Phosphorus-containing dendrimer composite nanomaterial, cell membrane bionic phosphorus-containing dendrimer composite nanomaterial as well as preparation and application of phosphorus-containing dendrimer composite nanomaterial and cell membrane bionic phosphorus-containing dendrimer composite nanomaterial

Abstract

The invention relates to a phosphorus-containing dendrimer composite nanomaterial, a cell membrane bionic phosphorus-containing dendrimer composite nanomaterial as well as preparation and application of the phosphorus-containing dendrimer composite nanomaterial. The preparation method comprises the following steps: combining a phosphorus-containing dendrimer terminated by sodium phosphite with superoxide dismutase, further loading manganese carbonyl to form a nano-composite, and wrapping the surface of the nano-composite with a macrophage membrane to construct the cell membrane bionic nano-drug. The preparation method has the advantages of being simple, easy to operate, good in biocompatibility and the like. The strategy provided by the invention has good development prospects and application values in treatment of type I diabetes mellitus or other autoimmune diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to a phosphorus-containing dendrimer composite nanomaterial, a cell membrane biomimetic phosphorus-containing dendrimer composite nanomaterial, and preparation and application thereof. Background Art

[0002] Diabetes is one of the leading causes of increased morbidity and mortality, currently affecting approximately 400 million people worldwide, of which approximately 5% suffer from type 1 diabetes (T1D). T1D is an autoimmune disease that causes excessive inflammatory responses due to immune dysregulation, ultimately leading to pancreatic dysfunction, apoptosis of pancreatic β cells and hyperglycemia (Nat. Rev. Drug Discovery 2014, 13, 278-289). Inflammatory responses promote the early induction and amplification of autologous T cell immune attacks on pancreatic β cells, and stabilize and maintain insulitis in the later stages. Current treatments for T1D mainly include insulin therapy, pancreas or islet transplantation, and immune interventions that seek to inhibit or regulate the activity of autoimmune T cells. However, these treatments face challenges such as severe hypoglycemia, insufficient islet donors, and off-target effects. Therefore, reducing excessive inflammatory responses in the body (such as by inhibiting excessive oxidative stress and the secretion of inflammatory factors) and inhibiting islet-specific autoreactive T cells to reverse β cell apoptosis are important strategies for the effective treatment of T1D.

[0003] In recent years, the delivery of related antioxidant drugs such as superoxide dismutase (SOD), CORM (carbon monoxide-releasing molecules) and catalase through nanotechnology can reduce inflammatory responses and inhibit cell apoptosis. Superoxide is one of the reactive oxygen species (ROS) produced in mammalian cells. When produced in excess, it can cause oxidative damage to various cellular components. SOD forms the body's first line of defense against oxidative stress and can effectively convert superoxide radicals into O2 and H2O2 to relieve oxidative stress. Lu et al. developed an antioxidant enzyme therapy based on a nanocomplex of SOD and CAT (SOD-CAT), which can effectively remove intracellular ROS and alleviate ROS-induced cell damage, thereby reducing ischemia-reperfusion injury and pathogen-induced liver damage (Adv. Mater. 2022, 34, 2105670). Carbon monoxide (CO) is an endogenous colorless gas produced by heme oxygenase (HO-1) by degrading heme molecules, which has anti-inflammatory and anti-apoptotic effects. CO can exert its anti-inflammatory effect by downregulating inflammatory cytokines and promoting the anti-inflammatory response of macrophages. Carbonyl manganese (MnCO), as a CO molecular prodrug, can release CO gas under the stimulation of ROS such as hydrogen peroxide and hydroxyl radicals. For example, Zhang et al. constructed a mesoporous silicon-based CO nanoplatform mimicking macrophage membranes, which can continuously release CO under high ROS levels in the pancreas of T1D mice, significantly improving the therapeutic effect of T1D by reducing inflammatory responses and inhibiting β-cell apoptosis (Biomaterials 2020, 245, 119986). Considering that a single therapeutic drug is difficult to cope with the complex pathogenic mechanism of T1D, increasing the dosage is usually accompanied by side effects such as digestive system problems, allergic reactions, liver or kidney damage, etc. Therefore, in order to solve these problems, it is usually necessary to combine multiple treatment methods or multi-drug therapy to achieve synergistic anti-inflammatory, antioxidant, anti-apoptotic and immunomodulatory treatment of T1D.

[0004] The immune checkpoint protein programmed death 1 (PD-1) is mainly expressed on activated T cells. It can inhibit the activity of T cells and maintain immune tolerance after interacting with programmed death receptor-ligand 1 (PD-L1). By utilizing the inhibitory immune checkpoint PD-1 / PD-L1, islet antigen-specific immune tolerance to autoreactive T cells can be achieved, preventing autoimmune attacks and thus reversing hyperglycemia. For example, Wang et al. reported that mesenchymal stem cell-derived extracellular vesicles (H@TI-EVs) loaded with 5-aminolevulinic acid hexyl ester and highly expressing PD-L1 have good inflammation targeting and immunosuppressive effects in T1D treatment. H@TI-EVs can reduce the activation of CD4+T cells through the PD-L1 / PD-1 immune checkpoint interaction and induce macrophages to switch from M1 to M2 to reshape the immune microenvironment, thereby efficiently treating T1D mouse models (Small 2023,19,2301019). Zhang et al. genetically engineered megakaryocyte-derived hematopoietic progenitor cells to produce PD-L1-overexpressing immunosuppressive platelets, which were used to effectively maintain normal blood sugar in newly developed hyperglycemic non-obese diabetic (NOD) mice. PD-L1 platelets can accumulate in the inflamed pancreas and inhibit the activity of pancreatic autoreactive T cells in NOD mice, increasing the percentage of regulatory T cells (Tregs), thereby maintaining pancreatic immune tolerance and protecting β cells from destruction (Adv. Mater. 2020, 32, 1907692). Macrophage membranes (PRMs) stimulated by IFN-γ have been shown to highly express PD-L1 protein. Hou et al. designed immunoengineered nanodecoys (PRM NDs) of PRM-encapsulated polylactic-coglycolic acid nanoparticles. In Zymosan A-induced arthritis mice and dextran sodium sulfate-induced ulcerative colitis mice, PRM NDs stimulated effective synergistic anti-inflammatory and immunosuppressive effects to reduce autoimmune damage (Adv. Mater. 2022, 34, 2108817). However, the currently developed nanoplatforms are still unable to achieve the purpose of efficient treatment of diabetes through synergistic anti-inflammatory / anti-oxidation / anti-apoptosis / immunomodulation. Therefore, the present invention provides a cell membrane biomimetic phosphorus-containing dendrimer composite nanomaterial to achieve efficient and comprehensive treatment of diabetes. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a phosphorus-containing dendrimer composite nanomaterial, a cell membrane biomimetic phosphorus-containing dendrimer composite nanomaterial and the preparation and application thereof.

[0006] The invention provides a phosphorus-containing dendrimer composite nano material, which comprises phosphorus-containing dendrimers and protein complexes capped with sodium phosphite and drugs; wherein the phosphorus-containing dendrimers and protein complexes capped with sodium phosphite are loaded with drugs.

[0007] The composite material uses phosphorus-containing dendrimers terminated with sodium phosphite as carriers, composites superoxide dismutase (SOD), and physically loads anti-inflammatory and antioxidant drugs inside.

