Puncture protein chimeric bionic cell membrane nano-vesicle and nano-vesicle with anti-cancer activity

Abstract

The invention belongs to the field of biomedical engineering materials, and particularly relates to a spike protein chimeric bionic cell membrane nano-vesicle and a nano-vesicle with anti-cancer activity. The invention provides a spike protein as shown in SEQ ID NO.1 (sequence identifier number 1). The invention further provides a nano-vesicle expressing the spike protein and a genetic engineering nano-vesicle which is prepared from the spike protein and is loaded with an active component. The spike protein and the nano vesicles provided by the invention can target tumor cells, can induce a membrane fusion process, and are beneficial to delivery of active ingredients, so that the prepared gene engineering nano particles have a better treatment effect on tumors. In addition, the spike protein disclosed by the invention can also be widely applied to tumor diagnosis, gene editing, mRNA delivery, macromolecular substance delivery and tumor immunotherapy, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical engineering materials, and particularly relates to a spike protein chimeric biomimetic cell membrane nanovesicle and a nanovesicle with anticancer activity. BACKGROUND

[0002] As a gene therapy drug, small interfering RNA (siRNA) has the characteristics of high precision, good safety and multiple drug targets. Therefore, siRNA drugs have clinical application prospects. In order to increase the circulation time in the body and improve the bioavailability of the drug, a series of nanomedicine delivery carriers have been developed in scientific research, such as nanoparticles (NPs), adeno-associated viruses (AAV), lipid nanoparticles (LNPs), cell membrane-coated nanoparticles (CNPs) and engineered virus-like particles (eVLPs). Among them, LNPs and AAV are two nanomedicine carriers widely used in clinical applications. In addition, cell membrane derivatives are a new type of biomimetic nanomaterial. The use of cell membrane coating technology to construct biomimetic nanoparticles for treatment has been extensively studied. This technology can greatly reduce the immune response of nanomedicines in the body, prolong the circulation time of drugs and improve the therapeutic efficacy.

[0003] However, the above-mentioned existing delivery carriers still have some problems in clinical applications, including low cytoplasmic delivery efficiency, poor tumor targeting specificity, especially for siRNA delivery. For example, lipid nanoparticles (LNPs) will naturally accumulate in liver cells when administered intravenously, and there are still obstacles in the application of anti-tumor therapy.

[0004] According to statistics, only a small part of nanoparticles entering the body can enter the target cells, and even fewer nanoparticles can enter the cytoplasm to exert drug efficacy. Therefore, improving the efficiency of drug entry into cells is of great significance to improve the therapeutic efficacy of drugs. However, there is little research on increasing the entry of nanomedicines into cells at present. How to obtain a gene carrier that is conducive to rapid gene delivery and easy to prepare is still a technical problem to be solved. SUMMARY

[0005] In view of the problems of the prior art, the present application provides a spike protein chimeric biomimetic cell membrane nanovesicle and a nanovesicle with anticancer activity.

[0006] A spike protein, the amino acid sequence of which is shown as SEQ ID NO. 1.

[0007] Preferably, the spike protein is expressed by a coding gene with a nucleotide sequence shown as SEQ ID NO. 2.

[0008] The application also provides a biomimetic cell membrane nanovesicle, which is obtained by micro-liposome extrusion on a cell membrane expressing the above spike protein.

[0009] Preferably, the cell membrane is a cell membrane of at least one of human renal epithelial cells, red blood cells, cancer cells or immune cells.

[0010] The application also provides a preparation method of the above biomimetic cell membrane nanovesicle, comprising the following steps:

[0011] Step 1, a coding gene of the spike protein is transfected into cells through a lentivirus transfection carrier system, and a cell line stably expressing the spike protein is screened out;

[0012] Step 2, the cell line screened out in step 1 is subjected to cell culture, and a cell membrane of the obtained cells is separated;

[0013] Step 3, the cell membrane is subjected to micro-liposome extrusion, and the biomimetic cell membrane nanovesicle is obtained.

[0014] Preferably, in step 1, the lentivirus transfection carrier system is pLVX-Hyg, psPAX2 and pMD2.G.

[0015] And / or, in step 2, the method for separating the cell membrane is differential centrifugation or sucrose gradient centrifugation.

