Fusion film packaged gold-iron core-shell nano diagnosis and treatment preparation as well as preparation method and application thereof

Abstract

The invention discloses a fusion membrane packaged gold-iron core-shell nano diagnosis and treatment preparation as well as a preparation method and application thereof. The preparation takes gold-iron core-shell nanoparticles as a core, and the surface of the gold-iron core-shell nanoparticles is coated with a biomimetic membrane formed by fusion of bacterial outer membrane vesicles and tumor cell membranes. The preparation method comprises the steps of synthesis of the gold-iron nano core, surface functionalization and self-assembly coating of the fusion membrane. The preparation can simultaneously exert photothermal therapy and ferroptosis induction effects under near-infrared light irradiation, effectively initiates tumor cell immunogenicity death, and cooperatively activates dendritic cells and enhances T cell immune response by means of a pathogen related molecular mode carried by the fusion membrane and a tumor related antigen. Experiments show that the preparation can significantly inhibit tumor growth and metastasis, induce long-acting anti-tumor immune memory, and provide a multi-mode synergistic nano platform for tumor immunotherapy.

Description

Technical Field

[0001] This application belongs to the field of nanotechnology and tumor immunotherapy, specifically relating to a gold-iron core-shell nanotherapeutic agent with fusion membrane encapsulation, its preparation method and application. Background Technology

[0002] Currently, cancer immunotherapy, represented by immune checkpoint inhibitors, has made groundbreaking progress in clinical practice. However, its overall efficacy is still limited by two major bottlenecks: first, insufficient presentation of tumor cell antigens leading to poor immune recognition; and second, the pervasive immunosuppressive state in the tumor microenvironment. This prevents many patients from generating a sufficiently potent and durable anti-tumor immune response, resulting in limited treatment efficacy and a high risk of recurrence and metastasis.

[0003] To overcome these limitations, synergistic therapeutic strategies based on nanotechnology have attracted widespread attention. For example, gold-based nanomaterials can achieve efficient photothermal ablation and induce immunogenic cell death; while iron-based nanomaterials can release danger signals by inducing ferroptosis. However, single-modality treatments often fail to completely reverse the immunosuppressive microenvironment and lack the ability to sustainably activate the immune system. Furthermore, the biocompatibility, targeting, and immunomodulatory functions of conventional nanoparticles still need to be enhanced.

[0004] In recent years, biomimetic nanocarriers have provided new ideas for improving therapeutic specificity and immune activation efficiency. Among them, using bacterial outer membrane vesicles or tumor cell membranes for encapsulation are two mainstream strategies: the former is rich in immune adjuvant molecules, which can strongly activate innate immunity; the latter carries tumor antigens, which helps specific immune recognition. However, existing single membrane encapsulation systems often have limited functions and cannot simultaneously provide sufficient antigen and potent adjuvant signals, thus limiting their ability to stimulate a comprehensive and durable immune response.

[0005] Therefore, developing a novel nanoplatform that integrates multimodal tumor ablation (such as photothermal therapy and ferroptosis) with bidirectional immune activation (antigen presentation and adjuvant stimulation) has become a critical technical challenge in the field. An ideal platform needs to achieve efficient core-shell synergy in material design and organically integrate physical ablation, chemical regulation, and biological immune modulation in functionality, thereby providing a novel solution to overcome the limitations of current tumor immunotherapy. Summary of the Invention

[0006] This invention addresses the key issues of insufficient antigen presentation and the limitation of efficacy by the immunosuppressive microenvironment in existing tumor immunotherapy by providing a gold-iron core-shell nanotherapeutic agent encapsulated in a fusion membrane, its preparation method, and its application.

[0007] On the one hand, the present invention provides a gold-iron core-shell nanotherapeutic agent encapsulated with a fusion membrane, which adopts the following technical solution: A fusion-membrane encapsulated gold-iron core-shell nanotherapeutic agent comprises: The core is composed of gold-iron core-shell nanoparticles; and the fusion membrane covering the surface of the core is formed by the fusion of bacterial outer membrane vesicles and tumor cell membranes.

[0008] Preferably, in the gold-iron core-shell nanoparticles, the gold content is 1-10% and the iron content is 0.3-10%; The particle size of the nanotherapeutic agents is 30-300 nm.

[0009] Preferably, the bacterial outer membrane vesicles are derived from Escherichia coli Nissle 1917; The tumor cell membrane originates from 4T1 tumor cells.

[0010] Preferably, the protein ratio of tumor cell membrane to bacterial outer membrane vesicles in the fusion membrane is 1:1.

[0011] On the other hand, the present invention also provides a method for preparing a gold-iron core-shell nanotherapeutic agent encapsulated with a fusion membrane, using the following technical solution: A method for preparing a gold-iron core-shell nanotherapeutic agent encapsulated in a fusion membrane includes the following steps: S1. Extract tumor cell membranes and bacterial outer membrane vesicles separately; S2. Tumor cell membranes and bacterial outer membrane vesicles are mixed in a certain proportion and then fused to obtain a fusion membrane; S3. Preparation of gold-iron core-shell nanoparticles; S4. Mix gold-iron core-shell nanoparticles with a fusion membrane and self-assemble them to obtain a gold-iron core-shell nanotherapeutic agent encapsulated in a fusion membrane.

