A cell bionic nanoparticle and its construction method and application

Abstract

The present invention belongs to the technical field of cell bionic nanomedicines, and specifically discloses a cell bionic nanoparticle and a construction method and application thereof. Cell bionic nanoparticles are obtained by simple ultrasonic fusion of the target cell membrane and the hydrophobic nanocore. The bionic nanocell can effectively inherit and simulate the functional proteins on the surface of the source cell membrane, and systematically replace the functions played by the source cell in the physiological and pathological process, such as adsorbing inflammatory factors, migration factors, etc., interfering with and inhibiting the disease process from the cellular mechanism, regulating the cell composition and immune microenvironment of the lesion site, without relying on additional drug delivery. The present invention innovatively proposes a therapeutic strategy for constructing immune tolerance in response to the related pathogenesis of immune tolerance deficiency. The homology and inherent immunosuppression of tumor cells to the lesion site of autoimmune diseases are reversely used to reconstruct the immune tolerance of the lesion site based on immune checkpoints, while replacing the target cells to adsorb autoimmune antibodies and reduce the level of immune response.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a cell bionic nanoparticle and a construction method and application thereof. Background Art

[0002] Cell bionic nanomedicines constructed using cell membrane fusion technology can achieve high-level functions such as active targeting and inflammation regulation by efficiently inheriting many functional molecules on the surface of source cells, such as signal receptors and surface antigens, which traditional nanomedicines need to go through a lot of complex chemical processes to achieve. At the same time, the inherent size effect of nanomedicines can achieve passive targeted enrichment of inflammatory areas. Therefore, a large number of excellent results have emerged in the field of inflammation treatment using immune cell membrane bionic nanomedicines.

[0003] However, this application is currently mainly focused on using cell membranes as functional auxiliary substances, and its therapeutic effect still mainly depends on the related drugs it carries. At the same time, the application of such nanomedicines in autoimmune diseases is still relatively lacking, and its main construction strategy focuses on carrying immunosuppressants and immune tolerance reconstruction substances, while few people pay attention to the inhibition of autoimmune antibody levels. More importantly, the process flow of these achievements is still complicated, which is not conducive to rapid and widespread clinical application, and the drug accessibility is poor.

[0004] Autoimmune diseases are a complex class of organ-specific or systemic inflammatory diseases that seriously affect the quality of life of patients. Existing nanomedicines for the treatment of autoimmune diseases mainly use complex chemical processes to co-construct carrier materials to carry the corresponding therapeutic drugs in order to regulate the level of inflammation and inhibit the autoimmune response in the lesion site. Even the newly born cell membrane bionic nanomedicines often use the cell membrane as an auxiliary substance. There is still a clear lack in current research to simply use the diverse physiological functions of the cell membrane to achieve the treatment of autoimmune diseases, especially to systematically achieve the regulation of inflammation levels, the reconstruction of immune tolerance, and the reduction of autoimmune antibody levels. Summary of the invention

[0005] The purpose of the present invention is to provide a cell bionic nanoparticle and its construction method and application in view of the above-mentioned deficiencies of the prior art, and to obtain a bionic nanocell by simply ultrasonically fusing the target cell membrane with the hydrophobic nanocore. The bionic nanocell can effectively inherit and simulate the functional proteins on the surface of the source cell membrane, systematically replace the functions played by the source cell in the physiological and pathological process, such as adsorbing inflammatory factors, migration factors, etc., interfering with and inhibiting the disease process from the cell mechanism, and regulating the cell composition and immune microenvironment of the lesion site without relying on additional drug delivery.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first object of the present invention is to provide a method for constructing cell bionic nanoparticles, comprising the following steps:

[0008] S1. Preparation of cell membranes,

[0009] Cultivating immune cells or tumor cells, and after ultrasonic disruption, removing the cell contents, taking the supernatant, centrifuging, and obtaining cell membranes; the immune cells include any one of lymphocytes and macrophages; the tumor cells are tumor cells that have the same autoimmune antigens as the pathological target cells;

[0010] S2. Preparation of cell-inspired nanoparticles.

[0011] Taking the cell membrane protein content as a standard, the cell membrane is mixed in double distilled water according to a preset ratio, and then hydrophobic nanoparticles are added, ultrasonically mixed, and then centrifuged to obtain cell bionic nanoparticles. The hydrophobic nanoparticles include any one of polylactic acid nanoparticles, polystyrene nanoparticles or polylactic acid-glycolic acid nanoparticles.

[0012] Furthermore, in step S1, the centrifugal speed is 10000-20000 rpm, and the centrifugal time is 1-60 min.

[0013] Furthermore, the mass ratio of cell membrane protein: hydrophobic nanoparticles is 10:1~1:10.

[0014] Further, ultrasonic mixing was performed for 3-20 min, and then centrifuged at 10,000-20,000 g.

