0040] Figure 14 Immunofluorescence staining of Bcl2 and Ki67 in the lungs. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0042] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0043] The present invention is described in further detail below with reference to the accompanying drawings:

[0044] 1. Specimen collection from colorectal cancer patients and miR21 in situ hybridization assay

[0045] Experimental Materials: Patients with colorectal cancer diagnosed by preoperative pathology in the Department of General Surgery, The First Affiliated Hospital of Xi'an Jiaotong University underwent radical surgery. Specimens were obtained from the resected tumor tissue, and normal adjacent tissue was identified by intraoperative frozen sections.

[0046] Experimental Methods: Tumor tissue and adjacent normal tissue were fixed in 10% neutral formalin at room temperature for at least 48 hours. Tissue blocks were placed in a dehydration basket and dehydrated in a graded alcohol solution. Tissue blocks were embedded using a Leica automated embedding machine, fixed on a Leica paraffin microtome, and sectioned to 6 μm thickness. Sections were unfolded in 37°C water, removed using adhesive slides, and air-dried at room temperature. Sections were sequentially placed in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, and anhydrous ethanol II for 5 minutes, air-dried, and then soaked in DEPC water. After air-drying, sections were placed in a retrieval chamber filled with antigen retrieval solution and boiled for 15 minutes. Digestion was performed with 22 μg / mL proteinase K in preheated 50 mM Tris solution for 15 minutes. 3% hydrogen peroxide solution was added dropwise to the tissue and blocked at room temperature for 10 minutes to remove endogenous catalase. Nucleic acid prehybridization solution was added dropwise and blocked at 37°C for 15 minutes. Pour off the pre-hybridization solution, add the probe has-mir-21-5p hybridization solution with a concentration of 1uM, and hybridize overnight in a constant temperature box at 37°C. Pour off the hybridization solution, wash with 2× sodium citrate buffer solution for 3 minutes, and wash three times with sodium citrate buffer solution, each time for 3 minutes. Add blocking serum rabbit serum at room temperature for 30 minutes. Pour off the blocking solution, add anti-DIG-HRP, and incubate at 37°C for 80 minutes. Wash three times with PBS, each time for 3 minutes. Add DAB color development solution to the tissue, observe the color development under a microscope, and stop the color development when brown positive cells appear. Stain with hematoxylin staining solution for 5 minutes and rinse with running tap water. Put the slices into 75% alcohol for 5 minutes-85% alcohol for 5 minutes-100% alcohol I for 5 minutes-100% alcohol II for 5 minutes-n-butanol for 5 minutes-xylene for 5 minutes in turn, and add sealing agent to seal the slices. Observe the positive cells under a microscope, count and count, and see the results. Figure 5 .

[0047] 2. Detection of miR21 expression

[0048] (1) For adherent cells: HCT-116, SW480, SW620, HT29, CCD-18Co, MC38, and CT26 cell lines, seed the cells in a 10 cm culture dish and change the medium every other day. When the cells have grown to 80%, pour out the culture medium, wash once with PBS, add 1 mL of RNAisoPlus, place horizontally, allow the lysis solution to evenly distribute on the cell surface and lyse the cells, then use a pipette to blow the cells off. Transfer the RNAisoPlus to a 1.5 mL centrifuge tube and let it stand at room temperature for 10 minutes.

[0049] For human or mouse tissue samples: Place the tissue in a mortar pre-cooled with liquid nitrogen and grind it with liquid nitrogen until the sample becomes powder. Centrifuge at 12500g at 4℃ for 10 minutes, aspirate the supernatant, and transfer it to a new 1.5mL centrifuge tube. (See Figure 5 )

[0050] (2) Add 1 / 5 of the RNAisoPlus volume of chloroform to the lysate from step (1) above, vortex vigorously for 15 seconds, and let the solution stand at room temperature for 5 minutes after it is fully emulsified.

[0051] (3) Centrifuge at 12500g at 4°C for 20 minutes.

[0052] (4) Carefully and gently remove the centrifuge tube. The solution will now separate into three layers: a colorless upper aqueous phase, a white protein layer, and a colored lower organic solution. Transfer the upper aqueous phase to another new 1.5 mL centrifuge tube.