[0008] Furthermore, the composite nanomaterial uses a phosphorus-containing dendrimer capped with sodium phosphite as a nano-delivery carrier, which is combined with superoxide dismutase through electrostatic forces, hydrogen bonds, cation-π, hydrophobic interactions and other forces, and further loads the drug carbonyl manganese inside to form a nano-complex.

[0009] Preferably, the sodium phosphite-terminated phosphorus-containing dendrimer is sodium phosphite-terminated phosphorus-containing dendrimer AK128, whose structural formula is The protein is superoxide dismutase SOD; the drug includes carbonyl manganese Mn2 (CO) 10 .

[0010] The present invention provides a method for preparing a phosphorus-containing dendrimer composite nanomaterial, comprising:

[0011] (1) mixing a phosphorus-containing dendrimer capped with sodium phosphite and a protein, and stirring at room temperature to obtain a phosphorus-containing dendrimer capped with sodium phosphite / protein complex;

[0012] (2) Mixing the drug and the phosphorus-containing dendrimer / protein complex capped with sodium phosphite, and stirring at room temperature to obtain a phosphorus-containing dendrimer composite nanomaterial.

[0013] Preferably, the reaction in step (1) is stirred at room temperature for 12 to 24 hours.

[0014] Preferably, in step (1), the mixture is stirred at room temperature and then centrifuged to collect the precipitate, wherein the centrifugation is performed at 8000-12000 rpm for 10-15 min.

[0015] Preferably, in the step (1), the mass ratio of the phosphorus-containing dendrimer capped with sodium phosphite to the protein is 2:1 to 10:1; and the protein is superoxide dismutase SOD.

[0016] The aqueous solution of phosphorus-containing dendrimers capped with sodium phosphite salt mixed in step (1) is added dropwise into the protein solution; wherein the solvent of the protein solution is phosphate buffer (PBS, pH=7.4).

[0017] Furthermore, the water is ultrapure water.

[0018] Preferably, in step (2), the mass ratio of the phosphorus-containing dendrimer / protein complex capped with sodium phosphite to the drug is 1:0.25 to 1:2.

[0019] The sodium phosphite-terminated phosphorus-containing dendrimer / protein complex is a sodium phosphite-terminated phosphorus-containing dendrimer and protein complex.

[0020] Preferably, the stirring time at room temperature in step (2) is 12 to 24 hours; after stirring at room temperature, centrifugation is performed to purify the mixture, and the supernatant is collected and centrifuged at 1000 to 1500 rpm for 10 to 15 minutes.

[0021] In the step (2), the drug, the phosphorus-containing dendrimer capped with sodium phosphite and the protein complex are mixed into a drug solution and then added dropwise into the phosphorus-containing dendrimer capped with sodium phosphite and the protein complex solution; the drug includes carbonyl manganese Mn2(CO) 10 ; The solvent of the drug solution is methanol; the solvent of the solution of the sodium phosphite-terminated phosphorus-containing dendrimer and protein complex is PBS buffer (pH=7.4).

[0022] The invention provides a cell membrane bionic material, wherein the cell membrane bionic material is a cell membrane coated with the phosphorus-containing dendritic macromolecular composite nano material.

[0023] Furthermore, the macrophage membrane stimulated by IFN-γ coats the phosphorus-containing dendrimer composite nanomaterial to construct a cell membrane bionic composite nanomedicine.

[0024] Furthermore, the cell membrane is a RAW264.7 cell membrane stimulated by IFN-γ.

[0025] The preparation of the RAW cell membrane comprises: mixing phenylmethylsulfonyl chloride (PMSF) and a hypotonic cell lysis solution evenly, then adding the cell lysis mixture to the RAW264.7 cell precipitation stimulated by IFN-γ, ice bathing, repeated freezing and thawing to break the cells, gradient centrifugation, precipitating macrophage cell membranes, and resuspending in a 1×PBS solution to obtain a cell membrane suspension.

[0026] The invention provides a cell membrane bionic material, comprising: mixing the phosphorus-containing dendrimer composite nanomaterial and a macrophage membrane suspension, and extruding the mixture by an extruder to obtain the cell membrane bionic material.

[0027] Preferably, the mass ratio of the phosphorus-containing dendrimer composite nanomaterial to the macrophage membrane is 1:0.5 to 1:2;

[0028] Preferably, the macrophage membrane is a macrophage membrane that highly expresses PD-L1 after IFN-γ stimulation.

[0029] The preparation of the RAW macrophage membrane suspension comprises: mixing phenylmethylsulfonyl chloride (PMSF) and a cell lysis solution evenly, then adding the mixture to a macrophage precipitate, freezing and thawing the cells, centrifuging, and resuspending in a PBS solution to obtain a macrophage membrane suspension.

[0030] Furthermore, the preparation of the macrophage membrane suspension includes: stimulating the original mouse macrophages (RAW264.7 cells) with IFN-γ, mixing phenylmethylsulfonyl chloride (PMSF) and hypotonic cell lysis solution, then adding the cell lysis mixture to the macrophage precipitate, ice bathing, repeatedly freezing and thawing the cells with liquid nitrogen and a water bath, and gradient centrifuging the treated cell suspension. The final precipitate is collected as the purified macrophage membrane and resuspended in a 1×PBS solution to obtain a macrophage membrane suspension.

[0031] The IFN-γ concentration for stimulating RAW264.7 cells was 50 ng / mL; the stimulation time was 24 to 48 h; the volume ratio of PMSF to hypotonic cell lysis solution was 1:100 to 150; the ratio of macrophage precipitation to cell lysis mixture was 4×10 7 Cells: 1-2mL. Ice bath time is 15-30min; parameters for repeated freezing and thawing are: freezing in liquid nitrogen, thawing at 36-38℃, repeated 3-6 times; parameters for gradient centrifugation are: centrifugation temperature is 4-10℃, centrifugation at 700-1000g for 10-15min, remove the precipitate, then centrifuge at 12000-14000g for 30-35min, remove the supernatant, and resuspend the precipitate in 100-500μL of 1×PBS solution.

[0032] Preferably, the extrusion is performed by repeatedly extruding 11 to 15 times using an Avanti micro extruder with a filter membrane pore size of 400 nm, and centrifuging after extrusion to collect the precipitate, wherein the centrifugation is performed at 8000 to 12000 rpm for 5 to 10 minutes.

[0033] The present invention provides an application of the phosphorus-containing dendrimer composite nanomaterial or the cell membrane bionic material in the preparation of anti-inflammatory / anti-oxidation / anti-apoptosis / immune tolerance maintenance drugs, such as anti-oxidation / anti-inflammatory / anti-apoptosis / immunomodulatory drugs for diabetes.

[0034] The present invention provides an application of the phosphorus-containing dendrimer composite nanomaterial or the cell membrane bionic material in preparing a drug for treating type I diabetes.

[0035] The present invention uses a phosphorus-containing dendrimer capped with sodium phosphite as a carrier, combines with SOD to self-assemble into a nanocomposite through physical interactions such as electrostatic force, hydrogen bond, cation-II, and hydrophobic interaction, and further physically loads Mn2(CO) 10; Extract the macrophage membrane that highly expresses PD-L1 after IFN-γ stimulation and wrap it on the surface of the nanocomposite to construct a cell membrane bionic nanodrug. The composite nanodrug provided by the present invention has the advantages of simple preparation method, easy operation, good biocompatibility, etc. The prepared cell membrane bionic nanodrug can preferentially target inflammatory cells and improve the delivery efficiency of proteins / drugs. On the one hand, it protects β cells from damage by alleviating oxidative stress and regulating mitochondrial membrane potential, while regulating macrophage phenotype and inflammation-related pathways to inhibit inflammatory response; on the other hand, the macrophage membrane that highly expresses PD-L1 gives the nanodrug the ability to regulate excessive immune response, which can effectively inhibit the activity of active T cells and maintain immune tolerance, thereby efficiently treating type I diabetes. The strategy provided by the present invention may have good development prospects and application value in the treatment of type I diabetes or other autoimmune diseases.