[0015] Furthermore, in step S2, the cell membrane may also be a hybrid cell membrane formed by the fusion of the cell membrane of an immune cell and the cell membrane of a tumor cell.

[0016] Furthermore, the tumor cells are liver cancer tumor cells that overexpress PD-L1 or lung cancer tumor cells that overexpress TSHR / IGF-1R / PD-L1.

[0017] Furthermore, the immune cells are macrophages.

[0018] The second object of the present invention is to provide a cell bionic nanoparticle constructed by the above method.

[0019] The third object of the present invention is to provide an application of the above-mentioned cell bionic nanoparticles in preparing drugs for treating autoimmune diseases.

[0020] Furthermore, the autoimmune disease includes autoimmune hepatitis or thyroid-related eye disease.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention provides a method for constructing cell bionic nanoparticles. Different from existing cell bionic nanomedicines, the present invention starts from the pathological mechanism of autoimmune diseases and obtains bionic nanocells by simply ultrasonically fusing the target cell membrane with the hydrophobic nanocore. The bionic nanocells can effectively inherit and simulate the functional proteins on the surface of the source cell membrane, systematically replace the functions of the source cells in the physiological and pathological processes, such as adsorbing inflammatory factors and migration factors, interfere with and inhibit the disease process from the cellular mechanism, and regulate the cell composition and immune microenvironment of the lesion site without relying on additional carrier drugs. Aiming at the related pathogenesis of immune tolerance deficiency, an innovative treatment strategy for constructing immune tolerance is proposed.

[0023] (2) The present invention provides reverse use of the homology and inherent immunosuppression of tumor cells to the lesions of autoimmune diseases to reconstruct immune tolerance based on immune checkpoints at the lesions, while replacing target cells to adsorb autoimmune antibodies and reducing the level of immune response.

[0024] (3) The present invention also creatively hybridizes the cell membrane as a modular functional set to construct hybrid bionic nanocells with dual functions, achieving simultaneous inhibition of immune response and inflammation, which has extremely important scientific research value and broad clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Comparison of Coomassie blue staining of biomimetic nano-lymphocyte surface proteins (LM&PLA-NPs) and lymphocyte membrane (LM);

[0026] Figure 2 This is a comparison of the particle sizes of PLA nanoparticles and bionic nanolymphocytes;

[0027] Figure 3 This is a graph of PD-L1 mRNA levels in PD-L1 overexpressing stable strains;

[0028] Figure 4 Figure 1 is a graph showing the expression level of PD-L1 protein in a PD-L1 overexpressing stable strain, where A is a fluorescence detection graph and B is a data statistics graph;

[0029] Figure 5 Western Blot was used to detect the bands of three target proteins expressed in macrophages and cell membranes with different pretreatments;

[0030] Figure 6 This is a comparison of the particle sizes of bionic nano lung cancer cells in pure water at different lung cancer tumor cell membrane / PLGA mass ratios;

[0031] Figure 7 This is a comparison of the zata potential of bionic nano lung cancer cells in pure water at different lung cancer tumor cell membrane / PLGA mass ratios;

[0032] Figure 8 The comparison of particle sizes of biomimetic nanohybrid cells in PBS and pure water obtained by different macrophage / tumor cell hybrid membranes and PS at different mass ratios;

[0033] Fig. 9 SDS-PAGE images of common polystyrene nanoparticles (PS), hybrid cell membranes (PHRM), biomimetic nano-hybrid cells (PHRM-NPs), macrophage membranes (PRM), tumor cell membranes (PHM), biomimetic nano-macrophages (PRM-NPs), and biomimetic nano-tumor cells (PHM-NPs) after Coomassie Brilliant Blue staining;

[0034] Fig.10 The graph shows the changes in serum AST levels in mice with autoimmune hepatitis treated with different doses of bionic nano-lymphocytes. In the graph, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001;

[0035] Fig.11 The figure shows the changes in serum ALT levels in mice with autoimmune hepatitis treated with different doses of bionic nano-lymphocytes. **, P < 0.01.

[0036] Fig.12 The figure shows the changes in the level of inflammatory factor TNF-α in mice with autoimmune hepatitis treated with different doses of bionic nano-lymphocytes. In the figure, *, P < 0.05, ****, P < 0.0001;

[0037] Fig.13 The graph shows the changes in the level of inflammatory factor IFN-γ in mice with autoimmune hepatitis treated with different doses of bionic nano-lymphocytes. In the graph, *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001;

[0038] Fig.14 The following are the gross specimens of the organs of mice with autoimmune hepatitis after treatment with different doses of biomimetic nanohybrid cells (PHRM-NPs);

[0039] Fig.15 Comparison of liver indexes of mice with autoimmune hepatitis after treatment with different doses of bionic nanohybrid cells (PHRM-NPs), **, P < 0.01, ***, P < 0.001, ****, P < 0.0001;

