Dual-targeting macrophage membrane nano system as well as preparation method and application thereof

Abstract

The invention discloses a dual-targeting macrophage membrane nano system and a preparation method and application thereof, and belongs to the field of biological medicine, MKA is wrapped in a macrophage membrane after AuNPs and mitochondrial targeting peptide KLA are connected. The preparation method comprises the following steps: firstly synthesizing AuNPs and modifying, and then wrapping with a macrophage membrane. In treatment, the compound can realize double targeting of tumor cells and mitochondria, enhance radiation-induced DNA damage, inhibit DDR, activate immune pathways and promote T cell infiltration, and can also consume glutathione and amplify oxidative stress to enhance immune effect, and the dose enhancement ratio of the compound in cooperation with 8GyX-rays reaches 3.1. In addition, the MKA is good in biocompatibility in vivo, long in blood circulation time, high in tumor targeting and capable of being quickly cleared through the kidney. The invention provides a new way for tumor radioimmunotherapy, and is helpful for promoting the progress of clinical tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a dual-targeted macrophage cell membrane nanosystem, its preparation method and applications. Background Art

[0002] Radiotherapy (RT), as one of the important means of tumor treatment, inhibits tumor growth by inducing nuclear DNA damage in tumor cells. However, clinical practice has found that the nuclear DNA damage caused by RT usually fails to trigger a sufficiently strong immune response. This is because DNA damage activates the DNA Damage Response (DDR), which inhibits anti-tumor immune inflammation, resulting in insufficient immune activation and greatly limiting the effectiveness of radiotherapy.

[0003] In recent years, studies have shown that the synergistic effect of mitochondrial DNA (mtDNA) and nuclear DNA damage can significantly enhance the immune response. 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 then stimulates immune cells and triggers a strong type I interferon response, contributing to enhanced anti-tumor immunity. However, in actual treatment, precisely delivering therapeutic drugs to tumor cells and mitochondria faces many challenges, such as low drug uptake efficiency by cells in tumor tissues, increased tumor interstitial pressure affecting the penetration of nanoparticles and drugs, etc., making it difficult for existing treatment methods to fully exert the potential of mitochondrial damage to enhance the immune response.

[0004] In the treatment of brain tumors, the blood-brain barrier (BBB) is a major obstacle. The BBB highly selectively protects the brain, but at the same time severely restricts the delivery of therapeutic drugs to brain tumors. Although the permeability of the BBB increases under pathological conditions such as inflammation or tumor growth, allowing some immune cells and nanoparticles to penetrate, how to effectively utilize this property to improve drug delivery efficiency remains a difficult problem.

[0005] In addition, subclinical inflammation in the tumor microenvironment is widespread. Immune cells such as macrophages, neutrophils, and dendritic cells are recruited to the tumor site, and they may create an immunosuppressive microenvironment, hindering the treatment effect. Although existing studies have shown that locally induced inflammation by low-dose X-rays can enhance immune cell recruitment and nanoparticle targeting efficiency, existing nanocarrier systems still have many deficiencies and cannot fully meet clinical needs.

[0006] Therefore, it is of great clinical significance and urgent need to develop a tumor radioimmunotherapy nanosystem that can effectively overcome the above problems, enhance mtDNA damage caused by radiotherapy, and improve the effect of immunotherapy. Summary of the Invention

[0007] The present invention aims to provide a dual-targeting macrophage cell 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 immune suppression in the tumor microenvironment, and improve the efficacy of tumor radioimmunotherapy.

[0008] In the first aspect of the present invention, it relates to a dual-targeting macrophage cell membrane nanosystem, including:

[0009] A macrophage cell membrane-coated KLA-Au nanoparticle system (MKA), which is formed by covalently connecting gold nanoparticles (AuNPs) and the mitochondrial targeting peptide KLA (KLAKLAKKLAKLAK) through a Schiff base reaction to form KLA-Au nanoparticles, and is encapsulated within the macrophage cell membrane. Macrophages have the ability of immune homing, and their cell membrane surfaces express receptors such as CCR2 and CX3CR1, which can specifically recognize and bind chemokines released from inflamed tumor sites, enabling MKA to accurately target tumor tissues and cells. At the same time, the mitochondrial targeting peptide KLA can guide AuNPs to specifically target mitochondria, achieving dual-targeting effects on tumor cells and mitochondria.

