Drug-loaded hybrid membrane system for targeting tumor-associated macrophages as well as preparation method and application of drug-loaded hybrid membrane system

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a drug-loaded hybrid membrane system for targeting tumor-associated macrophages as well as a preparation method and application of the drug-loaded hybrid membrane system. The invention discloses a hybrid membrane carrier for targeting tumor-associated macrophages. The hybrid membrane carrier is formed by fusing attenuated salmonella typhimurium ghost and an M2pep peptide modified macrophage membrane. The invention also discloses a drug-loaded hybrid membrane system for targeting tumor-associated macrophages. The drug-loaded hybrid membrane system comprises a hybrid membrane carrier and a drug which is loaded in the hybrid membrane carrier and is used for immunoregulation / tumor resistance. The drug-loaded hybrid membrane system provided by the invention integrates the advantages of active targeting, homologous targeting, macrophage specific targeting, biocompatibility, immune activation efficiency and the like, and develops a new treatment mode for malignant tumors with low immunotherapy effect rate.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug-loaded hybrid membrane system targeting tumor-associated macrophages, its preparation method, and its application. Background Technology

[0002] Current treatment options for lung cancer include surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and traditional Chinese medicine. However, traditional treatments often suffer from poor patient tolerance and severe side effects, and are limited by the immunosuppressive microenvironment, resulting in suboptimal efficacy.

[0003] Multiple factors in the tumor microenvironment (such as cytokines and metabolites secreted by tumor cells, and hypoxia) tend to polarize tumor-associated macrophages (TAMs) into the M2 type, which possesses immunosuppressive and pro-tumor properties. This polarization shift is accompanied by a significant weakening of TAMs' key anti-tumor functions, with a particularly pronounced decrease in phagocytic capacity. The weakened phagocytic function of TAMs directly impairs the body's ability to clear malignant cells, promotes tumor immune escape, and indirectly supports tumor growth, invasion, metastasis, and angiogenesis. Therefore, reversing the immunosuppressive phenotype of TAMs, especially restoring and enhancing their ability to phagocytose and clear tumor cells, is considered one of the key strategies for reshaping the anti-tumor immune response and overcoming current limitations in immunotherapy.

[0004] In lung cancer treatment, although existing drug delivery systems are used for the delivery of various drugs, they have significant drawbacks: poor targeting, mostly relying on the EPR effect for passive targeting, with a tumor accumulation rate of less than 5%, and unable to accurately target M2 type TAMs, making it difficult to reverse the immunosuppressive microenvironment; drug release is difficult to respond to lung cancer microenvironment signals, and is prone to premature leakage or incomplete release, affecting the TAMs regulatory effect.

[0005] Therefore, there is a need for a drug delivery system that can efficiently target tumors, reverse the phagocytic phenotype of TAMs, and effectively activate local tumor immunity. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a drug-loaded hybrid membrane system that targets tumor-associated macrophages, its preparation method and application, so as to solve the problems of poor targeting of the carrier system and low efficacy of existing tumor immunotherapy in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] This invention provides a hybrid membrane carrier that targets tumor-associated macrophages. The hybrid membrane carrier is composed of a shell formed by fusing attenuated Salmonella typhi and macrophage membranes modified with M2pep peptide.

[0009] The present invention also provides a method for preparing a hybrid membrane carrier targeting tumor-associated macrophages, comprising: mixing macrophage membranes modified with M2pep peptides with attenuated Salmonella typhi at a mass ratio of 1 to 2:1, and extruding the mixture through a filter membrane to obtain the hybrid membrane carrier.

[0010] In some embodiments of the present invention, the mass ratio of the M2pep peptide-modified macrophage membrane to the attenuated Salmonella typhi shadow is 1:1, 1.5:1, or 2:1.

[0011] In some technical solutions of the present invention, the preparation method of the macrophage membrane modified by M2pep peptide includes: mixing M2pep peptide and macrophage membrane at a mass ratio of 1:8~15, and then acting on it under an ultrasonic power of 100W~200W for 5min~15min to obtain the macrophage membrane modified by M2pep peptide.

[0012] The method for preparing the attenuated Salmonella typhi bacterial shadow includes: centrifuging attenuated Salmonella typhi at 5000 rpm to 7000 rpm for 8 min to 12 min and collecting the bacterial cells; then treating the bacterial cells with 0.5 to 1 mg / mL NaOH solution under shaking conditions at 150 rpm to 250 rpm for 3 to 5 hours; after treatment, centrifuging at 5000 rpm to 7000 rpm for 8 min to 12 min, collecting the precipitate, and washing it twice with sterile PBS to finally obtain the attenuated Salmonella typhi bacterial shadow.

[0013] In some embodiments of the present invention, the mass ratio of the M2pep peptide to the macrophage membrane is 1:8, 1:10, 1:12 or 1:15.

[0014] In some specific embodiments of the present invention, the method for preparing the attenuated Salmonella typhi bacterial shadow includes: centrifuging attenuated Salmonella typhi at 6000 rpm for 10 min and collecting the bacterial cells; then treating the bacterial cells with NaOH solution under shaking conditions at 200 rpm for 4 hours; after the treatment, centrifuging at 6000 rpm for 10 min, collecting the precipitate, and washing it twice with sterile PBS to finally obtain the attenuated Salmonella typhi bacterial shadow.

[0015] In some embodiments of the present invention, the attenuated Salmonella typhi is strain VNP20009.

[0016] In some technical solutions of the present invention, during the preparation process of the hybrid membrane carrier, a 500nm~800nm ​​filter membrane is extruded 20 to 40 times to obtain the hybrid membrane carrier.

[0017] The present invention also provides a drug-loaded hybrid membrane system for targeting tumor-associated macrophages, the drug-loaded hybrid membrane system for targeting tumor-associated macrophages comprising the hybrid membrane carrier and an immunomodulatory / antitumor drug loaded in the hybrid membrane carrier.