[0053] (5) Add the same volume of isopropanol to a 1.5 mL centrifuge tube, invert and mix thoroughly, and let it stand for 15 minutes.

[0054] (6) Centrifuge at 12500g for 15 minutes at 4°C. A white precipitate will appear at the bottom of the centrifuge tube.

[0055] (7) Carefully discard the supernatant, slowly add 75% ethanol, gently and slowly invert the tube to wash the white precipitate, centrifuge at 12500g at 4℃ for 5 minutes, and then carefully pour off the 75% ethanol.

[0056] (8) Repeat the steps in (7).

[0057] (9) Place the washed RNA in a fume hood, dry it at room temperature for 5 minutes, and add an appropriate amount of RNase-free water to dissolve it.

[0058] (10) The concentration and OD value of the dissolved RNA were measured using a Nanodrop UV spectrophotometer and stored at -80°C.

[0059] (11) The miR21 mature chain detection method was performed using Guangzhou Ruibo Biotechnology's Bulge-Loop miRNA qRT-PCR. The 10 μL reaction system is shown in Table 1 below.

[0060] Table 1 Main reagents used for miR21 reverse transcription

[0061]

[0062] (12) After the reaction system is mixed, centrifuge briefly and perform reverse transcription as follows: 42°C for 60 min, 70°C for 10 min. After the reaction, store the product at -20°C.

[0063] Detection of miR21 expression in colorectal cancer cell lines and tissues by qPCR

[0064] (13) Dilute the reverse transcription product obtained in step 2) 20-fold with DEPC water.

[0065] (14) Prepare 10 μL of the qPCR reaction system shown in Table 2 on ice:

[0066] Table 2 qRT-PCR reaction system

[0067]

[0068] (15) Gently mix the above reaction system and perform qPCR reaction using the reaction procedure shown in Table 3 below.

[0069] Table 3 qRT-PCR reaction procedure

[0070]

[0071] (16) The ΔΔCT method was used to analyze the data and obtain the relative expression level of miR21.

[0072] 3. Synthesis of ZnONPs Nanoparticles

[0073] 0.005 mol Zn(OAc)22H2O and 0.01 mol NaOH were dissolved in deionized water to prepare Zn(OAc)22H2O solution and NaOH solution, respectively. 25 mL of the Zn(OAc)22H2O solution was slowly added to 50 mL of the NaOH solution at room temperature with vigorous stirring to form a transparent white solution. After a large amount of white precipitate formed in the solution, the precipitate was collected and washed three times with anhydrous ethanol. The precipitate was dried to form a ZnO precursor. The ZnO precursor was heated to 250°C for 3 hours to obtain ZnO nanoparticles with uniform particle size. The resulting ZnO nanoparticles were modified with polyethylene terephthalate (PEI) to obtain ZnO-PEI nanocomposites (ZnONPs). A 20 μL sample of the ZnONPs suspension was pipetted onto a carbon-coated copper mesh and allowed to stand for 3-5 minutes before blotting out the excess liquid with filter paper. Drop 2% phosphotungstic acid onto a carbon-supported copper mesh and leave for 1-2 minutes. Remove excess liquid with filter paper and allow to dry at room temperature. Observe under a transmission electron microscope and collect images for analysis.

[0074] 4. Self-assembly and structural analysis of NPs / miR21antagomir nanocomplexes

[0075] The miR21 antagomir used in this example was synthesized by RiboBio. Its nucleotide sequence is shown in SEQ ID NO: 1 (5-UCAACAUCAGUCUGAUAAGCUA-3). It is a highly stable miR21 inhibitor. The sequence of the mature mmu-miR-21a-5p (target gene) it antagonizes is 5-UAGCUUAUCAGACUGAUGUUGA-3.

[0076] Maintaining the same volume of miR21 antagomiR (1 μL, 25 μM), 1 μL of miR21 antagomiR was mixed with different volumes of ZnONPs (0, 1, 2, 4, 6, 8, 10, 15, and 20 μL, 2 μg / mL) and vortexed for 20 seconds to mix thoroughly. The mixture was then disrupted and assembled using a QSONICA ultrasonic disruptor in pre-chilled 4°C water at an amplitude of 100 for 30 seconds. The water temperature was closely monitored during disruption, as high temperatures can affect the assembly of miRNA and ZnONPs. The resulting NPs / miR21 antagomiR containing varying concentrations were subjected to gel electrophoresis on 2% agarose gel. The optimal binding ratio of RNA to ZnONPs was determined based on the brightness and position of the bands. The optimal binding ratio determined was used for subsequent experiments to prepare new NPs / miR21 antagomiR for subsequent release experiments.

