Macrophage bionic vesicle nano preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a macrophage bionic vesicle nano preparation as well as a preparation method and application thereof. The invention provides a macrophage bionic vesicle nano preparation, which is characterized in that a tumor targeting peptide is used for modifying a macrophage bionic vesicle, and a targeting NSUN family methyltransferase siRNA (small interfering Ribonucleic Acid) is wrapped. The macrophage bionic vesicles modified by the tumor targeting peptide have good gastric cancer cell targeting property, the NSUN expression level of gastric cancer cells can be inhibited by wrapping targeted NSUN family methyltransferase siRNA, the abnormal modification level of gastric cancer m5C is reduced, and the effect of enhancing the treatment of the gastric cancer by the PD-1 antibody is achieved. The results of the embodiment show that the macrophage bionic vesicle nano preparation provided by the invention acts on gastric cancer cells, and can specifically target the gastric cancer cells, induce ferroptosis of the gastric cancer cells, improve the curative effect of a PD-1 antibody, prolong the survival time of mice and improve the tumor immune microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a macrophage biomimetic vesicle nanoformulation, its preparation method, and its application. Background Technology

[0002] Gastric cancer is the fifth most common cancer and the fourth leading cause of cancer death worldwide, with extremely poor prognosis for patients in advanced stages. Immune checkpoint inhibitors, especially PD-1 / PD-L1 antibodies, have shown significant efficacy in a variety of cancers, but the overall response rate in gastric cancer is only 10% to 20%, with most patients exhibiting primary or secondary resistance.

[0003] PD-1 antibodies restore T cell anti-tumor activity by blocking the binding of PD-1 to its ligand PD-L1. However, the gastric cancer tumor immune microenvironment is typically "immune-immune," characterized by significantly insufficient infiltration of cytotoxic T lymphocytes, accompanied by abnormal enrichment of immunosuppressive cells such as regulatory T cells and tumor-associated macrophages. This imbalance in the immune microenvironment, along with the enhanced immune escape capabilities of tumor cells through various mechanisms, collectively inhibits the therapeutic effect of PD-1 antibodies.

[0004] m 5 C(5-methylcytosine) modification is an important post-transcriptional RNA modification process that mainly occurs at the cytosine residues of mRNA molecules and is specifically catalyzed by NSUN family methyltransferases. This modification plays a crucial role in regulating mRNA stability, subcellular localization, and translation efficiency, and its aberrant expression is closely related to the occurrence and development of various diseases. Reports indicate that aberrant expression of NSUN family methyltransferases can promote M... 5 C-modification promotes gastric cancer development. Currently, there is no effective method to reverse NSUN family methyltransferase-mediated immunotherapy resistance. Therefore, developing a strategy that targets NSUN family methyltransferases and synergistically enhances the efficacy of PD-1 antibodies is of great clinical significance for improving gastric cancer immunotherapy. Summary of the Invention

[0005] The purpose of this invention is to provide a macrophage biomimetic vesicle nanoformulation, its preparation method and application. The macrophage biomimetic vesicle nanoformulation enhances the therapeutic effect of PD-1 antibody by targeting and inhibiting the expression of NSUN family methyltransferases, reshaping the tumor immune microenvironment.

[0006] This invention provides a macrophage biomimetic vesicle nanoformulation, which includes macrophage biomimetic vesicles, tumor-targeting peptides, and siRNA targeting NSUN family methyltransferases; The tumor-targeting peptide is modified on the surface of macrophage biomimetic vesicles; the siRNA is loaded into macrophage biomimetic vesicles.

[0007] As a preferred embodiment, the macrophages comprise RAW264.7 cells; the siRNA comprises targeting... NSUN2 Genetic si-NSUN2 .

[0008] As a preferred embodiment, the tumor-targeting peptide includes a targeting peptide that targets gastric tumors.

[0009] As a preferred embodiment, the siRNA comprises liposomes loaded with siRNA.

[0010] As a preferred embodiment, the mass ratio of the macrophage biomimetic vesicles to the siRNA-loaded liposomes is 1 × 10⁻⁶. 6 Quantity: 100~200μg.

[0011] As a preferred embodiment, the mass ratio of the macrophage biomimetic vesicles to the tumor-targeting peptides is 1×10⁻⁶. 6 ~5×10 6 100μg.

[0012] As a preferred embodiment, the macrophage biomimetic vesicle nanoparticle formulation has a particle size of 100~200nm.

[0013] The present invention also provides a method for preparing the macrophage biomimetic vesicle nanoformulation described above, comprising the following steps: modifying macrophage biomimetic vesicles with tumor-targeting peptides to obtain macrophage biomimetic vesicles modified with tumor-targeting peptides; The macrophage biomimetic vesicles modified with the tumor-targeting peptide were mixed with siRNA targeting NSUN family methyltransferases and extruded to obtain the macrophage biomimetic vesicle nanoformation.

[0014] The present invention also provides the application of the macrophage biomimetic vesicle nanoformulation described in the above scheme or the macrophage biomimetic vesicle nanoformulation prepared by the above preparation method in the preparation of gastric cancer targeted drugs.

[0015] The present invention also provides a gastric cancer targeted drug composition, comprising a tumor immune-related drug and the macrophage biomimetic vesicle nanoformulation described in the above-described scheme or the macrophage biomimetic vesicle nanoformulation prepared by the preparation method described above.

[0016] Beneficial Effects: This invention provides a macrophage-inspired biomimetic vesicle nanoformulation, comprising macrophage-inspired biomimetic vesicles, a tumor-targeting peptide, and siRNA targeting NSUN family methyltransferases; the tumor-targeting peptide is modified on the surface of the macrophage-inspired biomimetic vesicles; and the siRNA is loaded into the macrophage-inspired biomimetic vesicles. This invention uses a tumor-targeting peptide to modify macrophage-inspired biomimetic vesicles, resulting in macrophage-inspired biomimetic vesicles with good targeting ability against gastric cancer cells. Simultaneously, using macrophage-inspired biomimetic vesicles modified with the tumor-targeting peptide to encapsulate siRNA targeting NSUN family methyltransferases can inhibit NSUN expression levels in gastric cancer cells and reduce the m-cell expression of gastric cancer cells. 5 Abnormal C-level modification enhances the therapeutic effect of PD-1 antibody in gastric cancer. The results of the examples show that the macrophage-inspired biomimetic vesicle nanoformulation provided by this invention can specifically target gastric cancer cells, induce ferroptosis in gastric cancer cells, improve the efficacy of PD-1 antibody, prolong the survival time of mice, and improve the tumor immune microenvironment.

[0017] This invention also provides a method for preparing macrophage biomimetic vesicle nanoformulations, comprising the following steps: modifying macrophage biomimetic vesicles with tumor-targeting peptides to obtain tumor-targeting peptide-modified macrophage biomimetic vesicles; mixing the tumor-targeting peptide-modified macrophage biomimetic vesicles with siRNA targeting NSUN family methyltransferases and extruding the mixture to obtain the macrophage biomimetic vesicle nanoformulations. The method described in this invention is simple to operate, low in cost, and suitable for industrial-scale mass production. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 RGD-RNV@ in Example 1 si-NSUN2 Flowchart of the preparation process; Figure 2 For RGD-RNV@ si-NSUN2 Structural diagram and synergistic therapy diagram; the upper diagram is RGD-RNV@ si-NSUN2 Preparation process and structural diagram; the following diagram illustrates synergistic therapy. Figure 3 This is a transmission electron microscope (TEM) result image; Figure 4 For RGD-RNV@ si-NSUN2 Particle size distribution diagram; Figure 5 For RGD-RNV@ si-NSUN2 The effect of absorption; Figure 6This is a graph showing the effect of different treatment groups on ferroptosis induced in gastric cancer cells, where A represents Fe. 2+ A represents ROS fluorescence detection; B represents T-GSH level; C represents MDA level; D represents Fe. 2+ level; Figure 7 The results of the animal model experiments are shown in Figure 1; where A is a flowchart of the animal model treatment process; B is a graph of the tumor volume of mice in each group; and C is a graph of the tumors of mice in each group. Figure 8 Evaluation of RGD-RNV@ by flow cytometry si-NSUN2 The sensitization effect diagram; where A is the proportion of CD8 cells inside mouse tumors as assessed by flow cytometry; B is GZMB. + CD8 + T cells; C is CD8 + T cells; Figure 9 Evaluation of RGD-RNV@ by flow cytometry si-NSUN2 Sensitization effect diagram; where A is the proportion of CD206 and CD86 cells inside mouse tumors as assessed by flow cytometry; B is CD86 + Cells; C is CD206 + cell; Figure 10 For RGD-RNV@ si-NSUN2 Figure showing the results of the biosafety analysis. Detailed Implementation

[0020] This invention provides a macrophage biomimetic vesicle nanoformulation, which includes macrophage biomimetic vesicles, tumor-targeting peptides, and siRNA targeting NSUN family methyltransferases; The tumor-targeting peptide is modified on the surface of macrophage biomimetic vesicles; the siRNA is loaded into macrophage biomimetic vesicles.

