Ginseng exosome coated gold nanocluster compound and application thereof in preparation of medicine for treating lung cancer

Abstract

The invention discloses a ginseng exosome coated gold nano-cluster compound and application thereof in preparation of a medicine for treating lung cancer, and belongs to the field of biological medicine and nano-medicine preparations. The compound takes a gold nano-cluster loaded with siCD47 as an inner core and a ginseng exosome as a shell. The nano system is assembled by adopting a one-step micro-fluidic technology, the process is simple and convenient, and the repeatability is high. The gold nanoclusters in the core of the compound can effectively protect and deliver siCD47, efficiently silence CD47 genes of tumor cells and block immune escape signals; the ginseng exosome shell realizes the specific enrichment of the drug at the tumor site by utilizing the targeting of the natural lung, and can promote the phenotype polarization of macrophages to M1 and reprogram the tumor immune microenvironment. Through a synergistic immunotherapy mechanism of de-shielding and reactivation, the compound shows a remarkable inhibition effect on drug-resistant lung cancer in vivo and in vitro, is high in biological safety, and provides a new strategy and preparation for treatment of lung cancer, especially drug-resistant lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and nanomedicine formulation, specifically relating to a method and application for preparing a gold nanocluster lung cancer treatment drug based on a microfluidic chip, which encapsulates ginseng exosome nanovesicles loaded with interfering RNA. Background Technology

[0002] Lung cancer remains the leading cause of cancer incidence and mortality in China. In 2022, there were approximately 1.0606 million new cases of lung cancer in China, accounting for 22.0% of all new malignant tumor cases. Therefore, developing new therapeutic drugs to improve treatment outcomes is extremely urgent.

[0003] Small interfering RNA (siRNA), as a high-precision gene expression regulation tool, avoids the mutation and teratogenic risks associated with traditional gene therapy, showing significant promise in cancer treatment. Gold-based delivery platforms, through chemical or biological loading, can flexibly regulate size, surface charge, and function, facilitating the penetration of carried siRNA across cell membranes and enhancing intracellular accumulation, achieving efficient gene knockdown in cells and tissues. CD47 is frequently overexpressed in various malignant tumors. As a key "don't eat me" immune signal, CD47 helps tumor cells achieve immune escape by inhibiting the phagocytic activity of macrophages, thus being considered a highly promising target for tumor immunotherapy. Plant-derived exosomes, due to their rich content of various bioactive components, low immunogenicity, and preservation of natural pharmacological activity, have received widespread attention for their potential in targeted cancer therapy delivery. Furthermore, medicinal plant exosomes possess natural vesicular structures, allowing for more precise drug delivery to lesion sites according to the meridian tropism of traditional Chinese medicine, thereby enhancing efficacy, making them an ideal targeted delivery carrier.

[0004] Domestic and international scholars have explored the preparation and application of exosomes as antitumor carriers. Currently, a Chinese patent (publication number: CN119639677A) discloses tumor cell exosomes, an antitumor immunotherapy drug delivery system, its preparation method, and its applications. This technology utilizes the homing effect of tumor cell exosomes induced by starvation stimulation to construct a drug delivery platform that can improve targeting and enhance immune cell infiltration. However, the preparation process still requires dissolving the compound to be loaded with polylactic acid and polyethylene glycol amino in DMSO solution and loading it under high-speed vortex conditions. This process not only relies on organic solvents and synthetic polymers but also fails to eliminate the use of traditional chemical drugs. Therefore, incomplete impurity removal can damage normal tissues during application, posing toxicity risks and making it difficult to fundamentally prevent the development of tumor drug resistance.

[0005] Chinese patent (patent number: CN117582460B) discloses the application of *Brucea javanica* exosomes in the preparation of anti-breast cancer drugs and drugs that inhibit tumor angiogenesis. It focuses on the potential of *Brucea javanica* exosomes in inhibiting tumor angiogenesis; however, the prepared exosomes exhibit a wide range in particle size distribution and insufficient uniformity, which may affect their biological behavior and formulation stability. Furthermore, this technical solution does not evaluate its loading capacity as a drug delivery carrier, therefore its feasibility in functional delivery applications remains unclear.