[0018] In some technical solutions of the present invention, the drugs for immunomodulation / anti-tumor include: paeonol, curcumin, resveratrol, CSF-1R inhibitor, TLR agonist, CD40 agonist, CD47 inhibitor, PD-L1 inhibitor, doxorubicin, paclitaxel or gemcitabine.

[0019] Currently, there is a lack of simple and efficient formulations that can deliver drugs to tumor sites and effectively activate local tumor immunity. Therefore, this invention provides a drug-loaded hybrid membrane system targeting tumor-associated macrophages (TAMs). By combining M2pep peptide-modified macrophage membrane-mediated TAM targeting and the immunomodulatory effects of paeonol, the system significantly enhances drug accumulation at the tumor site and effectively promotes TAM phagocytosis, transforming it from an immunosuppressive to an anti-tumor agent. Simultaneously, *Bacterium acnes*, as a natural immune activator, further enhances the tumor immune response, forming a synergistic effect of targeted delivery, TAM reprogramming, and immune activation. This strategy provides a safe and efficient novel platform for lung cancer immunotherapy, with excellent clinical translational potential.

[0020] Previous studies have shown that paeonol can promote the phagocytic capacity of macrophages and has potential in immunomodulation and the treatment of related diseases. However, paeonol has a short biological half-life and low bioavailability, which are pharmacokinetic defects. In addition, it lacks tissue targeting and is prone to non-specific distribution. These characteristics seriously limit its clinical translation and application.

[0021] The drug-loaded hybrid membrane system for targeting tumor-associated macrophages constructed in this invention is used for the efficient loading of paeonol and the realization of tumor-targeted immunomodulation. This system integrates the natural tumor-homing properties of macrophage membranes, enabling precise recognition of tumor antigens and targeting of tumor sites, thus overcoming the weakness of bacterial shadow (BGs) targeting. BGs, as empty shell structures of Gram-negative bacteria, retain complete bacterial antigen and adjuvant properties, effectively activating immune responses and enhancing anti-tumor effects, while compensating for the weak immunogenicity of macrophage membranes. Furthermore, BGs have a large internal space, allowing for efficient loading of paeonol. Paeonol, as a lipophilic drug, can be efficiently encapsulated in this hybrid system and, by promoting the phagocytic function of tumor-associated macrophages (TAMs), induces their polarization from immunosuppressive (M2 type) to anti-tumor (M1 type), thereby directly reversing the immunosuppressive state of the tumor microenvironment (TME). To further improve targeting efficiency, this invention introduces the M2pep peptide, which, by leveraging its specific recognition ability of TAMs, enhances the enrichment of the system at the tumor site, ensuring that the drug and immunomodulatory components are precisely accumulated in the target area and exert a synergistic anti-tumor effect.

[0022] The present invention also discloses a method for preparing the drug-loaded hybrid membrane system targeting tumor-associated macrophages, comprising: mixing the drug for immunomodulation / antitumor with the hybrid membrane carrier at a mass ratio of 1:1 to 2, and extruding the mixture through a filter membrane to obtain the drug-loaded hybrid membrane system targeting tumor-associated macrophages.

[0023] In some embodiments of the present invention, the drug for immunomodulation / antitumor is mixed with the hybrid membrane carrier at a mass ratio of 1:1, 1:1.5 or 1:2.

[0024] In some embodiments of the present invention, in the preparation method of the drug-loaded hybrid membrane system targeting tumor-associated macrophages, a 500nm~800nm ​​filter membrane is squeezed 20~40 times to obtain the drug-loaded hybrid membrane system targeting tumor-associated macrophages.

[0025] In some embodiments of the present invention, after the drug for immunomodulation / antitumor is prepared into a dosage form that can effectively load hydrophobic drugs and form stable particles, it is assembled with a hybrid membrane carrier to prepare a drug-loaded hybrid membrane system targeting tumor-associated macrophages.

[0026] In some embodiments of the present invention, the drug for immunomodulation / antitumor includes paeonol; the paeonol is prepared into paeonol liposomes and then the drug delivery system is assembled.

[0027] In some embodiments of the present invention, the method for preparing the paeonol liposomes includes: dissolving the lipid components and paeonol in an organic solvent to form an oil phase; then removing the organic solvent from the oil phase to form a uniform film of lipids; and then adding an aqueous phase to allow the film to absorb water, swell, and disperse, ultimately forming a suspension of paeonol liposomes.

[0028] In some embodiments of the present invention, the paeonol liposomes have a drug loading of 10% to 15%, an encapsulation rate of 85% to 95%, and a particle size of 150 nm to 250 nm.

[0029] In this invention, the drug loading of paeonol in paeonol liposomes refers to the weight percentage of paeonol drug within the total weight of the liposomes themselves. The calculation formula is: (weight of paeonol in the liposomes / total weight of the liposomes) × 100%. Encapsulation efficiency refers to the percentage of drug successfully encapsulated within the liposomes relative to the initial total drug dosage. High encapsulation efficiency is crucial for ensuring drug delivery efficiency and reducing drug waste and toxic side effects.

[0030] In some embodiments of the present invention, the lipid components include soybean phospholipids and cholesterol.

[0031] In some embodiments of the present invention, the mass ratio of soybean phospholipids, cholesterol, and paeonol is 3~5:1~2:1.

[0032] In some embodiments of the present invention, the organic solvent is selected from low-toxicity and easily removable organic solvents, including but not limited to anhydrous ethanol and tert-butanol. Preferably, anhydrous ethanol is used as the organic solvent in the present invention. Firstly, anhydrous ethanol has a lower boiling point and better solubility at room temperature, allowing for faster and more complete removal without additional heating, thus improving preparation efficiency and avoiding high-temperature degradation of paeonol activity. Secondly, anhydrous ethanol does not solidify at low temperatures, making it easier to form a stable lipid film.

[0033] In some embodiments of the present invention, the preparation method of paeonol liposomes further includes a step of thoroughly removing organic solvents using rotary evaporation or vacuum drying to ensure that the final formulation meets the safety standards for residual solvents for injection. Specifically, the present invention uses vacuum drying to remove anhydrous ethanol.

