Dual-targeting macrophage membrane nanosystem and its preparation method and application

Abstract

The present invention discloses a dual-targeted macrophage membrane nanosystem, its preparation method and application, which belongs to the field of biomedicine. MKA is formed by connecting AuNPs with the mitochondrial targeting peptide KLA and then encapsulating it in the macrophage membrane. During preparation, AuNPs are first synthesized and modified, and then encapsulated with the macrophage membrane. During treatment, it can dual-target tumor cells and mitochondria, enhance radiation-induced DNA damage, inhibit DDR, activate immune pathways, promote T cell infiltration, and consume glutathione and amplify oxidative stress to enhance immune effects. The dose enhancement ratio synergistic with 8GyX-rays is 3.1. In addition, MKA has good biocompatibility in the body, a long blood circulation time, high tumor targeting, and can be quickly cleared through the kidneys. This invention provides a new approach for tumor radioimmunotherapy and helps promote progress in clinical tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a dual-targeting macrophage membrane nanosystem and a preparation method and application thereof. Background Art

[0002] Radiation therapy (RT), an important approach to cancer treatment, inhibits tumor growth by inducing nuclear DNA damage in tumor cells. However, clinical practice has shown that RT-induced nuclear DNA damage often fails to stimulate a sufficiently strong immune response. This is because DNA damage activates the DNA damage response (DDR), which suppresses anti-tumor immune inflammation, leading to insufficient immune activation and significantly limiting the effectiveness of radiotherapy.

[0003] In recent years, studies have shown that the synergistic effects of mitochondrial DNA (mtDNA) and nuclear DNA damage can significantly enhance immune responses. When mitochondrial function is abnormal, mtDNA and RNA leak into the cytoplasm, activating the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway, which in turn stimulates immune cells and triggers a strong type I interferon response, helping to enhance anti-tumor immunity. However, in actual treatment, the precise delivery of therapeutic drugs to tumor cells and mitochondria faces many challenges, such as low drug uptake efficiency by cells in tumor tissue and increased tumor interstitial pressure affecting the penetration of nanoparticles and drugs. These challenges make it difficult for existing treatments to fully realize the potential of mitochondrial damage to enhance immune responses.

[0004] The blood-brain barrier (BBB) ​​presents a significant obstacle in brain tumor treatment. While the BBB highly selectively protects the brain, it also severely limits the delivery of therapeutic drugs to brain tumors. Although BBB permeability increases in pathological conditions such as inflammation or tumor growth, allowing some immune cells and nanoparticles to penetrate, effectively leveraging this property to enhance drug delivery remains a challenge.

[0005] Furthermore, subclinical inflammation is prevalent in the tumor microenvironment, and immune cells such as macrophages, neutrophils, and dendritic cells are recruited to the tumor site, potentially creating an immunosuppressive microenvironment and hindering therapeutic efficacy. Although studies have shown that low-dose X-ray-induced local inflammation can enhance immune cell recruitment and nanoparticle targeting efficiency, existing nanocarrier systems still have many shortcomings and cannot fully meet clinical needs.

[0006] Therefore, the development of a tumor radioimmunotherapy nanosystem that can effectively overcome the above problems, enhance the mtDNA damage induced by radiotherapy, and improve the effect of immunotherapy has important clinical significance and urgent needs. Summary of the Invention

[0007] The present invention aims to provide a dual-targeted macrophage membrane nanosystem and its preparation method and application, so as to solve the problems existing in existing radiotherapy, such as insufficient immune activation, high tumor radiation resistance, low drug delivery efficiency and tumor microenvironment immunosuppression, and improve the efficacy of tumor radioimmunotherapy.

[0008] The first aspect of the present invention relates to a dual-targeting macrophage membrane nanosystem, comprising:

[0009] The macrophage membrane-encapsulated KLA-Au nanoparticle system (MKA) consists of gold nanoparticles (AuNPs) covalently linked to the mitochondrial-targeting peptide KLA (KLAKLAKKLAK) via a Schiff base reaction to form KLA-Au nanoparticles, which are then encapsulated within the macrophage membrane. Macrophages possess immune homing capabilities, expressing receptors such as CCR2 and CX3CR1 on their cell membranes. These receptors specifically recognize and bind to chemokines released by inflamed tumors, enabling MKA to precisely target tumor tissues and cells. Simultaneously, the mitochondrial-targeting peptide KLA guides the AuNPs specifically to the mitochondria, achieving dual targeting of both tumor cells and mitochondria.