[0036] The composite nanoplatform combines the phosphorus-containing dendrimers terminated by sodium phosphite with SOD through electrostatic forces, hydrogen bonds, cation-π, and hydrophobic interactions, and then physically loads the drug Mn2(CO) 10 , forming a nanocomposite material, and further coating the RAW264.7 cell membrane stimulated by IFN-γ on the surface of the nanocomposite by physical extrusion to construct the cell membrane bionic nanodrug ASM@PRM for the combined anti-inflammatory / anti-oxidation / anti-apoptosis / immunomodulatory treatment of type 1 diabetes.

[0037] The present invention uses Zeta potential and dynamic light scattering analysis (DLS), fluorescence spectrophotometer, transmission electron microscope (TEM), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and other means to characterize the physical and chemical properties of the prepared cell membrane bionic ASM@PRM nanomedicine. Then the CCK-8 method is used to analyze and evaluate the cytotoxicity of ASM@PRM and related control materials; the CCK-8 method and flow cytometry are used to analyze the cell protection ability of ASM@PRM and related control materials under oxidative stress environment; laser confocal microscopy and flow cytometry are used to detect the phagocytosis of macrophages on materials; flow cytometry is used to evaluate the effect of materials on intracellular ROS and mitochondrial membrane potential levels; flow cytometry is used to evaluate the effect of nanomaterials on macrophage typing; Western blot is used to detect the expression of HO-1, p38, p65 and p50 proteins in cells; and enzyme-linked immunosorbent assay (ELISA) kits are used to detect inflammation-related factors TNF-α and IL-1β expression levels; flow cytometry was used to detect the effects of ASM@PRM and related control materials on T cell maintenance of immune tolerance; a T1D mouse model was established, and the effect of the materials on the recovery of β-cell function was evaluated by monitoring blood glucose levels; ELISA was used to detect the levels of various proinflammatory factors (TNF-α, IL-6) and insulin levels to evaluate the anti-inflammatory effect and β-cell recovery effect of the materials in vivo; H&E staining was used to analyze the damage of pancreatic tissue after treatment; flow cytometry was used to analyze the proportion of various immune cells in the spleen (CD3+PD-1+T cells, CD3+C4+T cells, CD4+IFN-r+T cells, and CD4+Foxp3+T cells) to evaluate the effect of the materials on maintaining immune tolerance.

[0038] Beneficial Effects

[0039] (1) The process of the present invention is simple, the required reaction conditions are simple, and it is easy to operate and separate, and has good development prospects;

[0040] (2) The cell membrane biomimetic nanomedicine prepared by the present invention has good biocompatibility and can effectively improve the activity of SOD and Mn2(CO) 10The bioavailability of the drug is improved, and its intracellular delivery efficiency is enhanced, providing a new idea for the construction of safe and efficient nanomedicines; (3) The cell membrane biomimetic nanomedicine prepared by the present invention can preferentially target inflammatory cells. On the one hand, it protects β cells from damage by alleviating oxidative stress and regulating mitochondrial membrane potential, while regulating macrophage phenotype and inflammation-related pathways to inhibit inflammatory response; on the other hand, the macrophage membrane with high expression of PD-L1 gives the nanomedicine the ability to regulate excessive immune response, which can effectively inhibit the activity of active T cells and maintain immune tolerance, thereby effectively treating type I diabetes. The strategy provided by the present invention has good development prospects and application value in the treatment of type I diabetes or other autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A schematic diagram of the synthesis of the nanomaterial ASM@PRM in the present invention and its use in the treatment of type I diabetes;

[0042] Figure 2 The hydrated particle size and PDI diagram (A) and surface potential diagram (B) of AK128, SOD, AS, ASM and ASM@PRM prepared in Example 1;

[0043] Figure 3 This is a graph showing the change in hydrodynamic diameter of the ASM@PRM prepared in Example 1 in water, PBS, and DMEM culture media within one week;

[0044] Figure 4 TEM images of AS (A), ASM (B), and ASM@PRM (C) prepared in Example 1;

[0045] Figure 5 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) images of AK128, SOD, ASM, macrophage membrane suspension (RM), ASM@RM, macrophage membrane suspension (PRM) stimulated with IFN-γ, and ASM@PRM prepared in Example 1 (A) and Western blot results of protein expression on RM, ASM@RM, PRM, and ASM@PRM (B);

[0046] Figure 6 The cumulative CO release curves of ASM and ASM@PRM prepared in Example 1 at different H2O2 concentrations;

[0047] Figure 7 Cell viability graphs of ASM@PRM prepared in Example 1 at different concentrations co-incubated with unstimulated mouse pancreatic β cells (MIN6 cells) (A) and RAW264.7 cells (B) and with stimulated MIN6 cells (C) and RAW264.7 cells (D) for 24 hours;

[0048] Figure 8 MIN6 cells were treated with streptozotocin (STZ), and then incubated with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM for 12 hours, respectively, for ROS flow cytometric analysis (A) and fluorescence quantitative analysis (B). Fig. 9 Figure 1 shows the flow cytometric analysis of mitochondrial membrane potential in MIN6 cells treated with STZ and then co-incubated with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM for 12 h (A) and the quantitative analysis of the proportion of JC-1 polymers and monomers (B);

[0049] Fig.10 Figure 2 shows the flow cytometric analysis (A) and quantitative analysis (B) of apoptosis levels of MIN6 cells after they were treated with STZ and then co-incubated with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM for 12 hours.

[0050] Fig.11 The fluorescence intensity analysis of intracellular Cy5.5 after SOD, AS, ASM, ASM@RM, and ASM@PRM were co-incubated with M0 and M1 macrophages for 8 hours, respectively. PBS and SOD were used as controls. All experiments involving SOD used Cy5.5-labeled SOD.

[0051] Fig.12 The laser confocal microscopy analysis (A) and flow cytometry quantitative analysis (B) of intracellular ROS levels in RAW264.7 cells after LPS treatment and co-incubation with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM for 12 hours, respectively;

[0052] Fig.13 Figure 2 shows the flow cytometric analysis of CD86 and CD206 expression levels in RAW264.7 cells after they were treated with LPS and co-incubated with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM for 12 hours (A) and the quantitative results of M2 macrophages / M1 macrophages (B);

[0053] Fig.14 Western Blot detection results of HO-1, p38, p65, and p50 protein expression in RAW264.7 cells after LPS treatment and co-incubation of PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM with RAW264.7 cells for 24 hours (A) and corresponding quantitative graphs (BE);

[0054] Fig.15 Quantitative analysis of the expression levels of pro-inflammatory cytokines TNF-α (A) and IL-1β (B) in the cell supernatant after RAW264.7 cells were treated with LPS and co-incubated with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM for 24 hours, respectively;

[0055] Fig.16 After T cells were treated with IL-2, PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM were co-incubated with T cells for 48 h, and the gating strategy (A) of CFSE-labeled CD4+ T cells, representative flow cytometry analysis (B), and the proportion of dividing cells (C) calculated from (B);

[0056] Fig.17 The graphs of blood glucose changes in STZ-induced diabetic mice treated with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM for 42 days;

[0057] Fig.18 The results of the glucose tolerance test after intraperitoneal injection of glucose in STZ-induced diabetic mice at week 6 after treatment in each group (A) and the quantitative graph of the area under the curve calculated from (A) (B);

[0058] Fig.19 The levels of (A) TNF-α, (B) IL-6 in serum and (C) insulin in plasma of STZ-induced diabetic mice after 42 days of treatment with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM.

