Biomimetic iron-based nanoparticles, method of preparation and use

Abstract

The present application relates to a kind of bionic iron-based nanoparticles and preparation method and application, belong to cell membrane camouflage nanoparticle technical field.Bionic iron-based nanoparticles include: drug-loaded Fe3O4 Nanoparticle, and red blood cell membrane is compounded on the surface of drug-loaded Fe3O4 Nanoparticle, and red blood cell membrane is modified by galactose.Preparation method includes the following steps: S1, the ethanol solution of emodin is mixed with the ethanol dispersion liquid of hollow Fe3O4 Nanoparticle, centrifugal is discarded supernatant, and then vacuum drying;S2, red blood cell membrane is suspended in buffer, and incubation is added galactose;S3, drug-loaded Fe3O4 Nanoparticle and galactose-modified red blood cell membrane are mixed, and envelope is obtained bionic iron-based nanoparticles.The red blood cell membrane wrapped nanoparticle used in the present application can reduce the clearance of immune system to nanoparticle;Galactose-modified red blood cell membrane can effectively transport nanoparticle into M2TAMs, and emodin is released in M2TAMs, and cooperates with hollow Fe3O4 Nanoparticle to reprogram M2TAMs.

Description

Technical Field

[0001] This invention belongs to the field of cell membrane camouflage nanoparticle technology, specifically relating to a biomimetic iron-based nanoparticle, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cancer immunotherapy activates the host's immune system to fight cancer safely and effectively. 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 low number of patients responding to cancer immunotherapy (≈20%). Tumor-associated macrophages (TAMs) are typically 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-type tumor-associated macrophages (M2 TAMs), generated by the functional polarization of TAMs, are ubiquitous in the tumor microenvironment and play a crucial role in supporting tumor progression and metastasis. They participate in various processes such as providing nutritional support, promoting immune escape, promoting angiogenesis, and influencing tissue remodeling. Reprogramming M2-type tumor-associated macrophages (M2 TAMs) into M1-type tumor-associated macrophages (M1 TAMs) can increase the levels of interleukin-12 (IL-12), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) on 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 tumor microenvironments (TAMs) may also play a crucial role in macrophage polarization. For example, the iron supplement Ferumoxytol can induce a pro-inflammatory immune response through the polarization of M1 macrophages. Hollow iron oxide (Fe3O4) nanoparticles have been reported to promote the conversion of the M2 phenotype to the M1 phenotype in TAMs, leading to increased expression of Th1 cytokines such as IL-12, IFN-γ, and TNF-α, and reactivating the Th1 immune response. 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 hijack the function of tumor-infiltrating immune cells using their own metabolic byproducts, thus benefiting cancer cell invasion and metastasis. For example, during cancer cell glycolysis, ingested glucose undergoes a series of transformations, preferentially breaking it down into pyruvate, which is then catalyzed by lactate dehydrogenase (LDH) to produce lactate. The lactate secreted by cancer cell glycolysis accumulates and can be precisely sensed by tumor-associated macrophages (TAMs), leading to the functional polarization of TAMs and the production of M2 TAMs, which in turn contributes to tumor progression and metastasis. Emodin, a natural product rich in anthraquinones, has significant LDH inhibitory activity, capable of inhibiting and neutralizing extracellular acidification processes in tumors, thereby inhibiting the functional polarization of TAMs towards M2 TAMs. However, the limited solubility of emodin 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 towards the M1 type. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a biomimetic iron-based nanoparticle, its preparation method, and its application. A hollow Fe3O4 nanoplatform wrapped in a galactose-modified erythrocyte membrane is designed, with emodin loaded inside, to reverse the tumor immunosuppressive microenvironment and promote the effectiveness of anti-tumor immunotherapy.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] In a first aspect, a biomimetic iron-based nanoparticle includes: drug-loaded Fe3O4 nanoparticles and a red blood cell membrane composited on the surface of the drug-loaded Fe3O4 nanoparticles, the red blood cell membrane being modified with galactose.

[0010] The drug loaded in the Fe3O4 nanoparticles is emodin, and the mass of emodin accounts for 20-40% of the mass of the hollow Fe3O4 nanoparticles before drug loading.

[0011] The hollow Fe3O4 nanoparticles have a diameter of 180–220 nm.

[0012] The ratio of galactose to erythrocyte membrane cell origin is 100 μg: (2.68–6.25) × 10⁻⁶. 9 indivual.

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

[0014] Secondly, the preparation method of the above-mentioned biomimetic iron-based nanoparticles includes the following steps:

[0015] S1. Take emodin and hollow Fe3O4 nanoparticles with a mass ratio of (2-2.5):1. Mix the ethanol solution of emodin with the ethanol dispersion of hollow Fe3O4 nanoparticles, stir evenly at room temperature, centrifuge and discard the supernatant, and then vacuum dry to obtain drug-loaded Fe3O4 nanoparticles.

[0016] S2. Suspend the red blood cell membrane in buffer solution, add galactose in proportion, incubate for 25-35 min, and then squeeze it out 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 membrane 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 those with a mass ratio of (1.0–1.5):

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

[0020] Optionally, in the preparation method of hollow Fe3O4 nanoparticles, after separating the black precipitate, it is washed more than 3 times to remove unreacted substances.

[0021] Optionally, in the preparation method of hollow Fe3O4 nanoparticles, the drying method is vacuum drying.

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

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

[0024] Optionally, the preparation method of the red blood cell membrane in S2 includes: centrifuging whole blood at 3000-3500 rpm for 8-12 min at 4±2℃, discarding the supernatant, washing with PBS buffer, resuspending the obtained red blood cells in ultrapure water for 2-3 h to induce red blood cell lysis, 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 includes: mixing PBS buffer containing drug-loaded Fe3O4 nanoparticles and galactose-modified erythrocyte membranes and extruding through a 200–250 nm filter membrane.

