A cell membrane-biomimetic phosphorus-containing dendrimer loaded antibody nanomedicine and a preparation method thereof

Abstract

The present application relates to a kind of cell membrane biomimetic phosphorus-containing dendrimer loaded antibody nanomedicine and its preparation method, the nanomedicine is with sodium hypophosphite capped phosphorus-containing dendrimer as nanometer delivery carrier, combined with PD-1 antibody to form nanocomposite;Extraction M1 macrophage membrane is coated to the nanocomposite and obtains.This application can promote the specific proliferation of natural killer cells in peripheral blood mononuclear cells by using the immunoregulatory activity of phosphorus-containing dendrimer itself, while releasing PD-1 antibody can block the PD-1 receptor on the surface of NK cell and T cell, thereby blocking the PD-L1 / PD-1 immune checkpoint between tumor cells and immune cells, further stimulate immune response, to achieve the effect of enhanced inhibition of tumor growth, provide new strategy for efficient treatment of brain glioma.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomedicines, and specifically relates to a cell membrane-inspired phosphorus dendritic macromolecule-loaded antibody nanomedicine and its preparation method. Background Technology

[0002] Glioblastoma multiforme (GBM) is the most aggressive primary central nervous system tumor, with the shortest median survival and the lowest 5-year survival rate, accounting for approximately 80% of all central nervous system malignancies (Acta Neuropathologica, 2016, 131(6):803-820). Treatment of gliomas has always been one of the most challenging tasks in clinical oncology. Currently, various treatment methods exist, including surgery, chemotherapy, and radiotherapy. However, due to the tumor's unique location within the central nervous system, these conventional treatments offer little improvement in functional prognosis and quality of life for glioblastoma patients. More seriously, the presence of the blood-brain barrier (BBB) ​​restricts the entry of most macromolecular therapeutic drugs (antibodies, enzymes, etc.) (Pharmaceutics, 2022, 14(2):224), significantly reducing drug bioavailability. Although the tight junctions of the BBB are impaired in the later stages of brain tumor development, allowing for some drug penetration, the drug concentration reaching the tumor site is insufficient to inhibit rapid tumor growth, thus hindering highly effective glioma treatment.

[0003] Immune checkpoints are immunosuppressive pathways that regulate immune responses to maintain self-tolerance and protect surrounding tissues. Tumor cells utilize these pathways to evade attacks from immune cells such as cytotoxic T lymphocytes (CTLs) and NK cells, leading to low efficiency in tumor immunotherapy. In recent years, immune checkpoint blockade (ICB) therapy has been widely used as a revolutionary anti-tumor treatment method. These drugs inhibit the activity of immune checkpoints, reactivating the T-cell immune response against tumor cells, thereby enhancing anti-tumor effects (Front. Immunol., 2021, 12, 679425). The combination of emerging immunotherapies and nanomedicine delivery systems can effectively deliver drugs to gliomas, improving the immune microenvironment through biological functions, activating the immune cell response to gliomas, and thus inhibiting tumor growth and recurrence, thus showing promising research and application prospects (J. Control. Release, 2020, 320, 63-72). According to literature reports, the most common method for antibody delivery is to covalently link the antibody to a carrier via an indestructible chemical bond, enabling nanomedicines to penetrate the blood-brain barrier by binding to receptors on the BBB surface. However, this method involves chemical modification of proteins, which may partially affect the biological activity of functional proteins (such as antibodies) (Nat. Commun., 2019, 10(1):3850); at the same time, the efficiency of BBB penetration depends on the number of active groups on the designed carrier that can bind to transport receptors (ACS Nano, 2022, 16, 664-674).

[0004] To overcome the limitations of nanomaterials in penetrating the brain-body barrier (BBB), nanomedicines constructed using biomimetic cell membranes have shown great potential due to their excellent biocompatibility and brain-targeting properties (Small, 2019, 15, 1804105). For example, by further modifying biomimetic nanosuspensions that modify glioma cell membranes with targeting peptides, the nanomaterials can penetrate the BBB through the targeting action of specific proteins on the membrane, enabling highly effective treatment of gliomas (Bioact. Mater., 2021, 6, 4402-4414). Furthermore, immune cells such as macrophages possess unique antigens and receptors on their membrane surfaces, and their surfaces express highly expressed integrins β1 and α4, which can bind to vascular endothelial adhesion molecule-1 (VCAM-1) on the surface of endothelial cells, thus enabling them to cross the BBB (Adv. Mater., 2018, 30, 1804023; ACS Nano, 2021, 15, 20377-90).

[0005] Phosphorus-containing dendritic macromolecules are a class of nanoscale macromolecules with unique physicochemical properties. They possess highly branched three-dimensional structures and functionalizable surfaces, exhibit uniform molecular weight, and demonstrate good biocompatibility. Currently, phosphorus-containing dendritic macromolecules have been used as antiviral agents, natural killer (NK) cell activators, and anti-inflammatory agents, thus showing broad application prospects in the biomedical field. In recent years, researchers have discovered that modifying the surface of phosphorus-containing dendritic macromolecules with cyclic amines such as pyrrolidine, morpholine, methylpiperazine, and phenylpiperazine can serve as carriers for loading therapeutic genes for gene therapy of tumors (Biomacromolecules 2020, 21, 2502-2511). They can also be incorporated with metal ions to form nanomedicines with their own antitumor properties (NanoToday, 2020, 33, 100899). Phosphorus-containing dendritic macromolecules with surface-modified sodium phosphite play a key role in targeting and activating monocytes. They possess immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs), stimulate NK cell-specific proliferation, and thereby induce tumor death (Nanomedicine (NY, NY, US), 2016, 12, 2321-2330; Adv. Mater., 2023, 35, 2208277).

[0006] A search of relevant domestic and international literature and patents revealed that no reports have been found regarding the use of phosphate-containing dendritic macromolecules capped with sodium phosphite with immunomodulatory activity as carriers to load PD-1 antibodies and coat M1 macrophage membranes for the immunotherapy of gliomas in situ. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a cell membrane-mimetic phosphorus dendritic macromolecule loaded with antibody nanomedicine and its preparation method. This nanomedicine can, on the one hand, utilize the immunomodulatory activity of the phosphorus dendritic macromolecule itself to promote the specific proliferation of natural killer cells in peripheral blood mononuclear cells, and on the other hand, release PD-1 antibody to block PD-1 receptors on the surface of NK cells and T cells, thereby blocking the PD-L1 / PD-1 immune checkpoints of tumor cells and immune cells, further stimulating the immune response, so as to achieve an enhanced effect of inhibiting tumor growth and providing a new strategy for the efficient treatment of glioma.

