Engineering bionic nucleic acid nano diagnosis and treatment agent as well as preparation method and application thereof

Abstract

The invention discloses an engineered bionic nucleic acid nano diagnosis and treatment agent as well as a preparation method and application thereof, relates to the technical field of biological targeting, and aims to solve the problems that an engineered macrophage membrane modified bionic nucleic acid nano diagnosis and treatment agent for oral squamous cell carcinoma is lacked in the prior art, and the curative effect on invasive tumors of the oral squamous cell carcinoma is poor. According to the engineering bionic nucleic acid nano diagnosis and treatment agent, a cationic lipid nucleic acid medicine is wrapped with a macrophage membrane, and PD1 shown as SEQ.ID.NO.1 is stably expressed on the macrophage membrane; the cationic lipid nucleic acid medicine is prepared by loading Cip2a siRNA (small interfering Ribonucleic Acid) on a cationic liposome. The bionic nucleic acid nano diagnosis and treatment agent has a huge application prospect in preparation of oral squamous cell carcinoma diagnosis kits and medicines.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological targeting, in particular to an engineered biomimetic nucleic acid nanodiagnostic and therapeutic agent, a preparation method and application thereof. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in the head and neck, accounting for about 90% of the total number of oral malignant tumors. The comprehensive sequence treatment mainly based on surgical treatment has made great progress, but the overall five-year survival rate of patients is still about 50%. Therefore, exploring new highly effective, low-toxicity, and high-targeting drug preparations and methods has become a research hotspot.

[0003] With the continuous development of nanomedicine, gene therapy based on nanomedicine has attracted widespread attention in the immunotherapy of oral squamous cell carcinoma due to its targeted delivery, multi-drug co-delivery, and spatiotemporal controllable drug release. Among them, small nucleic acid drugs can expand the non-druggable protein target by forming a multi-dimensional structure, silence the target gene in a sequence-specific manner, and achieve precise regulation of disease proteins, showing great potential in the treatment of tumor-related diseases. Protein phosphatase 2A cancer inhibitor (Cip2a) as a key cancer protein is overexpressed in various human malignancies (such as breast cancer, lung cancer, gastric cancer, etc.). Its expression is usually associated with tumor, metastasis, chemotherapy resistance, and poor prognosis of patients, which makes it a key driver of tumor occurrence and a promising therapeutic target. The siRNA technology encapsulated by lipid nanoparticles (LNP) to inhibit Cip2a expression has great potential to reactivate the tumor suppressor function of protein phosphatase 2A (PP2A), degrade cancer proteins such as c-Myc, and activate pro-apoptotic signaling pathways, thereby inducing cell apoptosis and inhibiting tumor growth.

[0004] However, traditional small nucleic acid drug delivery carriers such as liposomes and polypeptides have low tumor targeting rate, body immune clearance, and tumor microenvironment physical barriers, which limit the safe and efficient targeting delivery of nucleic acid to the deep tumor.

[0005] Bionanotechnology, such as nanoparticles coated with engineered macrophage membrane, is considered as a promising method to reduce the immunogenicity of materials and improve the targeting specificity. Macrophages, as a kind of immune cells, have unique membrane protein composition, which makes the modified nanoparticles have good immune escape potential. On the other hand, the engineered bionic modified nanoparticles still have the complete "core-shell" structure, which can load certain therapeutic agents and specifically target tumor cells. Studies have shown that the OSCC tumor microenvironment has a higher expression level of programmed death receptor-ligand 1 (PD-L1) compared to other tumors. Therefore, blocking the binding of PD-L1 on the surface of OSCC cells in the tumor microenvironment to the programmed death receptor 1 (PD-1) on the surface of T cells can effectively reverse and enhance the function of exhausted T cells, and strengthen their anti-tumor effect. Therefore, the PD1-engineered macrophage membrane modified nucleic acid nanomedicine delivery system has great potential in safe drug delivery, precise targeting and multifunctionalization. Therefore, it is urgent to develop a PD1-engineered macrophage membrane modified bionic nucleic acid nanoparticle for treating oral squamous cell carcinoma. SUMMARY

[0006] The technical problem solved by the present application is:

[0007] There is a lack of engineered macrophage membrane modified bionic nucleic acid nanodiagnostic and therapeutic agent for oral squamous cell carcinoma in the prior art, and the effect on invasive oral squamous cell carcinoma is not good.

[0008] To solve the above problems, the inventors' team previously detected through a large number of clinical samples that the Cip2a gene was highly expressed in oral squamous cell carcinoma patients and was significantly related to disease progression, which seriously affected the prognosis of patients. Therefore, the Cip2a gene is a key indicator for detecting oral squamous cell carcinoma patients and an important target for treatment. Cip2a protein can stabilize c-Myc (Myelocytomatosis viral oncogene homolog) cancer protein and promote Akt (protein kinase B) signaling pathway, both of which are crucial for cell proliferation, survival and metabolism.

[0009] The technical solution adopted by the present application to solve the above technical problems is:

[0010] The present application provides an engineered bionic nucleic acid nanodiagnostic and therapeutic agent, which coats a cationic lipid nucleic acid drug with a macrophage membrane, wherein the macrophage membrane stably expresses PD1 based on SEQ.ID.NO. 1.

[0011] The cationic lipid nucleic acid drug is prepared by loading Cip2a siRNA with cationic liposomes.

[0012] Further, the PD1 stably expressed on the macrophage membrane is extracted after the macrophage is infected by the lentivirus and expression.

[0013] Puromycin is added during the infection process to kill the cells not effectively infected, and the stable strain of the PD1 stable expression is obtained under the maintenance of the Puromycin drug.