[0010] Properties of the nanosystem: After the successful binding of AuNPs and the mitochondrial targeting peptide KLA, detected by Fourier transform infrared spectroscopy (FTIR), there is a characteristic stretching vibration of the imine bond (C=N) at 1650 cm -1 ; the ultraviolet-visible spectrum shows that the plasmon absorption peak of AuNPs redshifts from 520 nm to 535 nm, indicating stable surface modification and no particle aggregation. The encapsulation of the macrophage cell membrane increases the hydrodynamic diameter of KLA-Au from 125.6 nm to 180.6 nm, and the zeta potential changes from 29.4 mV to -39.1 mV, simulating the properties of natural membranes and reducing non-specific interactions. At the optimal KLA-Au concentration of 1 mg / mL, the membrane encapsulation efficiency reaches 57.4%, and this nanosystem has good colloidal stability when stored at 4 °C for 14 days.

[0011] In the second aspect of the present invention, it relates to a preparation method of a dual-targeting macrophage cell membrane nanosystem, including the following steps:

[0012] 1. Synthesis of gold nanoparticles (AuNPs): AuNPs are synthesized by the citrate reduction method. A solution of chloroauric acid (HAuCl4) and a citrate solution are mixed under vigorous stirring and heated to boiling. When the color of the solution changes from yellow to dark red, it indicates the formation of AuNPs. Subsequently, AuNPs are purified by centrifugation and resuspended in deionized water.

[0013] 2. Functional modification of AuNPs with mitochondrial targeting peptide KLA: Using thiol-based reactions, the mitochondrial targeting peptide KLA was linked to the surface of AuNPs to prepare KLA-Au nanoparticles. The modified nanoparticles were purified by centrifugation and characterized using ultraviolet-visible spectroscopy.

[0014] 3. Preparation of MKA nanoparticles: Macrophages (RAW264.7) were cultured and harvested, and macrophage membrane vesicles were obtained by centrifugation using a mild cell lysis method. The KLA-functionalized AuNPs were incubated with macrophage membrane vesicles at room temperature, and the membrane vesicles were used to encapsulate the KLA-Au nanoparticles by continuous extrusion through a polycarbonate membrane to form MKA nanoparticles. The MKA nanoparticles were purified by centrifugation and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and Zeta potential measurement.

[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 for enhancing the effect of tumor radioimmunotherapy. During radiotherapy, the MKA nanosystem plays multiple roles. First, AuNP mediates nuclear DNA strand breaks (γH2AX foci > 25 per cell), and KLA induces the formation of mitochondrial permeability transition pores (mPTP) (ΔΨm > 80%). The two work together to damage the nuclear DNA and mitochondrial DNA of tumor cells, activating 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 cell infiltration into tumor tissues, enhancing the anti-tumor immune effect. Third, the MKA nanosystem can selectively accumulate in tumor mitochondria. The AuNP-mediated radiosensitization generates local ROS bursts, and the KLA-induced mPTP formation exacerbates mtDNA leakage and RIG-I / MAVS pathway activation. At the same time, the MKA nanosystem can also consume glutathione in tumors, amplifying oxidative stress (ROS increased 4.1-fold), forming a pro-oxidative tumor microenvironment conducive to immunogenic cell death.

[0017] Beneficial effects:

[0018] 1. Precise targeting and efficient treatment: The immune homing ability of the macrophage membrane and the specific binding of surface receptors to chemokines at the tumor inflammation site enable the MKA nanosystem to precisely target tumor cells, increase the drug concentration in tumor tissues, enhance the treatment effect, and reduce damage to normal tissues at the same time.