[0021] Unless otherwise specified, the raw materials described in this invention can all be obtained through conventional commercial methods in the field.

[0022] The macrophage biomimetic vesicle nanoformulation of the present invention comprises macrophage biomimetic vesicles modified with tumor-targeting peptides and siRNA encapsulated within the tumor-targeting peptide-modified macrophage biomimetic vesicles. As one embodiment, the particle size of the macrophage biomimetic vesicle nanoformulation is 100-200 nm. In specific embodiments of the present invention, the particle size of the macrophage biomimetic vesicle nanoformulation can be any value within the range of 100-200 nm, such as 100, 105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190, or 200 nm.

[0023] In one embodiment, the macrophages include RAW264.7 cells. The RAW264.7 cells of this invention highly express CD47, which can interact with signal regulatory protein α (SIRPα) on the surface of macrophages, forming a "don't eat me" signal, thus preventing macrophage phagocytosis and targeting delivery to tumor tissue.

[0024] In one embodiment, the tumor-targeting peptide includes a targeting peptide that targets gastric tumors. In another embodiment, the tumor-targeting peptide includes RGD cyclic peptide and / or EGFR polypeptide. Tumor-targeting peptides are a class of small molecule peptides capable of specifically recognizing and binding to tumor cells or the tumor microenvironment. In this invention, RGD cyclic peptide is used as an example. Although macrophage biomimetic vesicles possess certain natural chemotaxis, their targeting efficiency for specific tumors is limited and lacks specificity. RGD cyclic peptide (cRGD peptide) can specifically recognize and bind with high affinity to αvβ3 integrin receptors overexpressed on the surface of tumor vascular endothelial cells and various tumor cells. This receptor is significantly overexpressed in various malignant tumors such as glioma, breast cancer, and melanoma, but its expression is extremely low in normal tissues. This invention utilizes tumor-targeting peptides to modify macrophage biomimetic vesicles, endowing the vesicles with active targeting capabilities. The macrophage biomimetic vesicles modified with tumor-targeting peptides exhibit good targeting ability to gastric cancer cells and can efficiently accumulate from the circulatory system to the tumor site.

[0025] This invention modifies the surface of macrophage biomimetic vesicles with tumor-targeting peptides, laying the foundation for a multifunctional platform for subsequent drug delivery and therapy. The tumor-targeting peptide-modified macrophage biomimetic vesicles construct a universal tumor-targeting platform, which can be flexibly loaded with different therapeutic agents (such as chemotherapy drugs, siRNA, immunomodulators, etc.) to achieve integrated targeting and therapy. cRGD not only mediates tumor vascular endothelial adhesion but also enhances the vesicles' penetration ability into the tumor parenchyma (because αvβ3 integrin also participates in cell migration and invasion), potentially improving the problem of poor drug penetration in solid tumors. Simultaneously, macrophage-derived vesicles may carry immunomodulatory signals; after binding with cRGD, this may further enhance the regulation of the local tumor immune microenvironment, providing a new strategy for combined immunotherapy.

[0026] In one embodiment, the mass ratio of the macrophage biomimetic vesicles to the tumor-targeting peptide is 1×10⁻⁶. 6 ~5×10 6 Quantity: 100 μg. In a specific embodiment of the present invention, the mass ratio of the macrophage biomimetic vesicles to the tumor-targeting peptide can be 1 × 10⁻⁶. 6 ~5×10 6 Amount: Any ratio in 100 μg, for example, 1 × 10 6 100μg, 2×10 6 100μg, 2.5×106 100μg, 3×10 6 100μg, 4×10 6 100μg or 5×10 6 Quantity: 100 μg. The applicant's research found that the mass ratio of the macrophage-inspired biomimetic vesicles to the tumor-targeting peptides was 1 × 10⁻⁶. 6 ~5×10 6 At a concentration of 100 μg, it is beneficial for tumor-targeting peptides to modify the surface of macrophage biomimetic vesicles.

[0027] As one implementation, the siRNA includes a target NSUN2 Genetic si-NSUN2 NSUN family methyltransferases specifically catalyze M... 5 C(5-methylcytosine) modification promotes gastric cancer development; the siRNA described in this invention can inhibit tumor cells. NSUN2 Gene expression levels, thereby reducing gastric cancer m 5 Abnormal C modification levels enhance the therapeutic effect of PD-1 antibody in gastric cancer. In a specific embodiment of the present invention, the... si-NSUN2 Purchased from Suzhou Gemma Gene Co., Ltd., the aforementioned si-NSUN2 The nucleotide sequence of the sense strand is shown in SEQ ID NO.1 (5′-GCGAGAAGAUGAAGGUCAUTT-3′), and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2 (5′-AUGACCUUCAUCUUCUCGCTT-3′), wherein the TT at the end of the sense and antisense strands are dangling sequences.

[0028] In one embodiment, the siRNA comprises liposomes loaded with siRNA. This invention utilizes liposome-loaded siRNA to improve its stability, prevent degradation, and facilitate cellular absorption.

[0029] In one embodiment, the mass ratio of the macrophage biomimetic vesicles to the siRNA-loaded liposomes is 1 × 10⁻⁶. 6 Quantity: 100~200 μg. In a specific embodiment of the present invention, the mass ratio of the macrophage biomimetic vesicles to the siRNA-loaded liposomes can be 1×10⁻⁶. 6 Piece: Any value between 100 and 200 μg, for example, 1 × 10 6 100μg, 1×10 6 120μg, 1×10 6 130μg, 1×10 6 150μg, 1×10 6 160μg, 1×10 6180μg or 1×10 6 Quantity: 200 μg. The inventors discovered that the mass ratio of the tumor-targeting peptide-modified macrophage biomimetic vesicles to the siRNA-loaded liposomes is 1 × 10⁻⁶. 6 When the amount is 100~200μg, it is conducive to the fusion of the two, so that the liposomes can be loaded into macrophages to obtain macrophage biomimetic vesicle nanoformulation.

[0030] The present invention also provides a method for preparing the macrophage biomimetic vesicle nanoformation described above, comprising the following steps: modifying macrophage biomimetic vesicles with tumor-targeting peptides to obtain macrophage biomimetic vesicles modified with tumor-targeting peptides; The macrophage biomimetic vesicles modified with the tumor-targeting peptide were mixed with siRNA targeting NSUN family methyltransferases and extruded to obtain the macrophage biomimetic vesicle nanoformation.

[0031] In one embodiment, the macrophages are obtained by culturing in DMEM medium containing 10% fetal bovine serum at a temperature of 25–37°C for 6–12 hours. Preferably, the culture temperature is 28–32°C. In specific embodiments of the invention, the culture temperature can be any value within the range of 25–37°C, such as 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37°C. In another embodiment, the culture time is 8–10 hours. In specific embodiments of the invention, the culture time can be any value within the range of 6–12 hours, such as 6, 7, 8, 9, 10, 11, or 12 hours.

[0032] As one embodiment, the method for preparing macrophage biomimetic vesicles according to the present invention includes: subjecting macrophages to a first physical compression and a first density gradient separation to obtain macrophage biomimetic vesicles. As one embodiment, the density of the macrophages is ≥ (5~6) × 10⁻⁶. 6 / mL, this cell density is conducive to extrusion into biomimetic vesicles, obtaining a sufficient number of biomimetic vesicles. As one implementation, the first physical extrusion is performed using a mini-extrader extruder.

[0033] In one embodiment, the macrophages are subjected to a first physical compression to obtain physically compressed macrophages. In one embodiment, the pressure of the first physical compression is 500-1000 psi; in a preferred embodiment, the pressure of the first physical compression is 600-800 psi. In a specific embodiment of the invention, the pressure of the first physical compression can be any value within the range of 500-1000 psi, such as 500, 600, 700, 800, 900, or 1000 psi. In one embodiment, the temperature of the first physical compression is 50-60°C; in a specific embodiment of the invention, the temperature of the first physical compression can be any value within the range of 50-60°C, such as 50, 52, 55, 56, 58, or 60°C. In one embodiment, the duration of the first physical compression is 5-10 minutes; in a preferred embodiment, the duration of the first physical compression is 6-8 minutes; in a specific embodiment of the invention, the duration of the first physical compression can be any value within the range of 5-10 minutes, such as 5, 6, 7, 8, 9, or 10 minutes. In one implementation, the first physical compression is repeated 7 to 11 times; in a preferred embodiment, the first physical compression is repeated 8 to 10 times; in a specific embodiment of the present invention, the first physical compression can be repeated any value from 7 to 11 times, for example, 7, 8, 9, 10, or 11 times. The first physical compression of the present invention is performed under specific pressure, which can effectively break macrophages and protect the structure and function of membrane proteins; a suitable compression temperature can avoid protein thermal denaturation, reduce membrane fluidity, and achieve controllable macrophage rupture; the present invention, through repeated compression, can promote thorough mixing of membrane components and stabilization of vesicle structure, and make vesicle size uniform.