[0026] Thirdly, the above-mentioned biomimetic iron-based nanoparticles and / or the above-mentioned method for preparing biomimetic iron-based nanoparticles are used in at least one of the following a1)-a4):

[0027] a1) Reprogramming M2 TAMs into M1 TAMs or preparing products that reprogram M2 TAMs into M1 TAMs;

[0028] a2) Inhibit cancer cell proliferation or prepare products that inhibit cancer cell proliferation;

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

[0030] a4) Prepare 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 this invention are as follows:

[0034] 1. This invention can be used to remodel the tumor immune microenvironment. The erythrocyte membrane-encapsulated nanoparticles can "compensate" for the biological interface between the nanoparticles and erythrocytes, thereby reducing the clearance of nanoparticles by the immune system. Furthermore, due to the high expression of galactolectin in M2 TAMs, the galactose-modified erythrocyte membrane can effectively transport the nanoparticles to tumor-associated macrophages. The internally encapsulated emodin is released from within the M2 TAMs, significantly inhibiting the conversion of pyruvate to lactate, further inhibiting and neutralizing tumor acidification, reprogramming tumor-associated macrophages, and thus polarizing the M2 TAMs. Hollow Fe3O4 nanoparticles further convert M2 TAMs into M1 TAMs.

[0035] 2. This 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, and activating and recruiting CD8+. + and CD4 + T cells attack tumor cells, thereby improving the efficacy of tumor immunotherapy. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1 These are schematic diagrams of specific implementation methods. (A) represents the flowchart of the preparation method of biomimetic iron-based nanoparticles in Example 1, and (B) represents the schematic diagram of the principle of improving tumor metabolic immunotherapy.

[0038] Figure 2 The figures shown are the characterization results from Example 1. (A) shows the SEM image of hollow Fe3O4 nanoparticles, (B) shows the TEM image of hollow Fe3O4 nanoparticles, (C) shows the XRD pattern of hollow Fe3O4 nanoparticles, (D) shows the STEM and elemental mapping of hollow Fe3O4 nanoparticles, (E) shows the XPS spectrum of the Fe2p region of hollow Fe3O4 nanoparticles, (F) shows the N2 adsorption / desorption isotherm of hollow Fe3O4 nanoparticles, (G) shows the standard curve for establishing the high performance liquid chromatography method to evaluate the loading rate of emodin, (H) shows the SDS-PAGE analysis results, (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.

[0039] Figure 3 The diagrams shown in Example 1 illustrate the reprogramming capability. (A) is a schematic diagram of the reprogramming capability principle, and (B) shows the measurement of different doses. ER The graph shows the pyruvate content under the action of Fe3O4 NPs. (C) is a statistical graph of iron ion detection results in cells by ICP-MS, where asterisks indicate statistical significance compared with the control group: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (D) is a graph of M2 TAMs cell viability assessment. (E) is a statistical graph of CD16 / 32 expression level. (F) is a statistical graph of CD206 expression level. (G) is a statistical graph of TNF-α expression level after M2 TAMs were treated with different reagents, where asterisks indicate statistically significant differences in concentration compared with the control group, analyzed by one-way ANOVA and Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0040] Figure 4The diagrams shown are related to the in vitro antitumor evaluation in Example 1. (A) is a schematic diagram of the experimental setup. (B) is a diagram of 4T1 cell viability assessment. Statistical significance compared with the control group is indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001). (C) is a diagram of 3T3 cell viability assessment. (D) is a diagram of live and dead cell staining of 4T1 cell populations treated with different reagents. (E) is a diagram of flow cytometry analysis of 4T1 cell populations treated with different reagents.

[0041] Figure 5 This is a schematic diagram related to the in vivo antitumor evaluation in Example 1. (A) is a photograph of the hemolysis test sample and a quantitative statistical chart of the hemolysis rate (HR%). (B) is a fluorescence imaging image of the main organs and tumor. (C) is a graph of iron concentration measured by ICP-MS in the main organs and tumor. (D) is a timeline diagram of the tumor-bearing mouse model experiment. (E) is a tumor growth curve. (F) is a graph of mouse weight change. (G) is a H&E staining image of the tumor tissue. (H) is an immunofluorescence staining image of the tumor tissue. (I) is F4 / 80. + Expression level of CD16 / 32 in macrophages, (J) is F4 / 80 + Graph of CD206 expression levels in macrophages; in (A), (E) and (F), asterisks indicate statistical significance compared to the PBS group, determined by one-way ANOVA of Tukey post-hoc analysis, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0042] Figure 6 The images shown are the AST / ALT index detection results in Example 1. (A) is the AST index result image, and (B) is the ALT index result image.

[0043] Figure 7 The following are the results of the UREA / CREA index detection in Example 1: (A) is the result of the UREA index, and (B) is the result of the CREA index.

[0044] Figure 8 The following are the blood test results in Example 1: (A) is the MCV index result, (B) is the WBC index result, (C) is the MCHC index result, (D) is the HCT index result, (E) is the RBC index result, (F) is the MCH index result, (G) is the PLT index result, and (H) is the HGB index result.

[0045] Figure 9 This is a diagram showing the H&E staining results of the major organs in Example 1.

[0046] Figure 10 This is a photograph of the tumor-bearing mouse in Example 1.

[0047] Figure 11 This is a photograph of the state of the tumor in the mouse in Example 1.

[0048] Figure 12 It is CD4 in Example 1 + and CD8 + A graph showing the proportion of T lymphocytes. Detailed Implementation

[0049] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0051] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions. All raw materials and reagents used in the following examples are commercially available unless otherwise indicated.

[0052] The main materials and reagents used in this invention are listed below:

[0053] Sodium citrate, ferric chloride hexahydrate, polyacrylamide, and urea were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0054] 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 CD16 / 32 antibody, PE anti-mouse CD206 antibody and mouse TNF-α ELISA kit, from Biolegend (San Diego, CA, USA).

[0055] Apoptosis detection kit, from Solaibao (China).