[0019] 2. Synergistic radiosensitization: The radiosensitivity of AuNPs and the mitochondrial targeting effect of KLA are synergistic, amplifying the degree of DNA damage, continuously inhibiting DDR, and significantly reducing the radiation resistance of tumor cells. The dose enhancement ratio generated by the synergistic effect with 8 Gy X-rays is 3.1, effectively enhancing the radiotherapy effect.

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

[0021] 4. Good biosafety: Verified by in vivo experiments, after intravenous injection of MKA (10 mg Au / kg) into ICR mice, the platelet (PLT: 952 ± 67 × 10 9 / L) and white blood cell (WBC: 4.6 ± 0.8 × 10 9 / L) counts remained within the physiological range within 5 days, and there were no significant changes in serum biochemical markers (such as AST: 135 ± 4 U / L, ALT: 48 ± 3 U / L, BUN: 18 ± 2 mg / dL), and the ratio of organ weight to body weight was stable. MKA has a long blood circulation half-life (t1 / 2 = 8.2 ± 0.7 hours), can preferentially accumulate in tumor tissues (the accumulation amount in tumors reached 12.4 ± 1.8% ID / g at 24 hours, 4.7 times that of the main organs), and is rapidly cleared through the kidneys (68.3 ± 5.2% of the gold dose was excreted in urine within 48 hours), reducing the risk of non-target accumulation and long-term toxicity. 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 cytometry analysis of the change in CD11b expression after treating macrophage membranes with conditioned medium irradiated at a low dose.

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

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

[0027] Figure 6 : Determination of the macrophage membrane loading efficiency of different concentrations of KLA-Au using the polycarbonate membrane extrusion method.

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

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

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

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

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

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

[0034] Figure 13 : Representative fluorescence images (left) and quantitative analysis (right) of reactive oxygen species (ROS) in U87 cells after 8 Gy irradiation detected with the 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 consumption in U87 cells incubated with MKA within 30 hours.

[0037] Figure 16 : Mitochondrial membrane potential (ΔΨm) collapse in U87 cells measured by JC-1 staining in the control group, MKA-treated group, irradiated (IR, 8 Gy) group, or MKA + IR group ( <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 : Quantitative immunofluorescence of γ-H2AX for nuclear DNA double-strand breaks ( <0.001; Scale bar: 10 μm).

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

[0041] Figure 20 : Hematological parameters at 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 : Ratio of each organ (heart, liver, spleen, lung, kidney) to body weight at 0, 1, 3, 5, and 7 days after injection (n = 5).

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

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

[0046] Figure 25 : Distribution of MKA and control group in urine at 48 hours after injection. Detailed implementation manners

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

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

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

[0050] Functional modification of AuNPs with mitochondrial targeting peptide KLA: According to Figure 1 the Schiff base reaction principle, the mitochondrial targeting peptide KLA was linked to the surface of AuNPs. As can be seen from the Fourier transform infrared spectroscopy (FTIR), a characteristic peak of imine bond (C=N) appeared at 1650 cm -1 ; Figure 2 The ultraviolet-visible spectrum of

[0051] showed that the plasmon absorption peak red-shifted from 520 nm to 535 nm, confirming the successful connection of the mitochondrial targeting peptide KLA and the stable surface modification without particle aggregation. Figure 3 Preparation of MKA nanoparticles: RAW264.7 macrophages were cultured and pretreated with the conditioned medium of tumor cells irradiated with 8 Gy X-rays. As shown in Figure 4 the dynamic light scattering (DLS) measurement, its hydrodynamic diameter was 180.6 nm, Figure 5 indicating that the zeta potential was -39.1 mV, simulating the natural membrane properties to reduce non-specific interactions. As can be seen from Figure 6 , at the optimal KLA-Au concentration of 10 mg / mL, the membrane encapsulation efficiency reached 57.4%. Figure 7 showed that the size of this nanosystem only increased by about 30% after being stored at 4 °C for 14 days, with good stability.