[0034] After obtaining the physically squeezed macrophages, the present invention performs a first density gradient separation on the physically squeezed macrophages to obtain macrophage biomimetic vesicles. As one embodiment, the temperature of the first density gradient separation is 50-60°C. In a specific embodiment of the present invention, the temperature of the first density gradient separation can be any value within the range of 50-60°C, such as 50, 52, 55, 58, or 60°C. As one embodiment, the first density gradient separation includes sequential centrifugation and filtration. As one embodiment, the centrifugation force is 800-1000g, and the time is 5-10 min. In a specific embodiment of the present invention, the centrifugation force can be any value within the range of 800-1000g, such as 800, 900, or 1000g; the time can be any value within the range of 5-10 min, such as 5, 6, 7, 8, 9, or 10 min. As one embodiment, the filtration is sequentially passing through PC filter membranes of 2μm, 800nm, and 200nm. In this invention, macrophage samples subjected to physical compression still contain a large number of organelle fragments, incompletely broken cytoskeleton, protein aggregates, and other impurities, in addition to the target vesicles. First density gradient separation can efficiently remove cell debris and impurities, improving vesicle purity. First density gradient separation involves centrifugation (800-1000g) at a specific temperature (50-60℃) combined with multi-stage filtration (2μm→800nm→200nm), which removes impurities of different sizes step by step. At a relatively low centrifugal force of 800-1000g, larger cell debris is preferentially precipitated, avoiding mechanical damage to the vesicle structure caused by high-speed centrifugation. The multi-stage filtration sequentially traps particles from micrometers to submicrometers, ultimately allowing vesicles with a diameter of approximately 200nm or less to pass through efficiently, significantly improving the uniformity and purity of the product. Macrophage-derived vesicle membranes are rich in lipids and proteins, and their physical properties are significantly affected by temperature. In this invention, the first density gradient separation process employs a heating treatment at 50-60°C, close to the cell membrane phase transition temperature. This increases the fluidity of the lipid bilayer, facilitating the re-closure and structural stability of vesicles after compression. Furthermore, this temperature can partially inactivate residual proteases or nucleases in the vesicles, reducing the degradation of vesicle contents (such as proteins and RNA) and maintaining their biological activity. This temperature range also promotes the denaturation and precipitation of certain heat-labile proteins, making them easier to remove by centrifugation and further improving vesicle purity.

[0035] The preparation of macrophage biomimetic vesicles described in this invention maintains the natural biological characteristics and functional integrity of the vesicles. Compared to chemical extraction methods (such as surfactant treatment) or extreme physical methods (such as repeated freeze-thaw cycles and ultrasonic disruption), the first density gradient separation method used in this invention eliminates the need for detergents or organic solvents, thus avoiding damage to the vesicle membrane structure or the introduction of exogenous toxic substances. Multi-stage filtration maximizes the retention of specific antigens on the vesicle surface (such as macrophage marker proteins CD86 and MHC-II), which is beneficial for subsequent immune-targeted applications. The intact membrane structure ensures that the substances carried within the vesicles remain active during delivery. Furthermore, this method is highly reproducible, with ranges set for parameters such as centrifugation force (800~1000g), time (5~10min), temperature (50~60℃), and filter membrane pore size, facilitating laboratory replication and future clinical translation. The combination of centrifugation and filtration steps removes impurities while achieving precise screening of vesicle size, ensuring a highly uniform particle size distribution (concentrated below 200nm) in the resulting vesicle population. The macrophage biomimetic vesicles obtained in this invention remove most of the cell debris, which can reduce the risk of non-specific immune activation; structurally intact vesicles are more easily loaded with therapeutic molecules (such as siRNA) through electroporation or incubation; and retain the inherent chemokine receptors and adhesion molecules on the macrophage membrane surface, giving them the potential to hom to sites of inflammation or tumor.

[0036] After obtaining macrophage biomimetic vesicles, this invention washes, filters, centrifuges, and resuspends the macrophage biomimetic vesicles to obtain a macrophage biomimetic vesicle suspension. As one embodiment, the washing agent is PBS buffer solution; in this invention, the sieve residue is collected after filtration for later use. Filtration removes impurities, and this invention does not have special requirements for the filtration; any filtration method well-known to those skilled in the art can be used. As one embodiment, the centrifugal force is 800-1000g, and the centrifugation time is 5-10min. In a specific embodiment of this invention, the centrifugal force can be any value within the range of 800-1000g, such as 800, 900, or 1000g; the centrifugation time can be any value within the range of 5-10min, such as 5, 6, 7, 8, 9, or 10min. As one embodiment, the resuspension includes resuspending the bottom buffer pad using PBS buffer solution. This invention washes, filters, centrifuges, and resuspends the obtained macrophage biomimetic vesicles, removing substances such as nutrient solution and fetal bovine serum, avoiding any impact on subsequent compression. The macrophage biomimetic vesicles prepared in this invention express CD47, which can resist macrophage phagocytosis and better enrich tumor sites.

[0037] After obtaining the macrophage biomimetic vesicle suspension, the present invention mixes the macrophage biomimetic vesicle suspension with a tumor-targeting peptide and performs a first co-incubation to obtain a first co-incubation solution. As one embodiment, the tumor-targeting peptide includes an RGD cyclic peptide, which has been described above and will not be repeated here. In a specific embodiment of the present invention, the RGD cyclic peptide is DSPE-PEG2000-cRGD, purchased from MCE, Cat. No: HY-P0278. As one embodiment, the mass ratio of the macrophage biomimetic vesicle to the tumor-targeting peptide has been described above and will not be repeated here. The mass ratio of the macrophage biomimetic vesicle to the tumor-targeting peptide in the present invention ensures that each vesicle surface is modified with a sufficient amount of cRGD molecules to achieve effective targeting, while avoiding over-modification that could lead to vesicle membrane instability or aggregation. In one implementation, the temperature of the first co-incubation is 25-37°C; in a preferred embodiment, the temperature of the first co-incubation is 28-35°C; in a specific embodiment of the present invention, the temperature of the first co-incubation can be any value within 25-37°C, such as 25, 27, 28, 30, 32, 35, or 37°C. The first co-incubation of the present invention is carried out at a physiologically compatible temperature of 25-37°C. Within this temperature range, the vesicle lipid membrane maintains suitable fluidity, which is conducive to the spontaneous insertion of DSPE-PEG2000-cRGD molecules into the membrane, achieving uniform and stable surface modification. Simultaneously, it can avoid denaturation of key functional proteins such as CD47 on the vesicle surface due to high temperature, ensuring that their immune escape function is not affected. In one implementation, the time of the first co-incubation is 25-30 min; in a preferred embodiment, the time of the first co-incubation is 26-28 min. In a specific embodiment of the present invention, the time of the first co-incubation can be any value within 25-30 min, such as 25, 26, 27, 28, 29, or 30 min. The first co-incubation time of this invention is 25-30 minutes, which allows the cRGD peptide to fully bind to the vesicle membrane, achieving the optimal modification density. This avoids vesicle aggregation or leakage that may occur with prolonged incubation. Furthermore, this duration facilitates rapid, mass production, meeting the timeliness requirements of translational medicine and promoting large-scale preparation. After the first co-incubation, the CD47 protein ("Don't eat me" signal) expressed on the surface of the macrophage-inspired vesicles can bind to the SIRPα receptor on the macrophage surface, inhibiting the clearance of vesicles by the mononuclear-macrophage system (MPS) and significantly prolonging their circulating half-life. cRGD modification enables vesicles to actively seek out and anchor to tumor cells, forming a synergistic effect of "prolonged circulation + precise accumulation," significantly improving drug enrichment efficiency at tumor sites (enhanced EPR effect) while reducing off-target toxicity to normal tissues. This constructs an intelligent delivery system with both "stealth" and "homing" characteristics.