[0014] Further, the cationic lipid nucleic acid drug is LNP@Cip2a siRNA nanoparticles.

[0015] The mass ratio of the macrophage membrane stably expressing PD1 and the cationic liposome is 1: (2-5), and the wrapping is realized by co-extrusion through multiple layers of filter membranes in sequence by using an extruder.

[0016] The application further provides a preparation method of the engineered bionic nucleic acid nano diagnosis and treatment agent.

[0017] (1) Preparation of PD1-MM:

[0018] The macrophage is infected by the lentivirus, after the infection, Puromycin of (1-3) μg / ml is added to kill the cells not effectively infected, and finally the stable strain of the PD1 stable expression is obtained under the maintenance of the Puromycin drug.

[0019] The PD1-macrophage is collected for incubation, the cell suspension is broken and centrifuged, the cell membrane is collected, and the engineered macrophage membrane vesicle PD1-MM is obtained.

[0020] (2) Preparation of the cationic lipid nucleic acid drug:

[0021] The D-Lin-MC3-DMA, DOPE, Cholesterol and PEG2000-DMG are used to prepare an organic phase, the Cip2a siRNA based on SEQ.ID.NO.2 is dissolved in a buffer to prepare an aqueous phase; the organic phase is added dropwise into the aqueous phase by the organic phase injection method, and the lipid nanoparticles LNP@Cip2a siRNA are assembled.

[0022] (3) Preparation of PML@Cip2a siRNA:

[0023] The LNP@Cip2a siRNA and the PD1-MM are mixed in proportion, and the PML@Cip2a siRNA nanoparticle solution is obtained by co-extrusion through multiple layers of filter membranes in sequence by using an extruder; the organic solvent and the free small molecule compounds in the solution are removed, nuclease-free water is added, centrifuged, concentrated, and the physiological osmotic pressure is adjusted to obtain the PML@Cip2a siRNA.

[0024] Further, the lentivirus in step (1) is selected from HBLV-Pdcd1-3xflag-ZsGreen-PURO.

[0025] Further, the molar ratio of D-Lin-MC3-DMA, DOPE, Cholesterol, PEG2000-DMG in the organic phase in step (2) is (48-55):(8-12):(35-40):(0.5-2).

[0026] Further, the volume ratio of the water phase to the organic phase in step (2) is 3:1, and the molar ratio of the positive charge of D-Lin-MC3-DMA to the negative charge of Cip2a siRNA is 6:1.

[0027] Further, the mass ratio of LNP@Cip2a siRNA to PD1-MM in step (3) is (2-5):1.

[0028] Further, in step (3), the PML@Cip2a siRNA nanoparticle solution is obtained by co-extrusion through 800nm, 400nm and 200nm filter membranes in sequence by using an extruder.

[0029] The application of the above-mentioned engineered biomimetic nucleic acid nanodiagnostic and therapeutic agent in the preparation of an oral squamous cell carcinoma diagnostic kit and a drug also falls within the protection scope of the present application.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] The present application uses PD1-engineered macrophage membrane modified D-Lin-MC3-DMA cationic liposome nanoparticles loaded with Cip2a siRNA to construct a PML@Cip2a siRNA biomimetic nucleic acid nanodiagnostic and therapeutic system, which realizes the combination of biomimetic nanotechnology and genetic engineering technology; on the one hand, it has the biological functions of active targeting of deep tumor and inducing apoptosis; on the other hand, it can block the combination of PD-L1 on the surface of tumor cells and PD-1 on the surface of T cells, improve the tumor immune microenvironment, thereby realizing efficient deep delivery and precise tumor treatment of drugs, higher targeting into deep tumor, and enhancing the anti-tumor effect.

[0032] The PML@Cip2a siRNA nanodiagnostic and therapeutic agent of the present application can be applied to the field of tumor treatment or other disease diagnosis and treatment, and has great clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The present application is a PML@Cip2a siRNA biomimetic nucleic acid nanoparticle preparation method synthesis route;

[0034] Figure 2 WB detection and flow cytometry detection of PD1 level on the surface of PD1 engineered macrophages in the embodiments of the application;

[0035] Figure 3 Transmission electron microscopy (TEM) images of LNP@Cip2a siRNA, macrophage membrane vesicle PD1-MM with high expression of PD1, and PML@Cip2a siRNA nanoparticles in the embodiments of the application;

[0036] Figure 4 Hydrodynamic particle size (DLS) and Zeta potential images of LNP@Cip2a siRNA, PD1-MM, and PML@Cip2a siRNA nanoparticles in the embodiments of the application;

[0037] Figure 5 Flow cytometry detection images of PML@Cip2a siRNA biomimetic nucleic acid nanoparticle fusion in the embodiments of the application;

[0038] Figure 6 Laser confocal detection images of PML@Cip2a siRNA biomimetic nucleic acid nanoparticle fusion in the embodiments of the application;

[0039] Figure 7 Laser confocal microscope detection of SCC7 cell uptake (Figure A) and lysosome escape (Figure B) of PML@Cip2a siRNA in the embodiments of the application;

[0040] Figure 8 Flow cytometry detection of macrophage RAW264.7 cell uptake of PML@Cip2a siRNA in the embodiments of the application;

[0041] Figure 9 MTT experiment graph of PML@Cip2a siRNA inhibition of tumor cells in the embodiments of the application;

[0042] Figure 10 Flow cytometry analysis of PML@Cip2a siRNA-induced apoptosis in the embodiments of the application;

[0043] Figure 11 Cell scratch test analysis of PML@Cip2a siRNA inhibition of tumor cells in the embodiments of the application;