[0040] Fig.16The figure shows the comparison of spleen index of mice with autoimmune hepatitis after treatment with different doses of bionic nanohybrid cells (PHRM-NPs). In the figure, **, P < 0.01, ****, P < 0.0001;

[0041] Fig.17 The change of serum ALT level in mice with autoimmune hepatitis treated with different doses of bionic nanohybrid cells (PHRM-NPs), ****, P<0.0001;

[0042] Fig.18 The change of serum AST level in mice with autoimmune hepatitis treated with different doses of bionic nanohybrid cells (PHRM-NPs), ****, P<0.0001;

[0043] Fig.19 The graphs show the changes in serum inflammatory factors TNF-α and IFN-γ in mice with autoimmune hepatitis treated with different doses of bionic nanohybrid cells (PHRM-NPs). Figure A shows the changes in TNF-α, and Figure B shows the changes in IFN-γ. In the figure, ****, P<0.0001.

[0044] Fig. 20 The graphs are the levels of serum inflammatory factors TNF-α and IFN-γ in mice with autoimmune hepatitis treated with different doses of bionic nanohybrid cells (PHRM-NPs). Figure C is the level of IL-6, and Figure B is the level of IgG. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

[0045] Fig.21 This is a comparison of the changes in the levels of TNF-α, a serum inflammatory factor, in mice with autoimmune hepatitis treated with bionic nano lung cancer cells (LA-NPs). In the figure, ****, P<0.0001;

[0046] Fig. 22 This is a comparison chart of the changes in the levels of IFN-γ, a serum inflammatory factor in mice with autoimmune hepatitis treated with bionic nano lung cancer cells (LA-NPs). In the figure, ****, P<0.0001. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific test method, instrument or condition is specified in the examples, it is carried out according to the technology or condition described in the literature in this field or according to the product instructions. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0048] In the present invention, polylactic acid is abbreviated as PLA; polystyrene is abbreviated as PS; and polylactic-co-glycolic acid is abbreviated as PLGA.

[0049] The membrane protein in the present invention is measured by a commonly used BCA kit for quantification. The enhanced BCA protein concentration determination kit used in the implementation method is purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.

[0050] The preparation process of hydrophobic nanoparticles in the present invention is that the hydrophobic nanoparticles are prepared by emulsification / solvent volatilization method, 7.5mg of hydrophobic polymer is dissolved with 1mL of tetrahydrofuran, and 10mg of polyvinyl alcohol is added to 5mL of ultrapure water to prepare a 0.2% PVA solution. The PVA solution is placed on a magnetic stirrer, a magnetic stirrer is added, and under stirring, a 1mL syringe is used to draw the PLA solution, and it is slowly and uniformly added to the PVA solution. After the dropwise addition is completed, the probe is ultrasonicated for 5min (300w, open for 2s, close for 3s), and the rotary evaporator is rotary evaporated for 15min to remove most of the organic solvent THF. Freeze, freeze-dry, and store at -20°C for standby use. Hydrophobic nanoparticles include PLA nanoparticles, PS nanoparticles, and PLGA nanoparticles.

[0051] The cell bionic nanoparticles in the present invention are equivalent to bionic nanocells.

[0052] Unless otherwise indicated, in the practice of the present invention, conventional nanotechnology, nanoengineering, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology and pharmacology techniques within the skill of the art will be used. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual (4th Edition) (MR Green et al.); Animal Cell Culture: A Guide to Basic Techniques and Special Applications (Editor: RI Freshney, Seventh Edition); Enzymology Methods (published by Elsevier); Modern Molecular Biology: A Laboratory Manual (Editor: FM Ausubel et al.).

[0053] Mouse mononuclear macrophage cell line (RAW264.7) was purchased from Wuhan Pronocell Biotechnology Co., Ltd. and cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin mixture in a cell culture incubator at 37°C and 5% CO2. Mouse hepatocellular carcinoma cell line (Hepa1-6) was purchased from Wuhan Pronocell Biotechnology Co., Ltd. and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin mixture in a cell culture incubator at 37°C and 5% CO2. Mouse normal hepatocyte cell line (AML12) was cryopreserved by the Institute of Liver Diseases, Southwest Hospital Affiliated to Army Medical University. The culture medium was DMEM containing 10% fetal bovine serum (FBS), 0.5% ITS-G (100×), 40ng / ml dexamethasone, and 1% penicillin and streptomycin mixture; male 6-8 week old C57 / BL6J mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., weighing 18-20g. They were kept in the SPF animal room of the Basic Research Laboratory of Traditional Chinese Medicine, Nanqiao Temple District, Chongqing Hospital of Traditional Chinese Medicine, with free access to water. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Chongqing Hospital of Traditional Chinese Medicine.

[0054] Example 1

[0055] This embodiment provides a method for preparing lymphocyte biomimetic nanoparticles.