[0056] The culture dishes used for cell culture were from Cynetbio (China).

[0057] All chemicals are analytical grade and require no further purification.

[0058] A biomimetic iron-based nanoparticle includes: drug-loaded Fe3O4 nanoparticles and a red blood cell membrane composited on the surface of the drug-loaded Fe3O4 nanoparticles, wherein the red blood cell membrane is modified with galactose.

[0059] The drug loaded in the Fe3O4 nanoparticles is emodin, and the mass of emodin accounts for 20-40% of the mass of the hollow Fe3O4 nanoparticles before drug loading.

[0060] The hollow Fe3O4 nanoparticles have a diameter of 180–220 nm.

[0061] The ratio of galactose to erythrocyte membrane cell-derived galactose was 100 μg: (2.68–6.25) × 10⁻⁶. 9 mL -1 .

[0062] Optionally, the drug-loaded Fe3O4 nanoparticles have a diameter of 320–360 nm.

[0063] The preparation method of the above-mentioned biomimetic iron-based nanoparticles includes the following steps:

[0064] S1. Take emodin and hollow Fe3O4 nanoparticles with a mass ratio of (2-2.5):1. Mix the ethanol solution of emodin with the ethanol dispersion of hollow Fe3O4 nanoparticles, stir evenly at room temperature, centrifuge and discard the supernatant, and then vacuum dry to obtain drug-loaded Fe3O4 nanoparticles.

[0065] S2. Suspend the red blood cell membrane in buffer solution, add galactose in proportion, incubate for 25-35 min, and then squeeze it out through a filter membrane to obtain a galactose-modified red blood cell membrane of a set size.

[0066] S3. The drug-loaded Fe3O4 nanoparticles obtained in S1 and the galactose-modified erythrocyte membrane obtained in S2 are mixed and coated to obtain biomimetic iron-based nanoparticles.

[0067] Optionally, the raw materials for preparing the hollow Fe3O4 nanoparticles in S1 include those with a mass ratio of (1.0–1.5):

[0068] The preparation method includes: dispersing ferric chloride hexahydrate, sodium citrate, urea and polyacrylamide in distilled water in proportion (3.0~3.5):(0.8~1.0):(0.1~0.3); and dispersing ferric chloride hexahydrate, sodium citrate and urea in distilled water, then adding polyacrylamide, stirring to dissolve and reacting at 200±20℃ for 11~13h, cooling to room temperature and centrifuging to separate the black precipitate, drying the black precipitate at 60±5℃ overnight to obtain hollow Fe3O4 nanoparticles.

[0069] Optionally, in the preparation method of hollow Fe3O4 nanoparticles, after separating the black precipitate, it is washed more than 3 times to remove unreacted substances.

[0070] Optionally, in the preparation method of hollow Fe3O4 nanoparticles, the drying method is vacuum drying.

[0071] Optionally, in S1, the mixing method is ultrasonic dispersion for 25–35 min.

[0072] Optionally, in S1, the vacuum drying temperature is 60±5℃.

[0073] Optionally, the preparation method of the red blood cell membrane in S2 includes: centrifuging whole blood at 3000-3500 rpm for 8-12 min at 4±2℃, discarding the supernatant, washing with PBS buffer, resuspending the obtained red blood cells in ultrapure water for 2-3 h to induce red blood cell lysis, 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.

[0074] Optionally, in S3, the thin-film method includes: mixing PBS buffer containing drug-loaded Fe3O4 nanoparticles with galactose-modified erythrocyte membranes and extruding through a 200 nm filter membrane.

[0075] The above-mentioned biomimetic iron-based nanoparticles and / or the above-mentioned method for preparing biomimetic iron-based nanoparticles are used in at least one of the following a1)-a4):

[0076] a1) Reprogramming M2 TAMs into M1 TAMs or preparing products by reprogramming M2 TAMs into M1 TAMs;

[0077] a2) Inhibit cancer cell proliferation or prepare products that inhibit cancer cell proliferation;

[0078] a3) Activate anti-tumor immune responses based on macrophages and T cells or prepare products that activate anti-tumor immune responses based on macrophages and T cells;

[0079] a4) Prepare products for treating tumors.

[0080] Optionally, the cancer cells are breast cancer cells, specifically triple-negative breast cancer cells.

[0081] Optionally, the tumor is a breast cancer tumor, specifically a triple-negative breast cancer tumor.

[0082] Tumor-infiltrating immune cells typically experience metabolic stress due to dysregulation of tumor cell metabolic activity, leading to impaired anti-tumor immune responses. Abnormal bioenergetic activity of tumor cells utilizes large amounts of glucose to produce lactate through glycolysis. Numerous clinical data from patients show that serum LDH levels above 1000 IU / L predict advanced or metastatic disease. Glucose consumption and lactate accumulation disrupt the pro-inflammatory polarization capacity of macrophages, thereby promoting the growth of M2 TAMs. In tumor-bearing mice lacking lactate dehydrogenase (LDHA), the number of spleen myeloid-derived suppressor cells (MDSCs) is reduced, while the cytotoxic function of tumor-infiltrating natural killer (NK) cells is increased, indicating that lactate production promotes tumor growth and impairs innate immune responses. In mouse models, LDHA-mediated lactate production inhibits TFN-γ expression 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).

[0083] Emodin, an anthraquinone derivative extracted from rhubarb root, possesses various biological functions, including anti-inflammatory, antioxidant, anticancer, and neuroprotective effects. This invention utilizes magnetic hollow Fe3O4 NPs as carriers to deliver emodin into the tumor microenvironment, overcoming its hydrophobicity. The introduction of emodin successfully inhibited LDHA production, leading to a decrease in lactate levels and promoting the conversion of M2 TAMs to M1 TAMs. This reprogramming from immunosuppression to immunostimulation enhances the effectiveness of tumor immunotherapy.