[0059] Fig. 20 The following are representative H&E staining results of pancreatic tissues of STZ-induced diabetic mice after 42 days of treatment with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM. The arrows in the figure point to the pancreatic site.

[0060] Fig.21 Flow cytometric analysis and quantitative analysis of (A) CD3+PD-1+ T cells, (B) CD3+CD4+ T cells, (C) CD4+IFN-γ+ T cells, and (D) CD4+Foxp3+ T cells in the spleen tissue of STZ-induced diabetic mice after 42 days of treatment with PBS, AK128, AS, ASM, ASM@RM, and ASM@PRM. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0062] Unless otherwise stated, all chemical reagents were used directly without further purification.

[0063] Hydroxylated phosphorus-containing dendrimer AK128 comes from the research group of Professor JP Majoral of the French National Center for Scientific Research. SOD, Mn2(CO) 10 and streptozotocin (STZ) were from SigmaAldrich (St. Louis, MO). RAW264.7 cells (mouse macrophage cell line) were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. MIN6 cells (mouse insulinoma cell line) were from Yuchi Biotechnology Co., Ltd. (Shanghai). DMEM medium, fetal bovine serum, penicillin-streptomycin, and trypsin were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. JC-1 detection kit and ROS detection kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Cell apoptosis kit was purchased from Jiangsu Keygen Biotechnology Co., Ltd. Carbon monoxide probe was purchased from Xi'an Ruixi Biotechnology Co., Ltd. C57BL / 6 mice were purchased from Shanghai Slake Experimental Animal Center. Anti-CD86-PE, Anti-CD206-FITC, Anti-CD3-FITC, Anti-PD-1-PE, Anti-CD4-PE, Anti-CD4-FITC, Anti-IFN-γ-FITC, Anti-Foxp3-FITC and other antibodies were purchased from Thermo Fisher Scientific (Waltham, MA). Water with a resistivity higher than 18.2 MΩ.cm used in all experiments was purified by PURIST UV ultrapure water system (Shanghai Ruifulai Biotechnology Co., Ltd.).

[0064] Example 1

[0065] (1) 1.0 mg of sodium phosphite-terminated phosphorus dendrimer AK128 was dissolved in 250 μL ultrapure water, then added dropwise to a 0.25 mg / mL SOD aqueous solution (1 mL), and stirred at room temperature for 12 h until the solution turned milky white. The mixed solution was centrifuged at 12000 rpm and 4 °C for 10 min, and the precipitate was collected to obtain the AK128 / SOD (AS) nanocomposite. In addition, the encapsulation efficiency and loading efficiency of SOD were quantitatively determined by the BCA kit to be 75.18% and 15.82%, respectively.

[0066] (2) 1.0 mg Mn2(CO) 10 Dissolve in 200 μL methanol, add dropwise into a solution containing 2 mg / mL AS (2 mL), stir at room temperature for 24 h, transfer the mixture to a centrifuge tube after the reaction, centrifuge at 1500 rpm for 10 min at 4 °C, collect the supernatant to obtain AK128 / SOD / Mn2(CO) 10 Nanocomposite (ASM), precipitated as unloaded Mn2(CO) 10 At the same time, the Mn2(CO) in the precipitate was measured by ICP-OES. 10 The content of manganese element is calculated from the unloaded Mn2(CO) 10 The amount of Mn2(CO) can be calculated 10 The loading efficiency was 29.09wt% and the encapsulation efficiency was 82.03wt%.

[0067] (3) RAW264.7 cells were cultured in DMEM medium containing 50 ng / mL IFN-γ at 37°C and 5% CO2 for 48 h. RAW264.7 cells were centrifuged at 1000 g for 5 min to obtain a cell pellet. 30 μL of PMSF and 3 mL of hypotonic cell lysis buffer were mixed and evenly added to the cell pellet. The mixture was placed on ice for 15 min and repeated freeze-thaw method (freezing in liquid nitrogen, thawing at 37°C, repeated 3 times) was used. The pellet was centrifuged at 700 g for 10 min at 4°C and the pellet was removed. The pellet was centrifuged at 14000 g for 30 min and the supernatant was removed. The pellet was resuspended in 500 μL of 1× PBS solution to obtain a cell membrane suspension.

[0068] (4) 200 μg of ASM was mixed with 500 μL of cell membrane suspension (0.4 mg / mL) with a mass ratio of ASM to cell membrane of 1:1. The solution was extruded 13 times using an Avanti microextruder with a filter membrane pore size of 400 nm and centrifuged at 10,000 rpm for 6 min to remove excess cell membrane to obtain ASM@PRM.

[0069] Example 2

[0070] Appropriate amounts of AK128, AS, ASM, and ASM@PRM prepared in Example 1 were dissolved in ultrapure water to prepare solutions with a concentration of 0.5 mg / mL for measuring the hydrated particle size and surface potential. Figure 2As shown in the figure, the hydrated particle size of AK128 is 172.4±5.69nm and the potential is -20.1±3.84mV; after loading SOD, the hydrated particle size increases to 205.7±7.30nm and the potential changes to -27.6±0.37mV. The changes in hydrated particle size and potential prove the successful synthesis of AS. When AS is loaded with drug Mn2(CO) 10 After that, the hydrated particle size increased to 258.7±3.83nm, indicating the successful synthesis of the drug-loaded nanocomposite ASM. After further coating the IFN-γ stimulated macrophage membrane on the ASM surface by extrusion, the hydrated particle size of ASM@PRM increased to 293.5±3.98nm, and the potential increased to -16.8±0.20mV, which is similar to the pure cell membrane (PRM), proving the successful coating of the cell membrane. In addition, the hydrated particle size of ASM@PRM did not change significantly after standing in various solutions (water, PBS, DMEM culture medium) for one week ( Figure 3 ), which proves that ASM@PRM has good stability.

[0071] Example 3

[0072] The size and morphology of AS, ASM and ASM@PRM prepared in Example 1 were characterized. The TEM results of AS nanocomposites are shown in Figure 4 As shown in A, AS is uniformly spherical and has a particle size of approximately 193.3 nm. Figure 4 B shows that when AS is loaded with Mn2(CO) 10 After addition, the average particle size of the ASM composite increased to 226.6 nm. Figure 4 C shows that after wrapping the macrophage membrane, the average size of ASM@PRM is 239.2 nm, and the measured cell membrane thickness is about 20.3 nm.