[0077] The prepared NPs / miR21antagomir were mixed with different concentrations of sodium heparin solution (0, 0.5, 1, 2.5, 5, 10, 20 mg / mL) and incubated in a 37°C water bath for 30 min. The products were subjected to 2% agarose gel electrophoresis to test whether miR21antagomir could be successfully released. 1 μL of 25 μM miR21antagomir was used as a positive control to test the maximum efficiency of RNA release from NPs / miR21antagomir. (See Figure 3 )

[0078] 1 μL of 25 μM miR21 antagomir was mixed with 4 μL of 2 mg / mL ZnONPs and vortexed for 20 seconds to mix thoroughly. The mixture was then disrupted and assembled using a QSONICA ultrasonic disruptor in pre-chilled 4°C water at an amplitude of 100 for 30 seconds. The resulting product was placed on ice and examined by transmission electron microscopy.

[0079] 5. In vitro functional evaluation of NPs / miR21antagomir nanocomplexes

[0080] miR21 expression levels are significantly elevated in mouse colon cancer cell lines. To test the biological function of the NPs / miR21 antagomir nanocomplex, the MC38 mouse colon cancer cell line was first used in vitro to test whether the nanocomplex could inhibit tumor cell proliferation and promote apoptosis. Furthermore, the mouse fibroblast cell line L929 was used to verify the toxicity of the nanocomplex on normal somatic cells.

[0081] 1) MC38 and L929 cell recovery

[0082] Place the container containing the culture medium in an incubator for at least 15 minutes to rewarm. Thaw the cryovials of mouse colon cancer cell lines and mouse fibroblast cell lines purchased from the Chinese Academy of Sciences Cell Bank in a 37°C water bath with gentle shaking for 2-5 minutes. (Note: The lids should not be submerged in water.) When the liquid in the cryovials has dissolved, immediately remove them from the water bath and disinfect them by spraying with 70% ethanol. Transfer the contents of the cryovials to a 15 mL centrifuge tube containing 5 mL of complete culture medium. Centrifuge at 800 rpm for 5 minutes, discard the supernatant, resuspend the pellet in 1 mL of complete culture medium, and transfer it to a 100 mm culture dish. Change the culture medium the next day. When the cells reach 80% confluence, passage them once for subsequent experiments.

[0083] 2) Lipofectamine 2000 cell transfection experiment

[0084] One day before transfection, cells were digested with 0.15% trypsin, counted, and plated in 24-well plates to a density of approximately 70% on the day of transfection. 10 μL of 25 μM miR21 antagomiR was mixed with 100 μL of Opti-MEM Reduced Serum Medium to obtain Solution A. 3 μL of Lipofecamine 2000 was mixed with 100 μL of Opti-MEM Reduced Serum Medium to obtain Solution B. Solutions A and B were mixed and allowed to stand at room temperature for 20 minutes to obtain a mixed solution. This mixture was added to a 24-well plate that had been previously replaced with Opti-MEM medium. The 24-well plate was incubated in a 5% CO2, 37°C cell culture incubator for 24 hours, after which the medium was replaced with complete medium containing serum.

[0085] 3) NPs / miR21antagomir cell transfection experiment

[0086] One day before transfection, the cells were digested with 0.15% trypsin and counted, and the cells were seeded in a 24-well plate so that the density on the day of transfection was about 70%. 10 μL, 25 μM miR21antagomir was mixed with 4 μL, 40 μL 2 mg / mL ZnONPs, vortexed for 20 seconds to mix. The above mixture was broken and assembled using a QSONICA ultrasonic disruptor in pre-cooled 4°C water, with an amplitude of 100 and a breaking time of 30 seconds. It was then allowed to stand at room temperature for 30 minutes and then added to a 24-well plate that had been replaced with Opti-MEM culture medium in advance. The 24-well plate was placed in a 5% CO2 37°C cell culture incubator and incubated for 24 hours, and then the medium was replaced with complete culture medium containing serum. Fluorescently labeled cy3miRNA was assembled with nano zinc oxide to prepare fluorescently labeled NPs. After transfection of the cells, fluorescence detection demonstrated that nano zinc oxide had the ability to deliver miRNA to mammalian cells. The results are shown in Figure 3. Figure 4 .