[0044] Figure 12 Cell invasion and migration experiment analysis of PML@Cip2a siRNA inhibition of tumor cells in the embodiments of the application;

[0045] Figure 13Figure for the Western blot detection of the protein expression levels of Cip2a, Bax, Bcl-2, Caspase-3 and Cleaved Caspase-3 in the cells after PML@Cip2a siRNA treatment in the embodiments of the present application;

[0046] Figure 14 Flow chart for the treatment of the oral squamous carcinoma tumor-bearing mouse model by the PML@Cip2a siRNA biomimetic nucleic acid nanoparticles in the embodiments of the present application;

[0047] Figure 15 Therapeutic effect of the PML@Cip2a siRNA biomimetic nucleic acid nanoparticles on the oral squamous carcinoma tumor-bearing mouse model in the embodiments of the present application. In the figure, (A) tumor tissue diagram of each group after treatment; (B) analysis of tumor tissue weight after treatment; (C) tumor growth curve;

[0048] Figure 16 Tumor tissue hematoxylin-eosin staining (HE staining), immunohistochemical staining (IHC staining) and TUNEL staining (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) section diagrams of the PML@Cip2a siRNA treated oral squamous carcinoma tumor-bearing mouse model in the embodiments of the present application;

[0049] Figure 17 Body weight change diagram of the mice in each treatment group in the embodiments of the present application;

[0050] Figure 18 HE section of the main organs (heart, liver, spleen, lung, kidney) of the mice in each treatment group in the embodiments of the present application;

[0051] Figure 19 Detection diagram of the blood biochemical indicators (liver function indicators: AST and ALT), (kidney function indicators: UREA and CREA) of the mice in each treatment group in the embodiments of the present application;

[0052] Figure 20 Nanoparticle hemolysis experiment diagram in the embodiments of the present application;

[0053] Figure 21 Immune flow analysis diagram of the tumor tissue of the mice in each treatment group in the embodiments of the present application, including MHC II + Figure A) CD80 + CD86 + Figure B) CD4 + Figure C) CD8 + Figure D) T cell level;

[0054] Figure 22Figure A and B show ELISA detection of tumor tissues (Figure A) and serum (Figure B) of mice in each treatment group in the embodiment of the present application;

[0055] Figure 23 Figure A, B and C show volcano plots of differentially expressed genes of tumor tissues of mice in each treatment group in the embodiment of the present application; Figure D shows KEGG enrichment analysis of differentially expressed genes;

[0056] Figure 24 Figure shows GSEA enrichment analysis of RNA sequencing of tumor tissues of mice in each treatment group in the embodiment of the present application;

[0057] Figure 25 Figure shows RT-qPCR detection of tumor tissues of mice in each treatment group in the embodiment of the present application. DETAILED DESCRIPTION

[0058] In order to make the person skilled in the art better understand the present application, the exemplary embodiments or examples of the present application will be described in the following with reference to the accompanying drawings. Obviously, the described embodiments or examples are only a part of the embodiments or examples of the present application, but not all. Based on the embodiments or examples in the present application, all other embodiments or examples obtained by the person skilled in the art without making creative efforts should belong to the protection scope of the present application.

[0059] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0060] The beneficial effects of the present application will be described below in combination with specific embodiments.

[0061] Embodiment 1

[0062] The present embodiment provides a preparation method of a biomimetic nano nucleic acid system, as shown in the following steps: Figure 1

[0063] (1) Preparation of PD1-RAW264.7 cell membrane vesicle PD1-MM:

[0064] In the present application, Raw264.7 cells are infected with lentivirus HBLV-Pdcd1-3xflag-ZsGreen-PURO. After 48 h of infection, the cells not effectively infected are killed by adding and maintaining 2.0 μg / ml of Puromycin, and under the maintenance of Puromycin, a stable strain based on the PD1 stable expression shown in SEQ.ID.NO.1 is finally obtained.

[0065] ​PD1-RAW264.7 cells were collected, washed with phosphate buffer solution (PBS) three times, and then resuspended in Tris-HCl buffer (pH = 7.4) containing 1 mM CaCl2and 1x EDTA-free protease inhibitor, incubated at 4°C for 2 h, and then the cell suspension was placed in an ultrasonic cell disruptor under ice bath conditions at a power of 5% for 2 min. The ultrasonically treated cells were centrifuged at 4°C at 800 g for 10 min, and then the supernatant was collected and centrifuged at 4°C at 10,000 g for 10 min, and finally the supernatant was centrifuged at 4°C at 1.5x 10 5 g for 1 h. The cell membrane pellet was collected and resuspended after washing with cold PBS. After the cell membrane suspension was repeatedly extruded 10 times using an extruder, PD1-RAW264.7 cell membrane vesicles PD1-MM were obtained.

[0066] (2) Preparation of cationic lipid nucleic acid drugs:

[0067] 4-N,N-dimethylamino-butyric acid-dilinoleyl-methyl ester (D-Lin-MC3-DMA), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), cholesterol (Cholesterol), and dimyristyl glycerol-polyethylene glycol 2000 (PEG2000-DMG) were prepared into stock solutions with concentrations of 20 mg / ml, 10 mg / ml, 20 mg / ml, and 10 mg / ml, respectively, using ethanol.

[0068] The stock solutions were ultrasonically mixed according to the molar ratio of 4-N,N-dimethylamino-butyric acid-dilinoleyl-methyl ester (D-Lin-MC3-DMA), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), cholesterol (Cholesterol), and dimyristyl glycerol-polyethylene glycol 2000 (PEG2000-DMG) of 50: 10: 38.5: 1.5 to form an organic phase.