[0084] Tumor microenvironments (TMEs) contain key components of tumor-associated immunosuppressive mechanisms (TAMs), which are often associated with poor prognosis and drug resistance. Given the role of TAMs in promoting tumor growth, various strategies have been devised to counteract their effects. Iron-based nanoparticles can serve as key transformative entities against TAMs, promoting immune responses and safely and effectively sensitizing tumors to immunotherapy. Therefore, this invention employs a nanoplatform framework containing hollow iron oxide to synergistically induce the polarization of the M1-TAM phenotype. Another significant characteristic of cancer cells is the continuous production of lactate through aerobic glycolysis, which promotes TAM polarization towards the M2 phenotype, exacerbates immunosuppression within the TME, and leads to hypoxia. Therefore, scientists believe that reducing lactate production may restore the immunosuppressive tumor microenvironment.

[0085] Example 1

[0086] Preparation and identification of biomimetic iron-based nanoparticles.

[0087] 0.54 g of ferric chloride hexahydrate (also known as ferric chloride (III) hexahydrate, CAS No.: 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 stirred until it was completely dissolved into a solution. The solution was then reacted at 200 °C for 12 h. After cooling to room temperature, the solution was centrifuged to separate the black precipitate. The black precipitate was washed three times to remove any unreacted substances and then dried overnight under vacuum at 60 °C to obtain the final substance, namely hollow Fe3O4 nanoparticles (hollow Fe3O4 NPs).

[0088] like Figure 1 As shown in (A), 2 mg of emodin was added to 1 mL of anhydrous ethanol and sonicated for 10 min to obtain an ethanol solution of emodin; 1 mg of hollow Fe3O4 nanoparticles were added to 1 mL of anhydrous ethanol and sonicated for 30 min to obtain an ethanol dispersion of hollow Fe3O4 nanoparticles; the two were mixed and stirred continuously at room temperature for 24 h, centrifuged and the supernatant was discarded to obtain drug-loaded Fe3O4 nanoparticles. E The loading efficiency of emodin was calculated using Fe3O4 NPs; E Fe3O4 NPs were centrifuged and vacuum dried at 60°C for later use.

[0089] 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. The blood was then washed three times with cold phosphate-buffered saline (PBS, pH 7.4) to obtain red blood cells (RBCs). The RBCs were resuspended in 2 mL of ultrapure water for at least 2 h to induce erythrocyte lysis. The resuspension containing the ruptured RBCs was centrifuged at 9000 rpm for 10 min to remove the precipitate and released hemoglobin. The supernatant was then washed with 0.01 M PBS buffer until colorless to obtain red blood cell membranes (mRBCs). 6.25 × 10⁶ mRBCs were then centrifuged. 9 One mRBC was suspended in 1 mL of PBS buffer (PBS buffer concentration was 0.01 M), and 100 μg of galactose was added to 1 mL of buffer. After incubation for 30 min, galactose-modified red blood cell membranes (gRBCs) were formed. Then, the gRBCs were squeezed out through a 200 nm filter membrane to prepare 200 nm gRBCs.

[0090] Use in a dry state E Fe3O4 NPs were prepared at a concentration of 500 μg / mL. -1 of E Fe3O4 NPs dispersion, take 1 mL EFe3O4 NPs dispersion (dispersion medium: 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).

[0091] In this embodiment, hollow Fe3O4 NPs were prepared by treating FeCl3, polyacrylamide, citrate, and urea using a hydrothermal method. The characterization methods included:

[0092] Scanning electron microscope (SEM) images were obtained using a Zeiss Sigma 300 scanning electron microscope, such as... Figure 2 As shown in (A), the surface of hollow Fe3O4 NPs is rough.

[0093] The HT7800 performs transmission electron microscopy (TEM) imaging at an accelerating voltage of 120 kV, such as... Figure 2 As shown in (B), the monodisperse, spherical and hollow structures of Fe3O4 nanoparticles are displayed. The structures are uniform and the diameter is 206.5 ± 15.3 nm.

[0094] Crystal structure measurements were performed using an Ultima IV X-ray diffractometer (XRD, Bruker D8 ADVANCE / Ultima IV, Germany) at 8°min. -1 The following measurements were performed using Cu Kα radiation (λ = 0.15405 nm, 40 kV, 40 mA). The results for the hollow Fe3O4 NPs are as follows: Figure 2 As shown in (C), the diffraction patterns of these NPs are in very good agreement with the crystal structure of Fe3O4 NPs (JCPDS 75-0033).

[0095] 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 indicates that Fe and O elements are uniformly distributed.

[0096] Hollow Fe3O4 NPs were analyzed by X-ray photoelectron spectroscopy (XPS) 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 have binding energies of approximately 710.6 eV and 724.6 eV, respectively, which is consistent with the Fe3O4 reported in the literature.

[0097] N2 adsorption-desorption tests were performed on hollow Fe3O4 NPs. The N2 adsorption / desorption isotherms of hollow Fe3O4 NPs are as follows: Figure 2 (F) shows that the specific surface area and average pore size of the hollow Fe3O4 NPs are 229.2 m². 2 / g and 16.4nm confirm that hollow Fe3O4NPs are beneficial for encapsulating tumor therapeutic drugs.

[0098] In calculating the loading efficiency of emodin, a standard curve was established using high-performance liquid chromatography (HPLC) to evaluate the loading efficiency of emodin. Figure 2 As shown in (G), hollow Fe3O4 NPs can efficiently load emodin. Figure 2 As determined in (G), the loading ratio ([emodin] / [Fe3O4], w / w) in this embodiment is approximately 30.0%.

[0099] Analysis of gRBCs using SDS-PAGE R Fe3O4 NPs and marker proteins, samples stained with Coomassie Brilliant Blue, such 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 with galactose-modified erythrocytes, the average hydrodynamic diameter of Fe3O4 NPs increased from 205.7 ± 3.3 nm to ER The average hydrodynamic diameter of Fe3O4 NPs was 341.2 ± 5.7 nm, indicating successful encapsulation of the erythrocyte membrane. Figure 2 (I)).