[0006] Chinese patent (patent number: CN117224693A) discloses a novel method for preparing a tumor vaccine and its application. By modifying plant exosomes with solanine, the targeting ability of exosomes to dendritic cells is enhanced, and the overall immunotherapeutic effect of the vaccine is improved by promoting the proliferation of tumor-killing T lymphocytes. However, the modification of exosomes relies on a cumbersome process and introduces complex exogenous antigen complexes, which can easily lead to unpredictable and uncontrollable interactions between components in the system and their immunomodulatory effects. Summary of the Invention

[0007] The purpose of this invention is to address the problems of strong drug resistance, low targeting efficiency of delivery systems, and significant toxic side effects on normal tissues in existing antitumor drugs. This invention proposes a ginseng exosome-coated gold nanocluster complex and its application in the preparation of drugs for treating lung cancer. The synthesis process is simple, the antitumor mechanism is clear, and it has both high lung targeting efficiency and good biosafety.

[0008] This invention is achieved through the following technical solutions.

[0009] The present invention discloses a ginseng exosome-coated gold nanocluster complex, wherein the complex has a core-shell structure, wherein the core is a gold nanocluster (GC) loaded with siCD47, and the shell is a ginseng exosome (GE) encapsulating the core.

[0010] The siCD47 is a small interfering RNA sequence that targets the CD47 gene.

[0011] The present invention discloses a method for preparing a ginseng exosome-coated gold nanocluster complex, comprising the following steps:

[0012] S1. Provide gold nanocluster solution and SiCD47 solution;

[0013] S2. The gold nanocluster solution and the siCD47 solution are mixed through the first stage of a microfluidic chip, so that the gold nanoclusters and siCD47 self-assemble to form a gold nanocluster composite loaded with siCD47.

[0014] S3. The ginseng exosome solution and the complex solution obtained in step S2 are mixed through the second stage of the microfluidic chip, so that the ginseng exosomes encapsulate the gold nanocluster complex loaded with siCD47, to obtain the ginseng exosome-coated gold nanocluster complex.

[0015] The first stage of the microfluidic chip includes a three-inlet DC reaction channel; the second stage includes a symmetrical spiral structure mixing unit.

[0016] The present invention relates to the application of a ginseng exosome-coated gold nanocluster complex in the preparation of a drug for treating lung cancer.

[0017] GE not only endows the entire system with highly efficient lung-targeting capabilities but also promotes macrophage polarization towards the pro-inflammatory M1 phenotype and regulates the tumor immune microenvironment via the TLR4 signaling pathway. Meanwhile, the GC core significantly enhances siCD47 stability, efficiently knocks down the CD47 gene at lung tumor sites, and effectively blocks the "don't eat me" immune escape signal from tumor cells, thereby significantly enhancing the anti-tumor activity of tumor-associated macrophages (TAMs). In an orthotopic lung cancer mouse model, GE-GC significantly inhibited tumor progression by reprogramming the tumor immune microenvironment and demonstrated high biocompatibility.

[0018] Compared with existing technologies, the advantages of this invention are as follows: (1) The nanosystem is assembled using one-step microfluidic technology, which is simple and highly reproducible, providing a reliable foundation for high-quality, large-scale clinical translational production; (2) By utilizing the natural targeting properties of ginseng exosomes (GE) that are associated with the lung meridian, the nanosystem is specifically enriched at the site of lung tumors, significantly improving drug delivery efficiency and reducing non-specific exposure and potential toxicity to normal tissues; (3) The system has a clear structure and a well-defined anti-tumor mechanism. The gold nanocluster (GC) core effectively protects siCD47, achieving CD47 in tumor cells. 47-SIRPα “Don’t eat me” signal is efficiently silenced; at the same time, GE promotes macrophage polarization to pro-inflammatory M1 phenotype and participates in the reprogramming of immune microenvironment through TLR4 signaling pathway; (4) through the synergistic effect of macrophage phenotype reprogramming and immune checkpoint blockade, a combined immunotherapy mode of “deshielding + reactivation” is constructed, which significantly enhances the strength and persistence of anti-tumor immune response, improves the therapeutic effect, and effectively inhibits tumor recurrence and metastasis; (5) both in vivo and in vitro experiments show significant inhibitory effect on drug-resistant lung cancer models, providing a new strategy for overcoming tumor drug resistance in clinical practice. Attached Figure Description

[0019] Figure 1 Schematic diagram of microfluidic chip structure.