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

[0053] In vitro blood-brain barrier penetration experiment: A Transwell-based blood-brain barrier model ( Figure 8 ) was constructed, and human brain microvascular endothelial cells were seeded in the Transwell chamber. In the experimental group, MKA pretreated with the conditioned medium of tumor cells irradiated with 8 Gy (KLA-Au corresponding to the figure) was added, and in the control group, MKA treated with the non-irradiated conditioned medium (MKA corresponding to the figure) was added. As shown in Figure 9Data showed that compared with the untreated control group, the migration efficiency of MKA in the experimental group increased by 2.8-fold in the basolateral region (p<0.001). This was attributed to the activation of the TLR4 / NF-κB signaling pathway by DAMPs secreted by tumor cells upon radiation, upregulating the expression of CD11b and promoting the penetration of MKA through the blood-brain barrier.

[0054] 3D tumor spheroid penetration experiment: A 3D U87 glioblastoma spheroid model was constructed and co-incubated with MKA for 8 hours. It was clearly visible from the confocal laser scanning microscopy (CLSM) images that MKA achieved depth-dependent penetration, which was 4.1-fold higher than that of KLA-Au (p<0.001). Confocal Z-axis imaging showed its uniform distribution in each layer of the spheroid, which was due to the repeated CD11b-ICAM-1 binding at the irradiated tumor interface.

[0055] In vitro radiosensitization experiment: U87 glioblastoma cells and LO2 hepatocytes were used as the research objects. Figure 10 It was shown that after treating U87 cells with MKA+IR (200 μg / mL) for 6 hours, the survival rate decreased to 44.2% ( <0.001, compared with the control group), and the radiosensitivity was improved compared with free KLA-Au; Figure 11 It was indicated that MKA encapsulated by the prominent membrane had low toxicity to LO2 hepatocytes. After incubating at a concentration of 100 μg / mL for 48 hours, the cells still maintained 80% viability ( <0.001, compared with KLA-Au). Figure 12 Through high-resolution confocal laser scanning microscopy (CLSM) images, the co-localization of DiR-labeled KLA-Au nanoparticles (red fluorescence) and MitoTracker Green-labeled mitochondria (green fluorescence) was visually demonstrated. The results showed that KLA-Au was highly enriched at the junctions of mitochondrial cristae in tumor cells, with a Pearson correlation coefficient of r = 0.92 (scale bar: 20 μm), which was significantly better than that of commercial mitochondrial carriers. This indicated that the mitochondrial targeting peptide KLA successfully guided AuNPs to accurately localize in mitochondria, providing a spatial basis for subsequent induction of mitochondrial membrane potential collapse and mtDNA leakage.

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

[0057] Research on oxidative stress and apoptosis-related mechanisms: In the U87 cell experiment, Figure 13 The level of reactive oxygen species (ROS) in U87 glioblastoma cells was quantitatively analyzed using the DCFH-DA fluorescent probe. The left figure is a representative fluorescence image (scale bar: 50 μm), and the right figure is the quantitative data. The results showed that the ROS level in the MKA+8 Gy X-ray combined treatment group was 4.1-fold higher than that in the control group ( (<0.001). The ROS increases in the MKA-alone or irradiation-alone groups were 2.3-fold and 1.8-fold, respectively. These data confirm that MKA significantly exacerbates oxidative damage in tumor cells by depleting glutathione (GSH) and amplifying radiation-induced oxidative stress, creating key conditions for immunogenic cell death. Figure 14 and Figure 15 showed that MKA depleted intracellular glutathione (GSH) to 18.3 ± 2.4% of the baseline within 30 hours (p < 0.001). Annexin V / PI flow cytometry showed that MKA enhanced radiation-induced apoptosis, which was driven by mitochondrial targeting to activate caspase-9 / 3.