[0073] Example 4

[0074] The ASM@PRM prepared in Example 1 was subjected to SDS-PAGE characterization to verify the cell membrane coating and the retention of cell membrane proteins. The protein content in the macrophage membrane suspension and ASM@PRM prepared in Example 1 was determined by the 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, and then 15 μL of AK128 (the corresponding AK128 concentration when the protein concentration in ASM@PRM is 1 mg / mL), SOD, ASM, macrophage membrane suspension (RM), ASM@RM, macrophage membrane suspension (PRM) after IFN-γ stimulation, and ASM@PRM were added to the protein lanes respectively. The current was set to 100A and the time was set to 30 min. Figure 5As shown in A, no protein bands appeared in the AK128 group, while protein bands similar to SOD and pure PRM appeared in the ASM@PRM group, proving that the SOD protein was successfully loaded on the ASM@PRM, the PRM was successfully coated on the ASM@PRM surface, and the cell membrane protein was successfully retained. The Western blot experiment was used to detect whether the corresponding special protein was successfully retained on the cell membrane-coated material. The results are shown in Figure 5 As shown in B, representative pro-inflammatory cytokine binding receptors in PRM include CD120a that binds to TNF-α, CD121a that binds to IL-1β, and PD-L1 receptor protein that binds to PD-1. The expression level of these receptors on PRM is higher than that of natural RM, and they are still retained in ASM@PRM, which may be beneficial to improve the clearance ability of pro-inflammatory cytokines and combine with T cells to reduce their ability to attack β cells.

[0075] Example 5

[0076] In order to simulate the release of CO from ASM@PRM under high levels of hydrogen peroxide in vivo, PBS buffer containing different H2O2 concentrations (0, 0.1, 0.5, 1 mM) was prepared to analyze the Mn2(CO) 10 Redox responsive release performance. 1 mg / mL ASM@PRM was mixed with the above-mentioned different buffer solutions, and then a mixture of CO probe (10 μM) and PdCl2 (10 μM) was added to the above solution. At predetermined time points of 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 72 h, the excitation wavelength was set to 490 nm, the slit width was set to dex=dem=2.5 nm, and the fluorescence intensity at 527 nm in the range of 500-640 nm was tested by fluorescence spectroscopy. Calculation was performed based on the standard curve to plot the CO release of ASM@PRM under different conditions. Figure 6 As shown in the figure, ASM@PRM releases CO slowly in 0.1mM H2O2 buffer, with a drug release rate of 13.66% in 72h, while in 0.5mM and 1mM H2O2 buffers, the cumulative release of CO reaches 52.84% and 75.74% in 72h, while the release of ASM without macrophage membrane in 0.5mM H2O2 buffer is 57.80% in 72h. These results show that ASM@PRM nanoparticles have redox-sensitive CO release properties, and the cell membrane wrapping has little effect on the CO release effect.

[0077] Example 6

[0078] MIN6 and RAW264.7 cells were used as cell models to detect the cytotoxicity of ASM@PRM and related materials prepared in Example 1. MIN6 or RAW264.7 cells in the logarithmic growth phase were collected and 1×10 4 The cells were seeded at a density of 100 cells / mL in a 96-well plate. The culture medium used was a complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS, and incubated at 5% CO2 and 37°C for 12 hours. The original culture medium was discarded, and culture medium containing different concentrations of AK128, AS, ASM or ASM@PRM (AK128 concentration was 0, 12.5, 25, 50, 100, 200 μg / mL) was added to each well plate and co-cultured with the cells at 5% CO2 and 37°C for 24 hours. After that, the well plate was removed, the original culture medium was discarded, and the plate was washed three times with PBS. Fresh culture medium containing 10% (v / v) CCK-8 was added, and the cells were incubated in the incubator for another 3 hours. Finally, the absorbance of each well was tested at a wavelength of 450nm using a multifunctional microplate reader, and the cells treated with PBS were used as blank controls. The results are shown in Figure 2. Figure 7 As shown in the figure, within the experimental concentration range, with the increase of AK128 concentration, each group showed no significant cytotoxicity to MIN6 and RAW264.7 cells. It is worth noting that when macrophages stimulated by different concentrations of ASM@PRM and LPS (2μg / mL) were cultured for 24 hours, the cell viability was significantly improved. When the concentration of ASM@PRM was 750ug / mL, the macrophage viability reached 81.92%. At the same time, after MIN6 cells pretreated with STZ (10mM) were incubated with ASM@PRM for 24 hours, the cell viability increased from 58.07% to 89.72%. The results showed that the treatment of ASM@PRM can effectively protect MIN6 and RAW264.7 cells from oxidative stress damage and improve their cell viability and anti-apoptosis ability.

[0079] Example 7

[0080] MIN6 cells were used as a cell model to evaluate the ability of ASM@PRM to alleviate oxidative stress. In order to verify the effect of ASM@PRM nanocomplex on ROS scavenging in MIN6 cells, MIN6 cells in the logarithmic growth phase were collected and cultured at 1×10 5The cells were seeded at a density of 10 cells per well in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. The medium was replaced with RPMI-1640 medium containing STZ (at a concentration of 10 mM) and incubated with the cells for 16 hours. PBS was added to the control group (as the Control group). Subsequently, the medium was replaced with fresh DMEM medium containing PBS, AK128, AS, ASM, ASM@RM or ASM@PRM (AK128 concentration was 100 μg / mL) and incubated with the cells for 12 hours. The PBS-treated group served as the positive control group. After collecting the cells by trypsin digestion, the cell pellet was resuspended in 1 mL of PBS, and the ROS fluorescent probe DCFH-DA was added and incubated with the cells at 37°C for 20 minutes. After washing three times with PBS to remove the probes that were not bound to the cells, the cell pellet was resuspended in 300 μL of PBS and transferred to a flow tube. The effect of the nanomaterial on ROS clearance in MIN6 cells was evaluated by flow cytometry. The results are shown in Figure 8 As shown in the figure, compared with the positive control, AK128 did not show the ability to scavenge ROS. AS showed ROS scavenging ability by promoting the intracellular delivery of SOD. In addition, ASM can promote the intracellular delivery of SOD and improve the Mn2(CO) 10 The bioavailability of ASM@RM was enhanced, thereby showing enhanced ROS scavenging performance, thereby effectively alleviating excessive oxidative stress in macrophages. Compared with the ASM-treated group, ASM@RM showed a slightly enhanced ROS scavenging effect, while the ASM@PRM group had the most obvious scavenging effect on intracellular ROS. This is because PRM expresses higher levels of inflammatory cytokine receptors than RM, further targeting inflammatory MIN6 cells, enhancing the cell uptake of nanomedicines while synergistically exerting the antioxidant effects of each component, thereby alleviating oxidative stress.

[0081] Example 8

[0082] In order to verify the effect of ASM@PRM nanocomplex on mitochondrial membrane potential of MIN6 cells, MIN6 cells in logarithmic growth phase were collected and cultured at 1×10 5The cells were inoculated at a density of 10 cells per well in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. The cells were incubated with RPMI-1640 medium containing STZ (at a concentration of 10 mM) for 16 hours, and PBS was added to the control group (as the Control group). Subsequently, fresh DMEM medium containing PBS, AK128, AS, ASM, ASM@RM or ASM@PRM (AK128 concentration of 100 μg / mL) was replaced and incubated with the cells for 12 hours, and the PBS treatment group was used as the positive control group. After the cells were collected by trypsin digestion, 0.5 mL of JC-1 staining working solution was added, the cell pellet was resuspended, and incubated at 37°C in a cell culture incubator for 20 minutes. After the incubation, the cells were centrifuged at 1000 rpm for 5 minutes to precipitate the cells and the supernatant was discarded. After washing twice with JC-1 staining buffer, the cell pellet was collected and 500 μL of JC-1 staining buffer was added to resuspend the cells. The changes in mitochondrial membrane potential of different treatment groups were analyzed by flow cytometry. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers (J-aggregates) and produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is a monomer (J-monomer) and produces green fluorescence. The change of mitochondrial membrane potential can be detected by the change of fluorescence color. The results are as follows Fig. 9 As shown in the figure, the content of JC-1 monomer positive cells in the positive control group increased (57.39%), indicating that STZ can significantly induce a decrease in mitochondrial membrane potential, causing an imbalance in mitochondrial functional homeostasis. The content of green fluorescence positive cells in the AK128, AS, and ASM groups decreased compared with the positive control group, and the ASM@PRM group showed the least proportion of positive green fluorescence cells, proving that it has the strongest function in restoring mitochondrial membrane potential.