[0100] The size and zeta potential of nanoparticles were determined using a Nano-ZS zetaizer (Nanolink SZ901), such as... Figure 2 As shown in (J), the Zeta potential of Fe3O4 NPs significantly increased from -33.0 mV to... ER The Zeta potential of Fe3O4 NPs was -20.4 mV, close to that of galactose-erythrocyte membrane vesicles (gRBCs) (-8.5 mV), further confirming the formation of surface biomimicry.

[0101] The above results verify that ER The successful formation of Fe3O4 NPs indicates ER Fe3O4 NPs preserve a large amount of galactose-modified erythrocyte membranes to improve the biocompatibility of these nanoparticles and the biotargeting of M2 TAMs.

[0102] 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 The rapid proliferation of tumor cells is influenced by the active metabolism of glucose and glutamine. For example... Figure 3 As shown in (A), under the promotion of LDH activity, pyruvate is enzymatically converted into lactate, maintaining the NADH / NAD+ ratio and the cytoplasmic level of nicotinamide adenine dinucleotide (NAD+) to maintain glycolysis flux.

[0103] Evaluation of lactate dehydrogenase (LDH) inhibition.

[0104] The experiment used 40 μmol L -1 Reduced coenzyme I (NADH) and 200 μmol L -1 Pyruvate was added to a final volume of 200 μL with 0.66 ng or 0.38 mU of LDH enzyme (1 unit of enzyme was prepared at 37 °C and pH 8.55 using 1 μmol L...). -1 (Pyruvate was reduced to L-lactic acid); five different concentrations were used to generate concentration-response curves. ER Fe3O4 NPs dispersions, including 0, 25, 50, 100, and 200 μg mL. -1 Five different concentrations ER Fe3O4 NPs dispersion was added to each well of a 96-well plate at a rate of 8 μL. Subsequently, substrate and cofactor were added and dissolved in buffer (152 μL), followed by LDH enzyme solution (40 μL). The measurement procedure followed the guidelines provided by the pyruvate kit manufacturer.

[0105] At five different concentrations ER In addition to Fe3O4 NPs, zero concentration was added as a control, and the results were as follows: Figure 3 As shown in (B), with ER The increase in Fe3O4 NPs concentration gradually led to an increase in the accumulation of converted pyruvate, which further indicates ER Fe3O4 NPs can inhibit lactate dehydrogenase (LDH) activity, thereby inhibiting lactate production.

[0106] ER Fe3O4 NPs facilitate the transformation of M2 TAMs into M1 TAMs.

[0107] 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 RPMI-1640 medium containing fetal bovine serum (10%) and bispecific antibodies to penicillin and streptomycin (1%) (conditions: 37°C, 5% CO2) to obtain M0 macrophages. These cells were then incubated in a medium containing 20 ng / mL of... -1 IL-4 (interleukin-4) was incubated in 1640 medium for 12 h and then repolarized to M2 TAMs.

[0108] Iron uptake test:

[0109] The induced M2 TAMs were used at 1.6 × 10 5 Cells were seeded at a density of 1.6 mL / well in 6-well plates. The next day, the original cell culture medium was replaced with fresh culture medium containing 0, 25, 50, and 100 μg / mL, respectively. -1 ER Fe3O4 NPs (Fe3O4 NPs concentration). Incubate for another 6 hours, then wash 1-2 times with PBS, resuspend in PBS, and centrifuge. Finally, add 500 μL of aqua regia to dissolve the cells, bring the volume to 15 mL, and detect the iron ion concentration using inductively coupled plasma mass spectrometry (ICP-MS). The results are as follows: Figure 3 As shown in (C): With ER The increase in Fe3O4 NPs concentration and the dose-dependent increase in cellular iron levels indicate significant iron uptake in M2 TAMs.

[0110] Assessment via MTT ER Toxicity of Fe3O4 NPs:

[0111] M2 TAMs cells were then exposed to different concentrations of... R Fe3O4 NPs and ER M2TAMs cell viability was assessed after incubation with Fe3O4 NPs for 24 h; results are as follows: Figure 3 As shown in (D), different concentrations of R Fe3O4 NPs and ER Fe3O4 NPs did not show obvious toxicity after 24 hours of incubation.

[0112] CD16 / 32 and CD206 expression:

[0113] F4 / 80+ macrophages were sized at 1×10⁶ cells per well. 5 10 cells were seeded in a 24-well plate and seeded with 10 cells respectively. R Fe3O4 NPs and ERFe3O4NPs (equivalent to 100 μg mL) -1 After incubation with Fe3O4 for 24 h, the cells were washed twice with PBS and collected. Subsequently, the cells were redispersed in 500 μL of PBS containing either PE / Cy7 anti-mouse CD16 / 32 antibody or PE anti-mouse CD206 antibody, stained for 15 min, and the expression of the M1 phenotype-specific marker CD16 / 32 (PE / Cy7) and the M2 phenotype-specific marker CD206 (PE) was analyzed by flow cytometry. The results are as follows: Figure 3 As shown in (E), compared to the PBS group, ER Fe3O4 NPs and ER Fe3O4 NPs triggered increased CD16 / 32 expression, indicating increased M1 TAMs-induced expression; Figure 3 As shown in (F), R Fe3O4 NPs and ER The Fe3O4 NPs group significantly reduced CD206 expression, which may be due to... Figure 3 (F) is induced by M2 TAMs. Reprogramming M2TAMs into M1TAMs can trigger the release of pro-inflammatory factors such as TNF-α, which can be further detected by ELISA.