[0038] After obtaining the first co-incubation solution, the present invention washes, filters, ultracentrifuges, and resuspends the first co-incubation solution to obtain a suspension of macrophage biomimetic vesicles modified with tumor-targeting peptides. As one embodiment, the washing agent is PBS buffer solution; the present invention has no special requirements for the filtration, and any filtration method well known to those skilled in the art can be used. The reaction system after the first co-incubation of the present invention contains a large number of unbound free cRGD peptides (DSPE-PEG2000-cRGD). If not completely removed, these free peptides will prematurely saturate the αvβ3 integrin receptor at the tumor site, competitively occupying the tumor target and weakening the actual targeting efficiency of the modified vesicles; and will also produce non-specific binding in non-tumor tissues, triggering off-target effects and increasing the risk of potential side effects. Washing, filtering, ultracentrifuging, and resuspending the first co-incubation solution helps to completely remove unbound free peptides, ensuring targeting specificity and safety. Specifically, the washing step, through buffer replacement, can dilute and remove most of the free peptides and incubation byproducts. The filtration step can trap a small number of large-particle aggregates that may form, ensuring the homogeneity of the sample after subsequent centrifugation, and can remove impurities and endotoxins introduced during co-incubation, reducing immunogenicity. As one embodiment, the centrifugal force of the ultracentrifugation is 10000~12000g. In a specific embodiment of the present invention, the centrifugal force of the ultracentrifugation can be any value within the range of 10000~12000g, such as 10000, 11000, or 12000g. As one embodiment, the ultracentrifugation time is 60~80 min. In a specific embodiment of the present invention, the ultracentrifugation time can be any value within the range of 60~80 min, such as 60, 65, 70, or 80 min. Ultracentrifugation can separate targeted modified vesicles, optimizing formulation homogeneity and stability. Under the ultracentrifugation parameters of this invention (10000~12000g, 60~80min), cRGD-modified vesicles can be effectively precipitated to the bottom of the tube, forming a dense cushion layer. Simultaneously, smaller, unbound free peptide molecules are retained in the supernatant and discarded, resulting in a high-concentration, high-purity product. Furthermore, the 10000~12000g centrifugation force ensures separation efficiency while minimizing physical damage to the vesicle membrane structure from high-speed shear forces, protecting its integrity and bioactivity. As one embodiment, the resuspension includes resuspending the bottom buffer pad using PBS buffer solution. In this invention, the buffer pad is the bottom precipitate after centrifugation. The bottom precipitate (buffer pad) collected after ultracentrifugation is rich in intact and successfully modified vesicle populations, achieving the conversion from the reaction mixture to a high-purity targeted formulation. The resuspension using PBS buffer solution maintains a neutral pH and isotonicity, ensuring the stability of the vesicles before storage and administration.Furthermore, by resuspending the precipitate in a quantitative buffer solution, a standardized suspension with known concentration and volume can be obtained, facilitating precise quantitative dosing and downstream characterization analysis, ensuring the accuracy and repeatability of the dosage in animal experiments or subsequent treatments; and providing consistent samples for particle size analysis (DLS), concentration determination (BCA protein quantification or NTA nanoparticle tracking), in vitro activity assessment, and in vivo activity assessment.

[0039] After obtaining the macrophage biomimetic vesicle suspension modified with the tumor-targeting peptide, the present invention mixes and extrudes the RGD-modified macrophage biomimetic vesicles with siRNA targeting NSUN family methyltransferases to obtain the macrophage biomimetic vesicle nanoformation. As one embodiment, the siRNA comprises liposomes loaded with siRNA. The siRNA described in this invention has been described above and will not be repeated here.

[0040] As one embodiment, the method for preparing the siRNA-loaded liposomes includes: mixing phosphatidylcholine (SPC), phospholipid-polyethylene glycol-peptide, cholesterol, and an organic solvent to form a film, thereby obtaining a film-forming material; mixing the film-forming material with siRNA and extruding the mixture to obtain siRNA-loaded liposomes. As one embodiment, the mass fraction of phosphatidylcholine is 1-2 parts; the mass fraction of phospholipid-polyethylene glycol-peptide is 1-2 parts; the mass fraction of cholesterol is 1-2 parts; the mass fraction of siRNA is 1-2 parts; and the mass fraction of the organic solvent is 1-3 parts. SPC provides the main lipid backbone, forming a bilayer structure; cholesterol enhances membrane rigidity, reduces permeability, significantly improves the stability of liposomes in blood, and reduces leakage of contents; the DSPE-PEG-peptide's PEG chain prolongs circulation time, and the peptide portion provides additional functions, such as targeting and membrane penetration, achieving multifunctional synergy; the organic solvent effectively dissolves all lipid components, ensuring uniform mixing at the molecular level, which is the basis for forming a homogeneous film. In one specific embodiment of the present invention, the phosphatidylcholine comprises 2 parts by mass; the phospholipid-polyethylene glycol-peptide comprises 1 part by mass; the cholesterol comprises 1 part by mass; the siRNA comprises 1 part by mass; and the organic solvent comprises 2 parts by mass. A slightly higher amount of SPC (main lipid) ensures the complete formation of the lipid bilayer; the amounts of DSPE-PEG-2K and cholesterol are moderate to balance stability and stealth function. The introduction of DSPE-PEG-2K achieves a "long-cycle" effect while avoiding the "target masking" effect that may result from excessive PEGylation; cholesterol significantly enhances membrane rigidity, improves the stability of liposomes during subsequent mixing and extrusion with macrophage vesicles, and reduces siRNA leakage; the siRNA, as the core load, is fixed at 1 part; the amount of chloroform is sufficient to completely dissolve all lipids. This ratio achieves the best balance between structural stability, drug loading efficiency, and subsequent functional performance. In this invention, the preferred type of phospholipid-polyethylene glycol-peptide is DSPE-PEG-2K, purchased from Xi'an Ruixi Biotechnology Co., Ltd. In one embodiment, the organic solvent is chloroform.

[0041] In one embodiment, the film formation method is rotary evaporation. Film uniformity is a key prerequisite for controlling liposome encapsulation efficiency and particle size distribution, directly affecting subsequent drug loading efficiency and batch-to-batch consistency of the formulation. This invention forms an extremely thin and uniformly thick lipid film on the inner wall of the rotary flask through a rotary evaporation process, ensuring that during subsequent hydration, lipid molecules can be synchronously and uniformly dispersed in the aqueous phase, spontaneously assembling into liposomes with uniform particle size and regular structure. In one embodiment, the rotary evaporation temperature is 55~65℃; the rotary evaporation rate is 120~200 rpm. In specific embodiments of this invention, the rotary evaporation temperature can be any value within the range of 55~65℃, for example, 55, 57, 60, 62, or 65℃; the rotary evaporation rate can be any value within the range of 120~200 rpm, for example, 120, 130, 150, 160, 180, or 200 rpm. This invention limits the rotary evaporation temperature to 55-65°C, ensuring that the lipids remain in a liquid crystal state during organic solvent evaporation and film formation, exhibiting loose molecular arrangement and high fluidity. Furthermore, this temperature range promotes stable bilayer formation; under suitable fluidity, the components (SPC, cholesterol, DSPE-PEG-peptide) can mix more uniformly and embed into the lipid bilayer, forming a highly stable liposome precursor membrane with high encapsulation efficiency. The resulting lipid film can hydrate and swell more fully and rapidly in the hydration medium (containing siRNA), efficiently encapsulating siRNA through electrostatic interactions or gradient loading. Compared to direct mixing methods, this process significantly improves the encapsulation rate of siRNA and reduces the residue of free siRNA, which is crucial for expensive and easily degradable nucleic acid drugs. Furthermore, rotary evaporation is a mature process with scalable and highly repeatable operation. Parameters (temperature, rotation speed, vacuum) are easily and precisely controlled, with minimal batch-to-batch variation, making it suitable for laboratory-scale optimization and transition to pilot-scale and production-scale applications. Compared to complex equipment such as microfluidics, rotary evaporators are widely used, low-cost, and easy to operate, making them easier to implement in most research and production institutions. Simultaneously, the resulting multilayer liposome film, after hydration, can be easily extruded (through a fixed-pore-size filter membrane) to obtain small monolayer liposomes with uniform particle size and good single-compartment characteristics, meeting the stringent requirements for nanoparticle size (typically <200 nm) for intravenous injection. The siRNA-loaded liposomes described in this invention exhibit high consistency in particle size, surface charge, membrane fluidity, and drug loading, which is beneficial for subsequent extrusion with RGD-modified macrophage biomimetic vesicles.