[0008] This invention provides a cell membrane-inspired phosphorus dendritic macromolecule-loaded antibody nanomedicine, wherein the nanomedicine uses a sodium phosphite-terminated phosphorus dendritic macromolecule as a nanodelivery carrier, which binds to a PD-1 antibody to form a nanocomplex; the nanocomplex is obtained by extracting M1 macrophage membranes and coating the nanocomplex.

[0009] This invention provides a method for preparing cell membrane-inspired phosphorus dendritic macromolecules loaded with antibodies, comprising the following steps:

[0010] (1) Add the phosphite-terminated phosphate dendritic macromolecule AK128 dropwise to the PD-1 antibody solution, stir until the solution turns milky white, centrifuge to obtain the AK128-aPD1 complex;

[0011] (2) RAW264.7 cells were stimulated with LPS to generate M1 macrophages. Benzyl sulfonyl chloride was mixed with cell lysis buffer and then added to the M1 macrophage pellet. The cells were frozen and thawed repeatedly with liquid nitrogen and a water bath to break them up. The treated cell suspension was then centrifuged using a gradient centrifugation. The final precipitate was collected as the purified M1 macrophage membrane and resuspended in PBS solution to obtain the M1 macrophage membrane suspension.

[0012] (3) Mix the AK128-aPD1 obtained in step (1) with the M1 type macrophage membrane suspension obtained in step (2), extrude the mixture using an extruder, and centrifuge to obtain a cell membrane biomimetic phosphorus dendritic macromolecule loaded with antibody nanomedicine, denoted as AK128-aPD1@M1m.

[0013] The structural formula of AK128 in step (1) is:

[0014]

[0015] In step (1), the mass ratio of AK128 to PD-1 antibody is 4-5:1; the stirring time is 24-32h; and the centrifugation conditions are 12000-130000rpm for 10-15min.

[0016] The LPS concentration in step (2) is 2-5 μg / mL; the stimulation time is 24-32 h.

[0017] In step (2), the volume ratio of benzyl sulfonyl chloride to hypotonic cell lysis buffer is 1:100-200; the ice bath time is 15-30 min.

[0018] The ratio of M1 macrophage precipitate to cell lysis mixture in step (2) is 4-5 × 10⁻⁶. 7 Cells: 1 mL.

[0019] The parameters for repeated freeze-thaw breaking in step (2) are: freezing with liquid nitrogen, thawing at 37°C, and repeating 4-5 times.

[0020] The parameters for gradient centrifugation in step (2) are as follows: centrifugation temperature is 4℃, centrifuge at 700g for 10min to remove the precipitate, centrifuge at 14000g for 30min to remove the supernatant, and resuspend the precipitate in 200μL of 1×PBS solution.

[0021] In step (3), the mass ratio of AK128-aPD1 to M1 macrophage membrane is 1:1.

[0022] The present invention also provides a cell membrane-mimetic phosphorus dendritic macromolecule-loaded antibody nanomedicine for the immunotherapy of orthotopic glioma.

[0023] This invention first binds a sodium phosphite-terminated phosphorus-containing dendritic macromolecule to a PD-1 antibody through electrostatic forces, hydrogen bonds, cation-π interactions, and hydrophobic interactions. Then, it further encapsulates the M1 macrophage membrane onto the surface of the AK128-aPD1 nanocomposite via physical extrusion, constructing a cell membrane-inspired nanodrug, AK128-aPD1@M1m, for highly effective immunotherapy of orthotopic gliomas.

[0024] This invention characterizes the prepared cell membrane-inspired phosphorus-containing dendritic macromolecule / antibody nanomedicine using Zeta potential and dynamic light scattering analysis (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and SDS-PAGE. The cytotoxicity of AK128-aPD1@M1m and related control materials was analyzed and evaluated using the CCK-8 assay; the in vitro blood-brain barrier penetration performance of the nanomedicine was studied using Transwell assay; the effect of the complex on NK cell proliferation in PBMCs was detected using flow cytometry, and the expression of related cytokines such as IFN-γ was detected using ELISA; the effect of different material groups on cancer cell apoptosis after co-incubation with PBMCs was detected using flow cytometry, and related apoptosis proteins were detected using Western blotting (WB); the phagocytic effect of macrophages on the M1 macrophage membrane-coated nanomedicine and the targeting specificity of the nanomedicine to T cells were studied using laser confocal microscopy. An orthotopic glioma model was established to evaluate treatment efficacy; the effect of nanomedicines crossing the blood-brain barrier was evaluated using in vivo fluorescence imaging; tumor size was monitored using magnetic resonance imaging, and parameters such as glioma volume and mouse weight were statistically analyzed to assess in vivo treatment efficacy and biosafety; CD4+ in in vivo immune cells was analyzed using flow cytometry. + T cells, CD8 + Infiltration and distribution of T cells, Tregs, and NK cells.

[0025] Beneficial effects

[0026] (1) The process of this invention is simple, the required reaction conditions are simple, it is easy to operate and separate, and it has good clinical application prospects.

[0027] (2) The nanomedicine prepared by this invention has good stability, water solubility and biocompatibility, providing a new idea for constructing safe and efficient nanomedicines.

[0028] (3) The biomimetic nanomedicine prepared in this invention can significantly improve the bioavailability of PD-1 antibodies and enhance their intracellular delivery; after coating the macrophage membrane, it endows the nanomedicine with the ability to penetrate the blood-brain barrier. At the same time, on the one hand, it utilizes the immunomodulatory activity of the phosphorus-containing dendritic macromolecules to promote the specific proliferation of NK cells in peripheral blood mononuclear cells (PBMCs), and on the other hand, the released PD-1 antibody can block the PD-1 receptor on NK cells and T cells, thereby blocking the PD-L1 / PD-1 immune checkpoints of tumor cells and immune cells, further stimulating the anti-tumor immune response, so as to achieve the effect of enhanced inhibition of tumor growth, providing a new strategy for the efficient treatment of glioma. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the synthesis and application of the nanomedicine AK128-aPD1@M1m in this invention;

[0030] Figure 2 The hydrated particle size (a) and surface potential (b) of AK128, aPD1, AK128-aPD1, and AK128-aPD1@M1m in Example 2 are shown.