[0058] Study on the synergistic mechanism of mitochondrial DNA and nuclear DNA damage: U87 cells were treated with MKA and irradiation in combination. Figure 16 showed that the combined treatment of MKA and irradiation caused a severe collapse of the mitochondrial membrane potential, which decreased to 18.4 ± 2.1% of the control group (p < 0.001); Figure 17 showed that the release of mitochondrial RNA (mtRNA) increased by 4.8 - 7.3-fold (p < 0.001), activating the RIG-I / MAVS pathway and upregulating IFN-β transcription by more than 15-fold. Figure 18 The degree of nuclear DNA double-strand breaks was quantitatively evaluated by γ-H2AX immunofluorescence staining (scale bar: 10 μm). The results showed that the number of γ-H2AX foci in the MKA + 8 Gy X-ray group reached > 25 foci / cell , which was 3.5-fold higher than that of the control group ( <0.001). The number of foci in the MKA-alone or irradiation-alone groups was 12 foci / cell and 8 foci / cell, respectively. This result proves that AuNPs significantly amplify nuclear DNA damage through radiosensitization, synergistically with mitochondrial dysfunction induced by the mitochondrial-targeted peptide KLA, and jointly activate the cGAS-STING immune pathway, providing a molecular basis for enhancing the anti-tumor immune response. In addition, Figure 19 showed cell cycle arrest 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 the formation of micronuclei increased by 5.1-fold (p < 0.001).

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

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

[0061] In vivo pharmacokinetics and biodistribution study: By ICP-MS analysis ( Figure 23 ), the circulation time of MKA in the blood was prolonged, and the half-life (t1 / 2) was 8.2 ± 0.7 h, which was 2.3 times that of uncoated AuNPs (p < 0.001). Figure 24 showed the distribution of Au in tumors and major organs such as the liver and spleen; Figure 25 indicated rapid renal clearance of MKA, and 68.3 ± 5.2% of the injected gold dose was excreted via urine within 48 h (p < 0.001 compared with AuNPs).

[0062] In vivo anti-tumor efficacy evaluation: A tumor-bearing mouse model was established, and the mice were grouped and given different treatment regimens. By monitoring tumor volume and body weight, it was found that the tumor growth in the MKA + irradiation group was significantly inhibited and the survival period was prolonged. On the 8th day of treatment, samples were collected for detection to further verify the in vivo anti-tumor effect of MKA and its impact on immune parameters.

[0063] The above embodiments detailedly verified, with the aid of the attached drawings, the mechanism of the MKA nanosystem to significantly enhance the radioimmunotherapy effect through dual-targeted delivery (tumor microenvironment and mitochondria), synergistically damage nuclear DNA and mtDNA, and activate the RIG-I / cGAS-STING dual immune pathway, and it has good biocompatibility and clinical translation potential.

[0064] In summary, the present invention has successfully prepared and comprehensively characterized the MKA nanosystem. In in vitro experiments, MKA demonstrated good radiosensitizing effects, effectively inducing DNA damage and apoptosis in tumor cells; in in vivo experiments, MKA exhibited good biosafety, excellent tumor targeting, and significantly enhanced the efficacy of tumor radioimmunotherapy. The present invention provides an innovative and highly promising strategy for tumor radioimmunotherapy, which is expected to bring new breakthroughs and changes to clinical tumor treatment. In future research and applications, the preparation process and treatment regimen of MKA can be further optimized to achieve precise treatment according to different tumor types and individual patient differences, so as to achieve more efficient and safer tumor treatment goals. At the same time, the research results of the present invention also provide important references for in-depth research in related fields, contributing to the continuous progress of tumor treatment technologies. In terms of expanding the actual application scenarios, the possibility of combining this nanosystem with other treatment methods (such as chemotherapeutic drugs, immune checkpoint inhibitors, etc.) can be further explored, and the efficacy of the combined treatment regimen for different types of tumors can be evaluated to provide more options for clinical comprehensive tumor treatment. In optimizing the preparation process, attempts can be made to improve the synthesis method of nanoparticles, the encapsulation technology of macrophage cell membranes, and the modification strategy of targeting peptides, so as to improve the encapsulation efficiency, stability, and targeting of the nanosystem, reduce production costs, and promote its transformation from laboratory research to clinical application. For the individual differences of different tumor patients, personalized treatment research can be carried out. According to factors such as the patient's tumor type, stage, and gene expression profile, the dose and treatment regimen of MKA can be precisely adjusted to improve the treatment effect while reducing adverse reactions, achieving true precision medicine.