[0114] Assessment of TNF-α:

[0115] 1×10 per hole 4 M2 TAMs (100 μL) were incubated overnight in a 96-well plate. 0, 25, 50, and 100 μg mL were added, respectively. -1 of ER Fe3O4 NPs or R One of the Fe3O4 NPs, or added at concentrations of 0, 7, 15 and 30 μg mL respectively. -1 emodin (this is due to: ER Fe3O4 NPs or R In Fe3O4 NPs, the loading ratio of emodin was 30 wt%, which corresponds to emodin concentrations of 0, 7, 15, and 30 μg / mL. -1 The graph shows the second horizontal axis (PBS mixture; after 12 hours, the supernatant was collected, and TNF-α levels were detected using enzyme-linked immunosorbent assay (ELISA) (Biogend); the results are as follows). Figure 3 (G) shows the concentrations of emodin (from left to right) as 0, 7, 15, and 30 μg / mL. -1 (To facilitate comparison of the effects of the same emodin concentration), compared with PBS and a mixture of emodin (converted to the corresponding concentration), R Fe3O4 NPs andER The Fe3O4 NPs group significantly released TNF-α inflammatory factors, especially ER The Fe3O4 NPs group is associated with the synergistic release of emodin and Fe3O4 NPs.

[0116] These results confirm that ER Fe3O4 NPs possess the strongest M2 TAMs repolarization activity, laying the foundation for anti-tumor immunotherapy.

[0117] ER In vitro antitumor activity of Fe3O4 NPs

[0118] It has been reported that M1 TAMs with pro-inflammatory effects exert their antitumor activity by secreting pro-inflammatory cytokines, reactive nitrogen, and reactive oxygen species. To further confirm the antitumor potential of M2 TAMs converted to M1 TAMs, we used the 4T1-M2 TAMstranswell system for verification.

[0119] like Figure 4 As shown in (A), 1 × 10⁶ seeds were seeded per well in a Transwell co-culture 24-well plate. 5 M2 TAMs, at the same time, 2.5×10 4 4T1 cells were placed in the lower chamber and cultured overnight. The next day, they were treated with emodin, R Fe3O4 NPs and ER Fe3O4 NPs (equivalent to 100 μg mL) -1 Fe3O4 or 15μg mL -1 Cell viability was determined by MTT assay after culturing with emodin for 24 hours.

[0120] Assessment of 4T1 cell survival in the inferior vena cava:

[0121] Inoculate 1×10 at 100 μL per well 4 One TAMs or 3T3 cell line (derived from mouse embryonic fibroblasts) was incubated overnight in a 96-well plate; the original culture medium was discarded and the cells were washed 1-2 times with PBS, and then 0-200 μg / mL of the medium was added. -1 of R Fe3O4NPs and ER Fe3O4 NPs were treated. Then, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) was added and the mixture was incubated for 3.5 hours. The results were then detected using a microplate reader. The results are as follows: Figure 4 As shown in (B), due to Fe3O4 inducing M1 polarization and the release of inflammatory factors, RFe3O4 NPs can trigger 35.0% of 4T1 cell death, due to Fe3O4 inducing M1 polarization and the release of inflammatory factors. ER Fe3O4 NPs can trigger 54.7% cell death, which is related to the synergistic enhancement of M1 polarization by emodin; on the other hand, such as Figure 4 As shown in (C) R Fe3O4 NPs and ER Fe3O4 NPs in 0–200 μg mL -1 After incubation at the specified concentration for 24 hours, the cell viability of 3T3 cells remained above 90%, indicating that... ER Fe3O4 NPs are safe in vitro.

[0122] Apoptosis kit for detecting cell apoptosis:

[0123] To detect apoptosis, 4T1 cells in the lower chamber were collected using an EDTA-free trypsin collection device after the experiment. After washing with PBS, the cells were resuspended in binding buffer and stained with an apoptosis kit for 30 minutes. The stained cells were then analyzed by flow cytometry. Cells were cultured in calcein AM / PI working solution under a fluorescence microscope and incubated in the dark at 37°C for 30 minutes. The staining results were then observed under a fluorescence microscope. Figure 4 As shown in (D), it indicates that Fe3O4 induces M1 polarization and the release of inflammatory factors, and under the synergistic effect of emodin, it can significantly enhance the effect of M1 polarization, thereby leading to the most significant apoptosis in 4T1 cells.

[0124] Detection of 4T1 cell apoptosis using a double staining method for live / dead cells:

[0125] The killing effect of different reagents in the MTT assay on 4T1 cells was further verified by the cell viability / dead double staining method. The results are as follows: Figure 4 As shown in (E), the results are consistent with Figure 4 The trends of MTT test results in (D) are similar: Figure 4 In (E), the results of fluorescein-annexin V and propidium iodide (PI) staining analysis showed that emodin, R Fe3O4 NPs or ER The effect of Fe3O4 NPs on cell viability is mainly related to apoptosis. (Annexin V-FITC)- / PI+ represents necrotic cells, (Annexin V+FITC)+ / PI+ represents late-stage apoptotic cells, (Annexin V-FITC)+ / PI- represents viable cells, and (Annexin V-FITC)- / PI- represents early-stage apoptotic cells. Emodin, 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 repolarization activity of M2 TAMs by emodin and Fe3O4 NPs can induce significant apoptosis in 4T1 cells.

[0126] In summary, R Fe3O4 NPs or ER Fe3O4 NPs have good anti-tumor effects and low toxicity to normal cells.

[0127] Nanomedicines can only be further used in cancer treatment if they have low toxicity to humans. ER The in vitro cytotoxic effects of Fe3O4 NPs on 4T1 cells prompted the inventors to evaluate its potential for antitumor immunotherapy in vivo: toxicity was assessed through hemolysis tests, hematological analysis, and H&E staining of heart, liver, spleen, lung, and kidney tissues.

[0128] Hemolysis test:

[0129] Mouse red blood cells were washed with PBS to remove serum, and then resuspended in PBS (20 mL). ER Fe3O4 NPs were available in quantities 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. Following the above procedure, the hemolysis rate was assessed and calculated after incubation at 37°C for 3 hours. Figure 5 As shown in (A), different concentrations of ER The hemolysis rate of Fe3O4 NPs was all below 5%, indicating that the injection... ER Fe3O4 NPs are safe.