[0083] Example 9

[0084] In order to verify the protective effect of ASM@PRM nanocomplex on MIN6 cells, MIN6 cells in the logarithmic growth phase were collected and cultured at 1×10 5The cells were seeded at a density of 10 cells per well in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. The medium was replaced with RPMI-1640 medium containing STZ (at a concentration of 10 mM) and incubated with the cells for 16 hours. PBS was added to the control group (as the Control group). Subsequently, the medium was replaced with fresh DMEM medium containing PBS, AK128, AS, ASM, ASM@RM or ASM@PRM (AK128 concentration of 100 μg / mL) and incubated with the cells for 12 hours. The PBS-treated group served as the positive control group. The cells in the 12-well plate were then digested, collected by centrifugation and washed. The materials were used to detect the inhibition of apoptosis of the cells after STZ treatment according to the instructions of the Annexin V-FITC / PI Cell Apoptosis Detection Kit (KeyGen Biotechnology, China). The results are shown in Figure 2. Fig.10 As shown in the figure, compared with the positive control group, the prepared AK128, AS, ASM, and ASM@RM can reduce the apoptosis and necrosis rates of cells, while ASM@PRM shows the lowest apoptosis rate. This is mainly because the macrophage membrane after IFN-γ stimulation can target inflammatory MIN6 cells through highly expressed inflammatory cytokine receptors, enhance the cell uptake of nanomedicines, and synergistically exert the anti-inflammatory effects of AK128 and SOD, Mn2(CO) 10 Anti-inflammatory / antioxidant effects, thereby protecting cells from apoptosis caused by oxidative stress.

[0085] Example 10

[0086] RAW264.7 cells were used as a cell model to evaluate the phagocytic effect of cells on ASM@PRM. RAW264.7 cells were cultured at a rate of 2×10 5 The density of cells per well was inoculated in a 12-well plate. The culture medium used was DMEM complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS, and incubated for 24h at 5% CO2 and 37°C. The original culture medium was discarded and replaced with DMEM culture medium containing LPS (at a concentration of 2μg / mL) and incubated with the cells for 24h to induce M1 macrophages. Uninduced macrophages were M0 type. Subsequently, the culture medium was discarded, and the culture medium containing SOD, AS, ASM, ASM@RM or ASM@PRM ([SOD-Cy5.5] = 10μg / mL) was added and incubated with the cells at 5% CO2 and 37°C for 8h. The normal group was treated with PBS. The culture medium was discarded, the cells were washed three times with PBS, 1 mL of 2.5% glutaraldehyde was added to fix the cells at room temperature for 15 min, 1 mL of LDAPI was added to incubate the cells for 5 min, and the fluorescence intensity of the cells was observed under a laser confocal microscope after washing three times with PBS. Fig.11As shown in the figure, in M0 and M1 cells, compared with pure SOD, AS, ASM, and ASM@RM groups all showed higher fluorescence intensity, indicating that the intracellular delivery of SOD can be significantly enhanced under the mediation of nanomaterials. In addition, the fluorescence intensity of ASM@PRM in M1 macrophages is significantly higher than that in M0 macrophages. This is because the surface of PRM after IFN-γ stimulation highly expresses the receptor CD120a of inflammatory cytokines TNF-α and the receptor CD121a of IL-1β, so that the material can target TNF-α and IL-1β on the surface of inflammatory cells (M1 macrophages) after encapsulating PRM, thereby promoting the phagocytosis of ASM@PRM by cells and exerting the drug efficacy.

[0087] Embodiment 11

[0088] In order to verify the effect of ASM@PRM nanocomposite on alleviating oxidative stress in RAW264.7 cells, RAW264.7 cells were cultured at a rate of 1×10 5 The density of cells per well was inoculated in laser confocal culture dishes and 12-well plates, respectively. The culture medium used was DMEM complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS, and incubated for 24 hours at 5% CO2 and 37°C. The original culture medium was discarded and replaced with DMEM culture medium containing LPS (at a concentration of 2μg / mL) and incubated with cells for 24 hours. PBS was added to the control group (as the Control group). Subsequently, the culture medium was discarded, and the culture medium containing PBS, AK128, SOD, AS, ASM, ASM@RM or ASM@PRM (AK128 concentration of 100μg / mL) was added and incubated with the cells at 5% CO2 and 37°C for 12 hours. The PBS-treated group was used as the positive control group. For laser confocal microscopy observation, the cells were treated as follows: discard the culture medium, wash three times with PBS, add the ROS fluorescent probe DCFH-DA and incubate with the cells at 37°C for 20 minutes, wash three times with PBS, add 1mL 2.5% glutaraldehyde to fix at room temperature for 15 minutes, wash three times with PBS, add 1mL LDAPI and incubate with the cells for 5 minutes, wash three times with PBS, and observe the changes in cell fluorescence intensity under a laser confocal microscope. After trypsin digestion and collection of cells, flow cytometric analysis was performed, and the cell pellet was resuspended with 1mL PBS, and the ROS fluorescent probe DCFH-DA was added and incubated with the cells at 37°C for 20 minutes. After washing three times with PBS to remove the probe that was not bound to the cells, the cell pellet was resuspended with 300μL PBS and transferred to a flow tube. The effect of each nanomaterial on ROS removal in RAW264.7 cells was evaluated by flow cytometry. The results are shown in Fig.12As shown in A, the positive control group showed strong green fluorescence, while after treatment with AS, ASM, ASM@RM and ASM@PRM, the fluorescence intensity was weakened to varying degrees. Fig.12 As shown in B, the flow cytometry results showed that AK128 did not show ROS scavenging performance, while AS, ASM, ASM@RM and ASM@PRM could scaveng ROS. Compared with other treatment groups, ASM@PRM could target inflammatory macrophages on the one hand, and improve the intracellular delivery of SOD and Mn2(CO) on the other hand. 10 The bioavailability of this product shows the strongest ROS scavenging ability, effectively alleviating the excessive oxidative stress of macrophages.

