Bionic iron-based nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to bionic iron-based nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of cell membrane camouflage nanoparticles. The bionic iron-based nano-particles comprise drug-loaded Fe3O4 nano-particles and red cell membranes compounded on the surfaces of the drug-loaded Fe3O4 nano-particles, and the red cell membranes are modified by galactose. The preparation method comprises the following steps: S1, mixing an ethanol solution of emodin with an ethanol dispersion liquid of hollow Fe3O4 nanoparticles, centrifuging, discarding a supernatant, and carrying out vacuum drying; s2, suspending the erythrocyte membrane in a buffer solution, and adding galactose for incubation; and S3, mixing the drug-loaded Fe3O4 nanoparticles with the galactose modified erythrocyte membrane, and enveloping to obtain the bionic iron-based nanoparticles. According to the invention, the nanoparticles wrapped by the erythrocyte membrane can reduce the removal of the nanoparticles by an immune system; the nano-particles can be effectively transferred into the M2TAMs by the galactose modified erythrocyte membrane, the emodin is released in the M2TAMs, and the emodin and the hollow Fe3O4 nano-particles cooperate to reprogram the M2TAMs.

Description

Technical Field

[0001] The invention belongs to the technical field of cell membrane camouflage nanoparticles, and specifically relates to a bionic iron-based nanoparticle and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Cancer immunotherapy can activate the host immune system to fight cancer in a safe and effective manner. However, the highly complex tumor immunosuppressive microenvironment controlled by malignant cells, stromal cells, and infiltrating immune cells limits the response of solid tumors to immunotherapy, resulting in a small number of patients who respond to cancer immunotherapy (≈20%). Among them, tumor-associated macrophages (TAMs) are usually the most numerous and typical resident immunosuppressive cells in the tumor microenvironment, accounting for up to 50% in some solid tumors. In developing tumors, M2 tumor-associated macrophages (M2 TAMs) produced by functional polarization of TAMs are ubiquitous in the tumor microenvironment and play a key role in supporting tumor progression and metastasis. They participate in a variety of processes such as providing nutritional support, promoting immune escape, promoting angiogenesis, and affecting tissue remodeling. Reprogramming M2 tumor-associated macrophages (M2 TAMs) into M1 tumor-associated macrophages (M1 TAMs) can increase the expression of interleukin-12 (IL-12), gamma interferon (IFN-γ), and alpha tumor necrosis factor (TNF-α) in CD4 + It is expressed in T cells (Th1) and enhances the related Th1 immune response, thereby inhibiting tumor growth.

[0004] High concentrations of iron and reactive oxygen species in TAMs may also play a key role in macrophage polarization. For example, the iron supplement Ferumoxytol can induce a proinflammatory immune response through the polarization of M1 macrophages. It has been reported that hollow iron oxide (Fe3O4) nanoparticles can promote the transformation of M2 phenotype to M1 phenotype in TAMs, triggering increased expression of Th1 cytokines such as IL-12, IFN-γ, and TNF-α, and reactivating Th1 immune responses. However, nanoparticles are easily cleared by the reticuloendothelial system during long-term circulation, making it difficult for them to enter the tumor immune microenvironment.

[0005] On the other hand, cancer cells can use their own metabolic byproducts to hijack the functions of tumor-infiltrating immune cells to benefit cancer cell invasion and metastasis. For example, during cancer cell glycolysis, the ingested glucose is preferentially decomposed into pyruvate through a series of transformations, and finally catalyzed by lactate dehydrogenase (LDH) to produce lactate. After accumulation, the lactate secreted by cancer cell glycolysis can be accurately sensed by TAMs, leading to the functional polarization of TAMs and the production of M2 TAMs, which in turn leads to tumor progression and metastasis. Rhamnosin is a natural product rich in anthraquinones with significant LDH inhibitor activity, which can inhibit and neutralize the extracellular acidification process of tumors, thereby inhibiting the functional polarization process of TAMs to M2 TAMs. However, the limited solubility of rhamnosin greatly hinders its bioavailability, resulting in low tumor accumulation and low uptake by tumor-associated macrophages in vivo.

[0006] Therefore, there is an urgent need to develop a drug delivery system that can target tumor-associated macrophages (TAMs) in solid tumors and synergistically promote the polarization of TAMs to the M1 type. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a bionic iron-based nanoparticle, a preparation method and an application thereof, and a hollow Fe3O4 nanoplatform wrapped by a galactose-modified erythrocyte membrane is designed, which encapsulates emodin to reverse the tumor immunosuppressive microenvironment and promote the effectiveness of anti-tumor immunotherapy.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] In a first aspect, a biomimetic iron-based nanoparticle comprises: a drug-loaded Fe3O4 nanoparticle, and a red blood cell membrane composited on the surface of the drug-loaded Fe3O4 nanoparticle, wherein the red blood cell membrane is modified by galactose;

[0010] The drug encapsulated by the drug-loaded Fe3O4 nanoparticles is rheumatoid artemisia, and the mass of the rheumatoid artemisia accounts for 20-40% of the mass of the hollow Fe3O4 nanoparticles before drug loading;

[0011] The size of the hollow Fe3O4 nanoparticles is 180 to 220 nm in diameter;

[0012] The ratio of the number of galactose to the number of erythrocyte membrane cells is 100 μg: (2.68-6.25)×10 9 indivual.

[0013] Optionally, the drug-loaded Fe3O4 nanoparticles have a diameter of 320 to 360 nm.

[0014] In a second aspect, the method for preparing the above-mentioned biomimetic iron-based nanoparticles comprises the following steps:

[0015] S1, taking rheumatoid artemisia and hollow Fe3O4 nanoparticles in a mass ratio of (2-2.5):1, mixing the ethanol solution of rheumatoid artemisia and the ethanol dispersion of hollow Fe3O4 nanoparticles, stirring evenly at room temperature, centrifuging and discarding the supernatant, and then vacuum drying to obtain drug-loaded Fe3O4 nanoparticles;

[0016] S2, suspending the red blood cell membrane in a buffer, adding galactose in proportion, incubating for 25 to 35 minutes, and then extruding through a filter membrane to obtain a galactose-modified red blood cell membrane of a set size;

[0017] S3. The drug-loaded Fe3O4 nanoparticles obtained in S1 and the galactose-modified erythrocyte membranes obtained in S2 are mixed and coated to obtain biomimetic iron-based nanoparticles.