[0020] Figure 2 Physical images of GNC, GC, and GC-GE complex solutions.

[0021] Figure 3 Silencing efficiency of CD47 gene by different siRNA sequences.

[0022] Figure 4 Changes in GC complex potential under different GNC to siRNA concentration ratios.

[0023] Figure 5 TEM images of GNC, GC and GE-GC.

[0024] Figure 6 Fluorescence images of GNC, GC, and GE-GC complex solutions under 416 nm excitation light.

[0025] Figure 7 Particle size distribution of GE-GC solutions incubated for different times in serum-containing medium.

[0026] Figure 8 Drug release curves of GE-GC under different pH conditions.

[0027] Figure 9 Lysosomal escape at different times in GE-GC.

[0028] Figure 10 Effects of free siRNA, GC and GE-GC on CD47 mRNA expression in A549-TAX cells.

[0029] Figure 11 The killing effects of different concentrations of free siRNA, GC and GE-GC on A549-TAX cells.

[0030] Figure 12 Effects of free siRNA, GC and GE-GC on the expression of USP1 and p-ERK-related proteins in A549-TAX cells.

[0031] Figure 13 Polarization of macrophages by free siRNA, GC, and GE-GC.

[0032] Figure 14 The effects of free siRNA, GC, and GE-GC on the molecular characteristics of macrophage phenotypic transformation.

[0033] Figure 15 The activating effects of free siRNA, GC, and GE-GC on T cells.

[0034] Figure 16 Cellular safety evaluation of different concentrations of GNC, GC and GE-GC.

[0035] Figure 17 Evaluation of hemolysis by free siRNA, GC, and GE-GC.

[0036] Figure 18 Distribution of free siRNA, GC, and GE-GC in vivo at different times after tail vein injection.

[0037] Figure 19 Figure showing changes in body weight of mice in different treatment groups.

[0038] Figure 20 Tumor changes in mice in different treatment groups.

[0039] Figure 21 Anatomical images of the lungs of mice in different treatment groups, with the tumor site circled in red.

[0040] Figure 22 HE slices of the lungs of mice in different treatment groups.

[0041] Figure 23 Changes in blood IL-6 and TNF-α levels in mice from different treatment groups. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0043] (I) Fabrication of Microfluidic Chips

[0044] (1) The designed microfluidic chip structure is as follows Figure 1 As shown, it consists of two functional units connected in sequence: the first stage is a three-inlet liquid port and a direct-flow reaction channel with a channel width of 100μm and a height of 60μm; the core of the second stage is a mixing unit with a symmetrical spiral structure, with a channel width of 300μm and a height of 60μm. This unit consists of a spiral channel that rotates counterclockwise three times, an S-shaped connecting section, and a spiral channel that rotates clockwise three times, effectively enhancing fluid mixing by alternating the direction of the flow channels.

[0045] (2) Based on chip design, SU8-2075 silicon wafer molds were fabricated using photolithography. SU8-2075 photoresist was spin-coated onto a 4-inch silicon wafer at 4000 rpm for 60 seconds; then, a stepped pre-baking process was performed, heating at 65°C for 90 seconds and then at 95°C for 9 minutes. The silicon wafer was exposed to ultraviolet light (30 seconds) using a pre-designed pattern mask, followed by baking at 95°C for 7 minutes. The unexposed areas of the photoresist were removed using a developer, and then the wafer was baked at 150°C for 20 minutes to complete the pattern curing. Finally, the obtained SU8-2075 photolithography mold was subjected to silanization treatment.

[0046] (3) Mix the PDMS prepolymer and curing agent at a mass ratio of 10:1, and stir and degas in a mixing degassing machine for 3 minutes. Then, pour the uniform PDMS mixture onto the surface of the SU8-2075 silicon wafer mold, and transfer it to a vacuum dryer for 20 minutes to completely remove residual air bubbles. Place the mold on a 100℃ heating table for 15 minutes to cure. After the PDMS is fully cross-linked, cool it to room temperature and peel the PDMS substrate off the mold. Use a 0.75 mm diameter punch to prepare inlet and outlet ports at the corresponding positions. Then, place the PDMS substrate with microchannels and a clean glass substrate together in a plasma cleaner (170 mA, 700 V, 1 minute) for treatment. After surface activation, immediately perform bonding. Insert five plastic conduits with a length of about 18-20 cm into the liquid ports, fix them with sealant, and place the entire chip in an 80℃ oven for 1 hour to fully cure the sealant, thus completing the chip fabrication.