[0069] Cip2a siRNA based on SEQ.ID.NO.2 was dissolved in DEPC to prepare a 1 mg / mL stock solution.

[0070] The sequence of Cip2a siRNA is as follows:

[0071] Sense strand: CCCATATAGATGACTTAAT

[0072] Antisense strand: ATTAAGTCATCTATATGGG

[0073] According to the molar ratio of the positive charge (nitrogen atom, N) of D-Lin-MC3-DMA to the negative charge (phosphorus atom, P) of Cip2a siRNA, N / P=6:1, and the volume ratio of the organic phase to the aqueous phase, 1:3, the Cip2a siRNA mother liquor is dissolved in a sodium citrate aqueous solution (pH=4.5) to prepare an aqueous phase. The organic phase is added dropwise into the aqueous phase by using the organic phase injection method, vortexed for 1 min and left to stand for 10 min, to assemble the lipid nanoparticles LNP@Cip2a siRNA, which is loaded into a dialysis bag and placed in a PBS buffer (4°C) for dialysis for 12 h.

[0074] (3) Preparation of PML@Cip2a siRNA:

[0075] The LNP@Cip2a siRNA and the PD1-MM are mixed at a mass ratio of 3:1, and the co-extrusion is performed through the filter membranes (800 nm, 400 nm, and 200 nm) in sequence by using an extruder to obtain a PML@Cip2a siRNA nanoparticle solution. The obtained nanoparticle solution is transferred into a ready-to-use dialysis bag with a molecular weight cut-off (MWCO) of 3.5 kDa, and dialyzed in 1L of a PBS buffer with pH=7.4 for 10 h to remove the organic solvents and free small molecule compounds.

[0076] Concentration of the primary product: The obtained nanoparticles are transferred into an Amicon ultra-centrifugal filter tube (MWCO 3.5 kDa) and added with nuclease-free water, and then centrifuged at 2000 g at 4°C for concentration, and then added with PBS to adjust the physiological osmotic pressure.

[0077] Freeze-drying: After concentration, 20% sucrose aqueous solution is added, and the final product solution concentration is 10% sucrose aqueous solution, which is transferred into a freeze storage tube and frozen in a refrigerator at -80°C for 6 h, and then transferred into a freeze dryer, and freeze-dried overnight under the conditions of 4°C and 200 mTorr, and then packaged and stored in a refrigerator at -20°C or -80°C.

[0078] Comparative Example 1

[0079] The difference between this comparative example and Example 1 is that the RAW264.7 cell membrane vesicles MM are prepared according to the method of step (1), and the LNP@Cip2a siRNA and the MM are mixed at a mass ratio of 3:1 to prepare the ML@Cip2a siRNA.

[0080] Example 2

[0081] This embodiment provides the characterization of the biomimetic nanometer nucleic acid drug.

[0082] (1) The expression level of PD1 on the surface of engineered RAW264.7 cells was quantitatively analyzed using Western blot and flow cytometry. For example... Figure 2 As shown in Figure A, PD1 protein expression on the surface of PD1-RAW264.7 cells is increased, such as... Figure 2 As shown in Figure B, PD1 expression on the surface of PD1-RAW264.7 cells is increased.

[0083] (2) TEM detection method: A solution of PML@Cip2a siRNA nanoparticles was dropped onto a copper grid and incubated for 90 s. The droplet was then blotted dry with filter paper. Subsequently, the nanoparticles were stained with 0.2% phosphotungstic acid solution for 30 s, and the staining solution was carefully blotted dry. The morphology and particle size were observed under TEM. Figure 3 As shown, LNP@Cip2a siRNA is round, cell membrane extrusion vesicles PD1-MM are hollow and round, PML@Cip2a siRNA particles are round, cell membrane encapsulates lipid particles on the periphery, they are basically the same size and have good dispersibility.

[0084] (3) The particle size and potential detection methods were as follows: After diluting the PML@Cip2a siRNA nanoparticle solution, the hydrated particle size distribution and surface potential of each nanoparticle were measured using a Zetasizer Nano ZS (Malvern) nanoparticle size potentiostat. Figure 4 As shown, the hydrated particle size of PML@Cip2a siRNA detected by DLS was approximately 110 nm. The zeta potential was -5 mV.

[0085] (4) Detection of the fusion status of LNP@Cip2a siRNA with PD1-MM in PML@Cip2a siRNA: Liposomes were bound with the red fluorescent dye Dio and cell membranes were bound with the green fluorescent dye Dil. Figure 5 As shown, the fluorescence of liposome-labeled markers on the surface of enveloped liposomes binds to the fluorescence of cell membrane-labeled markers. (As illustrated...) Figure 6 As shown, LNP@Cip2asiRNA labeled with red fluorescence binds to PD1-MM labeled with green fluorescence to form orange-yellow fluorescence. The binding rate of the two exceeds 90%, with good stability and high drug encapsulation efficiency, which greatly improves the effectiveness and safety of drug delivery.

[0086] Example 3

[0087] This embodiment provides a cell uptake experiment.

[0088] (1) The laser confocal microscope was used to detect the uptake of PML@Cip2a siRNA by tumor cells SCC7 cells. In order to fully understand the uptake process of tumor cells, Cy5-labeled Cip2a siRNA was used instead of Cip2a siRNA in PML@Cip2a siRNA. LNP@Cip2a siRNA, ML@Cip2a siRNA and PML@Cip2a siRNA were incubated with SCC7 cells for 2 h, respectively. As shown in FIG. 1A, the PML@Cip2a siRNA group showed the highest fluorescence intensity, followed by the LNP@Cip2a siRNA group and the ML@Cip2a siRNA group. The results showed that the biomimetic liposome could effectively promote the uptake of siRNA by tumor cells. Figure 7 As shown in FIG. 1B, the biomimetic liposome could effectively promote the uptake of siRNA by tumor cells.