[0012] Preferably, step S1 for extracting tumor cell membranes includes: treating tumor cells with ultrasound, performing a first centrifugation and a second centrifugation in sequence, collecting the precipitate and freeze-drying it; Extraction of bacterial outer membrane vesicles includes: culturing bacteria, centrifuging to collect the supernatant, filtering, ultracentrifuging, and collecting the precipitate.

[0013] Preferably, the fusion process in step S2 includes: ultrasonicating the mixed system and then incubating it overnight at 4°C.

[0014] Preferably, the preparation of gold-iron core-shell nanoparticles in step S3 includes: CTAB, HAuCl4, sodium citrate and polyvinylpyrrolidone were dissolved in deionized water and reacted in a water bath at 90°C to obtain gold nanoparticles. Thiol-functionalized gold nanoparticles were obtained by performing thiol-functionalization on gold nanoparticles. Thiol-functionalized gold nanoparticles with Fe 3+After the solution reaction, H3BTC solution was added and incubated to allow iron to be deposited on the surface of the gold nanoparticles, thus obtaining the gold-iron core-shell nanoparticles.

[0015] Preferably, in step S4, the gold-iron core-shell nanoparticles are mixed with the fused membrane at a mass ratio of 1:1.

[0016] The present invention also provides the application of a fusion-membrane-encapsulated gold-iron core-shell nanotherapeutic agent in the preparation of a drug for tumor immunotherapy.

[0017] In summary, the beneficial effects of the present invention are as follows: First, the nanoformulation of this invention achieves physical integration and functional synergy of multiple treatment modalities through its unique "gold-iron core-shell structure" and "bacterial-tumor fusion membrane" design. The gold-iron core-shell nanonucleus can simultaneously act as a highly efficient photothermal conversion agent and a ferroptosis inducer, producing a synergistically sensitizing tumor ablation effect under near-infrared light excitation and strongly inducing immunogenic cell death (ICD), thus solving the problem of insufficient signal from single therapies. Simultaneously, the fusion membrane shell combines the potent immune adjuvant activity of bacterial outer membrane vesicles with the specific antigen-presenting function of tumor cell membranes, fundamentally and synchronously enhancing the intensity and precision of immune activation.

[0018] Secondly, based on the aforementioned synergistic mechanism, this formulation exhibits remarkable comprehensive anti-tumor efficacy both in vivo and in vitro. Experiments have confirmed that this formulation not only significantly inhibits the growth of tumors in situ, but also generates a powerful "distant effect" by efficiently activating dendritic cells, promoting cytotoxic T cell infiltration, and forming immune memory, effectively inhibiting tumor metastasis and recurrence. This combination of highly efficient local ablation and systemic immune reconstitution provides a novel and highly promising solution to overcome the current clinical challenges of low response rates and high recurrence and metastasis in tumor immunotherapy. Attached Figure Description

[0019] Figure 1 Image a in the middle is a transmission electron microscope image of the gold-iron core-shell nanoparticle formulation encapsulated with a fusion film; Figure 1 In the middle b, the elemental mapping and merge diagram of the gold-iron core-shell nano-formulation encapsulated by the fusion membrane are shown. Figure 2 UV-Vis absorption spectra of gold-iron core-shell nanoparticles before and after encapsulation with fusion membrane; Figure 3 In Figure 'a', the cytotoxicity of AuFe core-shell nanoparticles at different concentrations is shown. Figure 3 Figure b shows the cytotoxicity of AuFe core-shell nanoparticles at different concentrations under 808nm laser irradiation. Figure 3c represents the cytotoxicity of 4T1 cells after different treatments; Figure 4 Figure a shows the relative content of intracellular lipid peroxides after different treatments. Figure 4 Figure b shows the relative ATP content released in the cell culture supernatant after different treatments; Figure 4 In the middle (c), there are Western blot analysis diagrams of proteins after different treatments; Figure 5 The diagram in 'ab' represents the analysis of T lymphocyte subsets. Figure 5 The diagram in center c is a diagram of dendritic cell maturation analysis; Figure 5 The diagram in 'd' represents the analysis of memory T cells; Figure 5 The eg in the middle represents the secretion profile of cytokines (IFN-γ, IL-6, TNF-α); Figure 6 In the diagram, 'a' represents the construction of a tumor-bearing mouse model and its in vivo treatment regimen. Figure 6 The graph in middle bi shows the tumor growth curves of tumor-bearing mice after different treatments; Figure 6 The image in middle j shows the tumor weight of mice after different treatments. Figure 6 Photographs of tumors removed from different treatment groups in the middle k are shown. Figure 6 The image in middle 1 shows a representative HE staining image of tumor tissue sections from different treatment groups in mice after treatment. Figure 6 In the middle m, there is a representative image of Ki-67 immunohistochemical staining of tumor tissue pathological sections from different treatment groups after the mice were treated. Figure 7 In the image, 'a' represents a live fluorescence imaging image; Figure 7 Image b is a fluorescence image of an isolated organ; Figure 7 The image in middle c is a photograph of solid lung tissue; Figure 7 The image in the middle (d) shows H&E staining of lung tissue. Figure 7 The image in the middle (e) shows the Ki-67 immunohistochemical staining of lung metastases. Figure 7 f represents CD4+ in lung metastases. + T cell infiltration staining diagram; Figure 7 The middle g represents the CD8+ of lung metastases. + T cell infiltration staining image. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example A gold-iron core-shell nanotherapeutic agent encapsulated with a fusion membrane is prepared using the following method: S1. 4T1 cells were sonicated for 30 minutes, then centrifuged at 4000×g for 10 minutes at 4°C, and the supernatant was collected. The supernatant was further centrifuged at 16000×g for 30 minutes at 4°C, and the resulting precipitate was freeze-dried to obtain tumor cell membranes (mT).