[0016] The application also provides a use of the above biomimetic cell membrane nanovesicle in preparation of an anticancer drug, a tumor diagnosis reagent, a gene editing carrier, an mRNA delivery carrier, a macromolecular substance delivery carrier or a tumor immunotherapy drug.

[0017] The application also provides a nanoparticle with anticancer activity, which is prepared by assembling the above biomimetic cell membrane nanovesicle and an anticancer active ingredient.

[0018] Preferably, the anticancer active ingredient is a siRNA loaded with a cationic polymer complex; the use amount of the polymer complex and the siRNA is 1:1-10:1 according to the N / P ratio; and the use amount ratio of the siRNA and the biomimetic cell membrane nanovesicle is 4 micromoles:0.5 grams-4 micromoles:1.5 grams.

[0019] The application also provides a preparation method of the nanoparticles, which comprises the following steps: mixing the biomimetic cell membrane nanovesicle and the anticancer active ingredient, ultrasonic treatment, and filtration.

[0020] The application also provides a use of the nanoparticles in the preparation of an anticancer drug.

[0021] The application also provides an anticancer drug, which is prepared by adding the nanoparticles as an active substance into pharmaceutically acceptable excipients.

[0022] The "N / P ratio" or "N / P ratio" in the application refers to the ratio of the positive charge of the amine group and the negative charge of the phosphate group.

[0023] The application provides a new spike protein by modifying the sequence, which can regulate the membrane fusion process by enzymes and become a controllable membrane fusion. Compared with the traditional nanodrug which is taken up by endocytosis, the nanovesicle modified by the spike protein induces the membrane fusion process, can efficiently and quickly deliver siRNA and other drugs to the cytoplasm, and can improve the efficiency and treatment effect of the siRNA drug. Compared with the cell membrane nanovesicle without protein modification, the nanovesicle modified by the spike protein can interact with the receptors on the tumor cells, and relatively improve the targeting effect of the tumor. In addition, the nanovesicle and the nanoparticle of the application also have the advantages of simple preparation process and large-scale production. Therefore, the spike protein modified nanovesicle provided by the application can be applied to tumor drug delivery, tumor diagnosis, gene editing, mRNA delivery, macromolecular substance delivery and tumor immunotherapy, and has good application prospect.

[0024] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the application.

[0025] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the above subject matter of the application is limited to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Preparation flow chart of the nanoparticles;

[0027] Figure 2are the characterization of eS-BNVs nanovesicles and siRNA@eS-BNVs nanoparticles; wherein a is the average particle size of eS-BNVs nanovesicles, b is the negative staining electron micrograph of eS-BNVs nanovesicles, c is the sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting analysis of eS-BNVs nanovesicles, d is the average particle size of siRNA@eS-BNVs nanoparticles, and e is the negative staining electron micrograph of siRNA@eS-BNVs nanoparticles;

[0028] Figure 3 are the interaction of spike protein modified nanovesicles or nanoparticles with cells; wherein a is the nanovesicles and A549 ACE2+ cell mixing experiment fluorescence signal value change curve, b is the fluorescence signal value increment statistical analysis, c is the fluorescence signal value change curve of A549 ACE2+ cells, d is the flow cytometry detection of the presentation of siRNA-Cy5 drugs by different carriers, e is the fluorescence signal value change curve of A549 ACE2+ -Luc cells, and f is the fluorescence signal detection of the expression of luciferase (Luc) gene in different experimental groups.

[0029] Figure 4 are the distribution and delivery capacity of siRNA-Cy5@eS-BNVs in mice; wherein a is the accumulation of nanomedicines in the tumors of mice, b is the accumulation of nanomedicines in the blood of mice, and c is the knockdown of Luc gene in the tumors of mice by spike protein nanovesicles loaded with siLuc gene drugs.

[0030] Figure 5 are the anti-tumor experimental effects of siRNA@eS-BNVs nanoparticles in mice; wherein a is the tumor volume record of mice in different groups during the administration period, b is the body weight record of mice during the treatment period, and c is the fluorescence quantitative PCR (RT-PCR) quantitative analysis of EGFR gene on the tumors of mice in different groups after the treatment. DETAILED DESCRIPTION

[0031] In the following examples and experimental examples, the reagents and raw materials not specifically explained are commercially available.