[0047] (II) Construction of GC-GE System

[0048] (1) Chloroauric acid solution (HAuCl4, 100 mM, Sinopharm Chemical Reagent Co., Ltd.) and glutathione (GSH, 150 mM, Sigma-Aldrich) were added to a 100 mL round-bottom flask and placed on a magnetic stirrer. The mixture was stirred continuously at 70°C and 500 rpm and reacted in the dark for 24 hours. During the reaction, GSH reduced Au³⁺ to gold atoms, and the solution gradually changed from colorless to pale yellow, yielding a crude gold nanocluster solution (solution 1).

[0049] (2) Take a 2000 mL beaker, add an appropriate amount of ultrapure water and place a stir bar on a magnetic stirrer. Pour solution 1 into a pretreated dialysis bag (molecular weight cutoff 3500 Da, Solarbio), seal it, and place it in the beaker. Dialyze at 80 rpm under light-protected conditions for 24 hours to remove unreacted small molecule raw materials, obtaining a purified gold nanocluster solution (GNC). The solution is as follows: Figure 2 As shown.

[0050] (3) In the first stage of the microfluidic chip, GNC solution (1 mg / mL) and siRNA solution (3 mg / mL, Sangon Biotech (Shanghai) Co., Ltd.) were injected into the left and right feed ports at a flow rate of 2 mL / h, respectively. Simultaneously, ultrapure water was introduced into the central feed port at a flow rate of 20 mL / h as an auxiliary flow, allowing GNC and siRNA to self-assemble into a GC complex within the flow channel. In the second stage, ginseng exosome (GE) solution (10 mg / mL, Chongqing Aidi Mai Technology Co., Ltd.) was introduced into the chip to further mix with the GC complex. After collecting the mixture at the outlet, it was filtered through a 0.22 μm filter membrane (Biosharp) to obtain the GC-GE nanomedicine system. The solution is as follows: Figure 2 As shown, store at 4°C for later use.

[0051] (III) Preparation of GC Complex

[0052] (1) The preparation method of gold nanoclusters (GNC) solution is the same as that in Example 1.

[0053] (2) To screen for siRNA sequences that can efficiently silence CD47, we designed and purchased multiple candidate siRNA sequences targeting CD47, as shown in the table below:

[0054]

[0055] The gene knockdown efficiency was assessed using the Lipofectamine 2000 standard transfection method. Figure 3 As shown, compared with the untreated control group and the control group transfected with nonspecific siRNA (NC-siRNA), CD47 siRNA-1 exhibited the highest CD47 gene silencing efficiency among all tested sequences. Based on this result, we selected CD47 siRNA-1 for the subsequent construction of the GNC-siRNA complex (GC).

[0056] (3) In the first stage of the microfluidic chip, GNC solutions and siRNA solutions of different concentrations were injected into the left and right inlets at a flow rate of 2 mL / h, respectively. Simultaneously, ultrapure water was introduced into the central inlet and the second-stage channel at a flow rate of 20 mL / h as an auxiliary flow to promote the self-assembly of GNC and siRNA into the flow channel to form a GC complex. The zeta potential changes of the complex at different concentrations were monitored using a zeta potential analyzer (NanoBrook Omni, Brookhaven Instruments, USA) to determine the optimal ratio of GNC to siRNA. The results are as follows: Figure 4As shown, when the concentration of GNC solution is 1 mg / mL and the concentration of siRNA solution is 3 mg / mL, the Zeta potential tends to stabilize, indicating that the loading of siRNA on the GNC surface reaches saturation at this time, which is the optimal recombination condition.

[0057] (iv) Characterization of GC-GE system

[0058] (1) The morphology of the GNC, GC, and GE-GC complexes was characterized using transmission electron microscopy (TEM, Rigaku Corporation, Japan). The results are as follows: Figure 5 As shown, GNCs are spherical with an average diameter of about 2 nm and are well dispersed; the GC complex has a diameter of about 16.6 ± 3.0 nm, with an average of about 15 GNC particles per complex; the GE-GC complex has a diameter of about 50 nm and exhibits a complete core-shell structure.