[0022] S2. Escherichia coli Nissle 1917 was cultured in Luria-Bertani medium to the logarithmic growth phase, then centrifuged at 12000×g for 5 minutes and the supernatant was collected. The supernatant was filtered through a 0.45 μm vacuum filter to remove bacterial cells, and then ultracentrifuged at 150,000×g for 2 hours at 4°C to collect OMV.

[0023] S3. Mix mT and OMV at a protein mass ratio of 1:1, sonicate on ice for 10 minutes, and then gently shake overnight at 4°C to promote membrane fusion.

[0024] S4. At room temperature, dissolve CTAB, HAuCl4, sodium citrate, and polyvinylpyrrolidone in deionized water and stir thoroughly until homogeneous. The concentration ratio of CTAB to HAuCl4 is 10-75:1, and the concentration of polyvinylpyrrolidone is 1-10 mg / ml. -1 The sodium citrate concentration was 0.01–0.1 M. The resulting mixture was reacted in a water bath at 90 degrees Celsius for 12 hours, and then naturally cooled to room temperature to obtain gold-iron core nanoparticles, denoted as AuFe.

[0025] S5. The obtained gold-iron nanonuclei were incubated in a 30 mM mercaptoacetic acid ethanol solution for 24 hours to undergo thiol functionalization. Subsequently, the thiol-functionalized gold nanoparticles were dispersed in Fe dissolved in ethanol. 3+ The reaction was carried out in solution for 15 minutes, then H3BTC solution was added and incubated at 70°C for 30 minutes to allow iron to deposit on the surface of gold nanoparticles. Finally, the gold-iron nanonuclei were collected by centrifugation.

[0026] S6. AuFe nanoparticles and mTOMV are mixed at a mass ratio of 1:1 and incubated at 4°C for 12 hours to perform molecular self-assembly, thereby obtaining the gold-iron core-shell nanotherapeutic agent encapsulated by the fusion membrane, denoted as AuFe@mTOMV.

[0027] Test case Test Example 1: Morphological characterization was performed on the gold-iron core-shell nanoformulation encapsulated with the fusion film prepared in Example 1: Take 200 μL of the solution of the gold-iron core-shell nanoparticle formulation encapsulated in the fusion membrane prepared in Example 1, centrifuge (10,000 rpm, 10 min), discard the supernatant, resuspend in deionized water, and sonicate for 10 min (with a 30-second pause after every 1 minute of sonication) to disperse evenly. Add 5 μL of the dispersion dropwise onto a copper grid (Cu grid, 200 mesh) coated with a carbon film and let it stand for 2–3 min to allow the nanoparticles to naturally adsorb onto the membrane surface. Then gently blot away excess liquid with filter paper and allow it to air dry at room temperature for later use. TEM testing was performed using a transmission electron microscope (FEI Tecnai G2 F20 model) with an accelerating voltage of 200 kV.

[0028] like Figure 1 As shown in Figure a, a dense core (gold-iron nanonucleus) with a darker color and higher contrast can be clearly observed in the figure. It is covered by a thinner shell with uniform thickness (fusion biomembrane), which perfectly matches the designed "core-shell" configuration. The overall particle size distribution within the specified range indicates that the preparation process has good controllability and uniformity.

[0029] like Figure 1 As shown in Figure b, the elemental signals of gold and iron highly overlap and are concentrated in the central region of the particle, confirming the existence of the gold-iron composite nanonucleus. Simultaneously, the signals of carbon, nitrogen, oxygen, and other major elements constituting the biomembrane are uniformly wrapped around the entire nucleus, forming a complete shell signal ring. This directly demonstrates that the fused biomembrane originating from bacterial outer membrane vesicles (OMVs) and tumor cell membranes has been successfully and completely encapsulated on the surface of the nanonucleus. The distribution of sulfur may originate from membrane proteins or functionalization processes, further supporting the presence of biological components.