[0042] After obtaining the film-forming material, the present invention mixes the film-forming material with siRNA and extrudes it to obtain siRNA-loaded liposomes. As one embodiment, the mixing method is ultrasonic mixing; the ultrasonic mixing power is 50-100W, and the ultrasonic mixing time is 1-5 minutes. In a specific embodiment of the present invention, the ultrasonic mixing power can be any value within the range of 50-100W, for example, 50, 60, 70, 80, 90, or 100W; the ultrasonic mixing time can be any value within the range of 1-5 minutes, for example, 1, 2, 3, 4, or 5 minutes. The present invention uses ultrasonic mixing of the film-forming material and siRNA to ensure thorough mixing, which is beneficial for subsequent extrusion film formation. As one embodiment, the extrusion is performed using a liposome extruder.

[0043] After obtaining the liposomes loaded with siRNA, the present invention performs a second co-incubation with the tumor-targeting peptide-modified macrophage biomimetic vesicles and the siRNA-loaded liposomes to obtain a mixed suspension. As one embodiment, the mass ratio of the number of tumor-targeting peptide-modified macrophage biomimetic vesicles to the siRNA-loaded liposomes is 1 × 10⁻⁶. 6 Quantity: 100~200 μg. In a specific embodiment of the present invention, the ratio of the number of tumor-targeting peptide-modified macrophage biomimetic vesicles to the mass of siRNA-loaded liposomes is 1×10⁻⁶. 6 Pieces: Any proportion of 100~200μg, for example 1×10 6 100μg, 1×10 6 150μg or 1×10 6Quantity: 200 μg. The appropriate ratio of the number of tumor-targeting peptide-modified macrophage biomimetic vesicles to the mass of liposomes loaded with siRNA ensures that each multifunctional hybrid vesicle carries a sufficient and appropriately proportioned amount of siRNA therapeutic units, while maintaining optimal targeting ligand density on its surface, achieving a synergistic effect of 1+1>2. As one embodiment, the second co-incubation temperature is 25~37℃; as a preferred embodiment, the second co-incubation temperature is 28~35℃. In specific embodiments of the present invention, the second co-incubation temperature can be any value within the range of 25~37℃, for example, 25, 27, 28, 30, 32, 33, 35, or 37℃. As one embodiment, the second co-incubation time is 10~20 min; in specific embodiments of the present invention, the second co-incubation time can be any value within the range of 10~20 min, for example, 10, 12, 15, 17, 19, or 20 min. The 10-20 min incubation time of this invention is sufficient to complete effective pre-assembly, while avoiding particle aggregation or decreased stability that may be caused by excessively long incubation times. The temperature range of 25-37°C ensures that the membrane is in a suitable fluid state, which promotes interaction and forms a stable pre-fusion complex, while preventing protein denaturation or siRNA instability caused by high temperatures. The second co-incubation process can maximize the protection of the core activity of the two components and avoid damage to the modified RGD peptide and endogenous CD47 protein by drastic manipulation, ensuring the integrity of the dual functions of "active targeting" and "immune escape". It can also prevent siRNA from leaking or being degraded due to drastic environmental changes before fusion, ensuring the complete loading of siRNA. Furthermore, the well-defined incubation temperature, time, and ratio parameters can guarantee the performance consistency of different batches of nano-formulations.

[0044] After obtaining the mixed suspension, the present invention performs a second physical extrusion on the mixed suspension to obtain a physically extruded mixed suspension. In one embodiment, the pressure of the second physical extrusion is 500-1000 psi; in another embodiment, the pressure of the second physical extrusion is 600-800 psi. In a specific embodiment of the present invention, the pressure of the second physical extrusion can be any value within the range of 500-1000 psi, such as 500, 600, 700, 800, 900, or 1000 psi. In one embodiment, the temperature of the second physical extrusion is 50-60°C; in a specific embodiment of the present invention, the temperature of the second physical extrusion can be any value within the range of 50-60°C, such as 50, 52, 55, 56, 58, or 60°C. In one embodiment, the time of the second physical extrusion is 5-10 min; in a preferred embodiment, the time of the second physical extrusion is 6-8 min; in a specific embodiment of the present invention, the time of the second physical extrusion can be any value within the range of 5-10 min, such as 5, 6, 7, 8, 9, or 10 min. In one implementation, the second physical extrusion is repeated 7 to 11 times; preferably, it is repeated 8 to 10 times. In specific embodiments of the present invention, the number of repetitions of the second physical extrusion can be any value from 7 to 11, such as 7, 8, 9, 10, or 11 times. The second physical extrusion of the present invention involves forcing the mixed suspension formed in the second co-incubation through a filter membrane with a fixed pore size using a controllable mechanical force (500-1000 psi), thus completing the final membrane fusion and structural reorganization. At a temperature range of 50-60°C, the lipid bilayer is in a highly fluid state, significantly reducing the energy barrier required for membrane fusion. This allows for efficient fusion under relatively moderate pressure (600-800 psi), avoiding shear damage to siRNA or membrane proteins caused by excessive pressure, achieving gentle and thorough fusion. The present invention ensures that each nanoparticle in the suspension experiences equal mechanical force through repeated extrusion, resulting in a complete fusion reaction, a more concentrated particle size distribution in the final product, and avoiding the presence of unfused particles or excessively large aggregates. Compared to harsh methods such as ultrasound or repeated freeze-thaw cycles, the controllable extrusion shear force of this invention better preserves transmembrane proteins (such as CD47) and membrane anchoring peptides (cRGD), which is beneficial for maintaining their "don't eat me" signals and targeting functions. Furthermore, the extrusion process is completed rapidly in a closed system (total time 5-10 min), and temperature control avoids nuclease activation, minimizing siRNA leakage or degradation during fusion.The entire extrusion process can be carried out in a closed, disposable extruder or a strictly sterilized device, achieving aseptic operation and meeting the production requirements of injectable formulations. Furthermore, the novel hybrid vesicle structure formed by fusion is dense and has good membrane integrity. Its physical stability (such as anti-aggregation and anti-leakage) is superior to that of physical mixtures, which is more conducive to long-term storage and in vivo circulation. The process can be linearly scaled up for large-scale production.

[0045] After obtaining the physically squeezed mixed suspension, the present invention performs a second density gradient separation on the physically squeezed mixed suspension to obtain macrophage biomimetic vesicle nanoformulations. In one embodiment, the second density gradient separation includes sequential centrifugation and filtration. In one embodiment, the centrifugal force is 800-1000g, and the time is 5-10min; in a specific embodiment of the present invention, the centrifugal force can be any value within the range of 800-1000g, such as 800, 900, or 1000g; the centrifugation time can be any value within the range of 5-10min, such as 5, 6, 7, 8, 9, or 10min. In one embodiment, the filtration is sequentially passing through PC filter membranes of 2μm, 800nm, and 200nm. In the physically squeezed mixed suspension of the present invention, in addition to the target product, there may still be unfused free macrophage vesicles, unfused free drug-loaded liposomes, etc. The second density gradient separation can separate the macrophage biomimetic vesicle nanoformulations and remove unfused components and byproducts. Centrifugation, as a preliminary separation, precipitates the target product and removes impurities. A 2μm filter removes all residual debris of cellular size; an 800nm ​​filter removes any submicron-sized aggregates or larger, unfused vesicles; and a 200nm filter acts as a final check, ensuring that the nanoparticles passing through the filter are highly uniform in size (concentrated below 200nm), conforming to the optimal size range for intravenous injection, and maximizing impurity removal. Utilizing the second density gradient separation of this invention, particles >200nm, especially aggregates >1μm, completely eliminate the risk of capillary embolism that may arise from intravenous injection, effectively remove free liposome components or protein aggregates, reduce the potential immunogenicity of the formulation, and improve its in vivo tolerability. Furthermore, the entire separation process can be completed on a sterile operating table or in a closed system, and the final filtration step (using a sterile filter) itself is an effective sterilization filtration, directly yielding a sterile suspension suitable for in vivo experiments. The macrophage biomimetic vesicle nanoformulation prepared in this way has a high degree of consistency in particle size, drug loading, and surface target molecule density. Furthermore, the removal of impurities and aggregates greatly reduces the risk of further aggregation or instability.

[0046] As one implementation method, resuspending the macrophage biomimetic vesicle nanoformulation prepared in the above steps can yield a ready-to-use therapeutic product with a defined concentration, uniform particle size, sterility, and pyrogen-free properties. This product can be directly used in in vitro cell experiments and in vivo animal experiments, providing a unified and reliable starting point for subsequent research and development (such as stability studies, dosage exploration, and toxicology studies).