[0018] Optionally, the raw materials for preparing the hollow Fe3O4 nanoparticles in S1 include: a mass ratio of (1.0-1.5):

[0019] (3.0-3.5): (0.8-1.0): (0.5-0.9) of ferric chloride hexahydrate, sodium citrate, urea and polyacrylamide; the preparation method comprises: dispersing ferric chloride hexahydrate, sodium citrate and urea in distilled water according to a proportion, then adding polyacrylamide, stirring and dissolving, reacting at 200±20°C for 11-13h, cooling to room temperature, centrifuging to separate a black precipitate, and drying the black precipitate at 60±5°C overnight to obtain hollow Fe3O4 nanoparticles;

[0020] Optionally, in the method for preparing hollow Fe3O4 nanoparticles, after separating the black precipitate, washing it more than three times to remove unreacted substances;

[0021] Optionally, in the method for preparing hollow Fe3O4 nanoparticles, the drying method is vacuum drying.

[0022] Optionally, in S1, the mixing method is ultrasonic dispersion for 25 to 35 minutes.

[0023] Optionally, in S1, the vacuum drying temperature is 60±5°C.

[0024] Optionally, the method for preparing the red blood cell membrane in S2 includes: centrifuging whole blood at 3000-3500 rpm for 8-12 min at 4±2°C, discarding the supernatant, washing with PBS buffer, resuspending the obtained red blood cells in ultrapure water for 2-3 hours to induce red blood cell rupture, then centrifuging at 9000-9500 rpm for 8-12 min, taking the supernatant, washing with PBS buffer until colorless, and obtaining the red blood cell membrane.

[0025] Optionally, in S3, the thin film method comprises: mixing a PBS buffer solution containing drug-loaded Fe3O4 nanoparticles and galactose-modified red blood cell membranes and extruding the mixture through a 200-250 nm filter membrane.

[0026] In a third aspect, the application of the bionic iron-based nanoparticles and / or the method for preparing the bionic iron-based nanoparticles in at least one of the following a1)-a4):

[0027] a1) reprogramming M2 TAMs into M1 TAMs or preparing a product for reprogramming M2 TAMs into M1 TAMs;

[0028] a2) Inhibiting the proliferation of cancer cells or preparing products that inhibit the proliferation of cancer cells;

[0029] a3) Activating anti-tumor immune responses based on macrophages and T cells or preparing products that activate anti-tumor immune responses based on macrophages and T cells;

[0030] a4) Preparation of products for treating tumors.

[0031] Optionally, the cancer cells are breast cancer cells, specifically triple-negative breast cancer cells.

[0032] Optionally, the tumor is a breast cancer tumor, specifically a triple-negative breast cancer tumor.

[0033] The beneficial effects of the present invention are:

[0034] 1. The present invention can be used to reshape the tumor immune microenvironment. The nanoparticles wrapped in erythrocyte membranes can "compensate" for the biological interface between nanoparticles and erythrocytes, thereby reducing the clearance of nanoparticles by the immune system. In addition, due to the high expression of galactose lectins in M2 TAMs, the galactose-modified erythrocyte membrane can effectively transport nanoparticles into tumor-associated macrophages, and the internally encapsulated emodin is released inside M2 ​​TAMs, which can significantly inhibit the conversion of pyruvate to lactate, further inhibit and neutralize tumor acidification, reprogram tumor-associated macrophages, and polarize M2 TAMs. Hollow Fe3O4 nanoparticles further convert M2 TAMs into M1 TAMs.

[0035] 2. The present invention can convert M2 TAMs into M1 TAMs, thereby upregulating the secretion of tumor necrosis factor (TNF-α) and interleukin 12 (IL-12) by M1 TAMs, activating and recruiting CD8 + and CD4 + T cells attack tumor cells, thereby improving the effectiveness of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 It is a schematic diagram of a specific implementation method, (A) is a flow chart of the preparation method of biomimetic iron-based nanoparticles in Example 1, and (B) is a schematic diagram of improving tumor metabolic immunotherapy.

[0038] Figure 2 1 is a characterization result diagram in Example 1, (A) represents the SEM image of hollow Fe3O4 nanoparticles, (B) represents the TEM image of hollow Fe3O4 nanoparticles, (C) represents the XRD spectrum of hollow Fe3O4 nanoparticles, (D) represents the STEM and element mapping of hollow Fe3O4 nanoparticles, (E) represents the Fe2p region XPS spectrum of hollow Fe3O4 nanoparticles,

[0039] (F) shows the N2 adsorption / desorption isotherms of hollow Fe3O4 nanoparticles, (G) shows the established standard curve of HPLC for evaluating the loading rate of rhamnosine, (H) shows the results of SDS-PAGE analysis, (I) shows the statistical graph of the average hydrodynamic diameter of each sample, and (I) shows the statistical graph of the Zeta potential of each sample.

[0040] Figure 3 Schematic diagram of the reprogramming ability in Example 1, (A) is a schematic diagram of the reprogramming ability, (B) is a schematic diagram of the reprogramming ability at different doses ER The content of pyruvate under the action of Fe3O4 NPs, (C) is a statistical diagram of the results of ICP-MS detection of iron ions in cells, where the asterisk indicates that there is statistical significance compared with the control group: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, (D) is a graph of the viability evaluation of M2 TAMs cells, (E) is a statistical diagram of CD16 / 32 expression, (F) is a statistical diagram of CD206 expression, (G) is a statistical diagram of TNF-α expression after M2 TAMs were treated with different reagents, and the asterisk indicates that the concentration difference is statistically significant compared with the control group, after one-way analysis of variance and Tukey's post hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0041] Figure 4 Schematic diagrams of the in vitro anti-tumor evaluation in Example 1, (A) is a schematic diagram of the experimental device, (B) is a diagram of the 4T1 cell viability evaluation, and the statistical significance compared with the control group is indicated by asterisks (*p<0.05, **p<0.01,

[0042] ***p<0.001), (C) is the viability assessment of 3T3 cells, (D) is the live and dead cell staining of 4T1 cell populations treated with different reagents, and (E) is the flow cytometry analysis of 4T1 cell populations treated with different reagents.

[0043] Figure 5 : are schematic diagrams of the in vivo anti-tumor evaluation in Example 1, (A) is a photo of the hemolysis test sample and a quantitative statistical diagram of the hemolysis rate (HR%), (B) is a fluorescence imaging diagram of the main organs and tumors, (C) is a diagram of the iron concentration measured by ICP-MS of the main organs and tumors, (D) is a timeline diagram of the tumor-bearing mouse model test process, (E) is a tumor growth curve, (F) is a mouse weight change diagram, (G) is a H&E staining diagram of the tumor tissue, and (H) is an immunofluorescence staining diagram of the tumor tissue. (I) is F4 / 80 + The expression level of CD16 / 32 in macrophages, (J) is F4 / 80 + Figure 2 Expression levels of CD206 in macrophages; in (A), (E), and (F), asterisks indicate statistical significance compared with the PBS group, as determined by one-way ANOVA with Tukey's post hoc analysis, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0045] Figure 6 1 and 2 are graphs showing the results of AST / ALT index detection in Example 1, wherein (A) is a graph showing the results of AST index, and (B) is a graph showing the results of ALT index.