[0031] Figure 3 The graph shows the hydrodynamic diameter of AK128-aPD1@M1m prepared in Example 2 as a function of time in water, PBS, and DMEM medium.

[0032] Figure 4 TEM images of AK128-aPD1(a) and AK128-aPD1@M1m(b) prepared in Example 2;

[0033] Figure 5 The following is a Western blot result of M1 macrophages, M1 macrophage membrane suspension, and integrins α4 and β1 expressed on AK128-aPD1@M1m prepared in Example 2;

[0034] Figure 6 The images show SDS-PAGE images of AK128, aPD1, AK128-aPD1, M0 macrophage membrane suspension (M0m), AK128-aPD1@M0m, M1 macrophage membrane suspension (M1m), AK128-aPD1@M1m (a) and AK128, IgG, AK128-IgG, M0 macrophage membrane suspension (M0m), AK128-IgG@M0m, M1 macrophage membrane suspension (M1m), AK128-IgG@M1m (b) prepared in Example 2.

[0035] Figure 7 Cell viability graphs of AK128, AK128-aPD1, and AK128-aPD1@M1m prepared in Example 2 after co-incubation with C6 cells (a) and bEnd.3 cells (b) for 24 h;

[0036] Figure 8 The images shown are: (a) a laser confocal microscope image of RAW264.7 cells after co-incubation of Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m prepared in Example 2 with RAW264.7 cells for 6 hours; (b) a histogram of fluorescence intensity of RAW264.7 cells obtained by flow cytometry detection of Cy5.5 fluorescence; and (c) a quantitative analysis diagram of relative fluorescence intensity.

[0037] Figure 9 Flow cytometry analysis of NK cell proliferation after co-incubation with PBS, IL-2, IL-2+AK128, IL-2+AK128-aPD1, IL-2+AK128-aPD1@M1m and PBMCs cells (a) and analysis of relative NK cell proliferation ratio (b); Figure 10 Flow cytometry analysis of granzyme B expression levels in NK cells after co-incubation with PBS, IL-2, IL-2+AK128, IL-2+AK128-aPD1, and IL-2+AK128-aPD1@M1m and PBMCs (a) and quantitative analysis of IFN-γ (b) and perforin (c) expression levels detected by ELISA.

[0038] Figure 11 Laser confocal microscopy analysis of Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m after co-incubation with T cells for 6 hours;

[0039] Figure 12 A schematic diagram of the Transwell apparatus used in the in vitro blood-brain barrier penetration experiment of Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m (a), a diagram showing the change in TEER resistance of the endothelial cell layer before and after penetration (b), and an analysis of the penetration efficiency of different material groups (c).

[0040] Figure 13A schematic diagram (a) of the Transwell experimental setup for inducing C6 cell apoptosis after co-incubation of PBS, IL-2+PBS, IL-2+aPD1, IL-2+AK128, IL-2+AK128-aPD1 and IL-2+AK128-aPD1@M1m with PBMCs cells; a flow cytometry analysis (b) and a quantitative analysis (c) of the apoptosis effect in C6 cells;

[0041] Figure 14 The Western blot results show the effects of PBS, IL-2+PBS, IL-2+aPD1, IL-2+AK128, IL-2+AK128-aPD1 and IL-2+AK128-aPD1@M1m on the expression of Bax, Bcl-2, P53, PTEN and Caspase-3 proteins in C6 cells after incubation with PBMCs. (a) is the protein band image of the Western blot, and (bf) is the grayscale quantitative image of the protein bands.

[0042] Figure 15 In Example 14, after injecting PBS, aPD1, AK128, AK128-aPD1, and AK128-aPD1@M1m via the tail vein, the MRI images of mouse gliomas (a), the change in relative tumor volume (b), and the change in mouse body weight (c) were recorded over 11 days.

[0043] Figure 16 The images (a) and (b) show fluorescence imaging of the mouse brain at different time points after tail vein injection of Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m in Example 15.

[0044] Figure 17 In Example 14, after 11 days of treatment, the CD4 count in the spleen tissue of each experimental group was [data missing]. + T cells (a) and CD8 + Flow cytometry analysis of T cell (b) typing;

[0045] Figure 18 This is a flow cytometry analysis of Tregs typing in spleen tissue of each experimental group after 11 days of treatment in Example 14.

[0046] Figure 19 The image shows flow cytometry analysis of NK cell typing in PBMCs (a) and spleen (b) after 11 days of treatment in each experimental group in Example 14. Detailed Implementation

[0047] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0048] Unless otherwise specified, all chemical reagents can be used directly without further purification. The first-generation phosphorus-containing dendritic macromolecule AK128 was obtained from Professor JP Majoral's research group at the French National Centre for Scientific Research (CNRS). PD-1 and IgG antibodies were purchased from BioXcell. C6 cells (rat glioma cell line), mouse brain microvascular endothelial cell line (bEnd.3 cells), and RAW264.7 cells (mouse macrophage cell line) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin antibiotics, and trypsin were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. (Hangzhou, China). BCA assay kit, PMSF, cell lysis buffer, and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Mouse peripheral blood mononuclear cell separation kit was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). ICR mice were purchased from Shanghai Slack Laboratory Animal Center (Shanghai, China). Anti-CD8-PE, Anti-CD4-FITC, Anti-NK1.1-PE, Anti-CD3-FITC, Anti-GranzymeB-FITC were purchased from Thermo Fisher Scientific (Waltham, MA).