[0065] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A dual-targeting macrophage cell membrane nanosystem, characterized in that, It includes KLA-Au nanoparticles formed by covalently linking gold nanoparticles and the mitochondrial targeting peptide KLA through a Schiff base reaction, and macrophage cell membranes encapsulating the KLA-Au nanoparticles; the macrophage cell membranes are derived from the macrophage cell line RAW264.7, expressing CCR2 and CX3CR1 receptors on the surface, and can specifically target chemokines at the inflammatory tumor site. The extraction method of macrophage cell membrane vesicles includes: pretreating macrophages with the conditioned medium of tumor cells irradiated with 8 Gy X-rays to induce a 2.5-fold upregulation of CD11b receptor expression; separating cell membranes by hypotonic lysis and gradient centrifugation.

2. The dual-targeting macrophage cell membrane nanosystem according to claim 1, wherein The hydrodynamic diameter of the nanosystem is 180.6 ± 5.2 nm, and the zeta potential is -39.1 ± 2.3 mV.

3. The dual-targeting macrophage cell membrane nanosystem according to claim 1, characterized in that, The sequence of the mitochondrial targeting peptide KLA is KLAKLAKKLAKLAK, and its covalent connection with 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.

4. A method for preparing the dual-targeting macrophage membrane nanosystem according to any one of claims 1-3, characterized in that, It includes the following steps: 1) Synthesize gold nanoparticles by the citrate reduction method, and disperse them in deionized water after centrifugal purification; 2) Modify the mitochondrial targeting peptide KLA onto the surface of AuNPs through a thiol reaction to form KLA-Au nanoparticles; 3) Extract RAW264.7 macrophage cell membrane vesicles, and encapsulate KLA-Au nanoparticles within the membrane vesicles by the polycarbonate membrane extrusion method to form a dual-targeted nanosystem. The extraction method of the macrophage cell membrane vesicles includes: pretreating macrophages with the conditioned medium of tumor cells irradiated with 8 Gy X-rays to induce a 2.5-fold upregulation of CD11b receptor expression; separating cell membranes by hypotonic lysis and gradient centrifugation.

5. Use of the dual-targeted macrophage cell membrane nanosystem according to any one of claims 1-3 in the preparation of a drug for enhancing tumor radioimmunotherapy, characterized in that, The nanosystem exerts its function through the following mechanism: AuNPs mediate the breakage of nuclear DNA strands, resulting in >25 γH2AX foci per cell; The mitochondrial targeting peptide KLA induces the collapse of the mitochondrial membrane potential, leading to the release of mtDNA and mtRNA; Activate the RIG-I-MAVS and cGAS-STING immune pathways to promote CD8 + T cell infiltration.

6. The application according to claim 5, wherein When the nanosystem is used in combination with 8 Gy X-rays, the dose enhancement ratio ≥ 3.1, and it can deplete the glutathione in tumor cells to 18.3 ± 2.4% of the baseline, increasing the ROS level by 4.1 times.

7. The application according to claim 5, wherein The nanosystem can penetrate the blood-brain barrier, and the basolateral migration efficiency in the Transwell model is 2.8 times higher than that of the non-irradiated control group.

8. A tumor radioimmunotherapy composition, characterized in that, It contains the dual-targeted macrophage cell membrane nanosystem according to any one of claims 1 - 3 and a pharmaceutically acceptable carrier. The concentration of gold nanoparticles in the composition is 0.5 - 2 mg / mL, and the encapsulation efficiency of the macrophage cell membrane ≥ 57.4%.

9. The composition according to claim 8, characterized in that, After intravenous injection of the composition: The blood circulation half-life is 8.2 ± 0.7 hours; The tumor accumulation amount at 24 hours is 12.4 ± 1.8% ID / g, which is 4.7 times that of the main organs; 68.3 ± 5.2% of the gold dose is cleared through the kidneys within 48 hours.

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