[0056] The specific steps are:

[0057] (1) Extraction of lymphocyte membrane

[0058] Place the mouse spleen in 2% RPMI1640 medium; grind, pass through a 70μm cell strainer, and rinse with 2ml 2% RPMI1640 medium. Add an appropriate amount of 2% RPMI1640 medium, centrifuge at 4℃, 350g for 5 minutes, discard the supernatant, resuspend the cells with 1~2mL red blood cell lysis buffer, and incubate on ice for 5min. Add an appropriate amount of medium to terminate lysis, centrifuge at 4℃, 350g for 5 minutes, and discard the supernatant. Wash twice with PBS, centrifuge at 350g for 5 minutes, and discard the supernatant. Resuspend in an appropriate amount of PBS. Recorded as LM.

[0059] (2) Preparation of biomimetic nanolymphocytes

[0060] The prepared lymphocyte membrane was resuspended in PBS and the buffer was broken and separated using a probe ultrasonic probe on ice for 30 seconds (power 100W, on 2 seconds, off 3 seconds). 膜蛋白 :n PLA) were mixed, and the buffer solution was broken and separated using a probe ultrasonic probe on ice for 5 min (power 100 W, on 2 s, off 3 s), and then centrifuged at 12000 rpm for 20 min, and the supernatant was obtained. It was recorded as LM-NPs.

[0061] The membrane proteins of bionic nanolymphocytes were characterized and identified. Figure 1 As shown in the figure, the protein bands are the same, indicating that the bionic nano-lymphocyte membrane (LM&PLA-NPs) and the lymphocyte membrane (LM) express the same membrane proteins. Figure 2 As shown, the particle size of bionic nano-lymphocytes is between 250 and 300 nm.

[0062] Example 2

[0063] This embodiment provides a preparation of liver cancer tumor cell bionic nanoparticles.

[0064] The specific steps are:

[0065] (1) Construction of Hepa1-6 stable transgenic cell overexpressing PD-L1

[0066] Hepa1-6 cells in good growth condition and logarithmic growth phase were plated in 96-well plates and cultured in an incubator at 37°C and 5% CO2 overnight. Before infection, the virus was slowly thawed on ice, and the original cell culture medium was replaced with 50 μL of fresh culture medium. The values ​​of MOI were 1, 5, 10, and 20. The virus stock solution was added to the cells and gently mixed. Polybrene was added at a final concentration of 8 μg / ml. 50 μL of culture medium was added 4 hours after the addition of lentivirus infection. After about 24 hours of lentivirus infection, fresh complete culture medium was added and culture was continued in an incubator at 37°C and 5% CO2. Fluorescence was preliminarily observed under a microscope 48 hours after infection. After 72 hours of infection, the infection efficiency was about 80%. The infection conditions and MOI values ​​of the experimental group with good cell growth status can be used as the basis for subsequent infection experiments. 500 μL of Hepa1-6 cell suspension in good growth status and logarithmic growth phase was added to each well of a 24-well cell culture plate. A total of 9 wells were divided into three groups: PD-L1 overexpression group, PD-L1 vector control group, and blank control group. Three replicate wells in each group were placed in a 37°C, 5% CO2 incubator and cultured overnight. Before infection, the virus was slowly thawed on ice, the original culture medium was discarded, 250 μL of fresh culture medium was added to each well of the PD-L1 overexpression group, and the virus titer of 30 μL volume was 1×10 8 TU / mL of mouse PD-L1 overexpression lentivirus, 8 μL of polybrene virus infection enhancement solution, 250 μL of fresh culture medium was added to each well of the PD-L1 vector control group, and the virus titer of 30 μL was 1×10 8TU / mL of vector control lentivirus, 8μL of polybrene virus infection enhancement solution (purchased from Chengdu Yueyong Dajiang Technology Co., Ltd.), gently shake the culture plate to mix, and then move the cell culture plate into a cell culture incubator for continued culture. After 4 hours of lentivirus infection, the culture volume is supplemented to 500μL, and 500μL of fresh culture medium is added to each well of the blank control group; about 24 hours after lentivirus infection, the culture medium containing the virus in each well is replaced with fresh complete culture medium, and the cell culture plate is moved into a cell culture incubator for continued culture at 37℃ and 5% CO2; after continuing to culture for 1 day, discard the culture medium containing Add 1 ml of DMEM complete medium containing 10 μg / mL puromycin to the culture medium containing the virus for screening, and move the cell culture plate into a cell culture incubator to continue culturing at 37°C, 5% CO2; after 3 days of continued culture, replace the cell medium with DMEM complete medium containing 10 μg / mL puromycin; after 3 days of continued culture, take 2 wells from each group for overexpression and vector control PD-L1 stable transfectant verification. After verifying that the screening of lentiviral stable transfectants is successful, use DMEM complete medium containing 1 μg / mL puromycin for subsequent cell culture.