[0046] Figure 7 1 and 2 are graphs showing the results of the UREA / CREA index detection in Example 1, wherein (A) is a graph showing the results of the UREA index, and (B) is a graph showing the results of the CREA index.

[0047] Figure 8 These are graphs of blood test results in Example 1, (A) is a graph of MCV index results, (B) is a graph of WBC index results, (C) is a graph of MCHC index results, (D) is a graph of HCT index results, (E) is a graph of RBC index results, (F) is a graph of MCH index results, (G) is a graph of PLT index results, and (H) is a graph of HGB index results.

[0048] Fig. 9 This is a diagram of the H&E staining results of the main organs in Example 1.

[0049] Fig.10 This is a photo of the tumor-bearing mice in Example 1.

[0050] Fig.11 This is a photograph of the state of mouse tumor in Example 1.

[0051] Fig.12 CD4 in Example 1 + and CD8 + Ratio of T lymphocytes. DETAILED DESCRIPTION

[0052] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0053] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0054] The experimental methods in the following examples without specifying specific conditions are generally based on conventional conditions. All raw materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0055] The main materials and reagents used in the present invention are listed as follows:

[0056] Sodium citrate, ferric chloride hexahydrate, polyacrylamide and urea were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0057] FITC anti-mouse F4 / 80 antibody, FITC anti-mouse CD3 antibody, PE anti-mouse CD4 antibody, APC anti-mouse CD8 antibody, PE / Cy7 anti-mouse CD 16 / 32 antibody, PE anti-mouse CD 206 antibody and mouse TNF-α ELISA kit were from Biolegend (San Diego, CA, USA).

[0058] Apoptosis detection kit was from Solaibao (China).

[0059] The culture dishes used for cell culture were from Saining Biotechnology (China).

[0060] All chemicals were of analytical grade and used without further purification.

[0061] A bionic iron-based nanoparticle, comprising: a drug-loaded Fe3O4 nanoparticle, and a red blood cell membrane composited on the surface of the drug-loaded Fe3O4 nanoparticle, wherein the red blood cell membrane is modified by galactose;

[0062] The drug encapsulated by the drug-loaded Fe3O4 nanoparticles is rheumatoid artemisia, and the mass of the rheumatoid artemisia accounts for 20-40% of the mass of the hollow Fe3O4 nanoparticles before drug loading;

[0063] The size of the hollow Fe3O4 nanoparticles is 180 to 220 nm in diameter;

[0064] The ratio of the number of galactose to the number of cells from the red blood cell membrane is 100 μg: (2.68-6.25)×10 9 mL -1 .

[0065] Optionally, the drug-loaded Fe3O4 nanoparticles have a diameter of 320 to 360 nm.

[0066] The method for preparing the bionic iron-based nanoparticles comprises the following steps:

[0067] S1, taking rheumatoid artemisia and hollow Fe3O4 nanoparticles in a mass ratio of (2-2.5):1, mixing the ethanol solution of rheumatoid artemisia and the ethanol dispersion of hollow Fe3O4 nanoparticles, stirring evenly at room temperature, centrifuging and discarding the supernatant, and then vacuum drying to obtain drug-loaded Fe3O4 nanoparticles;

[0068] S2, suspending the red blood cell membrane in a buffer, adding galactose in proportion, incubating for 25 to 35 minutes, and then extruding through a filter membrane to obtain a galactose-modified red blood cell membrane of a set size;

[0069] S3. The drug-loaded Fe3O4 nanoparticles obtained in S1 and the galactose-modified erythrocyte membranes obtained in S2 are mixed and coated to obtain biomimetic iron-based nanoparticles.

[0070] Optionally, the raw materials for preparing the hollow Fe3O4 nanoparticles in S1 include: a mass ratio of (1.0-1.5):

[0071] (3.0-3.5): (0.8-1.0): (0.1-0.3) of ferric chloride hexahydrate, sodium citrate, urea and polyacrylamide; the preparation method comprises: dispersing ferric chloride hexahydrate, sodium citrate and urea in distilled water according to a proportion, then adding polyacrylamide, stirring and dissolving, reacting at 200±20°C for 11-13h, cooling to room temperature, centrifuging to separate a black precipitate, and drying the black precipitate at 60±5°C overnight to obtain hollow Fe3O4 nanoparticles;

[0072] Optionally, in the method for preparing hollow Fe3O4 nanoparticles, after separating the black precipitate, washing it more than three times to remove unreacted substances;

[0073] Optionally, in the method for preparing hollow Fe3O4 nanoparticles, the drying method is vacuum drying.

[0074] Optionally, in S1, the mixing method is ultrasonic dispersion for 25 to 35 minutes.

[0075] Optionally, in S1, the vacuum drying temperature is 60±5°C.

[0076] Optionally, the method for preparing the red blood cell membrane in S2 includes: centrifuging whole blood at 3000-3500 rpm for 8-12 min at 4±2°C, discarding the supernatant, washing with PBS buffer, resuspending the obtained red blood cells in ultrapure water for 2-3 hours to induce red blood cell rupture, then centrifuging at 9000-9500 rpm for 8-12 min, taking the supernatant, washing with PBS buffer until colorless, and obtaining the red blood cell membrane.

[0077] Optionally, in S3, the thin film method comprises: mixing a PBS buffer solution containing drug-loaded Fe3O4 nanoparticles and galactose-modified erythrocyte membranes and extruding the mixture through a 200 nm filter membrane.

[0078] Application of the bionic iron-based nanoparticles and / or the method for preparing the bionic iron-based nanoparticles in at least one of the following a1) to a4):

[0079] a1) reprogramming M2 TAMs into M1 TAMs or preparing a product for reprogramming M2 TAMs into M1 TAMs;

[0080] a2) Inhibiting the proliferation of cancer cells or preparing products that inhibit the proliferation of cancer cells;

[0081] a3) Activating anti-tumor immune responses based on macrophages and T cells or preparing products that activate anti-tumor immune responses based on macrophages and T cells;

[0082] a4) Preparation of products for treating tumors.

[0083] Optionally, the cancer cells are breast cancer cells, specifically triple-negative breast cancer cells.