[0047] After obtaining the macrophage-inspired biomimetic vesicle nanoformulation, the present invention further includes: washing, filtering, ultracentrifuging, and resuspending the macrophage-inspired biomimetic vesicle nanoformulation to obtain a macrophage-inspired biomimetic vesicle nanoformulation suspension. After the second density gradient separation, unbound free cRGD peptides, trace particles detached from the filter membrane or container, density gradient medium, or buffer salt crystals may still remain in the formulation. These process residues can be removed and the target product purified through subsequent steps. As one embodiment, the washing agent is a PBS buffer solution; as another embodiment, the filtration includes passing through a 0.22 μm filter membrane. The present invention does not have special requirements for the filtration; any filtration method well known to those skilled in the art can be used. Washing displaces the suspension environment, diluting and removing soluble small molecule impurities and ions. Filtration intercepts occasional large particles or fibers that may be introduced due to centrifugation or operation, ensuring continuous particle size uniformity. In one embodiment, the centrifugal force of the ultracentrifugation is 10000~12000g. In a specific embodiment of the present invention, the centrifugal force of the ultracentrifugation can be any value within the range of 10000~12000g, such as 10000, 11000, or 12000g. In another embodiment, the ultracentrifugation time is 60~80 min. In a specific embodiment of the present invention, the ultracentrifugation time can be any value within the range of 60~80 min, such as 60, 65, 70, or 80 min. Ultracentrifugation is a crucial purification step. Under suitable optimized centrifugal force (10000~12000g) and time (60~80 min), the target nano-formulation will form a dense and stable precipitate, while lighter unbound lipid or protein fragments will be retained in the supernatant and completely discarded, thereby obtaining a nanoparticle precipitate of extremely high purity and improving the colloidal stability of the formulation. In one embodiment, the resuspension is performed using PBS buffer solution. Resuspension allows for the redispersibility of macrophage-inspired biomimetic vesicle nanoformulations into a homogeneous, isotonic suspension. This facilitates precise quantification (concentration, volume) and provides optimal starting material for lyophilization or long-term cryogenic storage, ensuring consistency in formulation performance across different batches and at different usage points. The preparation method described in this invention meets the quality control requirements for preclinical and clinical studies. Ultracentrifugation combined with strict aseptic techniques significantly reduces the endotoxin level of the formulation; terminal sterilization filtration (0.22 μm) ensures sterility, laying the foundation for subsequent in vivo drug delivery experiments. The series of steps—washing, filtration, ultracentrifugation, and resuspension—results in nanoformulations with uniform concentration and high purity. This provides material support for reliable in vitro pharmacodynamics (e.g., cell killing, gene silencing efficiency) and in vivo pharmacokinetic / pharmacodynamic (PK / PD) evaluations, and also meets the basic requirements of regulatory agencies for critical quality attribute (CQA) characterization of nanomedicines.

[0048] The macrophage-inspired biomimetic vesicle nanoparticles prepared using the method described in this invention exhibit a bilayer lipid membrane structure with a particle size of approximately 100-200 nm. Furthermore, the preparation method described in this invention is simple to operate, low in cost, and suitable for industrial-scale mass production.

[0049] As one embodiment, the preparation method of the macrophage biomimetic vesicle nanoformulation includes the following steps: mixing and extruding macrophage biomimetic vesicles with siRNA targeting NSUN family methyltransferases to obtain siRNA-loaded macrophage biomimetic vesicles; incubating the siRNA-loaded macrophage biomimetic vesicles with tumor-targeting peptides to obtain the macrophage biomimetic vesicle nanoformulation. The parameter settings in this preparation method have been discussed above and will not be repeated here. A schematic diagram of this preparation method is shown below. Figure 2 As shown.

[0050] This invention also provides the application of the macrophage-inspired biomimetic vesicle nanoformulation described above in the preparation of targeted drugs for gastric cancer. This invention utilizes tumor-targeting peptide-modified macrophage-inspired biomimetic vesicles to encapsulate siRNA targeting NSUN family methyltransferases; the two work synergistically to target and deliver the siRNA to the tumor, inhibiting NSUN2 expression levels in gastric cancer cells and reducing the m... 5 Abnormal C modification levels enhance the therapeutic effect of PD-1 antibody in gastric cancer treatment.

[0051] This invention also provides a gastric cancer targeted drug composition, comprising a tumor immune-related drug and the macrophage biomimetic vesicle nanoformulation described in the above-described scheme or the macrophage biomimetic vesicle nanoformulation prepared by the above-described preparation method. As one embodiment, the tumor immune-related drug includes at least one of a PD-1 inhibitor and a PD-L1 inhibitor. As one embodiment, the PD-1 inhibitor includes a PD-1 antibody. In specific embodiments of this invention, a PD-1 antibody is used as an example, but this does not constitute a limitation on the scope of protection of this invention. This invention constructs a multifunctional therapeutic platform integrating active targeted delivery, induction of immunogenic cell death (ferroptosis), and reversal of immunosuppression by combining macrophage biomimetic vesicle nanoformulation with Si-NSUN2. This design precisely addresses two major challenges currently faced in gastric cancer immunotherapy: low drug accumulation efficiency at the tumor site and the inhibitory state of the tumor immune microenvironment. The results of the examples show that the RAW264.7 biomimetic vesicle nanoformulation provided by this invention can specifically target gastric cancer cells, induce ferroptosis in gastric cancer cells, improve the efficacy of PD-1 antibodies, prolong the survival time of mice, and improve the tumor immune microenvironment.

[0052] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1 The materials and instruments used in this embodiment are as follows: RAW264.7 cell culture reagents: DMEM (Bioind, USA), fetal bovine serum (Gibco, USA), carbon dioxide incubator (Forma), serum-free culture medium (Excell, China); clean bench, benchtop centrifuge (Eppendorf, Germany), ultracentrifuge (Beckman, USA).

[0054] RGD-RNV@ si-NSUN2 Extraction reagents for biological agents: RGD materials, encapsulation si-NSUN2 Nanocomposites (Xi'an Ruixi Biotechnology Co., Ltd.), Mini-extrader extruder (Avanti Polar Lipids, USA), PC filter membranes with different pore sizes of 2μm, 800nm, and 200nm (Xi'an Ruixi Biotechnology Co., Ltd.), transmission electron microscope (FEITecnai 12, Philips), ultracentrifuge (Beckman, USA), and nanoparticle tracking analysis (nanosighttracking analysis, UK).

[0055] 1. Preparation of RAW264.7 biomimetic vesicle nanoformulation, the flowchart is as follows: Figure 1 As shown, the steps are as follows: (1) RAW264.7 cells (5×10⁻⁶) 6 RAW264.7 cells were physically extruded using a mini-extrader extruder at a pressure of 700 psi, a temperature of 55 °C, and a time of 6 min, with the extrusion repeated 8 times. The extruded RAW264.7 cells were then centrifuged at 1000 g for 5 min, and the precipitate was sequentially passed through PC filter membranes with different pore sizes of 2 μm, 800 nm, and 200 nm to prepare RAW264.7 biomimetic vesicles, denoted as RNVs. (2) The RAW264.7 biomimetic vesicles prepared in step (1) were centrifuged at 1000g for 10min (Eppendorf Centrifuge 5804 centrifuge), the bottom buffer pad was collected, and the suspension was prepared by rinsing with PBS to obtain the RAW264.7 biomimetic vesicle suspension. (3) Mix RAW264.7 biomimetic vesicle suspension with RGD cyclic peptide at a ratio of 5×10 6The mixture was prepared at a ratio of 100 μg to 100 μg and co-incubated at 37 °C for 30 min to obtain RGD-modified RAW264.7 biomimetic vesicles, denoted as RGD-RNV. (4) The RGD-RNV prepared in step (3) was washed with PBS and filtered through a 0.22 μm filter membrane to obtain the filtered suspension. The filtered suspension was centrifuged at 11000g for 70 min and the bottom buffer pad (i.e. the bottom precipitate) was resuspended to obtain the RGD modified RAW264.7 biomimetic vesicle suspension, which was denoted as RGD-RNV suspension. (5) Dissolve phosphatidylcholine (SPC), phosphatidyl-polyethylene glycol-peptide (DSPE-PEG-2K, purchased from MCE, Cat. No: HY-142979), and cholesterol in 4 mL of chloroform, and evaporate the solution at 65 °C and 200 rpm in a flask to form a film at the bottom of the flask to obtain the film-forming material; then add 2 mL of 100 μg / mL chloroform. si-NSUN2 The solution (purchased from Suzhou Gemma Gene Co., Ltd.) was sonicated at 100W for 5 minutes to obtain a sonicated mixture. This mixture was then extruded using a liposome extruder (specific parameters set according to the instruction manual) to obtain CL- si-NSUN2 Liposomes, the CL- si-NSUN2 Liposome loading si-NSUN2 , denoted as CL- si-NSUN2 The suspension contained in which the mass ratio of SPC:DSPE-PEG-2K:cholesterol:siRNA:chloroform was 2:1:1:1:2.