[0087] 4) Cell immunofluorescence staining experiment

[0088] Discard the culture medium in the 24-well plate and wash the cells once with PBS. Apply 4% paraformaldehyde and fix at room temperature for 12 minutes, discard the paraformaldehyde, and wash three times with PBS, 5 minutes each time. Use a PBS solution containing 0.1% Triton-100 to perforate the cells and let them stand at room temperature for 20 minutes. Use a PBS solution containing 5% BSA to block at room temperature for 30 minutes. Dilute the corresponding primary antibody in the blocking solution, add it to the cells, place at 4°C overnight, and wash three times with PBS, 5 minutes each time. Place the corresponding secondary antibody dilution in PBS containing DAPI (2μg / mL), incubate at room temperature for 1 hour, wash three times with PBS, 5 minutes each time, and add enough new PBS solution to prevent the cells from drying. Take pictures under an inverted fluorescence microscope, and the results are as follows Figure 7 shown.

[0089] Among them, the antibodies used for immunofluorescence are shown in Table 4 below:

[0090] Table 4 Antibodies used in immunofluorescence experiments

[0091]

[0092] 5) CCK8 cell proliferation and cytotoxicity assay

[0093] Seed the cell suspension (5000 cells / 100 μL) in a 96-well plate and place the plate in an incubator for pre-incubation or for any other treatments. Add 10 μL of CCK8 to each well. (Note: Avoid creating bubbles in the wells.) Incubate the plate in the incubator for 1-4 hours, depending on the cell number. Measure the absorbance at OD450 using a microplate reader. (For results, see [Table 1]). Figure 1 and 6 )

[0094] 6) Live-Dead staining

[0095] Inoculate a cell suspension (5000 cells / 100 μL) in a 96-well plate and place the plate in an incubator for pre-incubation or related treatment. Prepare Live-Dead staining solution by adding 5 μL of calcein AM (Component A) and 20 μL of Lethidium homodimer-1 (Component B) to 10 mL of DPBS and gently mix. Discard the culture medium in the 96-well plate. Add 100 μL of the prepared Live-Dead staining solution to each well. Incubate at 20-25°C for 30 minutes. Photograph and count cells under an inverted fluorescence microscope. (For results, see [link to original text]). Figure 2 and 6 )

[0096] VI. Distribution of the membrane-camouflaged M@NPs drug delivery system after tail vein administration

[0097] After establishing the mouse colon cancer subcutaneous tumor model, the tumor volume reached 200 mm 3 Mice were randomly divided into four groups: PBS treatment group (PBS), DiR-labeled mixed cell membrane NPs treatment group (M@NPs / NC), NPs / Cy5miR21antagomir group (Cy5NPs), and DiR-labeled mixed cell membrane camouflaged NPs / Cy5miR21antagomir group (M@Cy5NPs).

[0098] According to the volume ratio of ZnO (2μg / μL) and miRNA21 (25μM) (v / v ratio = 4:1), the mixture was sonicated at 100 amplitude for 30 seconds (Q700, Qsonica, USA). After standing at room temperature for 5 minutes, the cell membrane was added and the mixture was allowed to stand at room temperature for 20 minutes. The DiR-labeled mixed cell membrane camouflaged NPs / Cy5-miR21antagomir nanocomplex was constructed. 60μL of ZnO (2μg / μL) was mixed with 15μL of Cy5-miR21antagomir (25μM). 75μL (300μg / mL) of cell membrane and other complexes were added in equal proportions to a final volume of 150μL. If the final volume of some experimental groups could not reach 150μL, PBS was added to 150μL to prepare M@NPs (see Figure 8 Mice were administered 150 μL of PBS, M@NPs / NC, Cy5NPs, and M@Cy5NPs, respectively, via the tail vein, once every 24 hours for two consecutive doses. Six hours after the second dose, the mice were euthanized using CO2, and the liver, spleen, kidney, lung, and subcutaneous tumor tissues were collected. Fluorescence detection and analysis of subcutaneous tumor tissues in each group of mice were performed using an OdysseyCLx dual-laser infrared imager. Fluorescence signals from tissues, organs, and subcutaneous tumor tissues in each group of mice were detected and analyzed using a PerkinElmer small animal imager.