[0059] (2) Since GNC, the GC complex, and the GE-GC system all contain GNC, they all produce orange fluorescence under 416 nm excitation light, as shown in the following results. Figure 6 As shown.

[0060] (3) The stability of GE-GC in cell culture medium containing 10% fetal bovine serum (FBS) was tested using a dynamic light scattering analyzer. The results are as follows: Figure 7 As shown, the particle size remained stable over 72 hours, indicating that the complex has good serum stability and is suitable for subsequent intravenous administration.

[0061] (4) To evaluate the drug release characteristics of GE-GC, GNCs, which possess fluorescence properties, were used to calculate the cumulative release amount by measuring the fluorescence intensity. The results are as follows: Figure 8 As shown, the release rate of GC was significantly higher at pH 6.5 than at pH 7.4, demonstrating that GE-GC exhibits pH-responsive release behavior. Specifically, over 60% of the GC was released within 48 hours at pH 6.5, compared to less than 30% at pH 7.4. This selective release to acidic environments enables it to specifically trigger drug delivery within the tumor microenvironment.

[0062] (v) Evaluation of the antitumor performance of the GC-GE system

[0063] (1) To facilitate observation, siRNA was labeled with Cy5 fluorescent markers (Cy5-siRNA). Since the lysosomal pathway is closely related to autophagy, successful lysosomal escape is crucial for the function of gene delivery systems. A paclitaxel-resistant human non-small cell lung cancer cell model (A549-TAX) was constructed by induction with gradient concentrations of paclitaxel (Selleck, Shanghai). Before all experiments, A549-TAX cells were stably cultured in drug-free medium for at least two weeks to eliminate interference from drug residues in subsequent experiments. A549-TAX cells were cultured at 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of / wells in confocal culture dishes and co-incubated for 1, 4, 8, and 12 hours with a GC-GE system containing Cy5-labeled siRNA (equivalent siRNA concentration of 100 nM). Subsequently, cells were washed with PBS, fixed with 4% paraformaldehyde, and the nuclei and lysosomes were stained sequentially with Hoechst (nuclear staining) and LysoTracker Green (DND-22, excitation wavelength 488 nm, ThermoFisher Scientific). Observation was performed using a confocal laser scanning microscope (CLSM, LSM810, Zeiss). The results are shown below. Figure 9 As shown, after 1 hour of incubation, the red Cy5 fluorescent signal (representing GC-GE) and the green LysoTracker signal highly overlapped, indicating that the system was mainly located in lysosomes; by 4 hours, the red signal and the green lysosomal marker showed obvious separation, indicating that GC-GE had successfully achieved lysosomal escape and effectively released siRNA into the cytoplasm, laying the foundation for the subsequent gene silencing process.

[0064] (2) To evaluate the gene knockdown efficiency of the GE-GC system, cells were co-incubated with free siRNA, GC complex, and the GE-GC system (equivalent siRNA concentration of 100 nM) for 24 hours, respectively. CD47 mRNA expression levels were then analyzed by RT-PCR. The results are as follows: Figure 10 As shown, GE-GC reduced CD47 mRNA expression by 78%, with a significantly better silencing effect than other groups; the free siRNA group only caused an 18% decrease, while the GE-GR (GE-GNC-nsRNA) complex loaded with meaningless sequences did not show a significant gene silencing effect.

[0065] (3) Further evaluation of the antitumor effect of GE-GC was conducted through cell viability and apoptosis analysis. A549-TAX cells were seeded in 96-well plates and co-cultured for 24 hours with free siRNA, GC complex, and GE-GC system at different concentrations (equivalent siRNA concentrations of 25, 50, 100, and 200 nM). Cell viability was detected using the CCK-8 assay. Cell viability was calculated by measuring the absorbance of the solution at 450 nm (OD450 nm) using an Infinite MPlex microplate reader (Tecan, Switzerland). The results are as follows: Figure 6 As shown, at the same concentration, GE-GC significantly inhibited cell viability and induced massive cell death, while free siRNA caused only minor cell damage. Apoptosis was analyzed using flow cytometry (CytoFLEX), and the results are as follows: Figure 11 As shown, GC and GE-GC induced approximately 30% and 50% of cell apoptosis, respectively, indicating that the introduction of the GE shell effectively enhanced the gene delivery efficiency and apoptosis-inducing ability of the system.