[0089] (2) After adding lysosome probe Lyso Tracker Deep Red (LTDR) and co-incubating for 2 h, as shown in FIG. 2A, the nano material was co-localized with LTDR, and the Pearson's correlation coefficient (PCC) reached 0.82. The co-localization of Cy5-Cip2a siRNA with lysosomes decreased after 4 h of incubation, indicating that siRNA was released from lysosomes. Figure 7 As shown in FIG. 2B, the biomimetic liposome could effectively promote the uptake of siRNA by tumor cells.

[0090] (3) Flow cytometry was used to detect the uptake of LNP@Cip2a siRNA, ML@Cip2a siRNA and PML@Cip2a siRNA by macrophage RAW264.7 cells. As shown in FIG. 3A, the PML@Cip2a siRNA group showed the highest fluorescence intensity, followed by the LNP@Cip2a siRNA group and the ML@Cip2a siRNA group. The results showed that the biomimetic liposome could effectively reduce the uptake of siRNA by macrophages. Figure 8 As shown in FIG. 3B, the biomimetic liposome could effectively reduce the uptake of siRNA by macrophages.

[0091] It can be seen that PML@Cip2a siRNA can actively target oral squamous cell carcinoma cell line SCC7 cells, release Cip2a siRNA to the cytoplasm through intracellular lysosome escape, and effectively reduce the phagocytosis of nano particles by macrophage RAW264.7 cells due to the wrapping effect of macrophage membrane.

[0092] Example 4

[0093] This example provides evaluation of the cytotoxicity of nano agents.

[0094] (1) MTT method was used to detect the toxicity of drugs on SCC7 cells.

[0095] Cells were seeded in 96-well plates at 3000 cells per well, and after 24 h, the original culture solution was removed, and the designed different concentrations of drugs were added into the cells, 100 μL per well, 4 parallel holes were set, and blank medium and cells without drugs were set as control holes. After 48 h of drug addition, 20 μL MTT was added per well, incubated at 37°C for 4 h, and then 150 μl DMSO was added to dissolve the formazan, and the absorbance value (OD) of each well was measured at 595 nm using a multifunctional enzyme label instrument. The cell survival rate calculation formula is: cell survival rate % = (experimental group OD value - blank group OD value) / (control group OD value - blank group OD value)

[0096] As shown in Figure 9 , the cell death rate of the biomimetic liposome treatment group increased, and the toxicity was in the order of: PML@Cip2a siRNA > ML@Cip2a siRNA > LNP@Cip2a siRNA.

[0097] (2) Flow cytometry was used to detect the apoptosis induction of drugs on SCC7 cells.

[0098] SCC7 cells in good growth state were seeded in 6-well plates at a cell density of 1×10 5 cells per well, and incubated in a 37°C incubator overnight to allow cell adhesion. A certain concentration of PML@Cip2a siRNA, ML@Cip2a siRNA and LNP@Cip2a siRNA was added, 3 parallel holes were set for each group, and incubated in a 37°C incubator for 48 h. PBS was used for washing, and the cells were digested with trypsin without EDTA for 2 min, and then the digestion was stopped by adding culture medium. The cell suspension was collected, centrifuged at 1000 rcf for 3 min, the supernatant was discarded, and the staining solution in the apoptosis kit was added for incubation in the dark for 30 min. Centrifugation was performed at 1000 rcf for 3 min, the supernatant was discarded, 500 μL Binding buffer was added to each tube, and the machine was detected. As shown in Figure 10 , PML@Cip2a siRNA induced the most cell apoptosis. The cell apoptosis rate was in the order of: PML@Cip2a siRNA > ML@Cip2a siRNA > LNP@Cip2a siRNA.

[0099] (3) Cell scratch test and Transwell test were used to detect the growth inhibition effect of biomimetic liposomes on SCC7 cells.

[0100] Cells were seeded at a density of 5×10 5Individuals were seeded in 6-well plates at a density to form a monolayer. Using a 200 μL sterile pipette tip, a straight line was drawn on the cell monolayer in each well perpendicular to the plate bottom. The cells were gently washed with PBS for 3 times to remove the cell debris. After 24 h, the medium was replaced with serum-free medium containing the corresponding drug group. Image acquisition was performed at 0 h and 48 h at the same position of the scratch under an inverted microscope. The cell migration rate was calculated according to the following formula: cell migration inhibition rate (%) = [1 - (area change of the experimental group / area change of the control group)] x 100%. As shown in FIG. 5, the biomimetic liposome treatment group significantly inhibited tumor cell migration. Figure 11

[0101] The polycarbonate membrane of the upper chamber (pore size 8 μm) of the Transwell was coated with Matrigel matrix glue (1:8 diluted with serum-free medium) and placed in a 37°C incubator for 1 h to solidify. After solidification, the chamber was hydrated with serum-free medium for 30 min. The cells were digested and resuspended, and the cell density was adjusted to 2.5 x 10 5 cells / mL with serum-free medium. 200 μL of the cell suspension (containing 5 x 10 4 cells) and the corresponding drug group were added to the upper chamber. 500 μL of medium containing 20% fetal bovine serum (FBS) was added to the lower chamber as a chemical attractant. The culture plate was incubated in a 37°C, 5% CO2 incubator for 24 h. The chamber was removed, and the Matrigel glue and uninvaded cells in the upper chamber were gently wiped off with a cotton swab. The chamber was fixed in 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 20 min. It was gently rinsed with PBS and air-dried. Cell invasion inhibition rate (%) = [1 - (number of cells penetrating the membrane in the experimental group / number of cells penetrating the membrane in the control group)] x 100%. As shown in FIG. 6, the biomimetic liposome treatment group significantly inhibited tumor cell invasion and migration. Figure 12

[0102] As can be seen, PML@Cip2a siRNA can significantly inhibit tumor growth, induce tumor cell apoptosis, and effectively kill oral squamous cell carcinoma cells SCC7.