[0067] (2) Extraction of PD-L1 highly expressed liver cancer cell membrane

[0068] Hepa1-6 cells overexpressing PD-L1 were cultured to about 80%, washed with 2 mL PBS, digested, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cell pellet was reserved for use; 1 mL of separation buffer prepared before use (225 mM mannitol, 75 mM sucrose, 0.5 mM EDTA, 30 mM Tris-HCl (pH 7.5), PMSF (1×) and protease / phosphatase inhibitors (1×)) was used to fully suspend the cells, broken with an ultrasonic probe on ice, centrifuged at 4°C, 1000 g for 10 min, and the supernatant was collected. The mitochondria were removed by further centrifugation at 4°C, 12000 g for 20 min to obtain Hepa1-6 cell membranes (Pretreated Hepa1-6 Membranes, PHM), which were freeze-dried and stored at -80°C for use. The cell membranes were recorded as PHM.

[0069] (3) Preparation of biomimetic liver cancer tumor cells

[0070] The prepared Hepa1-6 cell membrane was resuspended in PBS and the buffer was broken and separated using a probe ultrasonic probe on ice for 30 seconds (power 100W, on 2 seconds, off 3 seconds). The mass ratio of the membrane protein of the Hepa1-6 cell membrane to the PLGA nanoparticles was 1:1 (n 膜蛋白 :n PLGA) were mixed, and the buffer was broken and separated by ultrasonic probe on ice for 5 min (power 100 W, on 2 s, off 3 s), and then centrifuged at 12000 rpm for 20 min, and the supernatant was obtained. It was recorded as PHM-NPs.

[0071] like Figure 3 As shown, the PD-L1 mRNA level of the PD-L1 overexpressing stable strain is shown. + After Hepa1-6 cell line was transfected with PD-L1 overexpressing lentivirus, the PD-L1 mRNA level was significantly increased compared with the vector control group and the blank control group ( p <0.0001).

[0072] like Figure 4 As shown in the figure, A is the PD-L1 protein expression level diagram of the PD-L1 overexpression stable strain, and B is the data statistics diagram. It can be seen that Hepa1-6PD-L1 + After Hepa1-6 cell line was transfected with PD-L1 overexpressing lentivirus, the mean fluorescence intensity of PD-L1 protein detected by flow cytometry was significantly increased compared with the vector control group and blank control group ( p =0.0051), and the fluorescence intensity showed a single peak.

[0073] Example 3

[0074] This embodiment provides a preparation method of macrophage biomimetic nanoparticles.

[0075] (1) Extraction of macrophage membrane

[0076] RAW264.7 cells in good growth state and logarithmic growth phase were selected and cultured at 2×10 5Cells / well were seeded on 6-well plates and cultured for 24 hours. The cells were stimulated to differentiate into the M1 subtype for 48 hours in a cell culture incubator at 37°C and 5% CO2 using 100 ng / mL lipopolysaccharide (LPS) and different concentrations (25 or 50 ng / mL) of mouse IFN-γ. After the cells were cultured to about 80%, they were washed with 2 mL PBS, digested, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cell pellet was reserved for later use; 1 mL of separation buffer prepared before use (225 mM mannitol, 75 mM sucrose, 0.5 mM EDTA, 30 mM Tris-HCl (pH 7.5), PMSF (1×) and protease / phosphatase inhibitors (1×)) was used to fully suspend the cells, and the cells were broken with an ultrasonic probe on ice, centrifuged at 4°C, 1000 g for 10 min, and the supernatant was collected. The supernatant was further centrifuged at 4°C, 12000 g for 20 min and recorded as pretreated RAW264.7 cell membranes (Pretreated RAW264.7 Membranes, PRM), and freeze-dried and stored at -80°C for later use. The cell membranes were recorded as PRM.

[0077] (2) Preparation of bionic macrophages

[0078] The prepared RAW264.7 cell membrane was resuspended in PBS and the buffer was broken and separated using a probe ultrasonic probe on ice for 30 seconds (power 100W, on 2 seconds, off 3 seconds). The mass ratio of the membrane protein of the RAW264.7 cell membrane to the PLGA nanoparticles was 1:1 (n 膜蛋白 :n PLGA ) were mixed, and the buffer was broken and separated by ultrasonic probe on ice for 5 min (power 100 W, on 2 s, off 3 s), and then centrifuged at 12000 rpm for 20 min, and the supernatant was obtained. It was recorded as PRM-NPs.