[0066] (4) Phosphorylated extracellular signal-regulated kinase (p-ERK) plays a crucial role in the development and metastasis of lung cancer, while the deubiquitinating enzyme USP1 is an important regulator of the DNA damage repair pathway. Cells were co-incubated for 24 hours with free siRNA, GC complex, and GE-GC system (equivalent siRNA concentration of 100 nM each), and the expression levels of related proteins were detected by Western blot. The results are as follows: Figure 12 As shown, both the GC group and the GE-GC group downregulated the expression of p-ERK and USP1. GE-GC treatment resulted in the most significant decrease in p-ERK levels, with an inhibitory effect superior to GC alone. Free siRNA and the GE-GC group did not cause significant changes. This indicates that the GE-GC system has a synergistic enhancing effect in blocking key signaling pathways.

[0067] (vi) Tumor immune regulation role of the GC-GE system

[0068] (1) To evaluate the regulatory effect of GE-GC on macrophage polarization, RAW264.7 cells were treated with IL-4 (40 ng / mL) for 24 hours to induce differentiation from M0 phenotype to M2 macrophages. Subsequently, free siRNA, GC complex, and GE-GC system (equivalent siRNA concentration of 50 nM) were added to confocal culture dishes and the cells were treated for another 48 hours. The expression of the M1 marker CD80 and the M2 marker CD206 was detected by immunofluorescence staining. The results observed under a confocal microscope are shown below. Figure 13As shown, compared with the control group, free siRNA had no significant effect on the polarization state of macrophages, while both GC and GE-GC could effectively promote M1 phenotype polarization. Moreover, the changes in M1 / M2 marker expression caused by GE-GC were more significant than those caused by GC, indicating that the introduction of the GE shell can further enhance the polarization efficiency of M2 to M1 phenotype through a synergistic mechanism.

[0069] (2) To further elucidate the molecular characteristics of macrophage phenotypic transformation, the expression levels of M1-related cytokines (IL-6, TNF-α) and M2-related cytokines (IL-4, IL-10) were detected using ELISA. The results are as follows: Figure 14 As shown, IL-4 and IL-10 levels decreased in all treatment groups, while IL-6 and TNF-α increased, indicating that macrophages as a whole transitioned from an anti-inflammatory M2 state to a pro-inflammatory M1 state with tumor-killing capabilities. Compared with GC treatment alone, GE-GC induced a greater increase in IL-6 and a more thorough inhibition of IL-4.

[0070] (3) To evaluate the activation capacity of GE-GC for T cells, spleen cells were isolated from mouse spleens and cultured in RPMI-1640 medium containing IL-2 (20 ng / mL) to obtain mature T cells. A549-TAX cells were treated with free siRNA, GC complex, and the GE-GC system (equivalent siRNA concentration of 100 nM), and then co-cultured with the aforementioned T cells. The proportion of CD8⁺CD69⁺ T cells (CD69 is a marker of early T cell activation) was analyzed by flow cytometry. The results are as follows: Figure 15 As shown, compared with the untreated group, GC treatment increased the proportion of CD8⁺CD69⁺T cells to 24.8%, indicating that GC can effectively activate CD8⁺T cells; while in the GE-GC treated group, this proportion further increased to 47.4%. This further confirms the key role of GE in synergistically regulating the immune microenvironment.

[0071] (vii) Evaluation of the biosafety and organ distribution of the GC-GE system

[0072] (1) The toxicity of the GC-GE system to normal cells was evaluated using the CCK-8 assay. Mouse lung fibroblasts (MLF) were seeded in 96-well plates and co-cultured for 24 hours with free siRNA, GC complex, and the GE-GC system at different concentrations (equivalent siRNA concentrations of 25, 50, 100, and 200 nM). Absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The results are as follows: Figure 16 As shown, although cell viability showed a slight decreasing trend with increasing GE-GC concentration, it remained above 80% across the entire tested concentration range.