[0032] Example 1 Spike protein

[0033] The present application provides a new spike protein (hereinafter referred to as S protein), and the amino acid sequence (SEQ ID NO. 1) of the S protein is as follows:

[0034]

[0035] The nucleotide sequence of the S protein coding gene (SEQ ID NO. 2) is as follows:

[0036]

[0037] In this embodiment, the sequence of the S2' cleavage site of the S protein is leucine-valine-proline-arginine-glycine-serine (Leu-Val-Pro-Arg-Gly-Ser, LVPRGS). LVPRGS can be recognized and cleaved by Thrombin, causing a change in the conformation of the S protein. Through the above modification, the membrane fusion process can be regulated by enzymes, becoming a controllable membrane fusion.

[0038] Example 2: Biomimetic cell membrane nanovesicle

[0039] This embodiment provides a biomimetic cell membrane nanovesicle with strong targeting property for tumors with high expression of ACE2. It can be used as a good carrier for tumor treatment.

[0040] The specific preparation method of the biomimetic cell membrane nanovesicle is as follows:

[0041] 1. Plasmid construction and transfection

[0042] For the S protein of Example 1, primers for gene mutation were designed online using Vazyme software, and the primers were synthesized by Shengwo Bioengineering Co., Ltd. The plasmid was constructed as follows: the vector pLVX-Hyg was linearized by double digestion with Xhol and BamHI.

[0043] The constructed plasmid was sequenced and identified by Hangzhou Yikang Biotechnology Co., Ltd. The successfully sequenced plasmid was transfected into cells by a lentivirus transfection system, and a stable S protein-expressing cell line was selected using hygromycin.

[0044] The lentivirus transfection system involved in this embodiment is pLVX-Hyg, psPAX2 and pMD2.G, but the present application is not limited to the plasmid transfection system described in this embodiment. Any lentivirus transfection system that can achieve plasmid transfection function can be used.

[0045] Prepare the DNA and transfection reagent mixture in a sterile EP tube. The DNA mixture is pLK0.1puro 6 μg; psPAX2, 4.5 μg, pMD2.G, 1.5 μg and 10 μl-30 transfection reagent Lipo8000 diluted with 750 μl DMEM medium. After mixing evenly, add to the HEK293T cell culture dish. Continue to culture for 48 h, then use hygromycin complete medium to screen cells, and the concentration of hygromycin is 50-1000 μg / ml.

[0046] 2. Cell culture and cell membrane harvesting

[0047] Human epithelial kidney 293T cells expressing S protein were cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, sigma) and 1% penicillin-streptomycin (Invitrogen) added to the cell culture dish. When the cells grew to 90%, the cell membrane was harvested, the cells were washed with HEPES (25 mM HEPES, 150 mM NaCl, 0.1 mM EDTA) buffer, and the cells were separated and recovered from the cell culture dish and centrifuged at 500 x g for three times. The cells were broken by probe sonication, and the whole solution was rotated at 20,000 x g for 10 minutes. The supernatant was saved, and the supernatant was centrifuged at 100,000 x g for 60 min under ultracentrifugation. Then the final precipitate was collected with HEPES buffer as the purified human epithelial kidney cell membrane expressing S protein for subsequent experiments.

[0048] The above steps of the present embodiment obtain the cell membrane by differential centrifugation, and in other embodiments, the cell membrane can also be obtained by sucrose gradient centrifugation and the like.

[0049] 3. Preparation of nanovesicles (eS-BNVs)

[0050] The cell membrane obtained in step 2 was resuspended by HEPES and quantified by BCA protein quantification kit, so that the concentration of the cell membrane was 4 mg / ml. The purified cell membrane was filtered through a 0.22 μm filter membrane (Millipore), and was first pretreated by water bath ultrasonic method. Then it was co-extruded 31 times using a polycarbonate filter membrane (pore size of 800, 400 and 100 nm, Avanti polar lipids) micro-extruder to obtain nanovesicles with uniform particle size.

[0051] Experimental results Figure 2 a-c) show that in the present embodiment, the cell membrane is genetically modified to express the genetically modified spike protein, the cell membrane is extracted by differential centrifugation, and the spike protein modified nanovesicles are obtained by the combination of ultrasonic and extrusion. The experiment shows by TEM that the final obtained vesicle structure is a hollow structure, and the Malvern particle size instrument test result shows that the average particle size of the nanovesicles is 100-150 nm, which meets the effective range of penetrating into the tissue through the blood vessels. And the expression of S protein on the surface of the cell membrane is proved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting analysis.