[0079] like Figure 5The figure shows the band diagram of Western Blot detection of three target proteins expressed in macrophages and cell membranes with different pretreatments. It can be seen that GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal reference protein to balance the total protein levels among the groups. It can be seen that the expression of the three target proteins in macrophage subtypes with different pretreatments was different. (1) PD-L1 was expressed in all groups, especially in RAW264.7 cells stimulated with IFN-γ (50 ng / mL) for 48 hours and the PRM obtained had the highest expression; (2) Integrin β2 (β2 integrin) was highly expressed in the membrane of activated M1 phenotype macrophages; (3) Inflammatory factor receptors: TNF-α (tumor necrosis factor-α) receptor TNFR2 was expressed in all groups, especially in RAW264.7 cells stimulated with IFN-γ (50 ng / mL) for 48 hours and in the PRMs obtained, with the highest expression; IL-6 (interleukin-6) receptor IL-6R was highly expressed in the membrane of activated M1 phenotype macrophages; MCP-1 (monocyte chemoattractant protein-1) receptor CCR2 was highly expressed in RAW264.7 cells stimulated with IFN-γ (50 ng / mL) for 48 hours and in the PRMs obtained; IL-1 (interleukin-1) receptor IL-1R was highly expressed in RAW264.7 cells stimulated with IFN-γ (50 ng / mL) for 48 hours and in the PRMs obtained.

[0080] Example 4

[0081] This embodiment provides a preparation method of bionic nanoparticles of lung cancer tumor cells.

[0082] The specific steps are:

[0083] (1) Extraction of TSHR / IGF-1R / PD-L1 high-expressing lung cancer cell membrane

[0084] LA795 lung cancer cells with high expression of TSHR / IGF-1R / PD-L1 were cultured to about 80%, the culture medium was discarded, 2 mL of sterile PBS was added to wash the cells, PBS was discarded, and 2-3 mL of sterile PBS was added for digestion. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cell pellet was reserved for later use. 1 mL of separation buffer (225 mM mannitol, 75 mM sucrose, 0.5 mM EDTA, 30 mM Tris-HCl (pH 7.5), PM SF (1×) and protease / phosphatase inhibitors (1×)), gently and fully suspend the cells, break the separation buffer with a probe ultrasonic probe on ice for 5 min (power 100 W, on 2 s, off 3 s), then centrifuge at 4°C, 1000g for 10 min, collect the supernatant, further centrifuge at 4°C, 12000g for 20 min to remove mitochondria, etc., collect the supernatant and record it as the pretreated TSHR / IGF-1R / PD-L1 high-expressing LA795 lung cancer tumor cell membrane, and freeze-dried and stored at -80°C for later use.

[0085] (2) Preparation of biomimetic lung cancer tumor cells

[0086] The prepared lung cancer cell membrane was resuspended in PBS and the buffer was broken and separated using a probe ultrasonic probe on ice for 30s (power 100W, on 2s, off 3s). Then the membrane protein of the lung cancer cell membrane was mixed with PLGA nanoparticles at a mass ratio of (10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10) (n 膜蛋白 :n PLGA ) were mixed, and the buffer solution was broken and separated using a probe ultrasonic probe on ice for 5 min (power 100 W, on 2 s, off 3 s), and then centrifuged at 12000 rpm for 20 min, and the supernatant was obtained. It was recorded as LA-NPs.

[0087] like Figure 6 and Figure 7 As shown, the particle size and zeta potential statistics of bionic nano lung cancer cells in pure water at different lung cancer tumor cell membrane protein / PLGA mass ratios are shown. It can be seen that when the mass ratio of membrane protein to PLGA nanoparticles is 1:1, the absolute value of zeta potential is the largest, showing a stronger electrostatic repulsion, which helps the nanoparticles maintain their dispersed state and avoid aggregation.

[0088] Example 6

[0089] This embodiment provides a preparation of hybrid macrophage / tumor cell bionic nanoparticles.

[0090] (1) Preparation of hybrid cell membrane

[0091] The macrophage membrane of Example 3 and the tumor cell membrane PHM of Example 2 were selected at a ratio of 1:1.

[0092] The membrane fusion was completed by ultrasonic treatment (130 W, 42 kHz) at 37°C for 10 min, which was recorded as PHRM.

[0093] (2) Preparation of biomimetic nanohybrid macrophages / tumor cells

[0094] Hybrid cell membrane proteins in different ratios (cell membrane protein:PS=10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10) were mixed with PS nanoparticles, and the buffer was broken and separated using a probe ultrasonic probe on ice for 5 min (power 100 W, on 2 s, off 3 s), followed by centrifugation at 12000 g for 10 min to obtain biomimetic nano-hybrid macrophages / tumor cells, which were recorded as PHRM-NPs; only the cell membrane protein solution was used to break the buffer solution using a probe ultrasonic probe on ice for 5 min (power 100 W, on 2 s, off 3 s) to obtain PHRMVesicles.

[0095] like Figure 8 As shown in the figure, the particle size comparison of biomimetic nanohybrid cells obtained by different macrophage / tumor cell hybrid membranes and PS at different mass ratios in PBS and pure water shows that when the PHRM / PS weight ratio increases from 1:10 to 10:1, the particle size remains almost constant in pure water; while in PBS, the particle size increases with the increase, indicating that PRM is excessively added. The study found that PLA nanoparticles form aggregates in 1×PBS, and when the PHRM / PS mass ratio is ≥1 / 1, a uniform suspension is formed without obvious aggregation. The optimal PS / PHRM mass ratio is 1.