[0084] Optionally, the tumor is a breast cancer tumor, specifically a triple-negative breast cancer tumor.

[0085] Tumor-infiltrating immune cells often experience metabolic stress due to dysregulated metabolic activity of tumor cells, resulting in impaired antitumor immune responses. Abnormal bioenergetic activity of tumor cells utilizes large amounts of glucose to produce lactate through glycolysis; clinical data from a large number of patients show that serum LDH levels above 1000 international units (IU) / l indicate advanced or metastatic disease; glucose consumption and lactate accumulation interrupt the ability of macrophages to polarize pro-inflammatoryly, thereby promoting the growth of M2 TAMs. Tumor-bearing mice lacking lactate dehydrogenase (LDHA) have reduced numbers of splenic myeloid-derived suppressor cells (MDSCs) and increased cytotoxic function of tumor-infiltrating natural killer (NK) cells, indicating that lactate production promotes tumor growth and impairs innate immune responses; in mouse models, LDHA-mediated lactate production inhibits the expression of TFN-γ in tumor-infiltrating T cells and NK cells, thereby promoting tumor progression and immune escape. Therefore, drugs targeting tumor metabolism can synergistically enhance immunotherapy through metabolic reprogramming of the tumor microenvironment (TME).

[0086] Rhein is an anthraquinone derivative extracted from rhubarb roots, which has multiple biological functions, including anti-inflammatory, antioxidant, anti-cancer and neuroprotective. The present invention uses magnetic hollow Fe3O4 NPs as a carrier to deliver rhein into the tumor microenvironment to overcome the hydrophobicity of rhein. The introduction of rhein successfully inhibits the production of LDHA, resulting in a decrease in lactic acid levels and promoting the transformation of M2 TAMs to M1 TAMs. This reprogramming from immunosuppression to immunostimulation enhances the effectiveness of tumor immunotherapy.

[0087] The TME contains key components of TAMs, which are often associated with poor prognosis and drug resistance. Given the role of TAMs in promoting tumors, people have designed a variety of strategies to offset their effects; iron-based nanoparticles can become key transformation entities against TAMs, which can promote immune responses and safely and effectively sensitize tumors to immunotherapy. Therefore, the present invention uses a nanoplatform skeleton containing hollow iron oxide to synergistically induce polarization of the M1-TAM phenotype. Another notable feature of cancer cells is the continuous production of lactic acid through aerobic glycolysis, which promotes the polarization of TAMs to the M2 phenotype, exacerbating immunosuppression within the TME and causing hypoxia. Therefore, scientists believe that reducing the production of lactic acid may restore the immunosuppressive tumor microenvironment.

[0088] Example 1

[0089] Preparation and characterization of biomimetic iron-based nanoparticles.

[0090] 0.54 g of ferric chloride hexahydrate (also known as iron (III) chloride hexahydrate, CAS number: 10025-77-1), 0.865 g of sodium citrate and 0.36 g of urea were dispersed in 40 mL of distilled water; then 0.3 g of polyacrylamide was slowly added and completely dissolved into a solution by stirring, and then the solution was reacted at 200°C for 12 hours, and after cooling to room temperature, the solution was centrifuged to separate a black precipitate, and the black precipitate was washed three times to remove any unreacted substances, and then dried overnight under vacuum conditions at 60°C to obtain the final substance, namely hollow Fe3O4 nanoparticles (hollow Fe3O4 NPs).

[0091] like Figure 1 As shown in (A), 2 mg of rhein was added to 1 mL of anhydrous ethanol and ultrasonicated for 10 min to obtain a rhein ethanol solution; 1 mg of hollow Fe3O4 nanoparticles was added to 1 mL of anhydrous ethanol and ultrasonicated for 30 min to obtain an ethanol dispersion of hollow Fe3O4 nanoparticles; the two were mixed, stirred continuously at room temperature for 24 h, centrifuged and the supernatant was discarded to obtain drug-loaded Fe3O4 nanoparticles ( E Fe3O4 NPs) to calculate the loading efficiency of rhein; E The Fe3O4 NPs were centrifuged and dried under vacuum at 60 °C for later use.

[0092] like Figure 1 As shown in (A), whole blood was collected from healthy mice and centrifuged at 3000 rpm for 10 min at 4°C to remove plasma and buffer in the whole blood; then the whole blood was washed three times with cold phosphate buffered saline (PBS, pH 7.4) to obtain red blood cells (RBC); the obtained RBC was resuspended in 2 mL of ultrapure water for at least 2 h to induce red blood cell rupture; the resuspended liquid containing ruptured red blood cells was centrifuged at 9000 rpm for 10 min, the precipitate was discarded to remove the released hemoglobin, and then the supernatant was washed with 0.01 M PBS buffer until colorless to obtain red blood cell membrane (mRBC); 6.25×10 9 Each mRBC was suspended in 1 mL of PBS buffer (PBS buffer concentration was 0.01 M), and 100 μg of galactose was added to the 1 mL buffer and incubated for 30 min to form galactose-modified red blood cell membranes (gRBCs). The gRBCs were then squeezed out through a 200 nm filter membrane to prepare 200 nm gRBCs.

[0093] Use dry E The Fe3O4 NPs concentration was 500 μg mL -1 of E Fe3O4 NPs dispersion, take 1mL EThe Fe3O4 NPs dispersion (dispersion medium is PBS buffer) was mixed with 1 mL of gRBCs filtered through a 200 nm filter membrane, and then extruded through a 200 nm filter membrane to obtain biomimetic iron-based nanoparticles ( ER Fe3O4 NPs).

[0094] In this example, FeCl3, polyacrylamide, citrate and urea were treated by hydrothermal method to prepare hollow Fe3O4 NPs. The characterization method includes:

[0095] Scanning electron microscopy (SEM) images were obtained using a Zeiss Sigma300 scanning electron microscope. Figure 2 (A) shows that the surface of hollow Fe3O4 NPs is rough.

[0096] HT7800 performs transmission electron microscopy (TEM) imaging at 120kV accelerating voltage, such as Figure 2 (B) shows the monodisperse, spherical and hollow structure of Fe3O4 nanoparticles with uniform structure and a diameter of 206.5 ± 15.3 nm.

[0097] The crystal structure was measured using an Ultima IV X-ray diffractometer (XRD, Bruker D8 ADVANCE / UltimaIV, Germany) at 8° min -1 The measurement results of hollow Fe3O4 NPs were carried out using Cu Kα radiation (λ = 0.15405nm, 40KV, 40mA). Figure 2 As shown in (C), the diffraction patterns of these NPs are in good agreement with the crystal structure of Fe3O4 NPs (JCPDS 75-0033).