[0130] ER In vivo therapeutic effects of Fe3O4 NPs

[0131] 4T1 tumor-bearing mouse model experiment:

[0132] BALB / c mice (15–20 g, 6 weeks) were subcutaneously inoculated with 2 × 10⁻⁶ mol / L of mol / L. 6 4T1 cells were treated with 100 μL of PBS when the tumor volume reached 150–200 mm². 3 At that time, Cy5-loaded [product / method] was injected into the tail vein of mice. R Fe3O4 NPs 100μL (equivalent to 0.5mg Cy5 kg) -1Mice were used. After 24 hours, fluorescence imaging was performed on the heart, liver, spleen, lungs, kidneys, and tumors. Subsequently, major organs and tumors were harvested, thawed with 10 mL of concentrated nitric acid, diluted with ultrapure water, and ion concentrations were determined by ICP-MS. Results are as follows: Figure 5 As shown in (B), the load Cy5 ER Fe3O4 NPs showed significant accumulation in tumor tissue; ICP-MS ion concentration results were as follows: Figure 5 As shown in (C), it can be seen that 24 hours after injection, ER Fe3O4 NPs are mainly distributed in the spleen and lungs, and exhibit significant accumulation behavior in tumors.

[0133] BALB / c mice (15–20 g, 6 weeks) were subcutaneously inoculated with 2 × 10⁻⁶ mol / L of mol / L. 6 4T1 cells were treated with 100 μL of PBS when the tumor volume reached approximately 70 mm². 3 At times, such as Figure 5 As shown in (D), mice were randomly divided into four equal groups. On days 1, 3, and 5, each group received an intravenous injection of the following drugs: (a) PBS; (b) emodin; (c) ... R Fe3O4 NPs; (d) ER Fe3O4 NPs; the intravenous dose in mice is equivalent to 20 mg / kg of Fe3O4 NPs. -1 Or emodin 6mg / kg -1 .

[0134] During the treatment period, the body weight of the mice was continuously monitored, such as... Figure 5 As shown in (F), intravenous injection ER Fe3O4NPs had no significant effect on the body weight of mice during treatment. Tumor volume was continuously recorded during treatment, and the results were calculated using a formula (width) based on digital caliper measurements. 2 × length / 2) to assess tumor volume, such as Figure 5 As shown in (E), injection ER The group of mice with Fe3O4 NPs showed the slowest increase in tumor volume; the tumor status of the mice on day 18 was as follows: Figure 10 As shown, the removed tumor is in the following condition. Figure 11 As shown, with Figure 5 The tumor growth trend shown is consistent.

[0135] On day 8, tumor tissues from a subset of mice in each group were collected, washed with cold PBS, and then incubated in a solution containing collagenase IV and DNase I at 37°C for 45 minutes. Digestion was terminated on ice, and the digested tissues were filtered through a 70 μm nylon filter to obtain a single-cell suspension. Before flow cytometry antibody staining, tumor cells were treated with PE / Cy7 anti-mouse CD16 / 32 and PE anti-mouse CD206 to analyze the F4 / 80 ratio within the tumor tissue. + Expression levels of CD16 / 32 and CD206 in macrophages; and staining of lymph nodes with PE anti-mouse CD4 antibody and APC anti-mouse CD8 antibody.

[0136] (1) Flow cytometry analysis of CD16 / 32 and CD206 at F4 / 80 + The expression level in macrophages can serve as a reliable marker of macrophages within tumors; results showed that, compared with PBS, emodin, and... R Compared to Fe3O4 NPs, ER Fe3O4 NPs induce increased expression of CD16 / 32 and decreased expression of CD206, such as Figure 5 As shown in (I), the expression levels of CD16 / 32 after different treatments were 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 exhibit excellent TME reprogramming capabilities in vivo, enabling them to convert M2 TAMs into M1 TAMs.

[0137] (2) Flow cytometry detection of CD4 in tumor draining lymph nodes + or CD8 + The proportion of T lymphocytes, such as Figure 12 As shown, a large number of CD4 cells were observed in the tumor-draining lymph nodes. + and CD8 + T cells. PBS, emodin, R Fe3O4 NPs, ER Fe3O4 NPs group CD8 + The T lymphocyte infiltration rates were 10.3%, 11.2%, 18.0%, and 23.9%, respectively, and CD4+ infiltration rates were also observed. + The T lymphocyte infiltration rates were 24.5%, 37.0%, 44.1%, and 52.3%, respectively; these results confirm that... ER Fe3O4 NPs therapy can activate anti-tumor immune responses based on macrophages and T cells. Therefore, activating the immune-stimulating microenvironment can lead to the secretion of more inflammatory factors and promote CD8. + and CD4+ T cells infiltrate the tumor site, thereby enhancing the effectiveness of immunotherapy for tumors.

[0138] On day 18, mice were euthanized, and major organs (heart, liver, spleen, lung, and kidney) and tumors were removed. Samples were cut into 5μm sections, stained with H&E, and analyzed pathologically using a digital microscope. The pathological changes in the tumor sections after treatment were as follows: Figure 5 As shown in (G) ER Fe3O4 NPs showed good therapeutic effects under conditions of severe tumor cell apoptosis and necrosis; immunofluorescence staining of tumor tissue sections was performed: treatment with FITC anti-mouse CD80 antibody and PE anti-mouse CD86 antibody, followed by DAPI staining, and the results were as follows. Figure 5 As shown in H: In ER Under Fe3O4 NPs treatment, the fluorescence intensity of CD80 and CD86 was significantly higher than that of other treatment groups.

[0139] H&E staining results of major organs as follows Figure 9 As shown, no significant toxicity was observed in the H&E staining of major organs after intravenous injection in any group, further supporting this finding. ER Fe3O4 NPs exhibit good biocompatibility in vivo.