[0096] like Fig. 9 As shown, the SDS-PAGE images of ordinary polystyrene nanoparticles (PS), hybrid cell membranes (PHRM), bionic nanohybrid cells (PHRM-NPs), macrophage membranes (PRM), tumor cell membranes (PHM), bionic nanomacrophages (PRM-NPs), and bionic nanotumor cells (PHM-NPs) after Coomassie Brilliant Blue staining show that PHRM-NPs inherit the key biological characteristics of the source cells. After Raw264.7 cells are differentiated into M1 subtypes, M1 cell membranes are extracted to prepare PRM and Hepa1-6PD-L1. + The PHRM-NPs prepared by fusion of cell membranes extracted from Hepa1-6 cell lines obtained by transfecting the cell line with PD-L1 overexpression lentivirus still retained the high expression of membrane protein PD-L1, while very little cytoplasmic protein GAPDH remained, proving that the cell membrane protein extraction was relatively pure.

[0097] Characterization and stability testing of biomimetic nanohybrid macrophages / tumor cells revealed that the hydrodynamic diameter of PHRM-NPs was slightly larger than that of PLANPs, while its zeta potential was close to that of PRMNV. Meanwhile, transmission electron microscopy images intuitively observed the obvious "core-shell structure" of PHRM-NPs, indicating that PHRM was successfully coated on the surface of PSNPs.

[0098] Example 7

[0099] In vivo treatment of autoimmune hepatitis by biomimetic nanolymphocytes.

[0100] The study selected 6-8 week old male C57B6 mice as experimental subjects, and concanavalin A (ConA) was injected into the tail vein at a dose of 12 mg / kg to construct an animal model of mouse autoimmune hepatitis. 46 mice were randomly divided into 5 groups: Control group; ConA model group; ConA+1 mg / kg bionic nanolymphocyte group; ConA+2 mg / kg bionic nanolymphocyte group; ConA+4 mg / kg bionic nanolymphocyte group. Mice were killed 24 hours after tail vein injection to collect peripheral blood and required samples. The therapeutic effect was comprehensively evaluated from the aspects of organ appearance, inflammatory factor levels, inflammatory cell infiltration, etc. by serum detection of AST, ALT, TNF-α and IFN-γ, tissue section staining (pathological H&E staining, immunohistochemical staining), protein expression and other technical means.

[0101] like Fig.10 and Fig.11 As shown, compared with other groups, the liver function index transaminase levels AST and ALT in the ConA+4mg / kg bionic nanolymphocyte group were lower, and the AST result was roughly the same as the ALT result. Bionic nanolymphocytes can significantly reduce serum transaminase levels.

[0102] like Fig.12 and Fig.13 As shown, the levels of pro-inflammatory cytokines (TNF-α, IFN-γ) in serum decreased with increasing concentrations of therapeutic administration.

[0103] In summary, 4 mg / kg of bionic nanolymphocytes can alleviate liver damage in mice with autoimmune hepatitis caused by Con A modeling and administration, indicating that liver inflammation is alleviated.

[0104] Example 8

[0105] In vivo treatment of autoimmune hepatitis by biomimetic nanohybrid macrophage / tumor cells (PHRM-NPs).

[0106] Male C57B6 mice aged 6-8 weeks were selected as experimental subjects, and concanavalin A (ConA) was injected into the tail vein at a dose of 12 mg / kg to construct an animal model of mouse autoimmune hepatitis. 46 mice were randomly divided into 5 groups: Control group; ConA model group; ConA+1 mg / kg biomimetic nanohybrid cell group; ConA+2 mg / kg biomimetic nanohybrid cell group; ConA+4 mg / kg biomimetic nanohybrid cell group. Mice were killed 24 hours after tail vein injection to collect peripheral blood and required samples. The therapeutic effect was comprehensively evaluated from the aspects of organ appearance, inflammatory factor levels, inflammatory cell infiltration, etc. by serum detection of AST, ALT, TNF-α and IFN-γ, tissue section staining (pathological H&E staining, immunohistochemical staining), protein expression and other technical means.

[0107] like Fig.14 As shown in the figure, the liver of normal mice has a smooth surface, dark red color, clear pages and soft texture; after ConA treatment, the liver is dull in color, irregular in surface, granular, some tissues are adhered, and the edges of the leaves are dark and pale; correspondingly, the spleen of normal mice has a smooth surface, bright red color and soft texture; the spleen of the model group mice is dull in color, obviously enlarged, with uneven edges and partial necrosis; and the higher the dose, the more severe the lesions; after PHRM-NPs treatment, the volume of mouse liver tissue increased, the surface capsule was tense, moderately congested, red in color, smooth on the surface, and clear pages; the volume of the spleen decreased, necrosis decreased, and with the increase of the dosage, the appearance of the organ lesions improved.