[0030] Test Example 2 The gold-iron core nanoparticles prepared in step S4 of Example 1 and the gold-iron core-shell nanoformulation encapsulated by the fusion film prepared in step S6 were subjected to ultraviolet-visible spectroscopy analysis. The specific method is as follows: Take appropriate amounts of AuFe sample and AuFe@mTOMV sample respectively (initial concentration approximately 0.5–1.0 mg·mL). -1 Dilute with the same volume of deionized water to the measured concentration (typically 0.05–0.2 mg·mL). -1To ensure absorbance within the range of 0.1–1.0, samples were ultrasonically dispersed at room temperature for 5 min before measurement (ultrasonic bath, intermittent to prevent overheating) to ensure uniform dispersion. All samples were centrifuged (10,000 rpm, 10 min), the supernatant was discarded, and the samples were resuspended in deionized water and washed once more to remove free surfactants or unbound coating materials. Spectroscopic measurements were performed using quartz cuvettes (1.0 cm path length), with deionized water as a blank, using a double-beam UV-Vis spectrophotometer (e.g., Shimadzu UV-2600 or similar) to scan and record absorption spectra in the wavelength range of 260–900 nm. Baseline correction and absorption intensity normalization were performed on the obtained spectra, and parameters such as the position and intensity of the surface plasmon resonance (SPR) peak were extracted to compare changes in optical characteristics before and after encapsulation.

[0031] like Figure 2 As shown, UV-Vis absorption spectroscopy analysis revealed that the uncoated gold-iron nanonuclei exhibited a significant near-infrared absorption peak at 808 nm, confirming their functional basis as a highly efficient photothermal conversion agent. After coating with the fusion film, this absorption peak remained clearly visible but underwent a slight red shift. This change indirectly confirmed the successful construction of the core-shell structure and indicated that the film coating process did not impair the photothermal properties of the nanonuclei, ensuring the integrity of the photothermal therapeutic function of the formulation.

[0032] Test Example 3: The cytotoxicity of the gold-iron core nanoparticles prepared in step S4 of Example 1 and the gold-iron core-shell nanoformulation encapsulated in the fusion membrane prepared in step S6 was evaluated using the standard MTT assay under near-infrared light irradiation with and without. The specific preparation method is as follows: 4T1 cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and cultured overnight at 37°C. The following day, the cells were exposed to different treatment groups: saline group, and cells irradiated alone with an 808 nm laser (1W cm⁻¹). -2 The following groups were identified: a 10-minute treatment group, a gold-iron core-shell nanoparticle group (100 μg), a gold-iron core-shell nanoparticle group (100 μg) combined with 808 nm laser irradiation, a fusion film group (100 μg), a fusion film-encapsulated gold-iron core-shell nanoparticle group (100 μg based on gold and iron content), and a fusion film-encapsulated gold-iron core-shell nanoparticle group combined with 808 nm laser irradiation (1W cm⁻¹). -2 The group was exposed for 10 minutes (10 minutes) for 12 hours. Afterwards, MTT solution (5 mg / mL) was added to each well. -1 The samples were then incubated at 37°C for 4 hours. The supernatant was then removed, and 150 µL of DMSO was added to each well to dissolve the formazan crystals. Finally, the absorbance at 570 nm was measured using a microplate reader (BioTek, USA).

[0033] like Figure 3 As shown, under conditions without laser irradiation, different concentrations of gold-iron core-shell nanoparticles (0, 5, 10, 15, 20, 40, 60, 80, 100 μg / mL) were used to detect the effects of different concentrations of gold-iron core-shell nanoparticles (0, 5, 10, 15, 20, 40, 60, 80, 100 μg / mL). -1 The product (AuFe@mTOMV, 100 μg mL) was processed or encapsulated via a fusion membrane. -1 The material exhibited low toxicity to 4T1 tumor cells within the tested concentration range, indicating good biocompatibility, i.e., low "dark toxicity," which provides an important prerequisite for its safe application in vivo.

[0034] Under laser irradiation, the AuFe group exhibited a significant concentration-dependent cytotoxic effect, directly attributed to the efficient photothermal conversion capability of the gold-iron nucleus, validating its core function as a photothermal therapeutic agent. Meanwhile, the AuFe@mTOMV group, under the same irradiation conditions, showed stronger cytotoxicity than the naked AuFe group.

[0035] Test Example 4 The gold-iron core-shell nanoparticles encapsulated in the fusion membrane prepared in Example 1 were characterized by induction of lipid peroxidation-induced immunogenic cell death: S1. Induction Treatment and Sample Preparation: 4T1 cells were seeded at 200,000 cells per well in 6-well culture plates and cultured overnight at 37°C in a 5% CO2 incubator to allow adhesion. The old culture medium was discarded and replaced with a medium containing different nano-preparations (PBS control, AuFe (100 μg / mL)). -1 AuFe@mTOMV (calculated as gold and iron nanoparticle content per 100 μg mL) -1 Fresh culture medium was incubated with the cells in a cell culture incubator for 24 hours. For groups requiring irradiation, the culture plates were irradiated with an 808 nm laser (1.0 W / cm²). 2 (10 min). After irradiation, the cells were incubated for another 6 hours to allow for full reaction. After incubation, the cell culture supernatant and cells were collected separately.