[0140] Mice were observed after treatment, and the aspartate aminotransferase / alanine aminotransferase (AST / ALT) ratio in their blood was measured on day 18. The results are as follows: Figure 6 As shown; the urea / creatinine (UREA / CREA) ratio in mouse blood was measured, and the results are as follows. Figure 7 As shown; additionally, the following parameters were measured in mouse blood: 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 as follows. Figure 8 As shown above, the results indicate that the blood biochemistry and hematology analysis results of each group of mice are within the reference range, and the increase or decrease of each indicator is not significant, indicating good biocompatibility.

[0141] As can be seen from the above embodiments, this patent synthesizes an iron-based nanoplatform ( ER Fe3O4 NPs), including hollow Fe3O4 NPs of erythrocyte membrane cloaks loaded with natural products and modified with galactose. Figure 1 As shown in (B), it can not only efficiently assist in the delivery inhibition and neutralization of tumor acidification of emodin, but also synergistically convert M2 TAMs into M1 TAMs, thereby improving tumor metabolic immunotherapy.

[0142] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biomimetic iron-based nanoparticle, characterized in that, include: Drug-loaded Fe3O4 nanoparticles and a red blood cell membrane composited on the surface of the drug-loaded Fe3O4 nanoparticles, the red blood cell membrane being modified with galactose; The drug loaded in the Fe3O4 nanoparticles is emodin, and the emodin accounts for 20-40% of the mass of the hollow Fe3O4 nanoparticles before loading. The hollow Fe3O4 nanoparticles have a diameter of 180~220 nm. The ratio of galactose to erythrocyte membrane cell origin is 100 μg: (2.68 ~ 6.25) × 10⁻⁶. 9 indivual.

2. The biomimetic iron-based nanoparticles as described in claim 1, characterized in that, The mass of the emodin accounts for 30% of the mass of the hollow Fe3O4 nanoparticles before drug loading.

3. The biomimetic iron-based nanoparticles as described in claim 1, characterized in that, The hollow Fe3O4 nanoparticles have a diameter of 206.5 ± 15.3 nm.

4. The biomimetic iron-based nanoparticles as described in claim 1, characterized in that, The biomimetic iron-based nanoparticles have a diameter of 320~360nm.

5. The biomimetic iron-based nanoparticles as described in claim 4, characterized in that, The biomimetic iron-based nanoparticles have a diameter of 341.2 ± 5.7 nm.

6. A method for preparing biomimetic iron-based nanoparticles as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Take emodin and hollow Fe3O4 nanoparticles with a mass ratio of (2~2.5):

1. Mix the ethanol solution of emodin with the ethanol dispersion of hollow Fe3O4 nanoparticles, stir at room temperature for 24 hours, centrifuge and discard the supernatant, and then vacuum dry to obtain drug-loaded Fe3O4 nanoparticles. S2. Suspend the red blood cell membrane in buffer solution, add DSPE-PEG-Gala in proportion, incubate for 25~35 min, and then squeeze it out through a filter membrane to obtain galactose-modified red blood cell membrane of the set size. S3. The drug-loaded Fe3O4 nanoparticles obtained in S1 and the galactose-modified erythrocyte membrane obtained in S2 are mixed and coated to obtain biomimetic iron-based nanoparticles.

7. The method for preparing biomimetic iron-based nanoparticles as described in claim 6, characterized in that, The raw materials for preparing the hollow Fe3O4 nanoparticles in S1 include: ferric chloride hexahydrate, sodium citrate, urea, and polyacrylamide in a mass ratio of 0.54:0.865:0.36:0.

3. The preparation method includes: dispersing ferric chloride hexahydrate, sodium citrate, and urea in distilled water according to the proportion, then adding polyacrylamide, stirring to dissolve, reacting at 200±20℃ for 11~13h, cooling to room temperature, centrifuging to separate the black precipitate, drying the black precipitate at 60±5℃ overnight to obtain hollow Fe3O4 nanoparticles.

8. The method for preparing biomimetic iron-based nanoparticles as described in claim 7, characterized in that, In the preparation method of hollow Fe3O4 nanoparticles, after separating the black precipitate, it is washed more than three times to remove unreacted substances.

9. The method for preparing biomimetic iron-based nanoparticles as described in claim 8, characterized in that, In the preparation method of hollow Fe3O4 nanoparticles, the drying method is vacuum drying.

10. The method for preparing biomimetic iron-based nanoparticles as described in claim 6, characterized in that, In S1, the mixing method is ultrasonic dispersion for 25-35 minutes.

11. The method for preparing biomimetic iron-based nanoparticles as described in claim 6, characterized in that, In S1, the vacuum drying temperature is 60±5℃.

12. The method for preparing biomimetic iron-based nanoparticles as described in claim 6, characterized in that, The preparation method of red blood cell membrane in S2 includes: whole blood is centrifuged at 3000~3500 rpm for 8~12 min at 4±2℃, the supernatant is discarded, and after washing with PBS buffer, the obtained red blood cells are resuspended in ultrapure water for 2~3 h to induce red blood cell lysis. Then, the blood is centrifuged at 9000~9500 rpm for 8~12 min, the supernatant is collected, and the blood is washed with PBS buffer until colorless to obtain the red blood cell membrane.

13. The method for preparing biomimetic iron-based nanoparticles as described in claim 6, characterized in that, In S3, the coating method includes: mixing PBS buffer containing drug-loaded Fe3O4 nanoparticles with galactose-modified erythrocyte membranes and extruding through a 200 nm filter membrane.

14. The biomimetic iron-based nanoparticles prepared by the preparation method of any one of claims 1-5 or any one of claims 6-13, in the following a1) a2) Application of at least one of them: a1) Prepare products that inhibit the proliferation of cancer cells; a2) Prepare products for treating tumors.

15. The application as described in claim 14, characterized in that, The cancer cells are breast cancer cells; the tumor is a breast cancer tumor.

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