[0108] like Fig.15 and Fig.16 As shown in the figure, compared with the normal group mice, the liver and spleen indexes of mice increased significantly after ConA administration. After PHRM-NPs administration, the organ index gradually decreased with the increase of drug concentration. There was no statistical difference between the ConA+1 mg / kg PHRM-NPs group, the ConA+2 mg / kg PHRM-NPs group and the ConA+4 mg / kg PHRM-NPs group. These indicate that the inflammation of the liver and spleen has been alleviated.

[0109] like Fig.17 and Fig.18The results of mouse serum ALT showed that the ConA model group was significantly higher than the PBS control group (P < 0.001); the ConA + 1 mg / kg PHRM-NPs group, ConA + 2 mg / kg PHRM-NPs group and ConA + 4 mg / kg PHRM-NPs group were significantly lower than the Con A model group, and there was no statistical difference between the groups (P > 0.05). The AST results were roughly the same as the ALT results. PHRM-NPs can significantly reduce serum transaminase levels. This shows that 4 mg / kg of PHRM-NPs can reduce liver damage in mice with autoimmune hepatitis caused by ConA modeling and administration.

[0110] like Fig.19 As shown, Figure A is a comparison of the levels of inflammatory factor TNF-α, and Figure B is a comparison of the levels of inflammatory factor IFN-γ; Fig. 20 As shown in the figure, C is a comparison chart of the inflammatory factor IL-6 level, and D is a chart of the inflammatory factor IL-1β level. It can be seen that the production of proinflammatory factors after modeling and administration increased compared with the PBS control group, but after treatment with different doses of PHRM-NPs, the results of the enzyme-linked immunosorbent assay showed that since PHRM-NPs have the same inflammatory factor receptors as the source cells, they can adsorb proinflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-1β) in serum, and the level of proinflammatory cytokines will decrease with the increase of the therapeutic administration concentration. It shows that 4 mg / kg of PHRM-NPs can reduce liver damage in mice with autoimmune hepatitis caused by Con A modeling and administration, indicating that liver inflammation has been alleviated.

[0111] Example 9

[0112] In vivo treatment of thyroid-associated eye disease by biomimetic nanoparticles of lung cancer cells (LA-NPs).

[0113] The study selected 6-8 week old male SD rats as experimental subjects, and injected the retroocular tissue homogenate and Freund's adjuvant emulsified in a 1:1 / 2:1 ratio with a solvent at a dose of 1 mg / kg to construct mouse autoimmune thyroid-related eye disease. The therapeutic effect was comprehensively evaluated from the aspects of organ appearance, inflammatory factor levels, inflammatory cell infiltration, etc. through serum detection of TRAb, TSH, T3 and T4, tissue section staining (pathological H&E staining, immunohistochemical staining), protein expression and other technical means.

[0114] like Fig.21 and Fig. 22 As shown, LA-NPs can reduce autoimmune thyroid-associated eye disease caused by modeling administration, suggesting that the inflammation of thyroid-associated eye disease is alleviated.

[0115] For matters not mentioned above, the prior art applies.

[0116] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing cell biomimetic nanoparticles, characterized in that: The following steps are involved: S1. Preparation of hybrid cell membranes, The macrophage membrane and tumor cell membrane were ultrasonically treated at 37°C for 10 minutes at a mass ratio of 1:1 to complete membrane fusion. The ultrasonic conditions were 130W and 42kHz. The tumor cell membrane was extracted from liver cancer tumor cells that overexpressed PD-L1. S2. Preparation of cell-inspired nanoparticles. The hybrid cell membrane proteins were mixed with PS nanoparticles, and the separation buffer was broken with a probe ultrasonic probe on ice for 5 min, where the ultrasonic power was 100 W, on for 2 s, off for 3 s, and then centrifuged at 12000 g for 10 min to obtain biomimetic nanohybrid macrophages / tumor cells, which were recorded as PHRM-NPs; The mass ratio of the cell membrane protein to the PS nanoparticles is 1:10-1:

1.

2. The method according to claim 1, characterized in that The preparation process of the macrophage membrane is as follows: RAW264.7 cells in good growth state and logarithmic growth phase are selected and cultured for 24 hours, the cells are stimulated to differentiate into M1 subtype, and the cells are continued to be cultured until about 80%, the cells are washed, digested, centrifuged, the supernatant is discarded, and the cell pellet is reserved for use; the cells are fully suspended with a separation buffer, broken with an ultrasonic probe on ice, centrifuged at 4°C, 1000g for 10 minutes, the supernatant is collected, and then centrifuged at 4°C, 12000g for 20 minutes to collect the supernatant, which is the macrophage membrane.

3. A cell bionic nanoparticle constructed by the method according to any one of claims 1 to 2.

4. Use of the cell bionic nanoparticles as claimed in claim 3 in the preparation of a drug for treating autoimmune diseases, characterized in that: The autoimmune disease is autoimmune hepatitis.

Images