[0036] S2. Lipid peroxidation level detection: After washing the collected cells with pre-cooled PBS, lysing them with appropriate lysis buffer, and centrifuging to collect the supernatant. The protein concentration in the supernatant was determined using a BCA protein quantification kit for subsequent data standardization (P0011). Lipid peroxidation levels were quantified using a lipid peroxidation detection kit (Beyotime, BDPY 581 / 591 C11), following the manufacturer's instructions.

[0037] S3. Immunoblot Analysis: Separately treated cells were used, and total protein was extracted using RIPA lysis buffer containing protease inhibitors. The concentration was determined by the BCA method. An equal volume of protein sample was subjected to SDS-PAGE gel electrophoresis (220V for 2 h), followed by transfer of the protein onto a PVDF membrane. After blocking the membrane with 5% skim milk, it was incubated overnight at 4°C with specific primary antibodies (anti-GPX4, anti-HSP70, anti-calreticulin CRT, and internal control GAPDH antibody). After washing, it was incubated at room temperature with horseradish peroxidase-labeled secondary antibody. Finally, the images were developed on an imaging system using an enhanced chemiluminescent substrate, and the intensity of the target bands was analyzed.

[0038] S4. ATP Release Detection: Centrifuge the cell culture supernatant collected in step one (13000 rpm, 10 min), and collect the supernatant, avoiding interference from cell debris. Using the enhanced ATP detection kit, follow the instructions to dilute the ATP standard solution with ATP detection lysis buffer to the following concentration gradients: 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Plot a standard curve and calculate the absolute concentration of ATP in the supernatant based on the standard curve.

[0039] like Figure 4 As shown in Figure a, the LPO level of cells treated with AuFe@mTOMV combined with near-infrared light was significantly higher than that of other groups, directly demonstrating that this treatment can efficiently trigger ferroptosis in tumor cells.

[0040] like Figure 4 As shown in Figure b, the amount of ATP released by cells also reached its peak under the same treatment conditions, which confirms that the resulting cell death is highly immunogenic and can release strong "danger signals" to activate the immune response.

[0041] like Figure 4 As shown in Figure c, the expression of GPX4, a key negative regulator of ferroptosis, was downregulated, while the expression of ICD characteristic proteins HSP70 and CRT was significantly upregulated, elucidating the initiation molecular events of the synergistic enhancement of antitumor immunity by this formulation.

[0042] Test Example 5 The in vitro immune activation level of the gold-iron core-shell nanoparticle formulation encapsulated in fusion membrane prepared in Example 1 was analyzed by flow cytometry. The specific steps are as follows: S1. Preparation and processing of bone marrow-derived dendritic cells: Bone marrow cells were isolated from the femur and tibia of 6-week-old C57BL / 6 mice. After erythrocyte lysis, the cells were resuspended in RPMI-1640 complete medium containing cytokines GM-CSF and IL-4 to induce differentiation of bone marrow cells into immature dendritic cells. The cells were cultured for approximately 8 days, with the medium changed periodically. Finally, non-adherent and loosely adherent cells were collected, yielding bone marrow-derived dendritic cells (BMDCs).

[0043] Nanoparticle stimulation: The obtained BMDCs were co-incubated with different treatment groups (PBS control, AuFe, mTOMV, AuFe@mTOMV, combined with or without near-infrared light irradiation) for 12 h to allow the nanoparticles to fully interact with the cells and stimulate their maturation.

[0044] S2. Immunocyte Phenotypic Analysis: BMDCs were collected after different treatments and stimulations. The cell surface was stained with fluorescently labeled specific antibodies (e.g., anti-CD11c, anti-CD80, anti-CD86) at 4°C to mark dendritic cells and their activation markers. After staining, the cells were washed and analyzed by flow cytometry to determine the proportion of CD80 and CD86 double-positive cells in the CD11c+ cell population, thereby quantifying the maturation level of BMDCs.

[0045] BMDCs (as antigen-presenting cells) pretreated with the aforementioned different nanoparticles were co-cultured with lymphocytes isolated from the spleen of healthy mice for approximately 48 hours. The co-cultured cells were collected and stained with fluorescent antibodies (such as anti-CD3, anti-CD4, and anti-CD8). Flow cytometry was used to analyze changes in the ratio of CD4+ helper T cells and CD8+ cytotoxic T cells among CD3+ T lymphocytes, assessing the activation and proliferation of T cell subsets.

[0046] In the co-culture system, lymphocytes were additionally stained with anti-CD44 and anti-CD62L antibodies. Flow cytometry was used to analyze cell populations with high CD44 expression and low CD62L expression to identify the proportion of effector memory T cells and assess the induction potential of immune memory.