[0034] In some embodiments of the present invention, the drug loading of paeonol in the drug-loaded hybrid membrane system targeting tumor-associated macrophages is 3.5% to 4.5%, and the particle size of the drug-loaded hybrid membrane system targeting tumor-associated macrophages is 400 nm to 600 nm.

[0035] In this invention, the drug loading of paeonol in the drug-loaded hybrid membrane system targeting tumor-associated macrophages refers to the weight percentage of paeonol in the total weight of the entire drug-loaded hybrid membrane system targeting tumor-associated macrophages. The calculation formula is: (weight of paeonol in the entire system / total weight of the drug-loaded hybrid membrane system) × 100%.

[0036] The present invention also discloses the application of the drug-loaded hybrid membrane carrier targeting tumor-associated macrophages in the preparation of drugs for treating lung cancer.

[0037] The present invention also discloses the application of the drug-loaded hybrid membrane system targeting tumor-associated macrophages in the preparation of drugs for treating lung cancer.

[0038] In some embodiments of the present invention, the drug is a drug-loaded hybrid membrane system that targets tumor-associated macrophages as the sole active ingredient, and the drug also includes a pharmaceutically acceptable carrier.

[0039] The drug-carrying hybrid membrane system targeting tumor-associated macrophages carries paeonol.

[0040] In some embodiments of the present invention, the dosage form of the drug is selected from injections or emulsions; wherein, the injections include lyophilized powder injections and suspensions for injection.

[0041] Based on the technical solution of this invention, compared with the prior art, this invention has the following beneficial effects: Addressing the problem of poor targeting of free drugs in existing technologies, this invention fuses M2pep-modified macrophage membranes with Salmonella shadows to form a hybrid membrane carrier for targeted drug delivery. This hybrid membrane carrier integrates advantages such as active targeting, homologous targeting, macrophage-specific targeting, and consideration of both biocompatibility and immune activation efficacy, opening up a new treatment modality for malignant tumors with low immunotherapy response rates. The phospholipid bilayer of liposomes includes hydrophobic and hydrophilic ends; lipophilic drugs can be coated on the hydrophobic layer of the liposomes, thereby improving the drug's water solubility and enhancing its anti-tumor effect.

[0042] The drug-loaded hybrid membrane system provided in this invention exhibits excellent biocompatibility and stability, demonstrating superior targeting ability for tumor-associated immunotherapies (TAMs) both in vivo and in vitro. Through TAM-targeted recognition mediated by M2pep peptide-modified macrophage membranes and the immunomodulatory effects of paeonol, the system significantly enhances drug accumulation at tumor sites and effectively promotes TAM phagocytosis, transforming it from an immunosuppressive to an anti-tumor agent. Simultaneously, *Bacterium acnes*, as a natural immune activator, further enhances the tumor immune response, forming a synergistic effect of targeted delivery, TAM reprogramming, and immune activation. This strategy provides a safe and efficient novel platform for lung cancer immunotherapy, possessing significant clinical translational potential. Attached Figure Description

[0043] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0044] Figure 1 The preparation processes for mBG in Example 1 and mPAE@BG in Example 2 of this invention are shown below.

[0045] Figure 2 This is a characterization result diagram of the mBG obtained in Example 1 of the present invention; wherein, Figure 2 In the figure, A represents the agarose gel electrophoresis image of the lysed BG sample obtained after treating VNP20009 with 0.75 mg / mL NaOH for different times; Figure 2 In the image, B represents the representative scanning electron microscope (SEM) image (left) and transmission electron microscope (TEM) image (right) of BG. Figure 2 In the image, C represents the fluorescent colocalization image of BG (red) labeled with Salmonella-specific antibody and Dil labeled macrophage membrane (green); Figure 2 In this image, D represents a representative transmission electron microscope (TEM) image of mBG. Figure 2 In the figure, E represents the statistical results of dynamic light scattering (DLS), polydispersity index (PDI), and zeta potential of mBG.

[0046] Figure 3 This is a characterization result diagram of mPAE@BG in Embodiment 2 of the present invention; wherein, Figure 3 In the image, A represents a confocal laser scanning microscope (CLSM) image of mPAE@BG with Dil-labeled cell membrane (green) and C6-labeled liposomes (red); Figure 3 In this context, B represents a representative transmission electron microscope image of mPAE@BG; Figure 3 In the figure, C represents the statistical results of dynamic light scattering (DLS), polydispersity index (PDI), and zeta potential of mPAE@BG; Figure 3 In the diagram, D represents a representative high-performance liquid chromatography (HPLC) image of mPAE@BG. Figure 3 The "E" in the figure represents the statistical results of the relative cell viability of BMDM at different mPAE@BG concentrations as determined in the CCK8 cytotoxicity assay of mPAE@BG. Figure 3 The F in the figure represents the statistical results of hemolysis rate when mPAE@BG at different concentrations is co-incubated with red blood cells.

[0047] Figure 4 This is a graph showing the results of uptake efficiency testing during in vitro targeted function validation; where, Figure 4The "A" in the figure represents the fluorescence microscopy results of mPAE@BG, mBG, and BG labeled with Dil fluorescent dye and co-incubated with TAMs. The distribution of different formulations in TAMs can be seen intuitively (red fluorescent part). Figure 4 In the figure, B represents the statistical results of the average fluorescence intensity of cells in different treatment groups detected by flow cytometry.

[0048] Figure 5 This is a graph showing the detection results of TAMs phagocytic function in in vitro targeted functional validation; among them, Figure 5 In this context, A represents the result of flow cytometry analysis of TAMs treated with different formulations (PBS, BG, mBG, mPAE@BG) phagocytosing Lewis lung cancer cells labeled with CFSE fluorescent dye. + Peak diagram of cell fluorescence distribution; Figure 5 In the figure, B represents a bar chart showing the phagocytic capacity of TAMs against tumor cells (expressed as a percentage) under different formulation treatments.