[0103] Example 5

[0104] This example provides detection of the regulation of proteins in tumor cells by nanodrugs.

[0105] ​​After treatment, cells were collected, and RIPA protein lysis buffer was added to extract total intracellular protein. Protein quantification was performed using quinolinic acid (BCA) standards, and equal volumes of protein were separated by SDS-polyacrylamide gel electrophoresis. After electrophoresis, a PVDF membrane was transferred. The PVDF membrane was blocked with blocking buffer, and then incubated sequentially with primary antibodies (Cip2a, Bax, Bcl-2, Caspase-3, Cleaved Caspase-3, and β-tublin) and a horseradish peroxidase-conjugated secondary antibody. Finally, the PVDF membrane was soaked in ECL luminescent substrate solution and photographed using a gel imaging system. Figure 13 As shown, the biomimetic liposome PML@Cip2a siRNA significantly inhibited intracellular Cip2a protein expression and regulated apoptosis-related proteins, such as promoting Bax and Cleaved Caspase-3 protein expression and inhibiting Bcl-2 and Caspase-3 protein expression.

[0106] Example 6

[0107] This embodiment provides an evaluation of the in vivo antitumor effect of nanoparticles.

[0108] A CDX model of OSCC was established in tumor-bearing mice to evaluate the antitumor effect of PML@Cip2a siRNA. C57BL / 6 mice, 4-6 weeks old, were housed in an SPF-grade barrier system. Feed and water were sterilized and provided to the animals freely in the animal facility. A CDX model was established in C57BL / 6 mice when the tumor volume reached approximately 150 mm. 3 Mice were randomly divided into four groups: PBS group, LNP@NCsiRNA group (NC siRNA was a negative control small interfering RNA), LNP@Cip2a siRNA group, ML@Cip2a siRNA group, and PML@Cip2a siRNA group. Five mice were in each group. Administered the drug every three days at a dose of 400 mg / kg, with 150 μL injected intravenously into each mouse via the tail vein. Tumor volume and body weight were recorded every other day during the experiment. Mice were sacrificed after 21 days. The animal experimental procedure is as follows: Figure 14 As shown.

[0109] like Figure 15 As shown, there was no significant difference in tumor size between the PBS group and the LNP@NC siRNA group, while the ML@Cip2a siRNA group and the PML@Cip2a siRNA group showed a significant inhibitory effect on tumor growth compared to other groups, and no signs of tumor recurrence were observed during the 21-day monitoring period.

[0110] like Figure 16As shown, the PML@Cip2a siRNA group had more necrotic foci. IHC results showed that PML@Cip2a siRNA significantly reduced the positive rates of tumor ki67 and Cip2a, and PD-L1 was significantly downregulated. TUNEL results showed that the PML@Cip2a siRNA group had the highest proportion of apoptosis.

[0111] like Figure 17 As shown, the weight of mice in all groups did not change significantly during the treatment process, and there was no sharp decrease or increase in weight in a short period of time.

[0112] like Figure 18 As shown, no obvious toxic side effects were found in the nanomaterials, indicating that the drug dosage used in this study was reasonable and the various treatment methods had high biosafety.

[0113] like Figure 19 As shown, the nanomaterials did not cause any toxic side effects to liver or kidney function.

[0114] like Figure 20 As shown, the nanomaterials did not exhibit hemolysis.

[0115] Example 7

[0116] This embodiment provides an evaluation of the antitumor immune-enhancing effect of nanoparticles.

[0117] To investigate the role of PML@Cip2a siRNA in the tumor immune microenvironment, tumors from each treatment group were collected, digested into single cells, and subjected to staining for immune-related indicators, followed by flow cytometry analysis. Simultaneously, enzyme-linked immunosorbent assay (ELISA) was performed on tumor tissues and animal serum from each treatment group after treatment to detect the levels of TNF-α, IFN-γ, and IL-6.

[0118] The collection method is as follows: Tumor tissue was immersed in a six-well plate containing PBS, and the connective tissue surrounding the tumor was removed. The tissue was then washed twice with PBS and placed in the six-well plate. The tumor tissue was then cut into pieces approximately 1 mm in size using scissors. 3Small pieces of tissue were added to a six-well plate containing a solution of collagenase IV (200 U / mL) and DNase I (40 U / mL) in serum-free high glucose medium (DMEM), followed by incubation in a 37°C water bath for 1 h. A cell strainer was placed on top of a 50 mL centrifuge tube containing cells with a pore size of 100 μm, and the solution containing the digested tumor tissue pieces was slowly added to the strainer, while a solution of 2 mM EDTA in DMEM serum-free medium was added dropwise to stop the digestion. If large tissue pieces were still visible after filtration, the cell suspension was obtained by filtering again. The cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the pellet was obtained as the cells extracted from the tumor tissue. The cell pellet was resuspended in PBS buffer solution for subsequent antibody fluorescence staining.