[0098] The STEM and elemental mapping images of hollow Fe3O4 NPs were obtained using (TALOS F200, FEI, America), such as Figure 2 As shown in (D), energy dispersive spectroscopy (EDS) elemental mapping analysis shows that the distribution of Fe and O elements is uniform.

[0099] X-ray photoelectron spectroscopy (XPS) analysis of hollow Fe3O4 NPs was performed using a Thermo Scientific K-alpha X-ray photoelectron spectrometer (hv = 1486.6 eV). Figure 2 As shown in (E), the peaks corresponding to Fe2p3 / 2 and Fe2p1 / 2 in the XPS spectrum are at binding energies of approximately 710.6 eV and 724.6 eV, respectively, which is consistent with Fe3O4 reported in the literature.

[0100] The hollow Fe3O4 NPs were subjected to N2 adsorption and desorption tests. The N2 adsorption / desorption isotherms of the hollow Fe3O4 NPs are shown in Figure 2 (F) shows that the specific surface area and average pore size of hollow Fe3O4 NPs are 229.2 m 2 / g and 16.4nm, confirming that hollow Fe3O4NPs are beneficial for encapsulating tumor therapeutic drugs.

[0101] When calculating the uploading efficiency of rheum officinale, a standard curve was established by high performance liquid chromatography (HPLC) to evaluate the uploading efficiency of rheum officinale, such as Figure 2 As shown in (G), hollow Fe3O4 NPs can efficiently load rhein. Figure 2 As determined in (G), the loading ratio ([rhein] / [Fe3O4], w / w) in this example is approximately 30.0%.

[0102] Analysis of gRBCs by SDS-PAGE R Fe3O4 NPs and Marker protein, the samples were stained with Coomassie Brilliant Blue, as Figure 2 As shown in (H), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to confirm membrane proteins. The results showed that after encapsulation of galactose-modified red blood cells, the average hydrodynamic diameter of Fe3O4 NPs increased from 205.7±3.3nm to ER The average hydrodynamic diameter of Fe3O4 NPs was 341.2±5.7nm, indicating successful encapsulation of the erythrocyte membrane ( Figure 2 (I)).

[0103] Nano-zs zetasizer (Nanolink SZ901) was used to measure the size and zeta potential of nanoparticles. Figure 2 As shown in (J), the Zeta potential of Fe3O4 NPs increased significantly from -33.0 mV to ER The Zeta potential of Fe3O4 NPs was −20.4 mV, close to the value of galactose-red blood cell membrane vesicles (gRBCs) (−8.5 mV), which again confirmed the formation of surface biomimetic properties.

[0104] The above results verify that ER The successful formation of Fe3O4 NPs indicates ER Fe3O4 NPs preserved a large amount of galactose-modified erythrocyte membranes for improving the biocompatibility of these nanoparticles and the biotargeting of M2 TAMs.

[0105] In the prior art, inhibition of lactate and iron exposure has been shown to reprogram M2 TAMs to M1 TAMs. Under physiological conditions, the concentration of lactate in blood and healthy tissues is 1.5-3.0 mmol L -1 However, the lactic acid content in TME can reach 10-40 mmol L -1 , tumor cells are rapidly proliferating and are affected by the active metabolism of glucose and glutamine. Figure 3 As shown in (A), under the promotion of LDH activity, pyruvate is enzymatically converted to lactate, maintaining the NADH / NAD+ ratio and the cytoplasmic level of nicotinamide adenine dinucleotide (NAD+) to maintain glycolytic flux.

[0106] Evaluation of lactate dehydrogenase (LDH) inhibition.

[0107] The experiment used 40 μmol L -1 of reduced coenzyme I (NADH) and 200 μmol L -1 Pyruvate, add 0.66 ng or 0.38 mU of LDH enzyme (1 unit of enzyme converts 1 μmol L at 37°C and pH 8.55) to a final volume of 200 μL. -1 Pyruvate is reduced to L-lactate); to generate concentration-response curves, five different concentrations of ER Fe3O4 NPs dispersions, including 0, 25, 50, 100 and 200 μg mL -1 , five different concentrations ER The Fe3O4 NPs dispersion was added to 96-well plates, 8 μL per well; subsequently, substrate and cofactors were added, dissolved in buffer (152 μL), and LDH enzyme solution (40 μL); the measurement process complied with the guidelines provided by the manufacturer of the pyruvate kit.

[0108] In five different concentrations ER In addition to Fe3O4 NPs, zero concentration was added for comparison. The results are shown in Figure 3 As shown in (B), ER As the concentration of Fe3O4 NPs increased, the accumulation of converted pyruvate gradually increased, which further indicated that ER Fe3O4 NPs can inhibit the activity of lactate dehydrogenase (LDH) and thus inhibit the production of lactic acid.

[0109] ER Fe3O4 NPs have the ability to promote the reprogramming of M2 TAMs to M1 TAMs.

[0110] RAW 264.7 cells (mouse mononuclear macrophage leukemia cells) and 4T1 cells (mouse breast cancer cells, specifically triple-negative breast cancer cells) were incubated in 1640 medium (RPMI-1640 medium) containing fetal bovine serum (10%) and penicillin and streptomycin bispecific antibody (1%) (conditions were 37°C, 5% CO2 content) to obtain M0 macrophages, and then incubated in 20ng mL -1 The cells were incubated in 1640 medium containing IL-4 (interleukin-4) for 12 h to repolarize them into M2 TAMs.

[0111] Iron uptake test:

[0112] The induced M2 TAMs were divided into 1.6×10 5 The cells were inoculated into 6-well plates at a density of 10 cells / well (1.6 mL). The next day, the original cell culture medium was replaced with fresh culture medium containing 0, 25, 50, and 100 μg mL -1 ER Fe3O4NPs (Fe3O4NPs concentration). Incubate for another 6 hours, then rinse with PBS 1-2 times, resuspend with PBS, and then centrifuge; finally, add 500μL of aqua regia to dissolve the cells, and after the volume is fixed to 15mL, the iron ion concentration is detected by inductively coupled plasma mass spectrometry (ICP-MS). The results are as follows Figure 3 (C) As shown: ER As the concentration of Fe3O4 NPs increased, the iron level in the cells increased in a dose-dependent manner, indicating significant iron uptake in M2 TAMs.