[0049] Figure 6 This is a diagram showing the results of mPAE@BG targeting and in vivo biodistribution in C57BL / 6J tumor-bearing mice in this embodiment of the invention; wherein, Figure 6 In the image, A represents the result of in vivo fluorescence imaging; Figure 6 In the figure, B represents the statistical results of the average fluorescence intensity at different times during in vivo fluorescence imaging.

[0050] Figure 7 This is a diagram showing the results of fluorescence imaging analysis of ex vivo tumor tissue; among which, Figure 7 In the image, A represents the fluorescence imaging result of ex vivo tumor tissue; Figure 7 In the figure, B represents the statistical results of the average fluorescence intensity at different times during in vitro fluorescence imaging.

[0051] Figure 8 This is a graph showing the results of immunofluorescence analysis of tumor tissue; in which, Figure 8 In the image, A represents the IF staining result of a tumor section 24 hours after injection; Figure 8 In the figure, B represents the statistical results of quantitative analysis of mBG fluorescence signal labeled with DiR; Figure 8 The C in the figure represents the fluorescence gray value distribution curve of the BG group, which shows the co-localization analysis of the fluorescence signals of DiR (red) and anti-CD206 antibody (green); Figure 8 In the figure, D represents the fluorescence gray value distribution curve of the mBG group, which shows the co-localization analysis of the fluorescence signals of DiR (red) and anti-CD206 antibody (green).

[0052] Figure 9 A schematic diagram of the dosing regimen for systematically evaluating the antitumor efficacy of mPAE@BG.

[0053] Figure 10 The graph shows the tumor volume and weight of mice in different treatment groups after drug administration; among them, Figure 10 In the figure, A represents the tumor volume growth curve of mice implanted with tumors in different treatment groups; Figure 10 In the image, B represents a photograph of tumor tissue collected from mice treated with different methods on day 15 post-treatment. Figure 10 The figure shows the statistical results of tumor weight implanted in mice of different treatment groups on day 15 after treatment (C represents the tumor weight of mice implanted in different treatment groups).

[0054] Figure 11 Images showing the results of hematoxylin-eosin staining of tumor tissues from different treatment groups.

[0055] Figure 12 The graph shows the proportions of M1 and M2 macrophages in different groups, as well as the changes in the ratio of M1 to M2 macrophages; among them, Figure 12 In this context, A represents different groups of M1 macrophages (CD45). + CD11b + F4 / 80 + CD86 + The statistical results are shown in the graph. Figure 12 In this context, B represents different groups of M2 type macrophages (CD45). + CD11b + F4 / 80 + CD206 + The statistical results are shown in the graph. Figure 12 The "C" in the figure represents the bar chart showing the statistical results of the changes in the proportion of M1 and M2 macrophages in different groups.

[0056] Figure 13 In different groups (G1-G5), cytotoxic T lymphocytes (CTLs, CD3) + CD8 + The proportion of ) and helper T cells (CD3) + CD4 + The proportion of CD4 + / CD8 + The results of the ratio determination are shown in the figure; among them, Figure 13 In this context, A represents cytotoxic T lymphocytes (CTLs, CD3) in different groups. + CD8 + A bar chart showing the proportion of [missing information]. Figure 13 In this context, B represents the number of helper T cells (CD3+) in different groups. + CD4 + A bar chart showing the proportion of [missing information]. Figure 13 In this context, C represents different groups, CD4+ / CD8 + A bar chart showing the statistical results of the ratios.

[0057] Figure 14 This is a graph showing the results of measuring immunostimulatory cytokines in mouse serum using an enzyme-linked immunosorbent assay (ELISA); where, Figure 14 In the figure, A represents the statistical results of IFN-γ expression in serum; Figure 14 In the figure, B represents the statistical results of TNF-α expression in serum; Figure 14 The C in the figure represents the statistical results of IL-12p70 expression in serum. Detailed Implementation

[0058] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0059] The following description is based on specific embodiments.

[0060] Example 1: Preparation and characterization of a hybrid membrane carrier (mBG) targeting tumor-associated macrophages

[0061] 1. Preparation and characterization of M2pep peptide-modified macrophage membrane-bacterial hybrid membrane carrier (mBG)

[0062] Step 1: Preparation of macrophage membrane modified with M2pep peptide

[0063] (1) Extraction of Raw264.7 macrophage membrane components: The extraction of Raw264.7 macrophages was performed according to the instructions of the membrane protein extraction kit (manufacturer: Beyotime; catalog number: P0033). Specifically, Raw264.7 macrophages in good growth condition were collected. After the cells reached confluence, the cells were collected using a cell scraper and centrifuged at 600g and 4℃ for 5 min. The supernatant was discarded. The cells were then washed twice with pre-cooled PBS buffer (pH=7.4) and resuspended in membrane protein extraction reagent A containing 1mM PMSF. The mixture was incubated on ice for 15 min. The mixture was repeatedly frozen and thawed twice under liquid nitrogen and room temperature conditions. After that, it was centrifuged at 4℃ and 700g for 10 min, and the supernatant was collected. The supernatant was centrifuged again at 4℃ and 14000g for 30 min, and the supernatant was discarded to obtain the macrophage membrane protein components. Membrane protein concentrations were determined using the BCA protein quantification kit (manufacturer: Fodbio; catalog number: FD2001), and the results were recorded to determine the amount of reagents to be used subsequently.

[0064] (2) M2pep peptide-modified macrophage membrane: M2pep peptide (manufacturer: Xi'an Ruixi Biotechnology Co., Ltd.; product number: R-DS-0070) was mixed with membrane protein at a mass ratio of 1:10 and treated with ultrasonic sonicator (ultrasonic power 200W) for 5 min. After ultrasonic treatment, the mixture was stirred overnight at 4℃ to finally obtain M2pep-modified macrophage membrane.

[0065] Step 2: Preparation of Bacterial Shadow (BG)

[0066] Attenuated Salmonella VNP20009 (VNP20009) in the logarithmic growth phase was collected by centrifugation at 6000 rpm for 10 min. The cells were washed twice with sterile water and resuspended in sterile water to adjust the bacterial concentration to 10. 8 CFU / mL. Subsequently, the bacterial suspension was treated with 0.75 mg / mL NaOH solution (using the minimum inhibitory concentration of NaOH to prepare the bacterial shadow of VNP20009) at 37°C and 200 rpm for 4–5 hours. After the reaction, the sample was centrifuged at 6000 rpm for 10 min, the precipitate was collected, and washed twice with sterile PBS to obtain the bacterial shadow (BG).