[0099] Experimental results: The accumulation of M@NPs in tumor tissue was detected by intravenous injection. Figure 9 As shown, the near-infrared labeled cell membrane was assembled with cy5 fluorescently labeled cy5NPs and then injected intravenously into tumor mice. Fluorescence imaging was performed 10 hours later. Figure 9 The results in Figure A show that compared with the PBS control group, M@NPs are enriched in tumor tissue. The clearance of M@NPs by the body was compared at different time points. Figure 9 The results in Figure B show that compared with cy5NPs without cell membrane camouflage, membrane-camouflaged M@NPs can effectively escape the body's rapid clearance.

[0100] 7. M@NPs for the treatment of subcutaneous tumors of colorectal cancer

[0101] A subcutaneous transplanted tumor model of colon cancer in mice was established. The body weight and tumor volume of the mice were measured and recorded every day. Note: No measurement was performed when the tumor could not be touched by hand. 10 days after inoculation, the subcutaneous tumor of the mice was palpable, and all mice were randomly divided into 5 groups, namely: PBS group (PBS), mixed cell membrane fragment treatment group (M), ZnONPs group (NPs / NC), NPs / miR21antagomir group (NPs / miR21) and mixed cell membrane camouflaged NPs / miR21antagomir group (M@NPs / miR21). The first administration was performed on the tenth day after tumor cell inoculation, recorded as Day 1, and then the drug was administered once every 1 day by tail vein microinjection (see Figure 10 Center A). Tail vein administration is complex and can be facilitated by a mouse tail holder with venous visualization. Experienced personnel are required to ensure consistent dosing. After five consecutive doses, mice were euthanized, and kidney, lung, spleen, liver, and subcutaneous tumor tissues were collected. The collected tissues were divided equally, with one half frozen in liquid nitrogen and the other half fixed overnight in 4% paraformaldehyde for histological staining.

[0102] During the treatment process, no statistically significant difference in mouse body weight was observed, indicating that the mixed cell membrane-disguised NPs / miR21 antagomirs (M@NPs / miR21) does not cause liver toxicity and sudden weight loss in mice during colorectal cancer treatment. Tumor growth rates were significantly reduced in mice treated with the mixed cell membrane-disguised NPs / miR21 antagomirs (M@NPs / miR21). After euthanasia, subcutaneous tumors were weighed. Compared with the PBS-treated control group (PBS), the subcutaneous tumor weight of mice treated with the mixed cell membrane-disguised NPs / miR21 antagomirs (M@NPs / miR21) was only 1 / 5 of that of the PBS-treated group, indicating that the mixed cell membrane-disguised NPs / miR21 antagomirs exerted significant anti-tumor properties in mice.

[0103] 8. M@NPs for the Treatment of Pulmonary Metastases of Colon Cancer and Drug Accumulation in the Lungs

[0104] Experimental animals: 8-week-old wild-type C57 / BL6 female mice were purchased from the Animal Experiment Center of the School of Medicine of Xi'an Jiaotong University and housed in the SPF Animal Experiment Center.