[0010] Nanosystem characteristics: After AuNPs were successfully combined with the mitochondrial targeting peptide KLA, the Fourier transform infrared spectroscopy (FTIR) was used to detect the mitochondrial targeting peptide KLA. -1 The KLA-Au nanoparticles exhibit characteristic imine bond (C=N) stretching vibrations. UV-visible spectroscopy revealed a red-shift in the plasmon absorption peak of the AuNPs from 520 nm to 535 nm, indicating stable surface modification and the absence of particle aggregation. Macrophage membrane encapsulation increased the hydrodynamic diameter of the KLA-Au nanoparticles from 125.6 nm to 180.6 nm and shifted their zeta potential from 29.4 mV to -39.1 mV, mimicking natural membrane properties and reducing nonspecific interactions. At an optimal KLA-Au concentration of 1 mg / mL, the membrane encapsulation efficiency reached 57.4%. The nanosystem exhibited excellent colloidal stability when stored at 4°C for 14 days.

[0011] The second aspect of the present invention relates to a method for preparing a dual-targeting macrophage membrane nanosystem, comprising the following steps:

[0012] 1. Synthesis of Gold Nanoparticles (AuNPs): AuNPs were synthesized using the citrate reduction method. A gold chloride (HAuCl4) solution and a citrate solution were mixed with vigorous stirring and heated to boiling. The formation of AuNPs was indicated by a change in the solution color from yellow to deep red. The AuNPs were then purified by centrifugation and resuspended in deionized water.

[0013] 2. Functionalization of AuNPs with the mitochondrial targeting peptide KLA: The mitochondrial targeting peptide KLA was attached to the surface of AuNPs using a thiol-based reaction to prepare KLA-Au nanoparticles. The modified nanoparticles were purified by centrifugation and characterized by UV-visible spectroscopy.

[0014] 3. Preparation of MKA Nanoparticles: Macrophages (RAW264.7) were cultured and harvested, and macrophage membrane vesicles were obtained by gentle cell lysis and centrifugation. KLA-functionalized AuNPs were incubated with the macrophage membrane vesicles at room temperature. Polycarbonate membranes were then used to repeatedly squeeze the membrane vesicles to encapsulate the KLA-Au nanoparticles, forming MKA nanoparticles. The MKA nanoparticles were purified by centrifugation and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements.

[0015] The third aspect of the present invention relates to the use of the above-mentioned nanosystem in the preparation of anti-tumor drugs for radioimmunotherapy.

[0016] The MKA nanosystem of the present invention can be used to prepare drugs that enhance the effect of tumor radioimmunotherapy. During radiotherapy, the MKA nanosystem plays multiple roles. First, AuNP mediates nuclear DNA chain breakage (γH2AX foci>25 / cell), and KLA induces the formation of mitochondrial permeability transition pore (mPTP) (ΔΨm>80%). The two synergistically damage the nuclear DNA and mitochondrial DNA of tumor cells and activate the immune response. Second, the MKA nanosystem enhances mitochondrial dysfunction, promotes the leakage of mtDNA and mtRNA into the cytoplasm, activates the RIG-I-MAVS immune pathway, stimulates immune cells, triggers a strong type I interferon response, and promotes CD8 + T cells infiltrate tumor tissues, enhancing anti-tumor immunity. Thirdly, the MKA nanosystem can selectively accumulate in tumor mitochondria, where AuNP-mediated radiosensitization generates a localized ROS burst. KLA-induced mPTP formation exacerbates mtDNA leakage and RIG-I / MAVS pathway activation. Furthermore, the MKA nanosystem depletes glutathione within the tumor, amplifying oxidative stress (ROS increases 4.1-fold), creating a pro-oxidative tumor microenvironment that favors immunogenic cell death.