[0049] Example 1

[0050] Using BSA as a model protein, the binding ability of AK128 to proteins was verified. BSA and AK128 were dissolved separately in aqueous solutions. AK128 was added dropwise to the BSA solution at different mass ratios (AK128:BSA = 2:1, 4:1, 8:1, 12:1), and stirred at room temperature for 24 h until the solution turned milky white. The mixture was then transferred to centrifuge tubes and centrifuged at 12000 rpm for 15 min. The precipitate was collected and redissolved in 1 mL of ultrapure water to obtain the AK128-BSA nanocomposite. The surface potential, hydrodynamic diameter, and polymer dispersibility index (PDI) of AK128-BSA at different mass ratios were measured. Simultaneously, the residual protein content in the supernatant after centrifugation was detected using a BCA kit, and the encapsulation efficiency and loading rate of protein in AK128-BSA were calculated. As shown in Table 1, the hydrodynamic diameter of AK128 was 167.1 nm, and the hydrodynamic diameter of BSA alone was 3.86 nm. Compared to BSA, AK128-BSA exhibits a larger hydration kinetic size and a further decrease in surface potential, indicating that BSA and AK128 form a nanocomposite. Experimental results show that when the mass ratio of AK128:BSA is 4:1, the hydrated particle size of AK128-BSA is 172.01 nm, the potential is -28.7 mV, and the encapsulation efficiency and loading rate are 54.85% and 12.06%, respectively. Therefore, a mass ratio of AK128:BSA of 4:1 was ultimately selected as the ratio for subsequent experiments.

[0051] Table 1

[0052]

[0053] Example 2

[0054] (1) Dissolve aPD1 and AK128 separately in aqueous solutions. Add AK128 slowly dropwise to the aPD1 solution at a mass ratio of AK128:aPD1 = 4:1. Stir at room temperature for 24 hours until the solution turns milky white. Then transfer the mixture to a centrifuge tube, centrifuge at 12000 rpm for 15 minutes to collect the precipitate, resuspend the precipitate in an appropriate amount of ultrapure water, and centrifuge again. Finally, redisperse the precipitate in 1 mL of ultrapure water to obtain the nanocomposite AK128-aPD1, which was used to determine the surface potential and hydrodynamic diameter of AK128-aPD1. Simultaneously, the encapsulation efficiency and loading rate of aPD1 in AK128-aPD1 were measured using a BCA assay kit, finding to be 90.23% and 18.41%, respectively.

[0055] (2) RAW264.7 cells were stimulated with 2 μg / mL LPS to generate M1 macrophages. 30 μL of benzyl sulfonyl chloride (PMSF) was mixed with 3 mL of hypotonic cell lysis buffer. 5 × 10⁶ macrophages in the logarithmic growth phase were collected. 7Centrifuge at 1000 rpm for 5 min to obtain cell pellet. Add pre-mixed hypotonic cell lysis buffer to the cell pellet and incubate on ice for 15 min. Use a freeze-thaw cycle (freeze in liquid nitrogen, thaw at 37°C, repeat 3-5 times). Set the centrifugation temperature to 4°C and centrifuge at 700 g for 10 min to remove the pellet. Centrifuge at 14000 g for 30 min to remove the supernatant. Resuspend the pellet in 200 μL of 1×PBS solution to obtain macrophage membrane suspension.

[0056] (3) AK128-aPD1 was mixed with macrophage membrane suspension at a mass ratio of 1:1. The solution was extruded 15 times using an Avanti micro-extruder and centrifuged at 10,000 rpm for 6 min to remove excess cell membrane, yielding the AK128-aPD1@M1m complex. An appropriate amount of AK128-aPD1@M1m was diluted with ultrapure water to prepare a solution with a concentration of 1 mg / mL, which was used to determine the hydrated particle size and surface potential.

[0057] The results are as follows Figure 2 As shown, the hydration kinetic diameter of AK128 is 167.1 nm, and the potential is -20.8 mV; the hydration particle size of aPD1 alone is 6.34 nm, and the potential is -9.59 mV. The hydration particle size of AK128-aPD1 increases to 204.13 nm, and the potential is -25.30 mV, indicating that aPD1 successfully forms a nanocomposite with AK128. After coating the macrophage membrane by physical extrusion, the hydration particle size of AK128-aPD1@M1m increases to 228.7 nm, and the potential decreases to -10.3 mV, proving the successful coating of the cell membrane on the surface of AK128-aPD1. The hydration particle size of AK128-aPD1@M1m remains almost unchanged after one week of storage in various solutions (water, PBS, DMEM medium) (e.g., ...). Figure 3 This demonstrates that AK128-aPD1@M1m exhibits good material stability.

[0058] Example 3

[0059] The AK128-aPD1 and AK128-aPD1@M1m prepared in Example 2 were characterized for size and morphology. The TEM test results of AK128-aPD1 are as follows: Figure 4 As shown in (a), AK128-aPD1 is a uniformly spherical shape with a size of approximately 117.6 nm. Figure 4 (b) The particle size of AK128-aPD1@M1m is approximately 160.29 nm, and the thickness of the encapsulated cell membrane is approximately 14.2 nm.

[0060] Example 4

[0061] M1 macrophages, M1 macrophage membranes, and AK128-aPD1@M1m prepared in Example 2 were used. The cells were lysed on ice and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant protein solution was collected, and the protein concentration was measured. Subsequently, SDS-PAGE electrophoresis, membrane transfer, immunoassay, and ECL chemical contrast agent fixing were performed sequentially. The results are as follows: Figure 5 As shown, both macrophage membranes and AK128-aPD1@M1m possess characteristic protein bands of integrin α4 and β1, which can bind to the VCAM-1 (vascular cell adhesion molecule 1) receptor on endothelial cells, thereby delivering nanomaterials across the blood-brain barrier.