[0119] The staining method was as follows: in the immune cell analysis, the anti-CD16 / 32 antibody was first used to co-incubate with the cells (4°C, 30 min) to block the non-specific binding of Fc receptors. For the analysis of mature dendritic cells, the cell suspension was further stained with anti-CD45-FITC, anti-CD11c-PE-cy7, anti-MHC II-APC / Fire™750, anti-CD80-PE and anti-CD86-APC under light-protected conditions (4°C, 30 min). For the analysis of tumor-infiltrating lymphocytes, anti-CD45-FITC, anti-CD3-APC-cy7, anti-CD4-PE-cy7 and anti-CD8-APC were used for staining under the same conditions (light-protected, 4°C, 30 min). After staining was completed, the cells were centrifuged at 350 g for 5 min at 4°C with FACS buffer (1x PBS containing 3% FBS) for washing once, and finally 7-AAD-Percp-Cy5.5 reagent was added for flow cytometry detection.

[0120] ELISA detection as follows: tumor-bearing mice heart puncture blood, room temperature for 30 min, 4°C, 2000 g centrifugation 15 min. Take the supernatant, sub-packaging, -80°C preservation. Take the tumor tissue weighing, washing with pre-cooled PBS. According to the volume is 1:9 to join the PBS containing protease inhibitors, homogenate on ice. 4°C, 10000 g centrifugation 15 min. Take the supernatant, sub-packaging, -80°C preservation. Using mouse specific TNF-α, IFN-γ and IL-6 ELISA kit. The standard was serially diluted. The standard, sample (serum usually 1:2 dilution; tissue homogenate according to the pre-experiment dilution) was added to the pre-coated 96-well plate, 100 μL per well, setting 3 replicates. Incubate at room temperature for 2 h. Discard the liquid, wash the plate 4 times with washing solution, and pat dry. Add 100 μL biotinylated detection antibody per well, incubate at room temperature for 1-2 h. Wash the plate 4 times. Add 100 μL streptavidin-HRP per well, incubate at room temperature for 30 min in the dark. Wash the plate 4 times. Add 100 μL TMB substrate solution per well, develop at room temperature for 15-30 min in the dark. Add 50-100 μL stop solution per well. Immediately determine the absorbance value at 450 nm wavelength with a microplate reader, 570 nm or 540 nm as the reference wavelength.

[0121] As shown in Figure 21 , compared with the control group, PML@Cip2a siRNA can activate dendritic cells, promote antigen presentation, promote the infiltration of CD4 + and CD8 + T cells.

[0122] As shown in Figure 22 , PML@Cip2a siRNA promotes the secretion of anti-tumor factors TNF-α and IFN-γ in tumor tissue Figure 22 (A) and mouse serum Figure 22 (B), and down-regulates the level of pro-tumor factor IL-6, thereby improving the tumor immune microenvironment.

[0123] Example 8

[0124] To further explore the effect of PML@Cip2a siRNA on gene expression patterns, single cell sequencing RNA-seq was performed on the tumor of the control group and PML@Cip2a siRNA group mice.

[0125] Transcriptome analysis of 6 samples was completed by high-throughput sequencing technology, and a total of 35.90 Gb Clean Data was obtained, and the Clean Data of each sample reached 5.53 Gb, and the Q30 base percentage was 94.01% or more. The Clean Reads of each sample were respectively compared with the specified reference genome, and the comparison efficiency was more than 91.12%. Based on the comparison results, variable splicing prediction analysis, gene structure optimization analysis and new gene mining were carried out, 2067 new genes were mined, of which 534 were functionally annotated. In this embodiment, Fold Change≥2 and FDR<0.01 were used as the differential gene screening standard, and in each comparison group set, the results of differential expression gene list, differential expression gene function enrichment analysis, GSEA analysis and the like were obtained. The experimental process includes sample detection, library construction, quality control and machine sequencing.

[0126] As shown in Figure 23 As shown in A-C, after PML@Cip2a siRNA treatment, 2740 genes were up-regulated and 2313 genes were down-regulated in tumor tissues, and the expression of Cip2a gene in tumor tissues was significantly down-regulated.

[0127] As shown in Figure 23 As shown in D, after PML@Cip2a siRNA treatment, the pathways related to cell apoptosis and tumor immune response were activated.

[0128] As shown in Figure 24 As shown in E, after PML@Cip2a siRNA treatment, the pathways related to cell death and tumor immune response were activated.

[0129] As shown in Figure 25 As shown in the results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiment, the expression of representative genes of cell death and tumor immune response related pathways in tumor tissues of mice treated with control group and PML@Cip2a siRNA group was significantly increased.

[0130] The method of RT-qPCR experiment is as follows:

[0131] (1) RNA extraction: The frozen tissue was placed in a pre-cooled mortar and ground into fine powder with liquid nitrogen. The powder was quickly transferred to an EP tube containing 1 mL Trizol and allowed to stand at room temperature for 5 min for lysis. 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 s and allowed to stand at room temperature for 3 min. Centrifugation was performed at 4°C (12,000 g, 15 min), and the sample was divided into three layers (the upper colorless aqueous phase contained RNA, the middle layer was protein, and the lower organic phase). The upper aqueous phase was transferred to a new EP tube. An equal volume of isopropanol was added, and the mixture was gently inverted and mixed, and allowed to stand at room temperature for 10 min. Centrifugation was performed at 4°C (12,000 g, 10 min), and a white RNA precipitate was visible at the bottom of the tube. The supernatant was discarded, and the precipitate was washed with 1 mL of 75% ethanol (vortexed to suspend). Centrifugation was performed at 4°C (7,500 g, 5 min), and the ethanol was discarded, and the sample was allowed to air dry at room temperature for 5 min. 20 μL of RNase-free water was added to dissolve the RNA.