[0105] Experimental Procedure: MC38 mouse colon cancer cell lines were revived and cultured in 10 cm culture dishes. When the cell density reached 70%-80% and the cells were in the logarithmic growth phase, the cells were digested with 0.15% trypsin and washed twice with pre-chilled PBS. The cells were counted using a cell counter and the cell concentration was adjusted to 1×10 cells / mL with PBS. 7 The cells were aliquoted into 1.5 mL centrifuge tubes and placed on ice until ready to use. The mouse tail was disinfected with iodine and immobilized using a GEGD-Q9G intravenous visualization mouse tail injection fixture. Tumor cells were mixed using a pipette, and 100 μL of tumor cells were slowly injected into the tail vein of the mouse using a U100 1.0 mL insulin syringe. Hemostasis was achieved by pressing the injection site on the tail with a cotton swab. The mice were returned to SPF care for 3 weeks to allow the tumor cells to colonize the lungs through the circulatory system. Mice that received tail vein injections were randomly divided into four groups: PBS-treated group (PBS), mixed cell membrane-disguised ZnONPs group (M@NPs / NC), NPs / miR21 antagomiR (NPs / miR21), and mixed cell membrane-disguised NPs / miR21 antagomiR (M@NPs / miR21). Dosing was initiated on day 21 after tumor cell inoculation, with administration occurring every other day. After 15 consecutive administrations, the lung tissues of the mice were removed and the number and size of metastatic tumors in the lung tissues were counted under a stereomicroscope (see Figure 12 The lung tissue was divided into two equal parts, one half was placed in liquid nitrogen, and the other half was fixed with 4% paraformaldehyde overnight before subsequent experiments.

[0106] Experimental results: Lung fluorescence was detected 10 hours after administration. The results showed that the fluorescently labeled membrane-camouflaged M@NPs / miR21 could efficiently deliver miRNA to the lungs and effectively inhibit the occurrence and development of lung tumors (such as Figure 12 By HE staining of lungs (as shown). Figure 13 ) and Bcl2 and Ki67 immunofluorescence staining of the lungs (as Figure 14 The results showed that M@NPs could effectively inhibit the occurrence and development of lung tumors in lung metastasis model mice.

[0107] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A multi-cell membrane-mediated biomimetic nano / gene delivery platform, characterized in that: The ZnO-PEI nanocomposite and the miRNA21 antagonist were self-assembled in vitro to form NPs nanocomposites, which were then assembled with colon cancer cell membranes and macrophage membranes to construct a multi-cell membrane-mediated biomimetic nano / gene delivery platform M@NPs. The volume ratio of the ZnO-PEI nanocomplex to the miRNA21 antagonist is 4:1, and the volume ratio of the colon cancer cell membrane to the macrophage membrane is 2:1; the sequence of the miRNA21 antagonist is shown in SEQ ID NO: 1; the ZnO-PEI nanocomplex is prepared by modifying ZnO nanoparticles with PEI.

2. The multi-cell membrane-mediated biomimetic nano / gene delivery platform according to claim 1, characterized in that: The colon cancer cell membrane is MC38 tumor cell membrane.

3. The method for preparing the multi-cell membrane-mediated biomimetic nano / gene delivery platform according to claim 1 or 2, characterized in that: include: 1) Synthesis of ZnO-PEI nanocomposites; 2) ZnO-PEI nanocomplexes and miRNA21 antagonists were self-assembled in vitro to form NPs nanocomplexes; 3) The NPs nanocomplex was assembled with the colon cancer cell membrane and macrophage membrane to construct a multi-cell membrane-mediated biomimetic nano / gene delivery platform.

4. The method for preparing the multi-cell membrane-mediated biomimetic nano / gene delivery platform according to claim 3, wherein: The specific operations of step 1) are as follows: Dissolve Zn(OAc)2·2H2O and NaOH in deionized water to obtain Zn(OAc)2·2H2O solution and NaOH solution, respectively. Mix the Zn(OAc)2·2H2O solution and NaOH solution thoroughly, collect the precipitate, wash, and dry to obtain a ZnO precursor. The ZnO precursor was continuously heated at 250°C for 3 hours to obtain ZnO nanoparticles with uniform particle size. The ZnO nanoparticles were modified with PEI to prepare a ZnO-PEI nanocomposite.

5. The method for preparing the multi-cell membrane-mediated biomimetic nano / gene delivery platform according to claim 3, wherein: The ZnO-PEI nanocomplex and miRNA21 were mixed at a volume ratio of 4:1, and then ultrasonically treated at an amplitude of 100 for 90 seconds to assemble into NPs nanocomplex. After standing at room temperature for 5 minutes, the cell membrane was added at a volume ratio of 1:1 of NPs nanocomplex to cell membrane and allowed to stand at room temperature for 20 minutes.

6. Use of the multiple cell membrane-mediated biomimetic nano / gene delivery platform according to claim 1 or 2 in the preparation of drugs against colon cancer.

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