[0062] Example 5

[0063] The AK128, aPD1, AK128-aPD1, M0 macrophage membrane suspension (M0m), AK128-aPD1@M0m, M1 macrophage membrane suspension (M1m), and AK128-aPD1@M1m(a) prepared in Example 2, as well as the AK128, IgG, AK128-IgG, M0 macrophage membrane suspension (M0m), AK128-IgG@M0m, M1 macrophage membrane suspension (M1m), and AK128-IgG@M1m prepared in Example 2, were characterized by SDS-PAGE to verify the loading of antibodies aPD1 and IgG, cell membrane coating, and retention of cell membrane proteins. The protein content in the M0 macrophage membrane suspension, M1 macrophage membrane suspension, AK128-aPD1@M0m, and AK128-aPD1@M1m prepared in Example 2 was determined using a BCA protein quantification kit, and the protein content in each sample was adjusted to 1 mg / mL with PBS solution. 5 μL of protein marker was added to the first protein lane, followed by 15 μL of AK128, aPD1, AK128-aPD1 (concentrations corresponding to the protein concentration of each material when AK128-aPD1@M1m is 1 mg / mL), M0 macrophage membrane suspension (M0m), AK128-aPD1@M0m, M1 macrophage membrane suspension (M1m), and AK128-aPD1@M1m, respectively, in the protein lane. The current was set to 100 A, and the time was set to 30 min. Figure 6As shown in (a), the AK128 group did not produce similar protein bands, while the AK128-aPD1 group and the aPD1 group produced similar bands, proving that the antibody PD-1 was successfully loaded onto AK128. Simultaneously, the AK128-aPD1@M1m group, the M1 macrophage membrane suspension group, and the aPD1 group produced similar protein bands, demonstrating the successful coating of the M1 macrophage membrane on the AK128-aPD1 surface and the retention of macrophage membrane proteins. Similarly, protein markers, AK128, IgG, AK128-IgG, M0 macrophage membrane suspension (M0m), AK128-IgG@M0m, M1 macrophage membrane suspension (M1m), and AK128-IgG@M1m were added to the protein lane for SDS-PAGE experiments, as shown... Figure 6 As shown in (b), similar protein bands were observed in the AK128-IgG@M1m group, the M1 macrophage membrane suspension group, and the IgG group, demonstrating the successful coating of the M1 macrophage membrane on the surface of AK128-IgG and the retention of macrophage membrane proteins.

[0064] Example 6

[0065] The cytotoxicity of AK128-aPD1 and AK128-aPD1@M1m prepared in Example 2 was tested using C6 cells and bEnd.3 cells as models. C6 cells and bEnd.3 cells in logarithmic growth phase were collected and cultured at a ratio of 1 × 10⁻⁶ cells per well. 4 Cells were seeded at different densities in two 96-well plates and incubated at 37°C with 5% CO2 for 12 hours. The original culture medium was discarded, and each well was then incubated with culture medium containing different concentrations of AK128, AK128-aPD1, and AK128-aPD1@M1m (AK128 concentrations of 0, 20, 40, 80, and 160 μg / mL) for 24 hours at 37°C with the cells. Afterward, the plates were removed, the original culture medium was discarded, and the cells were washed three times with PBS. Fresh culture medium containing 10% (v / v) CCK-8 was added, and the cells were incubated for another 3 hours. Finally, the absorbance of each well was measured at 450 nm using a multi-sensor microplate reader. Cells treated with PBS served as a blank control, and cell viability was recorded as 100%. Results are as follows: Figure 7 As shown in ab, within the experimental concentration range, no significant cytotoxicity was observed in C6 cells and bEnd.3 cells with increasing AK128 concentration, demonstrating that AK128-aPD1 and AK128-aPD1@M1m have good cell compatibility.

[0066] Example 7

[0067] RAW264.7 cells were used as a model to evaluate their phagocytic capacity against Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m, and this was used to evaluate the immune evasion ability of AK128-Cy5.5-IgG@M1m. RAW264.7 cells were cultured at a rate of 1×10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 cells per well in 12-well plates using DMEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The plates were incubated at 37°C and 5% CO2 for 12 hours. The original medium was discarded, and cells were co-cultured with medium containing Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m ([Cy5.5-IgG] = 10 μg / mL) for 6 hours at 37°C and 5% CO2. The culture medium was discarded, and the cells were washed three times with PBS, fixed with glutaraldehyde (2.5%) for 15 minutes, washed three times with PBS, stained with DAPI for 15 minutes, washed three times with PBS, and then observed using laser confocal microscopy.

[0068] To quantitatively analyze the intracellular uptake of different materials, flow cytometry was used to monitor the fluorescence intensity of Cy5.5 in macrophages. RAW264.7 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density in 12-well plates. After overnight incubation, the medium in each well was replaced with 1 mL of fresh DMEM medium containing Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m. Cells were incubated for 6 h, washed three times with PBS, digested, centrifuged, and resuspended in 300 μL of PBS. Cells were analyzed by flow cytometry. Cells treated with PBS served as controls. Cells were counted at 1 × 10⁻⁶ cells per sample. 4 1 cell, measured 3 times. Results are as follows: Figure 8 As shown in Figure ac, macrophages phagocytosed significantly more AK128-Cy5.5-IgG than AK128-Cy5.5-IgG@M1m, indicating that coating M1 macrophages with a membrane can endow nanomaterials with immune evasion capabilities, reducing macrophage phagocytosis of AK128-Cy5.5-IgG@M1m. Therefore, macrophage membrane coating endows AK128-Cy5.5-IgG@M1m with immune evasion capabilities, potentially prolonging its blood circulation time and enhancing its accumulation at tumor sites.

[0069] Example 8

[0070] To evaluate the effect of nanomaterials on the proliferation of NK cells in peripheral blood mononuclear cells (PBMCs), peripheral blood was collected from 6-8 week old ICR mice. PBMCs were obtained using a mouse peripheral blood mononuclear cell isolation and culture medium kit. The culture medium was RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37°C with 5% CO2 and the medium was changed every three days. Cells were then cultured at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates, and culture medium containing IL-2, IL-2+AK128, IL-2+AK128-aPD1, and IL-2+AK128-aPD1@M1m (IL-2 concentration 400 U / mL, [AK128] = 40 μg / mL) was added. The cells were incubated with the medium at 5% CO2 and 37°C for 3 days. Subsequently, PBMCs were collected and incubated with Anti-CD3-FITC and Anti-NK1.1-PE antibodies on ice in the dark for 30 minutes. After incubation, the cells were washed twice with PBS and resuspended in 300 μL PBS for flow cytometry analysis. Results are as follows: Figure 9 As shown in ab, the proportion of NK cells in PBMCs of the AK128 group was higher than that in the PBS group and the IL-2 group, while the proliferation effect of NK cells in the AK128-aPD1 and AK128-aPD1@M1m groups was not significantly different from that in the AK128 group. This indicates that the phosphite-capped phosphorus-containing dendritic macromolecule AK128 can stimulate and activate the proliferation of NK cells. At the same time, loading PD-1 antibody and coating macrophage membrane did not affect the proliferation effect of AK128 on NK cells.