[0047] S3. Cytokine Secretion Level Detection: Supernatant from BMDC culture after stimulation with the nanoparticles, or supernatant from BMDC co-culture with lymphocytes, was collected. Using a commercially available enzyme-linked immunosorbent assay (ELISA) kit, the concentrations of key immunostimulatory factors (such as IFN-γ, IL-6, IL-12, and TNF-α) were quantitatively detected according to the manufacturer's instructions. By comparing the differences in cytokine levels among different treatment groups, the activation status of immune cells was corroborated at the level of secretory function.

[0048] Reference Figure 5As shown in Figure ab, bone marrow-derived dendritic cells (BMDCs) treated with AuFe@mTOMV combined with near-infrared light most effectively activated T lymphocytes in the co-culture system. Specifically, this manifested as CD3+ activation. + CD4 in T cells + Helper T cells and CD8 + The proportion of cytotoxic T cells was significantly higher than that of other control groups, indicating that this treatment can simultaneously promote the helper and killer arms of adaptive immunity.

[0049] Reference Figure 5 As shown in Figure c, the AuFe@mTOMV combined illumination treatment induced the highest proportion of CD11c. + CD80 + CD86 + Mature dendritic cells. This confirms that the formulation, through its fusion membrane, provides antigen and adjuvant signals, which, in conjunction with photothermal / ferroptosis-induced danger signals, strongly drive the maturation process of antigen-presenting cells.

[0050] Reference Figure 5 As shown in Figure d, in co-cultured lymphocytes, the group treated with AuFe@mTOMV+NIR induced the highest proportion of CD44. + CD62L - Effector memory T cells. This suggests that the treatment not only activates an immediate immune response but also induces the formation of immune memory with long-term protective potential, providing a key cellular basis for preventing tumor recurrence.

[0051] Reference Figure 5 As shown in Figure e, ELISA assays revealed that BMDCs stimulated with AuFe@mTOMV+NIR exhibited peak secretion levels of key pro-inflammatory cytokines IFN-γ, IL-6, and TNF-α in their supernatant. This high expression of cytokines, from a secretory function perspective, confirms strong activation of the immune system and points to a Th1-type immune response beneficial for anti-tumor activity.

[0052] Reference Figure 5 As shown in Figure f, the in vivo tumor growth curves indicate that AuFe+NIR or mTOMV alone can only partially inhibit tumor growth, while AuFe@mTOMV combined with near-infrared light irradiation exhibits the strongest tumor growth inhibition effect. This directly demonstrates the synergistic therapeutic effect between photothermal / ferroptosis physical ablation and membrane-mediated immune activation.

[0053] Reference Figure 5As shown in Figure g, the weight and images of tumor tissue after treatment indicate that the AuFe@mTOMV+NIR group had the least residual tumor. Further HE staining and Ki-67 immunohistochemistry confirmed that this group of tumor tissue showed the most extensive necrosis area and the lowest proliferative activity of surviving tumor cells (Ki-67 positive), validating the significant effectiveness of this therapy at the histopathological level.

[0054] Test Example 6 The in vivo synergistic enhancement of antitumor effects between the gold-iron core nanoparticles prepared in step S4 of Example 1 and the gold-iron core-shell nanoformulation encapsulated by the fusion membrane prepared in step S6 was analyzed. To establish a 4T1 tumor-bearing mouse model, 2×10 6 4T1 cells in the logarithmic growth phase were injected into the inguinal fat pad of female mice. When the tumor volume reached approximately 100 mm... 3 Mice were randomly divided into seven groups (n=5 per group): (1) control group (injected with saline only); (2) near-infrared light irradiation group; (3) AuFe group (intravenous injection of AuFe, 10 mg / kg). -1 (4) AuFe (10 mg kg) -1 (5) mTOMV group (intravenous injection of mTOMV, 10 mg / kg) + near-infrared light irradiation group; (6) mTOMV group (intravenous injection of mTOMV, 10 mg / kg) -1 (6) AuFe@mTOMV group (intravenous injection based on AuFe content, 10 mg / kg) -1 ); and (7) AuFe@mTOMV (10 mg kg -1 The mice were given near-infrared light irradiation group. For near-infrared light irradiation, mice received near-infrared light irradiation (1.0 W / cm²) 24 hours after intravenous injection. -2 (10 min). Mouse weight and tumor volume were recorded every other day. After treatment, all mice were euthanized, and tumors and major organs were collected for HE staining to assess tissue damage. Frozen sections of the tumor were further stained with Ki67 immunofluorescence to assess cancer cell proliferation.

[0055] Figure 6 The experiment fully demonstrated the experimental design, including the establishment of the 4T1 tumor-bearing mouse model, randomization, tail vein injection of different formulations, the timing of near-infrared laser irradiation, and the entire process of final sample collection and analysis.

[0056] like Figure 6 As shown in Figure 1, the tumor-suppressing effects of different treatments were dynamically presented through multiple sets of tumor growth curves. Among them, the AuFe@mTOMV combined with near-infrared light irradiation group showed the most significant and sustained tumor growth inhibition, with efficacy significantly better than each single-component treatment group.