[0056] (6) The RGD-RNV suspension prepared in step (4) is mixed with the CL- from step (5). si-NSUN2 The suspension was prepared at a ratio of 1×10 6 The mixtures were prepared at a ratio of 200 μg and incubated at 37 °C for 20 min to obtain a mixed suspension. The suspension was then subjected to high-temperature physical extrusion. The extruded suspension was centrifuged at 1000 g for 5 min. The precipitate was then passed sequentially through PC filter membranes with different pore sizes of 2 μm, 800 nm, and 200 nm to obtain RGD-modified RAW264.7 biomimetic vesicles. si-NSUN2 Nano-suspension; high-temperature physical extrusion temperature is 60℃, pressure is 800psi, and time is 8min; (7) Load the RGD-modified RAW264.7 biomimetic vesicles from step (6) onto the material. si-NSUN2 The nano-suspension was washed with PBS, filtered through a 0.22 μm filter membrane, and centrifuged at 12000 g for 60 min using a Beckman ultracentrifuge. The precipitate was collected from the bottom buffer pad (bottom precipitate) and resuspended in PBS to obtain a macrophage biomimetic vesicle nanoformation (RGD-modified RAW264.7 biomimetic vesicles loaded with CL-). si-NSUN2Liposomes, denoted as RGD-RNV@ si-NSUN2 .

[0057] The obtained RGD-RNV@ si-NSUN2 Structural diagram as follows Figure 2 As shown. By Figure 2 It can be seen that RGD-RNV@ si- NSUN2 RAW264.7 nanovesicles encapsulated and loaded with RGD-modified material si-NSUN2 The nanostructure of the formulation.

[0058] 2. Add the AW264.7 biomimetic vesicles (RNV) and CL- from the above steps. si-NSUN2 Liposomes (denoted as liposomes) si-NSUN2 -CL) and RGD-RNV@ si-NSUN2 (referred to as RNV@) si-NSUN2 The morphological characteristics were observed using transmission electron microscopy and nanoparticle tracking analysis, as follows: RGD-RNV@ si-NSUN2 20 μL of solution was mixed and dropped onto a 2 mm diameter copper grid. After standing for 5 min, residual liquid was absorbed with filter paper. The copper grid was then inverted onto a drop of 30 g / L phosphotungstic acid (pH 6.8) solution and negatively stained at 25 °C for 5 min. The grid was then dried under an incandescent lamp and observed and photographed under a transmission electron microscope. RNV and si-NSUN2 -CL operation is the same as RGD-RNV@ si-NSUN2 The obtained transmission electron microscope image is as follows: Figure 3 As shown. By Figure 3 It can be seen that RGD-RNV@ si-NSUN2 It has a bilayer lipid membrane structure.

[0059] AW264.7 biomimetic vesicles, RGD-RNV@ si-NSUN2 The particle size distribution diagram is as follows Figure 4 As shown. By Figure 4 It can be seen that RGD-RNV@ si-NSUN2 It is a vesicle-like structure with a particle size of about 180 nm, and has similar biological vesicle properties to the biomimetic vesicles of macrophages.

[0060] Example 2 RGD-RNV@ si-NSUN2 Effect of being taken up by tumor cells The materials and instruments used in this embodiment are as follows: 12-well cell culture plates (JET Biofil), RPMI1640 (Bioind, USA), trypsin (Sigma, USA), DIO dye (MCE, China), high-resolution rapid scanning imaging system (BZ-X810, United States), Cy5-si-NSUN2 (Genepharma, China), and DAPI (Beyotime, China).

[0061] RGD-RNV@Cy5- was prepared according to the method in Example 1. si-NSUN2 The difference is that si-NSUN2 in step (5) is replaced with Cy5-si-NSUN2.

[0062] RGD-RNV@Cy5- was stained with DIO dye at a ratio of 1 μg:100 μg. si-NSUN2, Obtain DIO-stained RGD-RNV@Cy5- si-NSUN2 , denoted as DIO-RGD-RNV@Cy5- si-NSUN2 Its internal Si-NSUN2 has Cy5 fluorescence.

[0063] MFC cells were digested with trypsin and centrifuged at 800 rpm for 5 min. The resulting MFC cells were seeded into 12-well cell culture plates. After the cells were fully adhered, 100 μg of DIO-RGD-RNV@Cy5- was added. si-NSUN2 Add DIO-RGD-RNV@Cy5- si-NSUN2 After 24 hours of incubation, unbound DIO-RGD-RNV@Cy5- was washed away with PBS buffer. si- NSUN2, The cell nuclei were then stained with DAPI, following the instructions in the DAPI manual, and observed using a fluorescence microscope.

[0064] An intake experiment was conducted to observe the MFC's intake of RGD-RNV@Cy5- si-NSUN2 The ability, the result is as follows Figure 5 As shown. By Figure 5 It can be seen that RGD-RNV@Cy5- is promoted through RGD targeting. si-NSUN2 Upon entering tumor cells, the tumor cells express two types of fluorescence: red (Cy5-si-NSUN2) and green (DIO-RGD-RNV).

[0065] Example 3 RGD-RNV@ si-NSUN2 Inducing ferroptosis in gastric cancer cells The materials and instruments used in this embodiment are as follows: 12-well cell culture plates (JET Biofil), RPMI 1640 (Bioind, USA), trypsin (Sigma, USA), Fe... 2+ Ion assay kit (MCE, China), MDA assay kit (Solarbio, China), T-GSH assay kit (Solarbio, China), ROS assay kit (Beyotime, China), high-resolution rapid scanning imaging system (BZ-X810, United States), microplate reader (FLX800, United States).

[0066] MFC cells were digested with trypsin, and the resulting pellet was seeded into 12-well cell culture plates. The MFC cells were randomly divided into four groups as follows: ① PBS group: 200 μL of PBS was added; ② RNV group: 200 μg of RAW264.7 biomimetic vesicles were added; ③ RNV@ si-NSUN2 Group: Added 200 μg of RNV@ without RGD cyclic peptide modification si-NSUN2 The RNV@ si-NSUN2 The preparation method is the same as in Example 1, except that the RAW264.7 biomimetic vesicles are not modified with RGD cyclic peptides; ④RGD-RNV@ si- NSUN2 Group: Add 200μg RGD-RNV@ si-NSUN2 The additives in all four treatment groups were prepared from the same batch as in Example 1.

[0067] After 24 hours of treatment in each group, unbound material was washed away with PBS buffer. ， Subsequently, Fe in MFC was detected according to the kit instructions. 2+ Changes in the expression of MDA, ROS, and T-GSH were observed, as shown in the following figures. Figure 6 As shown in Table 1, the results are all average values, n=3.

[0068] Table 1 Fe in MFC cells 2+ MDA and T-GSH expression results

[0069] Depend on Figure 6 It can be seen that MFC ingests RGD-RNV@ si-NSUN2 MFC's Fe will be adjusted later. 2+ It reduced the expression of ROS and MDA, and downregulated the expression of T-GSH.

[0070] Example 4 RGD-RNV@ si-NSUN2 In vivo animal models The materials and instruments used in this embodiment are as follows: cell culture dish (JET Biofil), RPMI1640 (Bioind, USA), trypsin (Sigma, USA), 615 mice (male, 6-8 weeks old) (Nanjing Mairuisi, China), and PD-1 antibody (BioLegend, USA).

[0071] MFC cells were digested with trypsin, and the precipitate was collected by centrifugation at a concentration of 2×10⁻⁶. 7 One MFC cell was inoculated into 615 mice, and the mice were randomly divided into 5 groups. The specific operation was as follows: ①PBS group: 5 days after MFC cell seeding, 100μL PBS was injected via the tail vein, once every 5 days for a total of 5 injections; 10 days after injection, 100μg IgG (control antibody of PD-1 antibody) was injected intraperitoneally, once every 5 days for a total of 4 injections. ②RNV group: 5 days after MFC cell seeding, 100μg of RAW264.7 biomimetic vesicles were injected via the tail vein, once every 5 days, for a total of 5 injections; 10 days after injection, 100μg of IgG (control antibody against PD-1) was injected intraperitoneally, once every 5 days, for a total of 4 injections. ③RGD-RNV@ si-NSUN2 Group: Five days after MFC cell seeding, 100 μg RGD-RNV@ was injected via tail vein. si- NSUN2 Administer 100 μg IgG (PD-1 control antibody) intraperitoneally every 5 days for a total of 5 injections. Ten days after the injection, administer 100 μg IgG (PD-1 control antibody) intraperitoneally every 5 days for a total of 4 injections. ④PD-1 antibody group: 5 days after MFC cell seeding, 100μL PBS was injected via the tail vein, once every 5 days for a total of 5 injections; 10 days later, 100μg of PD-1 antibody was injected intraperitoneally, once every 5 days for a total of 4 injections. ⑤PD-1 antibody + RGD-RNV@ si-NSUN2 Group: Five days after MFC cell seeding, 100 μg of RGD-RNV@ was injected via tail vein. si-NSUN2, Administer 5 injections every 5 days for a total of 5 times; 10 days after the initial injections, administer 100 μg of PD-1 antibody intraperitoneally every 5 days for a total of 4 times, as per the procedure below. Figure 7 As shown in A; RGD-RNV@ was observed in a subcutaneous tumor-bearing experiment in 615 mice. si-NSUN2 The inhibitory effect on mouse tumors and whether it enhances the therapeutic effect of PD-1 antibodies were investigated. Tumor volume was measured, and the results are as follows: Figure 7 As shown in B~C and Table 2, the results are all average values, n=5.