[0052] Example 3 Nanoparticles with anti-cancer activity

[0053] The embodiment provides a kind of nano-particle capable of targeting cancer cells, with anticancer activity.The anticancer active ingredient is polyethyleneimine (PEI) loaded siRNA, and in the embodiment, siRNA is the gene of interfering cell epidermal growth factor receptor (siEGFR).

[0054] Its preparation method is as shown in Figure 1 Including the following steps:

[0055] siRNA is mixed with PEI (20 μg, 25 kDa, N / P ratio is 5:1) in HEPES buffer (13.3 μg, final concentration is 4 μM), and incubated at room temperature for 15 minutes to form siRNA / PEI complex.Subsequently, the solution of siRNA / PEI complex is mixed with equal volume of the solution of eS-BNVs (4 mg / ml) prepared in embodiment 1, and ultrasonically treated at 4 DEG C for 1 hour to form S protein cell membrane coated nanoparticles.By 100 kDa concentration tube filtration (Millipore), S protein cell membrane coated siRNA nanoparticles (siRNA@eS-BNVs) are finally purified.

[0056] The particle size of prepared nanoparticle siRNA@eS-BNVs is measured by Malvern particle size instrument (Zetasizer Nano ZS90).The morphology of nanoparticles is photographed by using negative staining electron microscope (120 kV, JEM-1400, JEOL, Japan) after being stained with 1.5% (w / v) uranyl acetate.The spike protein on the surface of nanoparticles is detected by immunoblotting analysis (eBlot, China).

[0057] The experimental results Figure 2 d-e) show that the Malvern particle size instrument test results indicate that the average particle size of nano-vesicles is 100-150 nm, and the size meets the range of effectively penetrating into tissues through vascular permeation.The experiment shows that vesicle structure with hollow structure is finally obtained by TEM.

[0058] The nanoparticles prepared in the embodiment can be injected intravenously to achieve antitumor treatment.They can target tumor cells, thereby effectively accumulating in tumors, and can also induce membrane fusion process, can efficiently and rapidly deliver siRNA to cytoplasm, and can improve the efficiency and treatment effect of siRNA drugs.

[0059] The technical solutions of the application are further described by experiments.The samples used in the following experiments are prepared according to the method described in embodiments 2-3.Among them, siRNA-Cy5@eS-BNVs are also prepared according to the method described in embodiments 2-3, with the difference that siRNA is replaced by siRNA labeled with Cy5.

[0060] Experimental Example 1 In vitro membrane fusion experiment of nanovesicles loaded with sulforhodamine B (SRB@eS-BNVs)

[0061] I. Experimental Methods

[0062] The occurrence of membrane fusion events between spike protein nanovesicles and cells was verified by a liposome content mixing experiment. eS-BNVs nanovesicles and sulforhodamine B (SRB, 50 mM) fluorescent dye with self-quenching fluorescence characteristics were mixed in equal volumes, and the fluorescent dye was loaded into the eS-BNVs by low-power water bath ultrasonication at 4 degrees Celsius for 30 minutes. Free SRB fluorescent dye was filtered by ultrafiltration tube. Nanovesicles encapsulating SRB fluorescent dye (SRB@eS-BNVs) were prepared. Changes in fluorescent dye signal values were observed.

[0063] II. Experimental Results

[0064] Experimental Results Figure 3 a-b) show that nanovesicles and different cells mixed in equal volumes can observe an increase in fluorescence signal values under the induction of Thrombin, i.e., the occurrence of membrane fusion phenomena. The above results show that the spike protein provided by the present application can promote the occurrence of membrane fusion. The experimental results of the fluorescence quantification kinetics experiment prove that under the condition of enzyme cutting, S protein modified nanovesicles can fuse with A549 ACE2+ tumor cells.

[0065] Experimental Example 2 In vitro uptake of genetically engineered nanoparticles siRNA-Cy5@eS-BNVs and siLuc@eS-BNVs

[0066] I. Experimental Methods

[0067] The uptake of siRNA-Cy5 and siLuc genes encapsulated by spike protein nanovesicles was observed in A549 ACE2+ cells and A549 ACE2+ -Luc cells stably expressing luciferase (Luc) fluorescent protein. Experimental results were analyzed by cell flow cytometry and IVIS imaging.