[0071] Example 9

[0072] PBMCs treated in Example 8 were stained with Anti-granzyme B-FITC monoclonal antibody and analyzed by flow cytometry to detect granzyme B expression. Simultaneously, the culture supernatant was collected, and the levels of IFN-γ and perforin were detected using a mouse IFN-γ and perforin ELISA kit according to the manufacturer's instructions. Results are as follows... Figure 10 As shown in the figure, the levels of granzyme B, perforin, and IFN-γ in cells treated with AK128 were significantly higher than those in the PBS group, indicating that AK128 can effectively stimulate NK cells to secrete related anti-tumor cytokines, thereby inducing apoptosis. The loading of PD-1 antibody and the coating of macrophage membranes did not affect the expression of various related cytokines by AK128-stimulated cells.

[0073] Example 10

[0074] To evaluate the targeting ability of different materials to T cells, mouse spleens were harvested, and the spleen tissue was cut into small pieces, ground, and filtered through a 200-mesh sieve to obtain a cell suspension. T lymphocyte suspension was obtained through a nylon wool column, centrifuged (1000 rpm, 5 min), and resuspended in RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were then cultured for 1 day at 37°C with 5% CO2. T cells (2 × 10⁶ cells per well) were then... 5 Cells were seeded in six-well plates and cultured with fresh RPMI-1640 medium containing free Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m ([Cy5.5-IgG] = 10 μg / mL) at 5% CO2 and 37°C for 6 h. The original medium was discarded, and the cells were incubated with RPMI-1640 complete medium containing lysosomal green fluorescent staining solution at 37°C for 20 min. The cells were then centrifuged at 1700 rpm for 5 min, washed twice with PBS, stained with RPMI-1640 complete medium containing 10% Hoechst live cell nuclear staining reagent at 37°C for 20 min, centrifuged at 1700 rpm for 5 min, washed twice with PBS, and then 100 μL of PBS was added for observation under a laser confocal microscope. Results are as follows: Figure 11 As shown, the green fluorescence of lysosomes and the red fluorescence of Cy5.5-IgG hardly overlap, indicating that antibody IgG can target and bind to the surface of T cells. At the same time, coating the macrophage membrane does not affect the release and targeting of antibody IgG.

[0075] Example 11

[0076] The ability of the material to penetrate the blood-brain barrier was verified by simulating the in vitro blood-brain barrier using a Transwell model. bEnd.3 cells were introduced at a concentration of 2.5 × 10⁻⁶. 4 The cells were seeded at a density of [number] cells / well in the chambers of a 12-well Transwell plate. Each chamber had a polycarbonate membrane with 0.4 μm pores at the bottom. The chambers were then placed in the lower chamber of the Transwell plate and cultured in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2 for 5–7 days until the transendothelial resistance (TEER) was greater than 150 Ω·cm. 2A monolayer of cells resembling tight junctions in a BBB-like structure was formed, serving as an in vitro BBB model. Cy5.5-IgG, AK128-Cy5.5-IgG, and AK128-Cy5.5-IgG@M1m ([Cy5.5-IgG] = 10 μg / mL) nanomaterials were then added to the chambers of a Transwell system, with 1 mL of LDM-MEM complete medium added to the lower chamber. After 4 hours of incubation, TEER values ​​were analyzed, and the medium in the outer chamber was collected for fluorescence measurement. Results are as follows: Figure 12 As shown in Figure a, the change in TEER resistance before and after the material penetrates the bEnd.3 monocellular layer is negligible, indicating the integrity of the bEnd.3 monocellular layer. Figure 12 b). Meanwhile, AK128-Cy5.5-IgG@M1m exhibits higher penetration efficiency ( Figure 12 c) indicates that the macrophage membrane coating endows the nanomaterials with the ability to penetrate the BBB.

[0077] Example 12

[0078] Using C6 cells as a model, this study evaluated the effects of different materials on PBMCs cell apoptosis. PBMCs were cultured at 2 × 10⁶ cells per well. 5 C6 cells were seeded at a density of 2 × 10⁶ cells / well in six-well plates and incubated for 3 days with RPMI-1640 medium containing IL-2, IL-2+AK128, IL-2+AK128-aPD1, and IL-2+AK128-aPD1@M1m (IL-2 concentration 400 U / mL, [AK128] = 40 μg / mL) at 5% CO₂. C6 cells were then seeded at a density of 2 × 10⁶ cells / well. 5 PBMCs were seeded at a density of 10% in the lower chamber of a Transwell (6-well plate) using DMEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C for 24 h at 5% CO2. The co-incubated PBMCs and culture medium were then transferred to separate Transwell plate chambers. The old culture medium was removed from the lower layer, and 1 mL of RPMI-1640 medium was added. The cells were co-incubated for 12 h. Cells from each well were washed, collected, and resuspended in 195 μL of binding buffer. Annexin V-FITC and 7-AAD were added according to the kit instructions (5 μL each). Each sample was then incubated in the dark for 15 min and analyzed by flow cytometry. PBS-treated cells served as a control. Cells were counted at 1 × 10⁻⁶ cells per sample. 4 Each cell was measured three times. Results are as follows: Figure 13As shown in the figure, in the presence of IL-2 stimulating factor, PBMCs co-incubated with PD-1 antibody and AK128 can induce apoptosis of C6 cells to a certain extent. C6 cells treated with PBMCs co-incubated with IL-2+AK128-aPD1 produced the highest apoptosis and necrosis rates, while coating macrophage membrane did not significantly affect the apoptosis effect of nanomaterials on C6 cells.