[0072] Depend on Figure 7 It can be seen that mouse tumors take up RGD-RNV@ si-NSUN2 It will then inhibit tumor growth in mice and increase the therapeutic effect of PD-1 antibodies.

[0073] Table 2 Tumor volume in mice of each group

[0074] Example 5: Flow cytometry evaluation of RGD-RNV@ si-NSUN2 Sensitizing effect The raw materials and instruments used in this embodiment are as follows: CD45 antibody (BioLegend, USA), CD3 antibody (BioLegend, USA), CD8 antibody (BioLegend, USA), CD206 antibody (BioLegend, USA), CD86 antibody (BioLegend, USA), and F / 480 antibody (BioLegend, USA).

[0075] (1) Use scissors and tweezers to peel off the mouse tumor tissue, weigh it, and then cut it into pieces of 1-2 mm. 3 Small pieces of tumor tissue were collected and transferred to centrifuge tubes containing 1× collagenase. The tissue was digested in a 37°C water bath for 1 hour, shaking the tube every 10 minutes to ensure complete digestion. The tissue suspension was filtered through a 70μm cell sieve and transferred to a new centrifuge tube, where it was filtered again through a 30μm cell sieve. The resulting cell suspension was centrifuged at 500g for 5 minutes at 4°C. After discarding the supernatant, erythrocytes were lysed with 1mL ACK (Formax) for 5 minutes, followed by the addition of an equal volume of culture medium to terminate the lysis. The cells were then centrifuged at 300g for 8 minutes, the supernatant was discarded, and the cell pellet was resuspended in 10mL PBS. After counting, the cells were placed on ice for later use. (2) Count the single-cell suspension prepared in step (1), and count 2×10 6 Each cell was transferred to a 1.5 mL centrifuge tube and resuspended in 100 μL of PBS. The corresponding flow cytometry antibody was added to each cell suspension. RGD-RNV@ was evaluated using flow cytometry. si-NSUN2 The therapeutic effect of PD-1 antibody on mouse tumors. Results are as follows: Figures 8-9 As shown in Table 3, the experiment was repeated 3 times, and the results are all average values.

[0076] Table 3 Flow Cytometry Detection Results

[0077] Depend on Figures 8-9 It can be seen that mouse tumors take up RGD-RNV@ si-NSUN2 GZMB was added later. + CD8 +T cell and M1 macrophage infiltration were inhibited, while M2 macrophage infiltration was suppressed.

[0078] Example 6 RGD-RNV@ si-NSUN2 Biosafety Analysis The raw materials and instruments used in this embodiment are as follows: polyclonal antibody, biotin-labeled goat anti-rabbit IgG, SABC reagent, and DAB chromogenic solution (all purchased from Wuhan Sanying Biotechnology Co., Ltd., PK10006).

[0079] (1) Mouse tumor tissue was sent to Seville for embedding and sectioning to obtain paraffin sections; the paraffin sections were fixed in a 60℃ oven for 1-2 hours; the sections were then dewaxed by immersing them in the corresponding solutions in the following order: xylene 15 min, xylene 15 min, 100% ethanol 2 min, 95% ethanol 2 min, 85% ethanol 2 min, 75% ethanol 2 min, ddH2O 2 min; 3% H2O2, incubated at 37℃ for 20-30 min to inactivate endogenous enzymes in the tissue. After incubation, the H2O2 was removed, and the sections were washed three times with PBS for 5 min each time; (2) Prepare 0.01M citrate buffer. Place the paraffin sections from step (1) in a histochemistry cassette, pour in the citrate buffer, and boil in a water bath for 30 minutes for antigen retrieval. After natural cooling, wash twice with PBS for 5 minutes each time; spin dry the sections, circle them with a histochemistry pen, add 5% BSA, and block at room temperature for 30 minutes; remove the blocking solution, place the sections in a humidified chamber, and directly add polyclonal antibody diluted 1:50 by volume, and incubate overnight at 4°C. Wash three times with PBS for 5 minutes each time; spin dry the sections, add 1 drop of biotin-labeled goat anti-rabbit IgG, incubate at 37°C for 1 hour, and wash three times with PBS. Add SABC reagent and incubate at room temperature for 20 min. Wash with PBS 4 times, 5 min each time. After adjusting the white balance of the microscope, add DAB chromogenic solution and observe under the microscope. When the tissue block begins to turn yellow, immediately place it in water to stop the chromogenic process. Record the staining time to ensure that the staining time of all sections is consistent. Counterstain with hematoxylin for 30 s and rinse with running water. Dehydrate the sections in the reverse process of dewaxing. (3) Mount the slide with neutral resin and cover with a coverslip. After the slide is completely dry, observe the staining results under a microscope. The results are as follows: Figure 10 As shown.

[0080] according to Figure 10 It can be seen that RGD-RNV@ si-NSUN2 It does not harm the mice themselves while inhibiting tumor growth.

[0081] In summary, the RAW264.7 biomimetic vesicle nanoformulation provided by this invention can specifically target gastric cancer cells, induce ferroptosis in gastric cancer cells, improve the efficacy of PD-1 antibody, prolong the survival time of mice, and improve the tumor immune microenvironment.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A macrophage-inspired biomimetic vesicle nanoformulation, characterized in that, The macrophage biomimetic vesicle nanoformulation includes macrophage biomimetic vesicles, tumor-targeting peptides, and siRNA targeting NSUN family methyltransferases. The tumor-targeting peptide is modified on the surface of macrophage biomimetic vesicles; the siRNA is loaded into macrophage biomimetic vesicles.

2. The macrophage-inspired biomimetic vesicle nanoformulation according to claim 1, characterized in that, The macrophages include RAW264.7 cells; the siRNA includes targeted... NSUN2 Genetic si-NSUN2 .

3. The macrophage-inspired biomimetic vesicle nanoformulation according to claim 1, characterized in that, The tumor-targeting peptides include those targeting gastric tumors.

4. The macrophage-inspired biomimetic vesicle nanoformulation according to claim 1, characterized in that, The siRNA includes liposomes loaded with siRNA.

5. The macrophage-inspired biomimetic vesicle nanoformulation according to claim 4, characterized in that, The mass ratio of the macrophage biomimetic vesicles to the siRNA-loaded liposomes is 1 × 10⁻⁶. 6 Quantity: 100~200μg.

6. The macrophage-inspired biomimetic vesicle nanoformulation according to claim 5, characterized in that, The mass ratio of the macrophage biomimetic vesicles to the tumor-targeting peptides is 1×10⁻⁶. 6 ~5×10 6 100μg.

7. The macrophage-inspired biomimetic vesicle nanoformulation according to claim 1, characterized in that, The macrophage-inspired biomimetic vesicle nanoparticles have a particle size of 100-200 nm.

8. The method for preparing the macrophage biomimetic vesicle nanoformulation according to any one of claims 1 to 7, characterized in that, The process includes the following steps: modifying macrophage biomimetic vesicles with tumor-targeting peptides to obtain tumor-targeting peptide-modified macrophage biomimetic vesicles; The macrophage biomimetic vesicles modified with the tumor-targeting peptide were mixed with siRNA targeting NSUN family methyltransferases and extruded to obtain the macrophage biomimetic vesicle nanoformation.

9. The application of the macrophage biomimetic vesicle nanoformulation according to any one of claims 1 to 7 or the macrophage biomimetic vesicle nanoformulation prepared by the preparation method according to claim 8 in the preparation of gastric cancer targeted drugs.

10. A targeted drug composition for gastric cancer, characterized in that, This includes tumor immunotherapy-related drugs and macrophage biomimetic vesicle nanoformulations as described in any one of claims 1 to 7, or macrophage biomimetic vesicle nanoformulations prepared by the preparation method described in claim 8.

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