[0089] Example 12

[0090] In order to verify the effect of ASM@PRM nanocomplex on macrophage polarization, RAW264.7 cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were inoculated at a density of 10 cells per well in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. The cells were replaced with DMEM medium containing LPS (at a concentration of 2 μg / mL) and incubated for 24 hours. PBS was added to the control group (as the Control group). Subsequently, the culture medium was discarded, and culture medium containing PBS, AK128, SOD, AS, ASM, ASM@RM or ASM@PRM (AK128 concentration of 100 μg / mL) was added to the cells and incubated for 12 hours at 5% CO2 and 37°C. The PBS-treated group was used as the positive control group. Subsequently, the culture medium was discarded, washed twice with PBS, and the cells were collected by trypsin digestion. The cell pellet was resuspended with 200 μL PBS, and Anti-CD206-FITC and Anti-CD86-PE antibodies were added to the cells and incubated for 30 minutes on ice in the dark. After washing three times with PBS to remove the antibodies not bound to the cells, the cell pellet was resuspended with 300 μL PBS and transferred to a flow cytometry tube. The effect of the nanomaterial on macrophage polarization was evaluated by detecting the changes in the expression levels of CD86 and CD206 in the cells. Fig.13 As shown in the figure, after LPS treatment, the M2 / M1 ratio of macrophages was significantly downregulated compared with PBS, proving that LPS stimulation can effectively induce macrophages to M1 proinflammatory phenotype. The macrophages treated with ASM@PRM group had the highest M2 / M1 ratio, indicating that the material has excellent in vitro macrophage phenotype regulation performance, which can be achieved by combining SOD and Mn2(CO) 10 The biological function of AK128, combined with the immunomodulatory activity of the AK128 vector itself, promotes the polarization of the macrophage population from the M1 phenotype to the M2 phenotype.

[0091] Embodiment 13

[0092] In order to explore the molecular mechanism of ASM@PRM nanocomplex in regulating macrophages to fight against inflammation, RAW264.7 cells in the logarithmic growth phase were collected and cultured at 2×10 5 The cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 24 hours. The medium was replaced with DMEM medium containing LPS (at a concentration of 2 μg / mL) and incubated with the cells for 24 hours. PBS was added to the control group (as the Control group). Subsequently, the culture medium was discarded, and culture medium containing PBS, AK128, SOD, AS, ASM, ASM@RM or ASM@PRM (AK128 concentration of 100 μg / mL) was added to the cells and incubated at 5% CO2 and 37°C for 24 hours. The PBS-treated group served as the positive control group. Subsequently, the culture medium was collected, the cells were washed twice with PBS, and the cells were collected after trypsin digestion. The cells were washed twice with PBS to collect the cells. The protein concentration was determined, and then SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were performed in sequence. The HO-1, p38, p65, and p50 protein contents in the cells were studied, with β-actin used as an internal reference. The results are shown in Fig.14 As shown in the figure, under LPS stimulation, HO-1 protein expression was downregulated, while p38, p65, and p50 pro-inflammatory pathway-related proteins were upregulated, proving that macrophages were in a state of oxidative stress and severe inflammatory response under the induction of LPS. After ASM@PRM treatment, the HO-1 protein content in macrophages increased, while the content of p38, p65, and p50 pro-inflammatory pathway-related proteins decreased. This is because the PRM-encapsulated nanocomplex can actively target inflammatory macrophages and improve SOD and Mn2(CO) 10 The bioavailability of LPS was increased to enhance the anti-inflammatory and antioxidant activities of both, while combining the anti-inflammatory advantages of the carrier itself to relieve the oxidative stress induced by LPS and reduce the inflammatory response. The corresponding ELISA kits were used to detect the content of pro-inflammatory cytokines TNF-α and IL-1β in the collected culture medium. The results are as follows Fig.15 As shown in the figure, compared with the normal Control group, the levels of TNF-α and IL-1β proinflammatory cytokines in macrophages were significantly increased after LPS induction, and after treatment with each group of materials, the levels of both decreased to varying degrees, among which the ASM@PRM treatment group showed the lowest TNF-α and IL-1β levels, indicating that ASM@PRM can alleviate the inflammatory response by clearing ROS and promoting the polarization of M2 macrophages.

[0093] Embodiment 14

[0094] T cells were used as a cell model to evaluate the effects of different materials on maintaining immune tolerance. All animal experiments were conducted in strict accordance with the standards of the Animal Protection Association. Female C57BL / 6 black mice used in the experiment were purchased from Shanghai Slake Experimental Animal Center. The spleen tissue of the mouse was removed under sterile conditions, chopped and ground, and filtered through a 400-mesh filter to obtain a single-cell suspension, and the cells were centrifuged at 1500rpm for 10min. Then, the cells were treated with red blood cell lysis buffer and lysis was terminated with PBS supplemented with 1% FBS. Subsequently, lymphocytes were isolated using an animal tumor-infiltrating tissue lymphocyte isolation kit, and T lymphocyte suspension was obtained using a nylon wool column. IL-2 (10ng / mL) was added to the isolated T cells and incubated for 48h, and the group without IL-2 was used as the Control group. In order to evaluate the proliferation of CD4+T cells, the above-mentioned T cells (1×10 6 ) were stained with the cell division tracking dye CFSE (5 μM) at 37°C for 20 min, and then 5 volumes of cold culture medium containing 10% FBS were added to stop the reaction. The treated T cells were incubated with complete culture medium containing PBS, AK128, AS, ASM@RM or ASM@PRM ([ASM@PRM] = 375 μg / mL) for 48 h, and the cells were collected and labeled with anti-CD4-PE antibody, and then used for flow cytometry analysis. The results are shown in Fig.16 As shown, the effect of ASM@RM on the proliferation of CD4+T cells is negligible because the content of PD-L1 on the natural macrophage membrane is low. ASM@PRM can significantly inhibit the proliferation of CD4+T cells, as shown by a significant decrease in the proportion of dividing cells stained with CFSE. These results indicate that ASM@PRM can inhibit the activation of CD4+T cells by establishing the PD-1 / PD-L1 immune checkpoint (inhibitory axis), thereby inhibiting T cell proliferation.