[0079] Example 13

[0080] To further explore the molecular mechanism by which AK128-aPD1@M1m inhibits cancer cell growth, C6 cells were used as a model to evaluate the effects of different materials on the expression levels of intracellular apoptosis-related proteins (Bax, Bcl-2, P53, PTEN, and Caspase-3). PBMCs were cultured at 2 × 10⁶ cells per well. 5 C6 cells were seeded at a density of 2 × 10⁶ cells / well in six-well plates and incubated for 3 days with RPMI-1640 medium containing IL-2, IL-2+AK128, IL-2+AK128-aPD1, and IL-2+AK128-aPD1@M1m (IL-2 concentration 400 U / mL, [AK128] = 40 μg / mL) at 5% CO₂. C6 cells were then seeded at a density of 2 × 10⁶ cells / well. 5 PBMCs were seeded at a density of 10% fetal bovine serum and 1% penicillin-streptomycin in the lower chamber of a Transwell (6-well plate) and incubated at 37°C for 24 h with 5% CO2. The co-incubated PBMCs and culture medium were then transferred to separate Transwell plate chambers. The old culture medium was removed from the lower chamber, and 1 mL of RPMI-1640 medium was added. The cells were incubated together for 12 h. After incubation, the original culture medium was discarded, and the cells were washed three times with PBS. All cells were then digested, centrifuged, and collected. Cells were lysed on ice and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant protein solution was collected, and protein concentration was determined. Subsequently, SDS-PAGE electrophoresis, cell transfer, immunoassay, and ECL chemical contrast agent fixing were performed. The levels of Bax, Bcl-2, P53, PTEN, and Caspase-3 in the cells were analyzed. GAPDH was used as an internal control. The results are shown below. Figure 14 As shown in a. (As indicated by...) Figure 14In cells treated with AK128-aPD1, the intracellular Bax protein level significantly increased, while the Bcl-2 protein level significantly decreased. This result indicates that AK128-aPD1 can act on Bax and Bcl-2, inducing apoptosis via the mitochondrial pathway. However, after coating the cell membrane, the expression levels of Bax and Bcl-2 proteins in the AK128-aPD1@M1m group did not change significantly. To further verify the pro-apoptotic effect of AK128-aPD1@M1m, the expression levels of intracellular pro-apoptotic proteins p53, PTEN, and Caspase-3 were measured using Western blotting. Figure 14 df, AK128, and aPD1 can all increase the expression of P53, PTEN, and Caspase-3. The expression of P53, PTEN, and Caspase-3 in the AK128-aPD1 group is significantly increased, while the expression levels of P53, PTEN, and Caspase-3 proteins do not change significantly after coating the cell membrane.

[0081] Example 14

[0082] All animal experiments were approved by the Laboratory Animal Ethics Committee of Donghua University and conducted strictly in accordance with the standards of animal protection associations. Six- to eight-week-old female ICR mice used in the experiments were purchased from the Shanghai Slack Laboratory Animal Center (Shanghai, China). The orthotopic glioma model was established as follows: After anesthetizing the mice, their heads were fixed using a stereotaxic apparatus. The designated surgical area was disinfected, and a small hole was drilled in the skull after a light incision in the skin. Then, 5 μL of a solution containing 1×10⁻⁶ mmol / L of steroidal anti-inflammatory drugs (STIs) was injected. 5PBS cell suspension of C6 cells was slowly inoculated into the right striatum of each mouse over 5 minutes (2.0 mm dorsally from the anterior fontanelle, 2.0 mm lateral to the right sagittal suture, 3.0 mm depth). 5-7 days after inoculation, magnetic resonance imaging (MRI) was used to monitor and confirm the successful establishment of an orthotopic glioma model. The established orthotopic glioma model was used for subsequent in vivo MRI and antitumor therapy experiments. Healthy ICR mice (6-8 weeks old, 25-30 g) were used as model animals to construct an orthotopic glioma model to study the in vivo antitumor effect of AK128-aPD1@M1m. ICR mice with successfully constructed C6 orthotopic gliomas were randomly divided into 4 groups of 6 mice each: PBS, AK128, AK128-aPD1, and AK128-aPD1@M1m. During treatment, the drug was administered every 3 days, with the first injection defined as day 0, and treatment was administered on days 0, 3, and 6 for a total of 3 treatments. Each mouse was injected intravenously via tail vein with 100 μL of PBS, AK128, AK128-aPD1, and AK128-aPD1@M1m, respectively. The aPD1 dose (1.2 mg / kg) was maintained consistently for each experimental group mouse throughout each treatment. Simultaneously, tumor volume changes were monitored and recorded using magnetic resonance imaging (MRI) on days 0, 3, 6, and 10, with Magnevist (9.38 mg / mouse) used as the imaging agent. Furthermore, body weight changes were measured every other day for each mouse. Tumor volume (V) and relative tumor volume were calculated using the following formula: V = a × b 2 / 2, where a represents the measured length of the tumor and b represents the measured width of the tumor. Relative tumor volume = V / V0, where V0 and V represent the tumor volume before and after drug administration, respectively.

[0083] MRI images of brain tumors in each group and their relative tumor volumes are as follows: Figure 15 As shown in ab, the tumor volume in the PBS group mice increased rapidly over time, while the tumor growth in the AK128 group, aPD1 group, and AK128-aPD1 group mice was inhibited to varying degrees. Compared with the AK128-aPD1 group, the AK128-aPD1@M1m group showed better in vivo anti-tumor effects. This is because the prepared nanoparticles, coated with the M1-type macrophage membrane, can penetrate the blood-brain barrier more efficiently, delivering AK128-aPD1 to the tumor site. Furthermore, the synergistic effect of AK128 and aPD1 enhances the immunotherapy effect. Simultaneously, coating the M1-type macrophage membrane prolongs blood circulation time, demonstrating enhanced anti-tumor effects. Figure 15 As can be seen from c, the weight changes of mice in each group were not significant, indicating that AK128-aPD1@M1m has good biocompatibility in vivo.

[0084] Example 15

[0085] To verify the ability of synthesized nanomaterials to cross the blood-brain barrier in vivo, fluorescence imaging was performed on ICR mice with successfully modeled gliomas. Each mouse was injected via tail vein with 100 μL of Cy5.5-IgG, AK128-Cy5.5-IgG, or AK128-Cy5.5-IgG@M1m dissolved in PBS (Cy5.5-IgG concentration was 1.2 mg / kg per mouse). In vivo fluorescence imaging was performed at 1 h, 3 h, 6 h, and 12 h post-injection. The fluorescence imaging and quantitative analysis of Cy5.5-IgG in the brain for each group are shown below. Figure 16 As shown in ab, there was no significant difference in the aggregation amount of Cy5.5-IgG and AK128-Cy5.5-IgG in the brain. After coating the macrophage membrane, the fluorescence of AK128-Cy5.5-IgG@M1m in the brain was significantly enhanced and reached a peak at 6h, indicating that the prepared nanoparticles can efficiently penetrate the blood-brain barrier and reach the brain due to the coating of macrophage membrane.