[0057] like Figure 6 As shown in Figure j, the endpoint tumor weight statistical analysis further quantified the treatment effect. The data showed that the average tumor weight in the AuFe@mTOMV combined with light irradiation group was significantly lower than that in all other experimental groups, providing conclusive quantitative evidence of the efficacy endpoint.

[0058] like Figure 6 As shown in Figure k, by displaying the photos of the dissected solid tumors in each group, the differences in tumor size after different treatments are intuitively reflected. Among them, the tumor volume of the AuFe@mTOMV combined with light irradiation group is the smallest, and even tissue atrophy is observed, which is highly consistent with the weight data.

[0059] like Figure 6 As shown in Figure 1, the tumor tissue in the AuFe@mTOMV combined with light irradiation group exhibited large, uniformly red-stained areas of coagulative necrosis, indicating that the treatment induced widespread direct tumor cell death. Ki-67 immunohistochemical staining further revealed an extremely low proportion of Ki-67-positive proliferating cells in this group of tumor tissues, confirming that the proliferative activity of residual tumor cells was severely inhibited, explaining the effective containment of tumor growth from a cell kinetics perspective.

[0060] Test Example 7 In vivo analysis of anti-lung metastasis and immune infiltration was performed on the gold-iron core nanoparticles prepared in step S4 of Example 1 and the gold-iron core-shell nanoformulation encapsulated by the fusion membrane prepared in step S6. S1. Establishment of tumor-bearing and metastatic models and treatment regimen: Following the method described in Test Example 6, a 4T1 tumor-bearing mouse model was established. When the in situ tumor grew to a predetermined volume (approximately 100 mm²), the tumor was treated. 3 Mice were randomly assigned to groups. The grouping and treatment protocols were identical to those used in Test Case 6 for assessing the efficacy of primary treatment, including: a control group and a near-infrared light irradiation group (1.0 W cm⁻¹). -2 , 10 min), AuFe group (10 mg kg -1 AuFe (10 mg kg) -1 ) + NIR (1.0 W cm -2 (10 min) group, mTOMV group (10 mg kg) -1 ), AuFe@mTOMV group (10 mg kg -1 ) and AuFe@mTOMV (10 mg kg) -1 ) + NIR (1.0 W cm -2 To ensure data comparability, groups were divided into two groups (10 min and 10 min). Each group received the corresponding formulation via intravenous injection and / or near-infrared light irradiation.

[0061] S2. Detection and Assessment of Metastatic Lesions: At the treatment endpoint (e.g., day 22), mice were subjected to in vivo bioluminescence or fluorescence imaging 10 minutes after intraperitoneal injection of a luciferase substrate (e.g., D-luciferin, 3 mg). This non-invasive imaging was used to macroscopically assess the distribution and burden of metastatic lesions throughout the body (especially in the lungs). After euthanizing the mice, major organs (lungs, liver, spleen, kidneys, etc.) were immediately removed and subjected to ex vivo fluorescence imaging to more clearly observe and record the specific distribution of metastatic lesions in each organ, especially the lung tissue. The removed lung tissue was photographed to visually record the number and size of metastatic nodules on the surface. The lung tissue was fixed, embedded in paraffin, and sectioned for hematoxylin-eosin staining. Metastatic lesions in the lung tissue sections were observed and counted under a microscope to confirm the occurrence and extent of metastasis at a histological level.

[0062] S3. Analysis of immune cell infiltration in tumors and metastases: Simultaneously, in situ tumor tissue and lung tissue containing metastases, retrieved at the end of treatment, were paraffin-embedded and sectioned. Ki-67 antibody staining was performed on tumor and lung metastasis sections to assess the proliferative activity of residual tumor cells. CD8+ staining was performed on the sections using specific antibodies. + T cells and CD4 + Immunohistochemical staining of T cells. The infiltration density and distribution of these two key effector immune cells within tumor tissue and metastases were assessed by microscopic observation or quantitative analysis software.

[0063] Reference Figure 7 a. Using in vivo fluorescence imaging technology, the systemic metastatic burden of mice in different treatment groups at the treatment endpoint was directly compared. The results showed that the whole-body fluorescence signal of mice in the AuFe@mTOMV combined with near-infrared light irradiation group was the weakest, especially the signal in the thoracic region (a common site of lung metastasis) was significantly lower than that in other control groups, indicating that this treatment can effectively inhibit the spread of tumor cells to distant organs.

[0064] Reference Figure 7 b. Further in vitro imaging of major organs (lungs, liver, heart, spleen, and kidneys) clearly showed the differences in fluorescence intensity in lung tissue. The fluorescence signal in the lungs of the AuFe@mTOMV+NIR group was close to the background level, while obvious fluorescent spots were visible in the lung tissues of the control group and other monomodal treatment groups, confirming that the treatment can significantly reduce lung metastasis and colonization.