[0068] The specific operation is as follows:

[0069] Preparation of siLuc@eS-BNVs: siLuc was mixed with HEPES buffer (13.3 pg, final concentration 4 mM) and PEI (20 pg, 25 kDa, N / P ratio 5:1) at room temperature for 15 min to form siRNA / PEI complex. Then, the siLuc / PEI complex solution was mixed with equal volume of eS-BNVs (4 mg / ml) prepared in Example 1, and sonicated for 1 h at 4 °C to form S protein cell membrane-coated nanoparticles. The mixture was concentrated by filtration through 100 kDa concentrator tubes (Millipore) to remove free siLuc. Finally, S protein cell membrane-coated siLuc nanoparticles (siLuc@eS-BNVs) were purified.

[0070] Cellular uptake of siRNA-Cy5@eS-BNVs nanoparticles was observed in A549 ACE2+ Cellular uptake of siRNA-Cy5@eS-BNVs nanoparticles was observed in A549 ACE2+ Cellular uptake of different forms of siRNA-Cy5 by cells.

[0071] Luciferase (Luc) fluorescence protein in A549 ACE2+ Cellular uptake of siLuc nanoparticles was observed in A549 ACE2+ Luciferase (Luc) fluorescence protein in A549

[0072] II. Experimental results

[0073] The experimental results are shown in Figure 3As shown in Fig. c, from the fluorescence intensity of cell flow, the nano vesicles modified by spike protein in the thrombin enzyme cutting group have the highest fluorescence intensity, so the number of cell uptake of siRNA-Cy5 nanoparticles in this group is the largest. It is shown that A549 ACE2+ cells have enhanced cell uptake of siRNA-Cy5 modified by eS-BNVs. The experimental results show that eS-BNVs can enhance the cell uptake efficiency by enzyme cutting.

[0074] The experimental results are shown in Fig. Figure 3 d, A549 ACE2+ cells have enhanced cell uptake of siRNA-Cy5 modified by eS-BNVs. The experimental results show that eS-BNVs can enhance the cell uptake efficiency by enzyme cutting.

[0075] The above experimental results show that the spike protein modified nano vesicles of the present application can effectively combine with A549 ACE2+ tumor cells, achieve more efficient cell uptake and interfere with the expression of corresponding genes.

[0076] Experimental Example 3 Distribution and Efficacy of Genetically Engineered Nanoparticles siRNA-Cy5@eS-BNVs and siLuc@eS-BNVs in Mice

[0077] I. Experimental Methods

[0078] BALB / c nude mice were purchased from Jisui Yaoke Biotechnology Co., Ltd. All experiments were conducted in accordance with the experimental procedures and research procedures approved by the Sichuan University Animal Protection Committee.

[0079] First, a mouse tumor model was constructed by subcutaneously inoculating 2x10 6 A549 ACE2+ tumor cells in BALB / c nude mice. To verify the function of spike protein modified biomimetic nano vesicles mediated drug delivery, the drug distribution scheme of siRNA-Cy5 in mice was designed in this study. The experimental technical scheme related to drug distribution in this study is that the S protein modified biomimetic nano vesicles encapsulating siRNA-Cy5 are injected into the tail vein of subcutaneously inoculated A549 ACE2+Cellular mice (20 μg siRNA-Cy5 / each), to observe the distribution of siRNA-Cy5 in mice.

[0080] The study also designed an anti-tumor program for siRNA drugs in mice. The relevant cell uptake experimental technical scheme is that the S protein modified biomimetic nanovesicle encapsulates siLuc gene therapy to inoculate A549 ACE2+ -Luc cells in mice, to observe the tumor A549 ACE2+ -Luc cells in mice, to observe the tumor A549 ACE2+ -Luc cells in mice, to observe the tumor A549

[0081] II. Experimental results

[0082] As Figure 4 As shown in a-b, the S protein modified biomimetic nanovesicle encapsulating siRNA-Cy5 gene can better target tumor cells and prolong the circulation time in blood.

[0083] As Figure 4 c, to subcutaneously inoculate A549 ACE2+ tumor cells in mice, by tail vein injection of siRNA-Cy5 nanoparticles drugs encapsulated by spike protein modified nanovesicles, at different times, using IVIS to observe the accumulation of drugs in the tumor site.