[0086] Example 16

[0087] To verify the anti-tumor immunotherapy efficacy of AK128-aPD1@M1m in vivo, spleens were harvested from ICR mice in different treatment groups from Example 14 on day 11 under sterile conditions. The spleen tissue was cut into small pieces, ground, suspended in an RPMI-1640, and filtered through a 200-mesh sieve. The filtered suspension was centrifuged (1000 rpm, 5 min), resuspended in an RPMI-1640, and filtered 6-8 times through a nylon capillary column to obtain a T-cell suspension for further analysis. The obtained T cells were labeled with Anti-CD4-FITC and Anti-CD8-PE, respectively, and CD4+ in the spleen tissue was analyzed using flow cytometry. + T cells and CD8 + Quantitative analysis of T cells was performed; cell suspensions were stained with anti-CD4-PE, anti-CD25-FITC, and anti-Foxp3-APC antibodies to analyze regulatory T cells (Tregs). CD4+ levels in spleen tissues from each group were also analyzed. + T cells and CD8 + Quantitative analysis of T cells, such as Figure 17 As shown in ab, both aPD1 and AK128 can induce CD4. + T cells and CD8 + The increase in T cells indicates that both aPD1 and AK128 can restore the immune response function of T cells to varying degrees. The CD4 count in the AK128-aPD1@M1m group... + T cells and CD8 +The T cell counts were higher in the AK128-aPD1@M1m group than in the aPD1 group. This is because AK128 and aPD1 in AK128-aPD1@M1m play a joint regulatory role in the immune system, ultimately inducing CD4+. + T cells and CD8 + T-cell activation, coupled with the effective prolongation of nanomaterial circulation time in vivo after coating macrophage membranes, enhances the in vivo immunotherapy effect. For example... Figure 18 As shown, both aPD-1 and AK128 can inhibit Tregs to some extent. The AK128-aPD1@M1m group had the lowest Treg content. This means that the combined immunotherapy strategy developed by AK128-aPD1@M1m treatment can not only effectively distribute cytotoxic T cells in the spleen, but also promote the effective reduction of Tregs, thereby reversing the immunosuppressive TME and enhancing the effect of tumor immunotherapy.

[0088] Example 17

[0089] To verify the effect of AK128-aPD1@M1m on NK cell activation in vivo, peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood mononuclear cells (PBMCs) of ICR mice from different groups after treatment in Example 14 on day 11 under aseptic conditions. The mice were then sacrificed, and the spleens were harvested. PBMCs were isolated using a mouse peripheral blood mononuclear cell isolation medium kit and resuspended in intact RPMI-1640 medium. Simultaneously, the spleen tissue was cut into small pieces, ground, suspended in RPMI-1640, and filtered through a 200-mesh sieve. The filtered suspension was centrifuged (1000 rpm, 5 min) and resuspended in RPMI-1640. For the analysis of PBMCs and NK cells in the spleen, the cell suspensions were stained with anti-CD3-FITC and anti-NK1.1-PE antibodies, respectively. The results are as follows: Figure 19 As shown in ab, in the peripheral blood and spleen tissue of mice, the proliferation of NK cells in the AK128 group was higher than that in the PBS group and the aPD1 group. The AK128-aPD1@M1m group had the best activation effect on NK cells among all groups, indicating that AK128-aPD1@M1m can cross the blood-brain barrier, stimulate the proliferation of NK cells to the maximum extent, and enhance the anti-tumor immune response.

Claims

1. A cell membrane-mimetic phosphorus-containing dendritic macromolecule-loaded antibody nanomedicine, characterized in that: The nanomedicine uses sodium phosphite-terminated phosphorus-containing dendritic macromolecule AK128 as a nanodelivery carrier, which is then bound to a PD-1 antibody to form a nanocomplex; the nanocomplex is obtained by coating the membrane of M1 macrophages; the structural formula of AK128 is: 。 2. A method for preparing a cell membrane-mimetic phosphorus-containing dendritic macromolecule-loaded antibody nanomedicine, comprising the following steps: (1) Add the sodium phosphite-terminated phosphorus-containing dendritic macromolecule AK128 dropwise to the PD-1 antibody solution, stir until the solution turns milky white, centrifuge to obtain the AK128-aPD1 complex; wherein, The structural formula of the AK128 is as follows: ； (2) RAW264.7 cells were stimulated with LPS to generate M1 macrophages. Benzyl sulfonyl chloride was mixed with cell lysis buffer and then the cell lysis buffer was added to the M1 macrophage pellet. The cells were frozen and thawed repeatedly with liquid nitrogen and a water bath to break them up. The treated cell suspension was centrifuged in a gradient. The final precipitate was collected as the purified M1 macrophage membrane and resuspended in PBS solution to obtain the M1 macrophage membrane suspension. (3) Mix the AK128-aPD1 obtained in step (1) with the M1 type macrophage membrane suspension obtained in step (2), extrude the mixture using an extruder, and centrifuge to obtain a cell membrane biomimetic phosphorus dendritic macromolecule loaded with antibody nanomedicine, denoted as AK128-aPD1@M1m.

3. The preparation method according to claim 2, characterized in that: In step (1), the mass ratio of AK128 to PD-1 antibody is 4-5:1; the stirring time is 24-32 h; and the centrifugation conditions are 12000-130000 rpm for 10-15 min.

4. The preparation method according to claim 2, characterized in that: The LPS concentration in step (2) is 2-5 μg / mL; the stimulation time is 24-32 h.

5. The preparation method according to claim 2, characterized in that: In step (2), the volume ratio of benzyl sulfonyl chloride to hypotonic cell lysis buffer is 1:100-200; the ice bath time is 15-30 min.

6. The preparation method according to claim 2, characterized in that: The ratio of M1 macrophage precipitate to cell lysis mixture in step (2) is 4-5 × 10⁻⁶. 7 Cells: 1 mL.

7. The preparation method according to claim 2, characterized in that: The parameters for repeated freeze-thaw breaking in step (2) are: freezing with liquid nitrogen, thawing at 37°C, and repeating 4-5 times.

8. The preparation method according to claim 2, characterized in that: The parameters for gradient centrifugation in step (2) are as follows: the centrifugation temperature is 4℃, centrifuge at 700 g for 10 min to remove the precipitate, centrifuge at 14000 g for 30 min to remove the supernatant, and resuspend the precipitate in 200 μL of 1 × PBS solution.

9. The preparation method according to claim 2, characterized in that: In step (3), the mass ratio of AK128-aPD1 to M1 macrophage membrane is 1:1.

Images