[0017] Beneficial effects:

[0018] 1. Precise targeting and efficient treatment: The immune homing ability of macrophage membranes and the specific binding of surface receptors to chemokines at sites of tumor inflammation enable the MKA nanosystem to precisely target tumor cells, increase drug concentrations within tumor tissue, enhance therapeutic efficacy, and reduce damage to normal tissues.

[0019] 2. Synergistically enhances radiosensitization: The radiation sensitivity of AuNPs and the mitochondrial targeting of KLA synergize to amplify DNA damage, continuously inhibit DDR, and significantly reduce tumor cell radioresistance. The synergistic effect with 8Gy X-rays produces a dose enhancement ratio of 3.1, effectively enhancing the effectiveness of radiotherapy.

[0020] 3. Activate immune response: The mitochondrial and nuclear DNA damage induced by the MKA nanosystem continuously activates the immune response, promotes dendritic cell maturation, enhances antigen presentation and T cell activation, and attracts CD3 + 、CD4 + and CD8 + T cell infiltration and enhanced anti-tumor immune response.

[0021] 4. Good biosafety: In vivo experiments have shown that after intravenous injection of MKA (10 mg Au / kg) into ICR mice, platelet count (PLT: 952 ± 67 × 10 9 / L) and white blood cells (WBC: 4.6±0.8×10 9 Gold counts remained within the physiological range, serum biochemical markers (such as AST: 135±4 U / L, ALT: 48±3 U / L, and BUN: 18±2 mg / dL) showed no significant changes, and organ weight-to-body weight ratios remained stable. MKA has a long blood circulation half-life (t½ = 8.2±0.7 hours), preferentially accumulates in tumor tissues (accumulation in tumors reached 12.4±1.8% ID / g within 24 hours, 4.7 times that of major organs), and is rapidly cleared by the kidneys (68.3±5.2% of the gold dose was excreted in the urine within 48 hours), minimizing the risk of non-targeted accumulation and long-term toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Schematic diagram of the synthesis of gold nanoparticles (AuNPs).

[0023] Figure 2 : UV-visible spectra of different components in the MKA system.

[0024] Figure 3 : Flow cytometric analysis of changes in CD11b expression on macrophage membranes after treatment with conditioned medium from low-dose irradiated cells.

[0025] Figure 4 : Size distribution profile of MKA measured by dynamic light scattering (DLS).

[0026] Figure 5 : Zeta potential measurements of KLA-Au and MKA.

[0027] Figure 6 : The loading efficiency of KLA-Au at different concentrations onto macrophage membranes was determined using the polycarbonate membrane extrusion method.

[0028] Figure 7 : Size changes of MKA at 4°C for 14 days.

[0029] Figure 8 : Schematic diagram of the Transwell-based in vitro blood-brain barrier (BBB) ​​model.

[0030] Figure 9 : Distribution of MKA in the supernatant, intracellular compartments, and filtrate of the experimental and control groups after 3 h of incubation under basal conditions.

[0031] Figure 10 : Concentration-dependent viability of U87 glioblastoma cells after treatment with MKA (0-200 μg / mL) for 6 h.

[0032] Figure 11 : Cytotoxicity of KLA-Au and MKA in LO2 normal hepatocytes after 48 h exposure.

[0033] Figure 12 : High-resolution CLSM images confirmed the colocalization of DiR-labeled KLA-Au (red) with MitoTrackerGreen in mitochondria.

[0034] Figure 13 : Representative fluorescence images (left) and quantitative analysis (right) of reactive oxygen species (ROS) in U87 cells after 8 Gy irradiation using DCFH-DA probe (Scale bar: 50 μm; <0.001).

[0035] Figure 14 : Intracellular glutathione (GSH) levels in U87 cells treated with the control group, IR group, KLA-Au group, and MKA group (initial GSH: 20 μg / mL; <0.001).

[0036] Figure 15 : Time-dependent GSH depletion in U87 cells incubated with MKA for 30 h.