[0132] (2) cDNA synthesis: The PrimeScript RT Master Mix (TaKaRa, China) reverse transcription kit was used. The reverse transcription reaction was 500 ng of reverse RNA per 10 μL volume; the specific reaction system was as follows: sample RNA 500 ng, 5X PrimeScript RT Master Mix 2 μL, and finally ddH2O was added to a total volume of 10 μL; the PCR instrument was set as follows: 37°C for 5 min, 85°C for 15 s.

[0133] (3) RT-qPCR analysis was performed by SYBR Green Master Mix (11201ES08, Yeasen, China) reagent and LightCycler96 System (Roche, Germany) machine; the PCR reaction program was as follows: 95°C pre-denaturation for 5 min; second stage: 94°C denaturation for 30 s, 57°C annealing for 30 s, and 72°C extension for 45 s; 40 cycles; 72°C extension for 10 min, and a 30 s interval for melting curve drawing.

[0134] (4) After the reaction, the relative expression amount of each target gene was determined according to the Ct value change of each target gene.

[0135] Although the present application is disclosed as above, the protection scope of the present application is not limited to this. Various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the present application, and these changes and modifications shall fall within the protection scope of the present application.

Claims

1. An engineered biomimetic nucleic acid nanodiagnostic and nanotherapeutic agent, characterized in that: The macrophage membrane is wrapped with a cationic lipid nucleic acid drug, wherein the macrophage membrane stably expresses PD1 based on SEQ. ID. NO. 1; The cationic lipid nucleic acid drug is prepared by loading Cip2a siRNA in a cationic liposome.

2. The engineered biomimetic nucleic acid nanodiagnostic-nanod therapeutic agent of claim 1, wherein: The PD1 stably expressed on the macrophage membrane is obtained by extracting the cell membrane after the macrophage is infected by a lentivirus and expression. Puromycin is added during the infection process to kill the cells not effectively infected, and the stable strain of the PD1 stable expression is obtained under the maintenance of the Puromycin drug.

3. The engineered biomimetic nucleic acid nanodiagnostic-nanod therapeutic agent of claim 2, wherein: The cationic lipid nucleic acid drug is LNP@Cip2a siRNA nanoparticles. The mass ratio of the macrophage membrane stably expressing PD1 to the cationic liposome is 1: (2-5), and the wrapping is realized by co-extrusion through multiple layers of filter membranes in sequence by using an extruder.

4. A method for preparing an engineered biomimetic nucleic acid nanodiagnostic and nanotherapeutic agent, characterized in that: The method comprises the following steps: (1) Preparation of PD1-MM: The macrophage is infected by a lentivirus, and after the infection, the cells not effectively infected are killed by adding (1-3) μg / ml of Puromycin, and the stable strain of the PD1 stable expression is finally obtained under the maintenance of the Puromycin drug; The PD1-macrophage is collected for incubation, the cell suspension is broken and centrifuged, the cell membrane is collected, and the engineered macrophage membrane vesicle PD1-MM is obtained; (2) Preparation of a cationic lipid nucleic acid drug: D-Lin-MC3-DMA, DOPE, Cholesterol and PEG2000-DMG are used to prepare an organic phase, and Cip2a siRNA based on SEQ. ID. NO. 2 is dissolved in a buffer to prepare an aqueous phase; the organic phase is added dropwise into the aqueous phase by an organic phase injection method, and the lipid nanoparticles LNP@Cip2a siRNA are assembled; (3) Preparation of PML@Cip2a siRNA: LNP@Cip2a siRNA and PD1-MM are mixed in a certain proportion, and the PML@Cip2a siRNA nanoparticle solution is obtained by co-extrusion through multiple layers of filter membranes in sequence by using an extruder; the organic solvent and free small molecule compounds in the solution are removed, nuclease-free water is added, centrifuged, concentrated, and the physiological osmotic pressure is adjusted to obtain PML@Cip2a siRNA.

5. The preparation method of the engineered biomimetic nucleic acid nanotherapeutic agent according to claim 4, characterized in that: The lentivirus in step (1) is selected from HBLV-Pdcd1-3xflag-ZsGreen-PURO.

6. The preparation method of the engineered biomimetic nucleic acid nanotherapeutic agent according to claim 5, characterized in that: The molar ratio of D-Lin-MC3-DMA, DOPE, Cholesterol and PEG2000-DMG in the organic phase in step (2) is (48-55):(8-12):(35-40):(0.5-2).

7. The method for preparing engineered biomimetic nucleic acid nanotherapeutic agents according to claim 6, characterized in that: The volume ratio of the aqueous phase to the organic phase in step (2) is 3:1, and the molar ratio of the positive charge of D-Lin-MC3-DMA to the negative charge of Cip2a siRNA is 6:

1.

8. The method for preparing engineered biomimetic nucleic acid nanotherapeutic agents according to claim 7, characterized in that: The mass ratio of LNP@Cip2a siRNA to PD1-MM in step (3) is (2-5):

1.

9. The method for preparing the engineered biomimetic nucleic acid nanotherapeutic agent according to claim 8, characterized in that: The PML@Cip2a siRNA nanoparticle solution was obtained by co-extrusion through 800 nm, 400 nm and 200 nm filter membranes in sequence in step (3) using an extruder.

10. The use of the engineered biomimetic nucleic acid nanodiagnostic and nanotherapeutic agent of claim 1 in the preparation of an oral squamous cell carcinoma diagnostic kit or drug.

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