[0095] Embodiment 15

[0096] All animal experiments were conducted in strict accordance with the standards of the Animal Protection Association. Female SPF-grade 6-8-week-old C57BL / 6 mice were purchased from Shanghai Slake Experimental Animal Center. To induce β-cell damage and type I diabetes, C57BL / 6 mice were fasted overnight and given an intraperitoneal injection of freshly prepared streptozotocin STZ (10 mg / mL) in citric acid-sodium citrate buffer at a dose of 50 mg / kg body weight for 5 consecutive days. Healthy mice in the control group were injected with PBS (the negative control group was designated as the Healthy group). Subsequently, the blood glucose of mice was monitored for a long time. When the blood glucose level of C57 / BL6 mice was higher than 300 mg / dL, the mice were considered to have diabetes. The hyperglycemic mice were randomly divided into 7 groups (6 mice in each group) and injected with 100 μL PBS (the positive control group was designated as the T1D group), AK128, AS, ASM, ASM@RM, and ASM@PRM ([ASM@PRM] = 40 mg / kg) through the tail vein every 2 days. Blood glucose levels of diabetic mice were monitored once a week until the end point (42 days in total), and then the mice were sacrificed for further analysis. At the sixth week of treatment, three mice in each group underwent intraperitoneal glucose tolerance test (IPGTT). Briefly, when there was no significant change in blood glucose levels within 20 minutes, a glucose solution was injected intraperitoneally at a dose of 1.5 g / kg, and then blood glucose levels were recorded for the next 2 hours. The results of blood glucose testing of mice during treatment are shown in Figure 2. Fig.17 As shown in the figure, after STZ induction, the blood glucose level of mice gradually increased in the first three weeks and then stabilized. After STZ induction and ASM@PRM treatment, the rate of blood glucose increase was significantly inhibited. As a control, the blood glucose of healthy mice remained at a normal level. The IPGTT test results are shown in the figure. Fig.18 As shown in the figure, after the injection of glucose solution, the blood glucose level of healthy mice in the control group increased rapidly and returned to normal within 1.5 hours, indicating that healthy mice have the ability to secrete insulin rapidly and normally. At the same time, similar blood glucose change trends can be observed in mice treated with ASM@PRM after STZ induction. Fig.17 As shown, we calculated the area under the curve of each group to quantify the response of mice to glucose after different treatments. The responsiveness of diabetic mice treated with ASM@PRM group to IPGTT was significantly stronger than that of other treatment groups, and only slightly lower than that of healthy mice. The blood glucose levels of other diabetic mice increased rapidly within 15 minutes, and were still higher than the initial value even 2 hours after glucose injection. After the treatment, blood was collected from the eyeballs of diabetic mice after different treatments and centrifuged to obtain serum. The serum of healthy mice was used as a control. The ELISA method was used to detect the levels of serum inflammatory factors (TNF-α, IL-6) and plasma insulin in each group of mice. The ELISA test results are shown in Fig.19As shown in the figure, the content of inflammatory cytokines (TNF-α, IL-6) in the positive control group was significantly higher than that in healthy mice, while the inflammatory factors after ASM@PRM treatment reached the lowest level in all treatment groups except the negative control group. In addition, the insulin levels of all STZ-modeled diabetic mice were generally lower than those of healthy mice, but the insulin levels of mice in the ASM@PRM group were significantly higher than those of diabetic mice in other treatment groups. At the same time, the pancreatic islets of mice treated in different groups were stained with H&E to analyze the morphology and size of the islets. The results of histological analysis are shown in the figure. Fig. 20 As shown in the figure, compared with the islets in the negative control group, the islet structure in the positive control group was significantly abnormal, with relatively severe islet damage. Treatment of diabetic mice with ASM@PRM significantly improved islet structure and β-cell damage, which was attributed to the fact that ASM@PRM treatment could reduce the level of oxidative stress induced by STZ and reduce the production of inflammatory cytokines, thereby protecting the islet structure and maintaining insulin levels.

[0097] Example 16

[0098] In order to verify that ASM@PRM has the function of maintaining immune tolerance, immune cell typing analysis was performed on mice after different treatments. After 42 days of treatment, the spleen tissue of each group of mice was removed under sterile conditions, chopped and ground, and filtered through a 400-mesh filter to obtain a single cell suspension. The cells were centrifuged at 1500rpm for 10 minutes. Then, the cells were treated with red blood cell lysis buffer, and the lysis was terminated with PBS supplemented with 1% FBS. Subsequently, lymphocytes were separated using an animal tumor-infiltrating tissue lymphocyte separation kit, and T lymphocyte suspension was obtained using a nylon wool column. The cells were incubated with FITC-CD3 / PE-PD-1, FITC-CD4 / PE-CD3, FITC-CD4 / IFN-γ or FITC-CD4 / PE-Foxp3 antibodies at room temperature in the dark for 30 minutes. After washing with PBS three times, the cells were analyzed by flow cytometry. The results are shown in the figure. Fig.21 As shown in the figure, compared with the positive control group, the proportion of CD3+PD-1+T cells, CD3+CD4+T cells and IFN-γ+CD4+T cells in spleen tissue after ASM@PRM treatment was downregulated, indicating that ASM@PRM can reduce the activity of activated T cells and inhibit the attack of activated T cells on pancreatic β cells. Tregs cells (CD4+Foxp3+T cells), as inhibitors of T cell activation, can inhibit immune responses and maintain autoimmune tolerance. The results show that the proportion of Tregs cells in diabetic mice increased under the treatment of ASM@PRM, further promoting the protective effect on β cells.

Claims

1. A phosphorus-containing dendrimer composite nanomaterial, characterized in that: The composite nano material comprises phosphorus-containing dendrimers and protein complexes capped with sodium phosphite, and drugs; wherein the phosphorus-containing dendrimers and protein complexes capped with sodium phosphite are loaded with drugs.

2. The phosphorus-containing dendrimer composite nanomaterial according to claim 1, characterized in that: The structural formula of the sodium phosphite-terminated phosphorus-containing dendrimer The protein is superoxide dismutase SOD; the drug includes carbonyl manganese Mn2 (CO) 10 .

3. A method for preparing a phosphorus-containing dendrimer composite nanomaterial, comprising: (1) mixing a phosphorus-containing dendrimer capped with sodium phosphite and a protein, and stirring at room temperature to obtain a phosphorus-containing dendrimer capped with sodium phosphite / protein complex; (2) Mixing the drug and the phosphorus-containing dendrimer / protein complex capped with sodium phosphite, and stirring at room temperature to obtain a phosphorus-containing dendrimer composite nanomaterial.

4. The preparation method according to claim 3, characterized in that: In step (1), the reaction is stirred at room temperature for 12 to 24 hours; In the step (1), the mixture is stirred at room temperature and then centrifuged, wherein the centrifugation is performed at 8000 to 12000 rpm for 10 to 15 minutes; in the step (1), the mass ratio of the phosphorus-containing dendrimer capped with sodium phosphite to the protein is 2:1 to 10:1; and the protein is superoxide dismutase SOD; The aqueous solution of phosphorus-containing dendrimers capped with sodium phosphite salt mixed in step (1) is added dropwise into the protein solution; The solvent of the protein solution is phosphate buffer.

5. The preparation method according to claim 3, characterized in that: In the step (2), the mass ratio of the phosphorus-containing dendrimer / protein complex capped with sodium phosphite to the drug is 1:0.25 to 1:2; The stirring time at room temperature in step (2) is 12 to 24 hours; after stirring at room temperature, centrifugation is performed at 1000 to 1500 rpm for 10 to 15 minutes; in step (2), the drug, the phosphorus-containing dendrimer capped with sodium phosphite and the protein complex are mixed into a drug solution and added dropwise to the phosphorus-containing dendrimer capped with sodium phosphite and the protein complex solution; the drug includes carbonyl manganese Mn2(CO) 10 ; The solvent of the drug solution is methanol; the solvent of the solution of the sodium phosphite-terminated phosphorus-containing dendrimer and protein complex is PBS buffer.

6. A cell membrane bionic material, characterized in that: The cell membrane bionic material is a cell membrane coated phosphorus-containing dendrimer composite nanomaterial according to claim 1.

7. A cell membrane bionic material, comprising: The phosphorus-containing dendrimer composite nanomaterial according to claim 1 and a macrophage membrane suspension are mixed and extruded by an extruder to obtain a cell membrane bionic material.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the phosphorus-containing dendrimer composite nanomaterial to the macrophage membrane is 1:0.5 to 1:2; The macrophage membrane is a macrophage membrane that highly expresses PD-L1; The extruder extrusion is performed by repeatedly extruding 11 to 15 times using an extruder with a filter membrane pore size of 400 nm, and centrifuging after extrusion, wherein the centrifugation is performed at 8000 to 12000 rpm for 5 to 10 minutes.

9. Use of the phosphorus-containing dendrimer composite nanomaterial according to claim 1 or the cell membrane biomimetic material according to claim 6 in the preparation of anti-inflammatory / anti-oxidative / anti-apoptotic / immune tolerance maintenance drugs.

10. Use of the phosphorus-containing dendrimer composite nanomaterial according to claim 1 or the cell membrane biomimetic material according to claim 6 in preparing a drug for treating type I diabetes.

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