[0067] Step 3: Construction of Hybrid Carrier

[0068] M2pep-modified macrophage membranes and bacterial shadows were mixed at a mass ratio of 1:1 and extruded through an 800 nm polycarbonate membrane 20 times to obtain a preliminary mixed system formed by the physical extrusion and fusion of M2pep-modified macrophage membranes and bacterial shadows. Subsequently, the mixture was centrifuged at 4°C, 1000 rpm for 10 min, and the supernatant was collected to obtain the hybrid carrier (mBG).

[0069] 2. Characterization of the drug-loaded hybrid membrane carrier mBG targeting tumor-associated macrophages prepared above.

[0070] (1) Agarose gel electrophoresis analysis: Salmonella VNP20009 was treated with a 0.75 mg / mL NaOH solution for 0.5, 1, 2, 3, 4, and 5 hours, respectively. Genomic DNA was then extracted from the samples at each time point using a genomic DNA extraction kit (manufacturer: Tiangen; catalog number: DP302). The obtained DNA samples were electrophoresed on a 1% agarose gel prepared with 1×TAE buffer under constant voltage of 110 V for 50 min. After electrophoresis, the DNA bands of the samples at each treatment time were observed and compared under UV light to analyze the degree of DNA fragmentation.

[0071] (2) The morphology of BG was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0072] (3) Fluorescence colocalization analysis: M2pep-modified macrophage membranes were labeled with Dil fluorescent dye and then fused with bacterial shadows by extrusion to prepare a composite membrane structure. Subsequently, immunofluorescence staining was performed using Salmonella-specific antibodies. Finally, the labeled samples were imaged using a confocal laser scanning microscope (CLSM) to observe and analyze the spatial colocalization of different fluorescence signals.

[0073] (4) The morphology of mBG was characterized by transmission electron microscopy (TEM).

[0074] (5) Particle size and Zeta potential characterization: The hydrated particle size, polydispersity index (PDI) and Zeta potential of mBG were determined by dynamic light scattering (DLS) technology.

[0075] 3. Experimental Results

[0076] (1) Agarose gel electrophoresis analysis showed that genomic DNA degraded with increasing NaOH treatment time. When the treatment time reached 3-5 hours, only small fragments of degradation or no DNA bands were visible at the bottom of the lanes (e.g., Figure 2 (As shown in A in the figure). This result indicates that treating VNP20009 with 0.75 mg / mL NaOH for at least 3 hours ensures that its genomic DNA is lysed and removed, thus successfully obtaining the empty shell bacterial shadow (BG) structure with missing contents.

[0077] (2) Morphological characterization of BG: Scanning electron microscopy (SEM) results showed that BG had an elliptical vesicle structure with a smooth surface and clear lysis pores. Transmission electron microscopy (TEM) images further verified the empty vesicle structure of BG from the cross-section, showing that its internal electron density was low, confirming that the cytoplasmic contents had been successfully removed. These results directly demonstrate the successful preparation of BG.

[0078] (3) Fluorescence colocalization analysis: Confocal laser scanning microscopy (CLSM) images (e.g.) Figure 2 As shown in Figure C), there is significant spatial overlap between Salmonella-specific antibody-labeled BG (red fluorescence) and Dil-labeled macrophage membrane (green fluorescence), and a clear yellow co-localization signal is observed in the merging channel. This result confirms that the physical fusion of M2pep-modified macrophage membrane and bacterial shadow was successfully achieved by extrusion, forming a structurally complete hybrid membrane carrier (mBG).

[0079] (4) Morphological characterization of mBG: Transmission electron microscopy (TEM) results are as follows Figure 2As shown in D in the image, an additional thin-film structure with a different electron density than the bacterial shadow is visible on the exterior of the mBG, which is attributed to the fusion of the macrophage membrane. This image provides direct morphological evidence for the successful construction of the hybrid membrane.

[0080] (5) Dynamic light scattering (DLS) analysis results showed that the particle size of mPAE@BG was approximately 620 nm (PDI≈0.126), and the Zeta potential was approximately -20.16 mV (e.g., ...). Figure 3 (as shown in C).

[0081] Example 2: Preparation and characterization of a hybrid membrane system (mPAE@BG) loaded with paeonol

[0082] 1. A method for preparing a drug-loaded hybrid membrane system (mPAE@BG) targeting tumor-associated macrophages (e.g.) Figure 1 As shown in the figure, the specific details are as follows:

[0083] Step 1: Preparation of Paeonol Liposomes

[0084] Paeonol liposomes were prepared using a typical thin-film hydration method, specifically: 12 mg of soybean lecithin, 3 mg of cholesterol, and 3 mg of paeonol were dissolved in anhydrous ethanol to form the oil phase. The oil phase was then vacuum-evaporated in a water bath at 37°C (vacuum degree 0.06-0.09 MPa, 37°C) for 30 min to remove the organic solvent (anhydrous ethanol), allowing the lipid components to form a uniform thin film on the bottle wall. Subsequently, 6 mL of PBS was added as the aqueous phase, and the mixture was hydrated in a shaker at 37°C and 200 rpm for 1 h until a homogeneous liposome suspension was formed. The resulting liposome suspension was sonicated using a probe sonar (300 W, 10 min), and then filtered sequentially through 0.45 μm and 0.22 μm microporous membranes to obtain paeonol liposomes (PAEs) with uniform particle size.

[0085] Step 2: Assembly of Paeonol Liposomes with Hybrid Membrane Carriers

[0086] Paeonol liposomes and hybrid carriers were freeze-dried separately using a freeze dryer. The freeze-dried powders were then mixed in PBS at a mass ratio of 1:1 and extruded through a 600nm polycarbonate membrane 20 times to obtain a drug-loaded hybrid membrane system targeting tumor-associated macrophages.