[0113] Through MTT assessment ER Toxicity of Fe3O4 NPs:

[0114] M2 TAMs cells were incubated with different concentrations of R Fe3O4 NPs and ER The viability of M2TAMs cells was evaluated after incubation with Fe3O4 NPs for 24 h; the results are shown in Figure 3 (D) shows that different concentrations of R Fe3O4 NPs and ER Fe3O4 NPs showed no obvious toxicity after incubation for 24 h.

[0115] CD16 / 32 and CD206 expression:

[0116] F4 / 80+ macrophages were cultured at 1×10 5 Cells were seeded in 24-well plates and R Fe3O4 NPs and ERFe3O4NPs (equivalent to 100μg mL -1 After incubation with 5% Fe3O4 for 24 h, the cells were washed twice with PBS and collected. Subsequently, the cells were redispersed in 500 μL of PBS containing PE / Cy7 anti-mouse CD16 / 32 antibody or PE anti-mouse CD206 antibody and stained for 15 min. The expression of M1 phenotype-specific marker CD16 / 32 (PE / Cy7) and M2 phenotype-specific marker CD206 (PE) were analyzed by flow cytometry. The results are shown in Table 2. Figure 3 (E) Compared with the PBS group, ER Fe3O4 NPs and ER The Fe3O4 NPs group triggered an increase in CD16 / 32 expression, indicating an increase in the induction of M1 TAMs; Figure 3 As shown in (F), R Fe3O4 NPs and ER The Fe3O4 NPs group significantly reduced the expression of CD206, which may be Figure 3 (F) M2 TAMs induced by reprogramming M2TAMs into M1TAMs can trigger the release of pro-inflammatory cytokines such as TNF-α, which was further detected by ELISA.

[0117] Assessment of TNF-α:

[0118] 1×10 per well 4 M2 TAMs (100 μL) were added overnight in a 96-well plate. 0, 25, 50, and 100 μg mL -1 of ER Fe3O4 NPs or R Fe3O4 NPs or 0, 7, 15, and 30 μg mL -1 Rheum officinale (this is due to: ER Fe3O4 NPs or R In Fe3O4 NPs, the loading ratio of rheum officinale was 30w%, and the corresponding concentrations of rheum officinale were 0, 7, 15 and 30μg mL -1 ), which is indicated on the second horizontal axis in the figure (PBS mixture, supernatant was collected after 12 hours, and TNF-α level was detected by enzyme-linked immunosorbent assay (ELISA) (Biogend); the results are shown in Figure 3 (G) The concentrations of Emodin from left to right are 0, 7, 15 and 30 μg mL -1 , in order to compare the effects of the same concentration of emodin components), compared with the mixture of PBS and the corresponding concentration of emodin, R Fe3O4 NPs andER The Fe3O4 NPs group could significantly release TNF-α inflammatory factor, especially ER Fe3O4 NPs group, which was related to the synergistic release of rhamnosin and Fe3O4 NPs.

[0119] These results confirm that ER Fe3O4 NPs have the strongest M2 TAMs repolarization activity, laying the foundation for anti-tumor immunotherapy.

[0120] ER In vitro antitumor effect of Fe3O4 NPs

[0121] It is reported that pro-inflammatory M1 TAMs exert anti-tumor activity by secreting pro-inflammatory cytokines and reactive nitrogen and oxygen species. To further confirm the anti-tumor potential of M2 TAMs converted into M1 TAMs, we used the 4T1-M2 TAMs transwell system for verification.

[0122] like Figure 4 (A) 1×10 5 M2 TAMs, meanwhile, will have 2.5×10 4 4T1 cells were placed in the lower chamber and cultured overnight. The next day, emodin, R Fe3O4 NPs and ER Fe3O4 NPs (equivalent to 100 μg mL -1 Fe3O4 or 15μg mL -1 The cells were cultured with emodin for 24 h, and the cell viability was determined by MTT method.

[0123] Assessment of 4T1 cell survival in the lower chamber:

[0124] 1 × 10 cells were seeded in 100 μL per well. 4 TAMs or 3T3 cells (derived from mouse embryonic fibroblasts) were incubated in 96-well plates overnight; the original culture medium was discarded and washed 1-2 times with PBS, and then 0-200 μg mL -1 of R Fe3O4NPs and ER Fe3O4 NPs were treated. Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and cultured for 3.5 hours, and then detected by microplate reader; the results are shown in Figure 4 (B) As shown, Fe3O4 induces M1 polarization and the release of inflammatory factors. RFe3O4 NPs triggered 35.0% of 4T1 cell death, which was due to Fe3O4-induced M1 polarization and the release of inflammatory factors. ER Fe3O4 NPs could trigger 54.7% cell death, which was related to the synergistic enhancement of M1 polarization by rhamnosin; on the other hand, Figure 4 (C) R Fe3O4 NPs and ER Fe3O4 NPs in the range of 0 to 200 μg mL -1 After incubation at 40% RT-PCR for 24 h, the cell survival rate of 3T3 cells was above 90%, indicating that ER Fe3O4 NPs are safe in vitro.

[0125] Apoptosis kit to detect cell apoptosis:

[0126] To detect cell apoptosis, 4T1 cells in the lower chamber of the device were collected using trypsin without ethylenediaminetetraacetic acid (EDTA) after the experiment, washed with PBS, suspended in binding buffer, stained with an apoptosis kit for 30 minutes, and then analyzed by flow cytometry. Under a fluorescence microscope, cells were cultured with calcein AM / PI experimental working solution, incubated in the dark at 37°C for 30 minutes, and the staining results were observed under a fluorescence microscope. Figure 4 As shown in (D), it shows that Fe3O4 induces M1 polarization and the release of inflammatory factors, and under the synergistic effect of rhamnosus, the effect of M1 polarization can be significantly enhanced, resulting in the most significant apoptosis of 4T1 cells.

[0127] Cell live / dead double staining method to detect 4T1 cell apoptosis:

[0128] The cell live / dead double staining method was used to further verify the killing effect of different reagents in the MTT method on 4T1 cells. Figure 4 (E) shows the result Figure 4 The trend of MTT assay results in (D) is similar: Figure 4 In (E), the results of fluorescein-annexin V and propidium iodide (PI) staining analysis showed that rhamnosin, R Fe3O4 NPs or ER The effect of Fe3O4 NPs on cell viability is mainly related to apoptosis. (AnnexinV-FITC)- / PI+ represents necrotic cells, (AnnexinV+FITC)+ / PI+ represents late apoptotic cells, (AnnexinV-FITC)+ / PI- represents living cells, and (AnnexinV-FITC)- / PI- represents early apoptotic cells. R Fe3O4 NPs and ERThe proportions of apoptotic cells treated with Fe3O4 NPs were 14.8%, 27.7% and 42.4%, respectively, indicating that the synergistic M2 TAMs repolarization activity of emodin and Fe3O4 NPs could induce significant 4T1 cell apoptosis.