[0037] Figure 16 : Mitochondrial membrane potential (ΔΨm) collapse in U87 cells was measured by JC-1 staining in the control, MKA-treated, irradiated (IR, 8 Gy) or MKA+IR groups ( <0.001 compared with the control group; scale bar: 20 μm).

[0038] Figure 17: qPCR analysis of mitochondrial RNA (mtRNA) release (12S, COX1, D-loop, ND1).

[0039] Figure 18 : γ-H2AX immunofluorescence quantification of nuclear DNA double-strand breaks ( < 0.001; scale bar: 10 μm).

[0040] Figure 19 : Statistical graph of the percentage of cells in each cell cycle.

[0041] Figure 20 : Hematological parameters 1, 3, and 5 days after intravenous injection of MKA (10 mg Au / kg) or PBS.

[0042] Figure 21 : Serum biochemical markers within 5 days after injection.

[0043] Figure 22 : Ratios of various organs (heart, liver, spleen, lung, and kidney) to body weight at 0, 1, 3, 5, and 7 days after injection (n=5).

[0044] Figure 23 :The blood circulation half-life of MKA and the urinary gold excretion kinetics of MKA and free AuNPs within 48 hours were determined by ICP-MS ( <0.001).

[0045] Figure 24 : Biodistribution of MKA in major organs and tumors 24 hours after injection, expressed as percentage of injected dose per gram (%ID / g) ( <0.001, tumor compared with organ).

[0046] Figure 25 : Distribution of MKA and control group in urine 48 hours after injection. DETAILED DESCRIPTION

[0047] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are intended only to explain the present invention, and the scope of protection of the present invention should include the entire contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement the entire contents of the claims of the present invention.

[0048] Example 1: Preparation and characterization of MKA nanoparticles

[0049] Synthesis of gold nanoparticles (AuNPs): Reference Figure 1Using the citrate reduction method, 100 mL of an aqueous solution containing 1 mM HAuCl₄ was heated to boiling with vigorous stirring. 10 mL of a 1% sodium citrate solution was quickly added and refluxed for 30 minutes. The solution color changed from yellow to red, indicating the formation of AuNPs. The solution was purified by centrifugation (12,000 rpm, 20 minutes) and resuspended in deionized water.

[0050] Functional modification of AuNPs with mitochondrial targeting peptide KLA: Based on Figure 1 The mitochondrial targeting peptide KLA was attached to the surface of AuNPs based on the Schiff base reaction principle. From the Fourier transform infrared spectroscopy (FTIR), it can be seen that the mitochondrial targeting peptide KLA is attached to the surface of AuNPs. -1 The characteristic peak of imine bond (C=N) appears at Figure 2 The UV-visible spectrum showed that the plasmon absorption peak red-shifted from 520 nm to 535 nm, confirming that the mitochondrial targeting peptide KLA was successfully connected and the surface modification was stable without particle aggregation.

[0051] Preparation of MKA nanoparticles: RAW264.7 macrophages were cultured and pretreated with conditioned medium of tumor cells irradiated with 8 Gy X-rays. Figure 3 As shown in Figure 3, CD11b expression was upregulated by 2.5-fold by flow cytometry. Cell membrane vesicles were extracted and incubated with KLA-functionalized AuNPs at room temperature to prepare MKA nanoparticles. Figure 4 Dynamic light scattering (DLS) measurements showed that its hydrodynamic diameter was 180.6 nm. Figure 5 The zeta potential is -39.1mV, which simulates the natural membrane properties and reduces nonspecific interactions. Figure 6 It can be seen that when the optimal KLA-Au concentration is 10 mg / mL, the membrane encapsulation efficiency reaches 57.4%. Figure 7 It was shown that the nanosystem only increased in size by about 30% when stored at 4°C for 14 days, indicating good stability.