[0087] 2. The hybrid membrane system (mPAE@BG) loaded with paeonol prepared above was characterized.

[0088] (1) The morphology of mPAE@BG was characterized by transmission electron microscopy (TEM).

[0089] (2) Particle size and Zeta potential characterization: The hydrated particle size, polydispersity index (PDI) and Zeta potential of mPAE@BG were determined by dynamic light scattering (DLS) technology.

[0090] (3) Determination of drug loading and loading efficiency: The actual drug loading of paeonol in mPAE@BG was analyzed by high performance liquid chromatography (HPLC), and its loading efficiency was calculated.

[0091] (4) Cell compatibility evaluation: The cytotoxicity of mPAE@BG was assessed using the CCK-8 assay. Bone marrow-derived macrophages (BMDM) were co-incubated with different concentrations (12.5~200 μg / mL) of mPAE@BG for 24 hours, and cell viability was calculated.

[0092] (5) Hemolytic test: mPAE@BG was co-incubated with red blood cells, the absorbance of the supernatant was measured at 540 nm, the hemolysis rate was calculated, and its blood compatibility was evaluated.

[0093] 3. Experimental Results

[0094] (1) Morphological characterization: Using coumarin 6 instead of paeonol as the indicator, confocal laser scanning microscopy (CLSM) images showed significant colocalization between the hybrid membrane and liposomes, indicating successful assembly of the drug delivery system (e.g. Figure 3 (As shown in A). Furthermore, transmission electron microscopy revealed that the mPAE@BG particles exhibit a typical "core-shell" structure: the core is an electron-dense liposome core, and the outer shell is a continuous and rough membrane structure, derived from the macrophage membrane and its surface M2pep modification (e.g., ...). Figure 3 (As shown in B in the diagram).

[0095] Therefore, this invention provides a drug-loaded hybrid membrane system targeting tumor-associated macrophages. This system has a core-shell structure, with a core consisting of a paeonol liposome (PAE); the outer shell is composed of an M2pep peptide-modified Raw264.7 macrophage membrane and a live attenuated Salmonella typhi shadow (BG), together forming a complete paeonol-loaded shadow-macrophage hybrid membrane delivery system (mPAE@BG).

[0096] (2) Dynamic light scattering (DLS) analysis results showed that the particle size of mPAE@BG was approximately 516 nm (PDI≈0.076), and the Zeta potential was approximately -17.36 mV (e.g., ...). Figure 3 (as shown in C).

[0097] (3) Drug loading capacity: High performance liquid chromatography analysis showed that the loading efficiency (LE%) of paeonol in mPAE@BG was 3.6% (e.g., Figure 3 (D in the middle).

[0098] (4) Cell compatibility: The cell compatibility of mPAE@BG with BMDM was assessed using the CCK-8 assay. BMDM exposure to mPAE@BG drug concentrations increased from 12.5 μg / mL to 200 μg / mL for 24 hours did not induce significant cytotoxicity (e.g., ...). Figure 3 (As shown in E).

[0099] (5) Hemolysis test: The hemolysis test showed that when the concentration of mPAE@BG did not exceed 20 mg / mL, the hemolysis ratio was less than 5% (e.g., Figure 3 As shown in F in the figure, the good biocompatibility of mPAE@BG is revealed.

[0100] Example 3: Functional validation of a drug-loaded hybrid membrane system targeting tumor-associated macrophages:

[0101] 1. In vitro targeting and efficacy validation

[0102] (1) Intake efficiency detection

[0103] Lewis cells were cultured in serum-free DMEM (manufacturer: Gibco, catalog number: C11965500BT / formulation: containing 4.5g glucose, containing glutamine, and free of sodium pyruvate) for 24 hours. The cell supernatant was collected, centrifuged at 3000 rpm for 20 min at 4°C, and the supernatant was collected again. 10% fetal bovine serum was added to obtain tumor supernatant conditioned medium. Tumor-associated macrophages (BMDMs) were treated with this tumor supernatant conditioned medium for 48 hours to construct a tumor-associated macrophage (TAM) model.

[0104] mPAE@BG, mBG, and BG were labeled with Dil fluorescent dye and co-incubated with TAMs for 4-6 hours (specifically, 4 hours in this example). The uptake was observed by fluorescence microscopy to preliminarily determine the uptake of different carriers by TAMs. Then, the fluorescence intensity of each group of cells was detected by flow cytometry (FCM) to calculate the uptake efficiency.

[0105] (2) Detection of phagocytic function of TAMs

[0106] To evaluate the regulatory effect of drug delivery systems on TAMs function, TAMs were treated with different formulations for 24 hours and then co-incubated with Lewis lung cancer cells labeled with CFSE fluorescent dye for 4 hours. Subsequently, the phagocytic efficiency of TAMs on tumor cells was analyzed by FCM.

[0107] (3) Experimental results

[0108] 1) Intake efficiency test results showed that, compared with the unmodified group, TAMs significantly increased the uptake rates of mBG and mPAE@BG by 2.45 times and 2.53 times, respectively (e.g., ...). Figure 4 As shown in A and B in the figure, the modification of the macrophage membrane by M2pep conjugation significantly promotes the uptake of mPAE@BG by TAMs.

[0109] 2) Flow cytometry results showed that the ability of TAMs to phagocytose CFSE-labeled tumor cells was significantly activated after treatment with mPAE@BG. The phagocytic efficiency of TAMs in the mPAE@BG group was as high as (59.9±2)%, significantly higher than that in the PBS control group (23.5±0.7)%. Figure 5 (As shown in A and B in the figure). These data indicate that BG-targeted delivery of paeonol liposomes modified with M2pep conjugated macrophage membranes can effectively promote the ability of TAMs to phagocytose tumor cells and restore their anti-cancer activity.