[0129] In summary, R Fe3O4 NPs or ER Fe3O4 NPs have good anti-tumor effects and low toxicity to normal cells.

[0130] Nanomedicines can be further used in cancer treatment only if they are less toxic to humans. ER The cytotoxic effect of Fe3O4 NPs on 4T1 cells in vitro prompted the inventors to evaluate their antitumor immunotherapeutic potential in vivo: their toxicity was evaluated by hemolysis assay, hematological analysis, and H&E staining of heart, liver, spleen, lung, and kidney tissues.

[0131] Hemolysis test:

[0132] Mouse erythrocytes were washed with PBS, serum was removed, and then suspended in PBS (20 mL). ER Fe3O4 NPs were prepared at concentrations equivalent to 0, 25, 50, 100, 200, 400, and 800 μg mL -1 Fe3O4 NPs. The positive control was a red blood cell suspension diluted with deionized water, and the negative control was a red blood cell suspension diluted with PBS. According to the above procedure, the hemolysis rate after incubation at 37 °C for 3 h was evaluated and calculated, as Figure 5 (A) shows that different concentrations of ER The hemolysis rate of Fe3O4 NPs was below 5%, indicating that the injection ER Fe3O4 NPs are safe.

[0133] ER In vivo therapeutic effects of Fe3O4 NPs

[0134] 4T1 tumor-bearing mouse model experiment:

[0135] BALB / c mice (15-20 g, 6 weeks old) were subcutaneously inoculated with 2×10 6 100 μL of PBS for 4T1 cells. When the tumor volume reaches 150-200 mm 3 The Cy5-loaded R Fe3O4 NPs 100μL (equivalent to 0.5mg Cy5 kg -1Mice), and after 24 hours, the heart, liver, spleen, lung, kidney and tumor were imaged with fluorescence. Then, the main organs and tumors were taken, the acid was thawed with 10 mL of concentrated nitric acid, diluted with ultrapure water, and the ion concentration was determined by ICP-MS; the results are shown in Figure 5 (B) shows that Cy5-loaded ER Fe3O4 NPs accumulate significantly in tumor tissues; the results of ICP-MS determination of ion concentrations are shown in Figure 5 As shown in (C), it can be seen that 24 hours after injection, ER Fe3O4 NPs were mainly distributed in the spleen and lungs and showed obvious accumulation behavior in tumors.

[0136] BALB / c mice (15-20 g, 6 weeks old) were subcutaneously inoculated with 2×10 6 100 μL of PBS for 4T1 cells, when the tumor volume reaches about 70 mm 3 When Figure 5 As shown in (D), the mice were randomly divided into four groups of equal number. On the 1st, 3rd and 5th days, the groups were intravenously injected with the following drugs: (a) PBS; (b) emodin; (c) R Fe3O4 NPs; (d) ER Fe3O4 NPs; intravenous dose in mice is equivalent to 20 mg kg Fe3O4 NPs -1 or rheum officinale 6mg kg -1 .

[0137] The weight of mice during treatment was continuously recorded. Figure 5 (F) As shown, intravenous injection ER Fe3O4NPs had no significant effect on the body weight of mice during treatment. The tumor volume of mice was continuously counted during treatment, and the formula (width 2 × length / 2) to assess tumor volume, such as Figure 5 (E) As shown, injection ER The tumor volume of mice in the group treated with Fe3O4 NPs increased the slowest; the tumor status of mice on day 18 was as follows: Fig.10 As shown, the status of the removed tumor is as follows Fig.11 As shown, Figure 5 The tumor growth trend shown is consistent.

[0138] On the 8th day, tumor tissues of a portion of mice in each group were collected, rinsed with cold PBS, and then incubated in a solution containing collagenase IV and DNase I. Digestion was performed at 37°C for 45 minutes, terminated on ice, and the digested tissues were filtered through a 70 μm nylon filter to obtain a single cell suspension. Before cell flow cytometry antibody staining, tumor cells were treated with PE / Cy7 anti-mouse CD16 / 32 and PE anti-mouse CD206 to analyze F4 / 80 in tumor tissues. + The expression levels of CD16 / 32 and CD206 in macrophages; lymph nodes were stained with PE anti-mouse CD4 antibody and APC anti-mouse CD8 antibody.

[0139] (1) Flow cytometric analysis of CD16 / 32 and CD206 in F4 / 80 + The expression level in macrophages can be used as a reliable marker of macrophages in tumors; the results showed that R Compared with Fe3O4 NPs, ER Fe3O4 NPs induced an increase in the expression of CD16 / 32 and a decrease in the expression of CD206, e.g. Figure 5 As shown in (I), the expression of CD16 / 32 after different treatments was 22.4%, 27.8%, 30.0% and 54.8%, respectively; Figure 5 As shown in (J), the expression levels of CD206 after different treatments were 16.9%, 13.2%, 5.9% and 3.5%, respectively, indicating that ER Fe3O4 NPs have excellent TME reprogramming ability in vivo and can convert M2 TAMs into M1 TAMs.

[0140] (2) Flow cytometry detection of CD4 + or CD8 + The proportion of T lymphocytes, such as Fig.12 As shown, a large number of CD4 + and CD8 + T cells. PBS, emodin, R Fe3O4 NPs, ER Fe3O4 NPs group CD8 + The infiltration rates of T lymphocytes were 10.3%, 11.2%, 18.0%, and 23.9%, respectively. + The infiltration rates of T lymphocytes were 24.5%, 37.0%, 44.1%, and 52.3%, respectively; these results confirmed that ER Fe3O4 NPs treatment can activate anti-tumor immune responses based on macrophages and T cells. Therefore, activation of the immune stimulatory microenvironment can secrete more inflammatory factors and promote CD8 + and CD4+ T cells infiltrate the tumor site, thereby enhancing the effect of immunotherapy on tumors.

[0141] On the 18th day, the mice were euthanized, and the major organs (heart, liver, spleen, lung, and kidney) and tumors were removed. The samples were cut into 5 μm sections, stained with H&E, and pathologically analyzed using a digital microscope: After the treatment, the pathological changes of the tumor sections were as follows: Figure 5 As shown in (G), ER Fe3O4 NPs showed good therapeutic effects when tumor cell apoptosis and necrosis were the most serious. Immunofluorescence staining was performed on tumor tissue sections: FITC anti-mouse CD80 antibody and PE anti-mouse CD86 antibody were used for treatment, followed by DAPI staining. The results are shown in Figure 2. Figure 5 H: ER Under Fe3O4 NPs treatment, the fluorescence intensity of CD80 and CD86 was significantly higher than that in other treatment groups.