[0052] Example 2: In vitro performance verification of MKA nanoparticles

[0053] In vitro blood-brain barrier penetration experiment: Construction of a blood-brain barrier model based on Transwell ( Figure 8 ), human brain microvascular endothelial cells were seeded in Transwell chambers. The experimental group was added with MKA pre-treated with conditioned medium of tumor cells irradiated with 8Gy (corresponding to KLA-Au in the figure), and the control group was added with MKA pre-treated with conditioned medium of non-irradiated cells (corresponding to MKA in the figure). Figure 9The data showed that compared with the non-irradiated control group, the basolateral migration efficiency of MKA in the experimental group increased by 2.8 times (p<0.001), thanks to the DAMPs secreted by tumor cells induced by radiation, which activated the TLR4 / NF-κB signaling pathway, upregulated CD11b expression, and promoted MKA to penetrate the blood-brain barrier.

[0054] 3D tumor spheroid penetration assay: A 3DU87 glioblastoma spheroid model was constructed and incubated with MKA for 8 hours. Confocal laser scanning microscopy (CLSM) images clearly demonstrated depth-dependent penetration of MKA, 4.1-fold greater than that of KLA-Au (p<0.001). Confocal z-axis imaging revealed uniform distribution across all layers of the spheroid, attributed to repeated CD11b-ICAM-1 binding at the radiation-pretreated tumor interface.

[0055] In vitro radiosensitization experiment: U87 glioblastoma cells and LO2 hepatocytes were used as research objects. Figure 10 It was shown that after treatment of U87 cells with MKA+IR (200 μg / mL) for 6 hours, the survival rate dropped to 44.2% ( <0.001, compared with the control group), the radiosensitivity was improved compared with free KLA-Au; Figure 11 This indicates that the protruding membrane-coated MKA has low toxicity to LO2 hepatocytes. After incubation at a concentration of 100 μg / mL for 48 hours, the cells still maintained 80% viability ( <0.001, compared with KLA-Au). Figure 12 High-resolution confocal laser scanning microscopy (CLSM) images visually demonstrate the colocalization of DiR-labeled KLA-Au nanoparticles (red fluorescence) and MitoTracker Green-labeled mitochondria (green fluorescence). The results showed that KLA-Au was highly enriched at the cristae junctions of tumor cell mitochondria, with a Pearson correlation coefficient of r=0.92 (scale bar: 20 μm), significantly superior to commercial mitochondrial vectors. This demonstrates that the mitochondrial-targeting peptide KLA successfully guides the precise localization of AuNPs to mitochondria, providing a spatial basis for the subsequent induction of mitochondrial membrane potential collapse and mtDNA leakage.

[0056] Example 3: Study on the therapeutic mechanism of MKA nanoparticles

[0057] Study on the mechanism of oxidative stress and apoptosis: In the U87 cell experiment, Figure 13 Reactive oxygen species (ROS) levels in U87 glioblastoma cells were quantitatively analyzed using the DCFH-DA fluorescent probe. Representative fluorescence images (left) (scale bar: 50 μm), and quantitative data (right). Results showed that ROS levels in the MKA+8Gy X-ray combined treatment group increased 4.1-fold compared to the control group ( <0.001), with ROS increases of 2.3-fold and 1.8-fold in the MKA-only or irradiation-only groups, respectively. These data confirm that MKA significantly exacerbates oxidative damage in tumor cells by depleting glutathione (GSH) and amplifying radiation-induced oxidative stress, creating critical conditions for immunogenic cell death. Figure 14 and Figure 15 Results showed that MKA depleted intracellular glutathione (GSH) to 18.3±2.4% of baseline within 30 hours (p<0.001). Annexin V / PI flow cytometry revealed that MKA enhanced radiation-induced apoptosis, driven by mitochondrial-targeted activation of caspase-9 / 3.