[0110] 2. Tumor-targeted delivery efficacy and in vivo biodistribution of mBG

[0111] (1) Establishment of Lewis lung cancer tumor-bearing mouse model

[0112] Lewis lung cancer cells at a density of 1 million cells / 100 μL were subcutaneously in the right back of C57BL / 6J wild-type mice (male, 6-8 weeks old, 18-22g). The inoculation continued until the tumor volume reached 100-150 mm². 3 At that time, a Lewis lung cancer subcutaneous xenograft mouse model was successfully constructed and can be used for experiments.

[0113] (2) In vivo and in vitro fluorescence imaging

[0114] DiR fluorescently labeled BG (bacterial shadow) or mBG (macrophage membrane-modified bacterial shadow) was injected via the tail vein into Lewis lung cancer subcutaneous xenograft mice (BG 20 mg / kg / mouse, 100 μL). The distribution and accumulation of fluorescence signals in vivo were observed at different time points (6 h, 12 h, 24 h, 48 h) using a small animal in vivo imaging system. Twenty-four hours after injection, the mice were sacrificed, tumor tissue was isolated, and in vitro fluorescence imaging was performed to quantitatively analyze the fluorescence intensity in the tissue, in order to assess the tumor-targeting efficiency of the carrier.

[0115] (3) Immunofluorescence colocalization of tumor tissue

[0116] Frozen sections of tumor tissue were prepared, stained with immunofluorescence (IF) (anti-CD206 antibody labeled TAMs), and observed under a confocal microscope to analyze the co-localization of mBG and TAMs.

[0117] (4) Experimental results

[0118] 1) In vivo fluorescence imaging results showed that, compared with unmodified BG, mBG accumulation at the tumor site was 1.6 times higher at 12 hours post-injection (e.g., ...). Figure 6 (As shown in A and B in the diagram).

[0119] 2) In ex vivo tumor tissue, the fluorescence intensity of DiR in the mBG group was higher than that in the BG group, confirming the good tumor targeting efficiency of mBG (e.g., Figure 7 (As shown in A and B in the figure). Immunofluorescence analysis of ex vivo tumor tissue showed that the red fluorescence signal of mBG was stronger than that of the BG group, and the red fluorescence signal of mBG significantly overlapped with the green fluorescence of anti-CD206 (as shown in the figure). Figure 8 As shown in Figures A through D), this demonstrates that mBG is effectively phagocytosed by TAMs under the guidance of the M2 peptide. These results confirm that mBG prepared through biomimetic engineering, modified with macrophage cell membranes and the M2 peptide, can achieve tumor homing and TAM targeting in vivo.

[0120] 3. Systematically evaluate the antitumor efficacy of mPAE@BG.

[0121] (1) Experimental grouping and drug administration

[0122] A Lewis lung cancer subcutaneous xenograft model was established using the method described above (the same method was used for 2. Tumor-targeted delivery efficacy and in vivo biodistribution of mBG). Patients were randomly divided into 5 groups (n=7): PBS group, BG group, paeonol liposome group, PAE@BG group, and mPAE@BG group. The drugs were administered via tail vein on days 0, 2, 4, and 6. Tumor volume was measured periodically and calculated using the following formula: Tumor volume = (length × width) 2 ) / 2).

[0123] Administration: PBS group, i.e. G1: administered via tail vein injection, PBS buffer, 100 μL per mouse.

[0124] Group BG, or G2: Bacterial spores (BG) were dissolved in sterile PBS and injected via the tail vein at a dose of 20 mg / kg, with an injection volume of 100 μL per mouse.

[0125] Paeonol liposome group (PAE-lipo group), namely G3: Paeonol liposomes were dissolved in sterile PBS and injected via tail vein at a dose of 3 mg / kg of paeonol, with an injection volume of 100 μL per mouse.

[0126] PAE@BG group, or G4: Paeonol-loaded bacterial shadow (PAE@BG) was dissolved in sterile PBS and injected via tail vein at a dose of 20 mg / kg BG and 3 mg / kg paeonol, with an injection volume of 100 μL per mouse.

[0127] The mPAE@BG group, or G5, involved dissolving the macrophage membrane-bacterial hybrid vector (mPAE@BG) loaded with paeonol in PBS-free M2pep and then injecting it via the tail vein at a dose of 20 mg / kg BG and 3 mg / kg paeonol, with an injection volume of 100 μL per mouse.

[0128] All groups were administered the drug continuously for 15 days, after which the inhibitory effect on tumor growth was observed, and pathological analysis and tumor microenvironment immune cell analysis were performed on the tumor tissue. Simultaneously, mouse serum was collected for cytokine detection.

[0129] (2) Experimental results

[0130] 1) Tumor growth inhibition effect

[0131] Dosing regimen such as Figure 9 As shown in the figure. All mice in all groups were sacrificed on day 15 after drug administration, and tumors were isolated and weighed. Results are as follows. Figure 10 A to Figure 10 As shown in Figure C: Compared with the control group and the BG group, the PAE, PAE@BG, and mPAE@BG treatment groups all showed significant tumor growth inhibition effects, specifically manifested as tumor volume reduction and weight reduction. Notably, the mPAE@BG group showed the best anti-tumor effect, with significantly better efficacy than the PAE@BG group. This result confirms that cell membrane modification and M2pep functionalization can significantly enhance the tumor targeting and therapeutic efficacy of PAE@BG.

[0132] 2) Tumor histopathological analysis

[0133] The results of hematoxylin-eosin (HE) staining of tumor tissue showed the following: Figure 11 As shown: Histopathological analysis of HE staining showed that the tumor tissue in the mPAE@BG group exhibited the largest areas of apoptosis and necrosis.

[0134] 3) Tumor microenvironment immune cell analysis

[0135] Flow cytometry was used to analyze the infiltration of immune cells in the tumor microenvironment (TME), including M1 macrophages (CD45). + CD11b + F4 / 80 + CD86 + M2 macrophages (CD45) + CD11b + F4 / 80 + CD206 + The proportion of M1 and M2 ratios; tumor-infiltrating lymphocytes (CD3+) + ), cytotoxic T cells (CTLs, CD3)+ CD8 + ), helper T cells (CD3) + CD4 + The proportion of CD4 and CD4 + / CD8 + ratio.