[0084] The above experimental results show that the spike protein modified nanovesicle has good tumor targeting effect in mice.

[0085] Experimental example 4 Anti-tumor effect of genetically engineered nanoparticles siRNA@eS-BNVs in mice

[0086] I. Experimental method

[0087] The expression of siEGFR was used to interfere with the expression of epidermal growth factor receptor to experiment the anti-tumor effect. A549 ACE2+ tumor carrying mice were given siRNA@eS-BNVs treatment (20 μg siRNA-Cy5 / each), including spike protein modified nanoparticles and a series of control group experiments. The treatment course was to give drug once every 2-3 days, for two weeks, through tail vein administration. The anti-tumor effect of the drug was observed by measuring the size of the tumors of different groups of mice.

[0088] II. Experimental results

[0089] Experimental results ( Figure 5 This study demonstrated that delivering siEGFR to the cytoplasm using nanoparticles siRNA@eS-BNVs successfully inhibited tumor growth. Detection of EGFR gene expression in tumor cells revealed decreased EGFR mRNA expression. Simultaneously, monitoring mouse body weight during the treatment period showed no significant changes, indicating the safety of the spike protein-modified nanovesicles.

[0090] As can be seen, siRNA@eS-BNV nanoparticles can treat mice subcutaneously inoculated with human non-small cell lung cancer, significantly increasing drug accumulation at the tumor site and inhibiting tumor growth. They exhibit good biocompatibility and safety.

[0091] As can be seen from the above embodiments and experimental examples, the present invention provides a novel spike protein, spike protein-modified nanovesicles, and nanoparticles loaded with active ingredients using these nanovesicles. The spike protein and nanovesicles of the present invention can target tumor cells and induce membrane fusion, which is beneficial for the delivery of active ingredients. Therefore, the genetically engineered nanoparticles made from them have better therapeutic effects on tumors. Furthermore, the spike protein of the present invention can also be widely used in tumor diagnosis, gene editing, mRNA delivery, macromolecule delivery, and tumor immunotherapy. Therefore, the present invention has broad application prospects.

Claims

1. A spike protein, characterized in that, The amino acid sequence of the spike protein is shown as SEQ ID NO.

1.

2. The Spike protein according to claim 1, characterized in that: The spike protein is expressed by a coding gene with a nucleotide sequence shown as SEQ ID NO.

2.

3. A biomimetic biological cell membrane nanovesicle, characterized in that: It is to construct a nano-capsule by using the cell membrane expressing the spike protein of claim 1 or 2.

4. The biomimetic biological cell membrane nanovesicle according to claim 3, characterized in that: The cell membrane is the cell membrane of at least one of human renal epithelial cells, red blood cells, cancer cells or immune cells.

5. The method for preparing biomimetic biological cell membrane nanovesicles according to claim 3 or 4, characterized in that, It comprises the following steps: Step 1, the coding gene of the spike protein is transfected into cells by a lentivirus transfection carrier system, and a cell line stably expressing the spike protein is screened out; Step 2, the cell line screened in step 1 is cultured, and the cell membrane of the obtained cells is separated; Step 3, the cell membrane is prepared by ultrasonic, self-assembly, extrusion or microfluidic method.

6. The use of the biomimetic cell membrane nano-capsule of claim 3 or 4 in the preparation of an anticancer drug, a tumor diagnosis reagent, a gene editing carrier, a nucleic acid and nucleic acid fragment carrier, a macromolecular substance delivery carrier or a tumor immunotherapy drug.

7. A nanoparticle having anticancer activity, characterized in that: It is to assemble the biomimetic cell membrane nano-capsule of claim 3 or 4 with an anticancer active ingredient.

8. The nanoparticle according to claim 7, wherein: The anticancer active ingredient is a cationic polymer complex loaded siRNA; the amount of the polymer complex and siRNA is 1:1-30:1 according to the N / P ratio; the amount ratio of siRNA and biomimetic cell membrane nano-capsule is 4 μmol:0.5 g-4 μmol:1.5 g.

9. The use of the nanoparticle of claim 7 or 8 in the preparation of an anticancer drug.

10. An anticancer drug, characterized in that: It is to add a pharmaceutically acceptable adjuvant to the nanoparticle of claim 7 or 8 as an active substance.

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