[0058] Study on the synergistic mechanism of mitochondrial DNA and nuclear DNA damage: U87 cells treated with MKA combined with irradiation, Figure 16 It showed that the combined treatment of MKA and irradiation caused a severe collapse of mitochondrial membrane potential, which decreased to 18.4±2.1% of the control group (p<0.001); Figure 17 It showed that the release of mitochondrial RNA (mtRNA) increased by 4.8-7.3 times (p<0.001), activated the RIG-I / MAVS pathway, and upregulated IFN-β transcription by more than 15 times. Figure 18 The degree of nuclear DNA double-strand breaks was quantitatively assessed by immunofluorescence staining of γ-H2AX (scale bar: 10 μm). The results showed that the number of γ-H2AX foci in the MKA+8Gy X-ray group was 1.37 × 10 nm. >25 / cell , increased by 3.5 times compared with the control group ( <0.001), the number of foci in the MKA or irradiation groups was 12 / cell and 8 / cell, respectively. This result demonstrates that AuNPs significantly amplify nuclear DNA damage through radiosensitization, synergize with mitochondrial dysfunction induced by the mitochondrial targeting peptide KLA, and jointly activate the cGAS-STING immune pathway, providing a molecular basis for enhancing anti-tumor immune responses. In addition, Figure 19 The cells were shown to be arrested in the G2 / M phase (62.3±3.8% in the MKA+IR group and 22.1±2.4% in the control group, p<0.001), and micronucleus formation increased 5.1-fold (p<0.001).

[0059] Example 4: In vivo performance evaluation of MKA nanoparticles

[0060] In vivo biocompatibility assessment: ICR mice were intravenously injected with MKA (10 mg Au / kg). Figure 20 It showed that platelet (PLT) and white blood cell (WBC) counts were within the physiological range within 5 days, and there was no significant hematological abnormality compared with the PBS group (p>0.05); Figure 21 It showed that there was no statistically significant difference in the levels of serum biochemical markers (AST, ALT, BUN) compared with the baseline; Figure 22 The results showed that the ratios of various organs to body weight remained stable over 7 days. These results indicate that MKA has good biocompatibility.

[0061] In vivo pharmacokinetics and biodistribution studies: ICP-MS analysis ( Figure 23 ), the circulation time of MKA in the blood was prolonged, with a half-life (t1 / 2) of 8.2±0.7 hours, which was 2.3 times that of uncoated AuNPs (p<0.001). Figure 24 It shows the distribution of Au in tumors and major organs such as liver and spleen; Figure 25 The results showed that MKA was rapidly cleared by the kidneys, with 68.3±5.2% of the injected gold dose excreted in the urine within 48 hours (p<0.001 compared with AuNPs).

[0062] In vivo anti-tumor efficacy evaluation: A tumor-bearing mouse model was established, and mice were divided into groups and given different treatment regimens. Monitoring of tumor volume and body weight revealed significant tumor growth inhibition and prolonged survival in the MKA + irradiation group. Samples were collected and tested on day 8 of treatment to further verify the in vivo anti-tumor efficacy of MKA and its impact on immune parameters.

[0063] The above examples, with the help of the accompanying drawings, demonstrate in detail that the MKA nanosystem significantly enhances the effect of radioimmunotherapy through dual-targeted delivery (tumor microenvironment and mitochondria), synergistic damage to nuclear DNA and mtDNA, and activation of the RIG-I / cGAS-STING dual immune pathway, and has good biocompatibility and clinical translation potential.

[0064] In summary, the present invention successfully prepared and fully characterized the MKA nanosystem. In vitro, MKA demonstrated excellent radiosensitization, effectively inducing DNA damage and apoptosis in tumor cells. In in vivo, MKA exhibited good biosafety, excellent tumor targeting, and significantly enhanced the efficacy of tumor radioimmunotherapy. This invention provides an innovative and highly promising strategy for tumor radioimmunotherapy, potentially bringing new breakthroughs and revolutions in clinical cancer treatment. Future research and applications will allow for further optimization of MKA preparation processes and treatment protocols, enabling targeted treatment tailored to different tumor types and individual patient differences, to achieve more efficient and safer cancer treatment. Furthermore, the research findings of this invention provide important insights and references for further research in related fields, contributing to the continuous advancement of tumor treatment technologies. Regarding practical application, this nanosystem can be further explored in combination with other therapeutic approaches (such as chemotherapeutic drugs and immune checkpoint inhibitors), evaluating the efficacy of combined treatments for different tumor types, and providing more options for comprehensive clinical cancer treatment. In terms of process optimization, efforts can be made to improve nanoparticle synthesis methods, macrophage membrane encapsulation techniques, and targeting peptide modification strategies to enhance the encapsulation efficiency, stability, and targeting of the nanosystem, reduce production costs, and promote its transition from laboratory research to clinical application. Based on the individual differences of different cancer patients, personalized treatment research can be conducted. Based on factors such as tumor type, stage, and gene expression profile, the MKA dosage and treatment regimen can be precisely adjusted to improve therapeutic efficacy while minimizing adverse reactions, thus achieving true precision medicine.