[0136] The results are as follows Figure 12 A to Figure 12 As shown in C, compared with the control group, mPAE@BG treatment significantly increased M1 macrophages (CD45). + CD11b + F4 / 80 + CD86 + The proportion of M2 macrophages (CD45) was reduced, while the proportion of CD45 macrophages was decreased. + CD11b + F4 / 80 + CD206 + The proportion of M1 to M2 cells was significantly increased. This finding indicates that mPAE@BG can effectively promote the phenotypic polarization of tumor-associated macrophages (TAMs) from M2 to M1.

[0137] In terms of adaptive immune response, such as Figure 13 A to Figure 13 As shown in C, mPAE@BG treatment significantly enhanced cytotoxic T lymphocytes (CTLs, CD3+). + CD8 + The proportion of CD3+ cells increased significantly, while the proportion of helper T cells (CD3+) increased significantly. + CD4 + The proportion of CD4 decreased, leading to a decrease in CD4 concentration. + / CD8 + The decrease in the ratio indicates that mPAE@BG can simultaneously activate local adaptive immune responses.

[0138] 4) Serum cytokine detection

[0139] Mouse serum was collected, and the concentrations of IFN-γ, TNF-α, and IL-12p70 in mouse serum were detected using an IFN-γ ELISA kit (manufacturer: Elabscience; catalog number: E-EL-M0048), a TNF-α ELISA kit (manufacturer: Elabscience; catalog number: E-EL-M3063), and an IL-12p70 ELISA kit (manufacturer: Xinbosheng; catalog number: EMC006.96).

[0140] Serum cytokine test results as follows Figure 14 A to Figure 14As shown in Figure C, the concentrations of key immunomodulatory factors such as IFN-γ, TNF-α, and IL-12p70 in the mPAE@BG treatment group were significantly higher than those in the control group. This indicates that mPAE@BG can effectively activate the anti-tumor immune response in mice, which is mainly based on the Th1 immune response. Among these factors, IFN-γ can enhance the recognition and killing of tumor cells by immune cells and inhibit the immunosuppressive microenvironment; TNF-α can induce tumor cell apoptosis and recruit immune cells; and IL-12p70 can regulate the initiation and amplification of the anti-tumor immune response. This result further confirms that mPAE@BG can not only target and regulate the function of TAMs, but also systematically activate the body's anti-tumor immunity by upregulating the above-mentioned cytokines, providing immune mechanism support for inhibiting tumor growth and improving prognosis.

[0141] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A hybrid membrane carrier targeting tumor-associated macrophages, characterized in that, The hybrid membrane carrier is formed by fusing attenuated Salmonella typhi shadow and M2pep peptide-modified macrophage membrane.

2. The method for preparing the hybrid membrane carrier as described in claim 1, characterized in that, include: M2pep peptide-modified macrophage membranes were mixed with attenuated Salmonella typhi at a mass ratio of 1 to 2:1, and then extruded through a filter membrane to obtain a hybrid membrane carrier.

3. The method for preparing the hybrid membrane carrier targeting tumor-associated macrophages as described in claim 1, characterized in that, The method for preparing the M2pep peptide-modified macrophage membrane includes: mixing M2pep peptide with macrophage membrane at a mass ratio of 1:8~15, and then treating it under an ultrasonic power of 100W~200W for 5min~15min to obtain the M2pep peptide-modified macrophage membrane. The method for preparing the attenuated Salmonella typhi bacterial shadow includes: centrifuging attenuated Salmonella typhi at 5000 rpm to 7000 rpm for 8 min to 12 min and collecting the bacterial cells; then treating the bacterial cells with 0.5 to 1 mg / mL NaOH solution under shaking conditions at 150 rpm to 250 rpm for 3 to 5 hours; after treatment, centrifuging at 5000 rpm to 7000 rpm for 8 min to 12 min, collecting the precipitate, and washing it twice with sterile PBS to finally obtain the attenuated Salmonella typhi bacterial shadow.

4. A drug-loaded hybrid membrane system targeting tumor-associated macrophages, characterized in that, The drug-loaded hybrid membrane system targeting tumor-associated macrophages includes the hybrid membrane carrier as described in claim 1 and the immunomodulatory / antitumor drug component carried in the hybrid membrane carrier.

5. The drug-loaded hybrid membrane system as described in claim 4, characterized in that, The drugs used for immunomodulation / antitumor include: paeonol, curcumin, resveratrol, CSF-1R inhibitors, TLR agonists, CD40 agonists, CD47 inhibitors, PD-L1 inhibitors, doxorubicin, paclitaxel, or gemcitabine.

6. The method for preparing the drug-loaded hybrid membrane system targeting tumor-associated macrophages as described in claim 4, characterized in that, include: The drug for immunomodulation / antitumor is mixed with the hybrid membrane carrier at a mass ratio of 1:1 to 2, and then extruded through a filter membrane to obtain the drug-loaded hybrid membrane system targeting tumor-associated macrophages.

7. The method for preparing the drug-loaded hybrid membrane system targeting tumor-associated macrophages as described in claim 4, characterized in that, The drug for immunomodulation / antitumor includes paeonol; the paeonol is prepared into paeonol liposomes and then assembled into a drug delivery system.

8. The use of the hybrid membrane carrier targeting tumor-associated macrophages as described in claim 1 in the preparation of a medicament for treating lung cancer.

9. The application of the drug-loaded hybrid membrane system targeting tumor-associated macrophages as described in claim 4 in the preparation of a medicament for treating lung cancer, characterized in that... The drug has a drug-loaded hybrid membrane system that targets tumor-associated macrophages as its sole active ingredient, and the drug also includes a pharmaceutically acceptable carrier. The drug-carrying hybrid membrane system targeting tumor-associated macrophages carries paeonol.

10. The application of the drug-loaded hybrid membrane system targeting tumor-associated macrophages as described in claim 9 in the preparation of a medicament for treating lung cancer, characterized in that... The dosage form of the drug is selected from injections or emulsions; wherein, the injections include lyophilized powder injections and suspensions for injection.

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