[0142] H&E staining results of major organs are as follows Fig. 9 As shown in the figure, H&E staining of major organs in each group showed no obvious toxicity after intravenous injection. These data further support ER Fe3O4 NPs have good biocompatibility in vivo.

[0143] After treatment, the mice were observed and the aspartate aminotransferase / alanine aminotransferase (AST / ALT) indexes in the blood of the mice were measured on the 18th day. Figure 6 As shown; the urea / creatinine (UREA / CREA) index in the mouse blood was detected, and the results were as follows Figure 7 In addition, the following indicators were detected in the blood of mice: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC), red blood cells (RBC) and hematocrit (HCT). The results are shown in Figure 8 As shown; the above results show that the blood biochemistry and hematology analysis results of each group of mice are within the reference range, the increase or decrease of each index is not large, and it has good biocompatibility.

[0144] From the above examples, it can be seen that this patent synthesizes an iron-based nano-platform ( ER Fe3O4 NPs), where the natural product is loaded with galactose-modified erythrocyte membrane capes of hollow Fe3O4 NPs. Figure 1 As shown in (B), it can not only efficiently assist the delivery of emodin to inhibit and neutralize tumor acidification, but also synergistically convert M2 TAMs into M1 TAMs, improving tumor metabolic immunotherapy.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A biomimetic iron-based nanoparticle, It is characterized in that include: Drug-loaded Fe 3 O 4 Nanoparticles, and the composite drug-loaded Fe 3 O 4 an erythrocyte membrane on the surface of the nanoparticle, wherein the erythrocyte membrane is modified by galactose; The drug-loaded Fe 3 O 4 The drug encapsulated in the nanoparticles is rhamnosine, and the mass of rhamnosine accounts for 1.3% of the hollow Fe 3 O 4 20-40% of the mass of the nanoparticles; The hollow Fe 3 O 4 The size of the nanoparticles is 180 to 220 nm in diameter; The ratio of the number of galactose to the number of erythrocyte membrane cells is 100 μg: (2.68-6.25)×10 9 indivual.

2. The biomimetic iron-based nanoparticles according to claim 1, It is characterized in that The mass of rhamnosine accounts for the hollow Fe 3 O 4 30% of the mass of the nanoparticles; Optionally, the hollow Fe 3 O 4 The size of the nanoparticles was 206.5 ± 15.3 nm in diameter; Optionally, the size of the biomimetic iron-based nanoparticles is optional, and the drug-loaded Fe 3 O 4 The size of the nanoparticles is 320-360 nm in diameter, preferably 341.2±5.7 nm in diameter.

3. A method for preparing the biomimetic iron-based nanoparticles according to any one of claims 1 to 2, It is characterized in that The following steps are involved: S1, take the mass ratio of (2-2.5):1 of rhubarb and hollow Fe 3 O 4 Nanoparticles, ethanol solution of rhein and hollow Fe 3 O 4 The ethanol dispersion of the nanoparticles was mixed, stirred at room temperature for 24 h, centrifuged and the supernatant was discarded, and then vacuum dried to obtain the drug-loaded Fe 3 O 4 Nanoparticles; S2, suspending the red blood cell membrane in a buffer, adding galactose in proportion, incubating for 25 to 35 minutes, and then extruding through a filter membrane to obtain a galactose-modified red blood cell membrane of a set size; S3, the drug-loaded Fe obtained in S1 3 O 4 The nanoparticles are mixed with the galactose-modified erythrocyte membrane obtained in S2 and coated to obtain biomimetic iron-based nanoparticles.

4. The method for preparing the biomimetic iron-based nanoparticles according to claim 3, It is characterized in that Hollow Fe in S1 3 O 4 The raw materials for preparing the nanoparticles include: ferric chloride hexahydrate, sodium citrate, urea and polyacrylamide in a mass ratio of (1.0-1.5): (3.0-3.5): (0.8-1.0): (0.5-0.9); the preparation method includes: dispersing ferric chloride hexahydrate, sodium citrate and urea in distilled water according to a proportion, then adding polyacrylamide, stirring and dissolving, reacting at 200±20°C for 11-13h, cooling to room temperature, centrifuging to separate a black precipitate, and drying the black precipitate at 60±5°C overnight to obtain a hollow Fe 3 O 4 Nanoparticles.

5. The method for preparing the biomimetic iron-based nanoparticles according to claim 4, It is characterized in that Hollow Fe 3 O 4 In the method for preparing the nanoparticles, after the black precipitate is separated, it is washed more than three times to remove unreacted substances; Optional, hollow Fe 3 O 4 In the method for preparing nanoparticles, the drying method is vacuum drying.

6. The method for preparing the biomimetic iron-based nanoparticles according to claim 3, It is characterized in that In S1, the mixing method was ultrasonic dispersion for 25 to 35 min; Optionally, in S1, the vacuum drying temperature is 60±5°C.

7. The method for preparing the biomimetic iron-based nanoparticles according to claim 3, It is characterized in that The preparation method of the red blood cell membrane in S2 includes: centrifuging whole blood at 3000-3500 rpm for 8-12 minutes at 4±2°C, discarding the supernatant, washing with PBS buffer, resuspending the obtained red blood cells in ultrapure water for 2-3 hours to induce red blood cell rupture, then centrifuging at 9000-9500 rpm for 8-12 minutes, taking the supernatant, washing with PBS buffer until colorless, and obtaining the red blood cell membrane.

8. The method for preparing the biomimetic iron-based nanoparticles according to claim 3, It is characterized in that In S3, the thin film method comprises: 3 O 4 The nanoparticles were mixed with galactose-modified erythrocyte membrane in PBS buffer and extruded through a 200 nm filter membrane.

9. Use of the bionic iron-based nanoparticles according to any one of claims 1 to 2 and / or the method for preparing the bionic iron-based nanoparticles according to any one of claims 3 to 8 in at least one of the following a1) to a4): a1) reprogramming M2 TAMs into M1 TAMs or preparing a product for reprogramming M2 TAMs into M1 TAMs; a2) Inhibiting the proliferation of cancer cells or preparing products that inhibit the proliferation of cancer cells; a3) Activating anti-tumor immune responses based on macrophages and T cells or preparing products that activate anti-tumor immune responses based on macrophages and T cells; a4) Preparation of products for treating tumors.

10. The use according to claim 9, It is characterized in that The cancer cells are breast cancer cells; Optionally, the tumor is a breast cancer tumor.

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