[0065] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. Dual-targeting macrophage membrane nanosystem, characterized in that: The invention comprises KLA-Au nanoparticles formed by covalently linking gold nanoparticles and mitochondrial targeting peptide KLA through a Schiff base reaction, and macrophage membranes encapsulating the KLA-Au nanoparticles; the sequence of the mitochondrial targeting peptide KLA is KLAKLAKKLAK, and the covalent linking to the AuNPs is achieved through a thiol reaction; the particle size of the AuNPs is 15-25 nm, and the surface plasmon absorption peak is 535±3 nm; the macrophage membranes are derived from the macrophage cell line RAW264.7, express CCR2 and CX3CR1 receptors on the surface, and can specifically target chemokines at inflammatory tumor sites; the method for extracting macrophage membrane vesicles comprises: pretreating the macrophages with tumor cell conditioned medium containing 8Gy X-ray irradiation to induce a 2.5-fold increase in CD11b receptor expression; and separating the cell membranes by hypotonic lysis and gradient centrifugation.

2. The dual-targeting macrophage membrane nanosystem according to claim 1, characterized in that: The hydrodynamic diameter of the nanosystem was 180.6±5.2 nm and the zeta potential was -39.1±2.3 mV.

3. A method for preparing the dual-targeting macrophage membrane nanosystem according to any one of claims 1-2, characterized in that: The following steps are involved: 1) Gold nanoparticles were synthesized by citrate reduction, purified by centrifugation, and dispersed in deionized water; 2) The mitochondrial targeting peptide KLA was modified onto the surface of AuNPs via a thiol reaction to form KLA-Au nanoparticles; 3) extracting RAW264.7 macrophage membrane vesicles, and encapsulating KLA-Au nanoparticles within the membrane vesicles via a polycarbonate membrane extrusion method to form a dual-targeted nanosystem. The macrophage membrane vesicle extraction method comprises: pretreating the macrophages with conditioned medium from tumor cells containing 8 Gy X-ray irradiation to induce a 2.5-fold upregulation of CD11b receptor expression; and isolating the cell membranes via hypotonic lysis and gradient centrifugation.

4. Use of the dual-targeting macrophage membrane nanosystem according to any one of claims 1-2 in the preparation of drugs for enhancing tumor radioimmunotherapy, characterized in that: The nanosystem functions through the following mechanisms: AuNPs mediated nuclear DNA strand breaks, resulting in γH2AX foci numbers >25 / cell; The mitochondrial targeting peptide KLA induces the collapse of mitochondrial membrane potential, leading to the release of mtDNA and mtRNA; Activate the RIG-I-MAVS and cGAS-STING immune pathways and promote CD8 + T cell infiltration; The tumor is a glioblastoma.

5. The use according to claim 4, characterized in that The nanosystem had a dose enhancement ratio of ≥3.1 when combined with 8Gy X-rays, and could deplete glutathione in tumor cells to 18.3±2.4% of the baseline, increasing ROS levels by 4.1-fold.

6. The use according to claim 4, characterized in that The nanosystem was able to penetrate the blood-brain barrier, and its basolateral migration efficiency in the Transwell model was 2.8 times higher than that of the non-irradiated control group.

7. A tumor radioimmunotherapy composition, characterized in that: The invention comprises the dual-targeting macrophage membrane nanosystem according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier, wherein the gold nanoparticle concentration of the composition is 0.5-2 mg / mL and the macrophage membrane encapsulation efficiency is ≥57.4%.

8. The composition according to claim 7, characterized in that After intravenous injection, the composition: The blood circulation half-life is 8.2±0.7 hours; The 24-hour intratumoral accumulation was 12.4 ± 1.8% ID / g, which was 4.7 times that of major organs; 68.3±5.2% of the gold dose was eliminated by the kidneys within 48 hours.

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