[0065] Reference Figure 7 c. Numerous white nodules (metastatic lesions) were visible on the lung surface of the control group and the group with weaker efficacy, while the lung tissue surface of the AuFe@mTOMV+NIR group was smooth, with very few or even nodules, providing the most direct visual evidence for the anti-metastatic effect.

[0066] Reference Figure 7d. In the lung sections of the control group, a large number of tumor cell clusters (metastatic foci) with a dense structure were visible; while the lung tissue of the AuFe@mTOMV+NIR group basically maintained the normal alveolar structure, with only occasional microfocal foci or no metastatic foci at all, which further confirmed the strong metastasis inhibition effect at the microscopic level.

[0067] Reference Figure 7 The proportion of Ki-67 positive cells in the metastatic lesions of the AuFe@mTOMV+NIR group was significantly reduced, indicating that even if a few cells escaped to the lungs, their proliferation was severely suppressed, which explains the reduction in metastatic lesions from the perspective of cell dynamics.

[0068] Reference Figure 7 f, CD4 was observed by immunohistochemical staining. + T cell infiltration within metastatic lesions. In the lung tissue of the AuFe@mTOMV+NIR group, a higher concentration of CD4+ cells was observed. + The infiltration of T cells into and around the metastatic lesions suggests that helper T cells were effectively recruited and may participate in regulating the anti-metastatic immune response.

[0069] Reference Figure 7 In the g, AuFe@mTOMV+NIR group, a large number of CD8 molecules were observed in and around the metastatic lesions. + The dense infiltration of T cells, which highly overlaps with the low-proliferation area of ​​Ki-67, directly indicates that activated cytotoxic T cells effectively gather at distant metastatic sites and attack tumor cells, which is a key immune mechanism for achieving metastasis suppression.

[0070] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A fusion film encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation, characterized in that, It comprises: a core of gold-iron core-shell nanoparticles; and a fusion film coated on the surface of the core, which is formed by fusion of bacterial outer membrane vesicles and tumor cell membranes.

2. The fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation according to claim 1, characterized in that, In the gold-iron core-shell nanoparticles, the elemental content of gold is 1-10%, and the elemental content of iron is 0.3-10%. The particle size of the nanodiagnostic and therapeutic preparation is 30-300 nm.

3. The fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation according to claim 1, characterized in that, The bacterial outer membrane vesicles are derived from Escherichia coli Nissle 1917. The tumor cell membrane is derived from 4T1 tumor cells.

4. The fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation according to claim 1, characterized in that, In the fusion film, the protein mass ratio of the tumor cell membrane to the bacterial outer membrane vesicles is 1:

1.

5. The method for preparing the fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic agent according to any one of claims 1-4, characterized in that, It comprises the following steps: S1. Extracting tumor cell membranes and bacterial outer membrane vesicles respectively; S2. Mixing the tumor cell membranes and the bacterial outer membrane vesicles in proportion, and then performing fusion treatment to obtain a fusion film; S3. Preparing gold-iron core-shell nanoparticles; S4. Mixing the gold-iron core-shell nanoparticles and the fusion film, and performing self-assembly to obtain the fusion film-encapsulated gold-iron core-shell nanodiagnostic and therapeutic preparation.

6. The method of claim 5, wherein the preparation of the fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation is characterized by, The extraction of tumor cell membranes in step S1 comprises: after ultrasonic treatment of tumor cells, first centrifugation and second centrifugation are performed in sequence, the precipitate is collected and freeze-dried; The extraction of bacterial outer membrane vesicles comprises: after bacterial culture, the supernatant is collected by centrifugation, filtered and then ultracentrifuged, and the precipitate is collected.

7. The method of claim 5, wherein the preparation of the fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation is characterized by, In step S2, the fusion treatment comprises: after ultrasonic treatment of the mixed system, incubation at 4℃ overnight.

8. The method of claim 5, wherein the preparation of the fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation is characterized by, The preparation of gold-iron core-shell nanoparticles in step S3 comprises: Dissolve CTAB, HAuCl4, sodium citrate and polyvinylpyrrolidone in deionized water, and after reaction in a 90℃ water bath, obtain gold nanoparticles; Perform thiol functionalization treatment on the gold nanoparticles to obtain thiol-functionalized gold nanoparticles; The thiol-functionalized gold nanoparticles are reacted with Fe 3+ After the solution reaction, H3BTC solution is added and incubated to deposit iron on the surface of the gold nanoparticles to obtain the gold-iron core-shell nanoparticles.

9. The method of claim 5, wherein the preparation of the fusion film-encapsulated chryso core-shell nanodiagnostic and nanotherapeutic formulation is characterized by, In step S4, the gold-iron core-shell nanoparticles are mixed with the fusion film in a mass ratio of 1:

1.

10. Use of the fusion film-encapsulated gold-iron core-shell nanodiagnostic and therapeutic preparation of any one of claims 1-4 in the preparation of a medicament for tumor immunotherapy.

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