[0073] (2) Blood compatibility of the GC-GE system was evaluated by hemolysis test. Fresh mouse blood was collected, allowed to stand for 3 hours, centrifuged at 1500 rpm for 10 minutes, and the supernatant plasma was removed. A 4% red blood cell suspension was prepared by resuspending the blood in PBS. Free siRNA, GC complex, and the GE-GC system (equivalent siRNA concentration of 200 nM) were added separately. PBS and ultrapure water were used as negative and positive controls, respectively. After standing at room temperature for 2 hours, the supernatant was collected by centrifugation. The absorbance at 540 nm was measured using an ELISA reader, and the hemolysis rate was calculated. The results are as follows: Figure 17 As shown, the hemolysis rates of the GC and GE-GC groups were similar to those of the PBS negative control group, and no detectable hemolysis was induced even at high test concentrations, indicating that the system has good blood biocompatibility.

[0074] (3) To evaluate the in vivo distribution and lung targeting performance of the GE-GC system, female BALB / c nude mice (6 weeks old) were randomly divided into three groups (n=3 per group). Free siRNA, GC complex, and the GE-GC system (each mouse was injected with 15 μg Cy5-siRNA) via tail vein injection, respectively. The IVIS Spectrum in vivo imaging system was used to dynamically monitor the different groups at 2, 6, 12, and 24 hours. The results are as follows: Figure 18 As shown, fluorescence appeared in the kidneys of mice in all groups 2 hours after injection, indicating that the metabolic pathway of the nanodelivery system is mainly cleared by the kidneys. By 6 hours, the GE-GC group showed significant fluorescence accumulation in the lungs, demonstrating excellent lung targeting ability. This characteristic is closely related to the targeting function of the GE shell. At 12 hours, the signal of the free siRNA group had weakened significantly, the GC group still had residues in the kidneys, while the GE-GC group still maintained high fluorescence intensity in the lungs, indicating that the GE-GC system has better stability and lung retention characteristics. By 24 hours, GE-GC still showed obvious fluorescence in the lungs, further confirming its persistent accumulation ability in target tissues.

[0075] (viii) Evaluation of the efficacy of GC-GE systemic therapy for lung cancer

[0076] (1) To construct a drug-resistant tumor cell model stably expressing luciferase, A549-TAX cells were infected with luciferase lentivirus (GEnechem, Shanghai). Cells were then cultured at a rate of 2 × 10⁶ cells / year. 5 The cells were seeded at a density of 2 μg / mL in 12-well plates. After 24 hours, lentivirus transduction was performed at an MOI of 10 for 72 hours. The cells were then selected with 2 μg / mL puromycin for 3 days to obtain the A549 / TAX-Luc cell line that stably expresses luciferase.

[0077] (2) Establish an orthotopic lung cancer model to evaluate the antitumor activity of the GE-GC system. BALB / c nude mice underwent lateral thoracotomy under isoflurane (2–3%) anesthesia to expose the fourth intercostal space. 50 μL of A549 / TAX-Luc cell-Matrix gel suspension (1×10⁻⁶ cells / mL) was injected using a microsyringe. 6 Cells were injected into the left lung parenchyma (1–2 mm below the pleura) and fed for 7 days post-surgery to allow for the formation of a stable tumor.

[0078] (3) Tumor-forming mice were randomly divided into five groups (n=3 / group): saline control group, free siRNA group, GC group, GE-GC group, and GE-GR (significant siRNA) group. Each group received an intravenous injection of the corresponding drug (siRNA content 15 μg / mouse) every 48 hours, for a total of 5 administrations. Throughout the experiment, changes in body weight were recorded for each group, such as… Figure 19 As shown, there were no significant fluctuations in body weight in all treatment groups, indicating that the drugs used did not cause significant systemic toxicity.

[0079] (4) Tumors were monitored using in vivo bioluminescence imaging. D-fluorescein (150 mg / kg) was injected intraperitoneally before each imaging session, and images were acquired using the IVIS Spectrum system under 2% isoflurane anesthesia. Results are as follows: Figure 20 As shown, the free siRNA group only showed slight tumor growth inhibition for the first 4 days after administration, after which the tumor grew rapidly, possibly due to its rapid degradation in the blood. The tumor growth trend of the GE-GR group and the saline group was similar. Both the GC and GE-GC groups could continuously inhibit tumor growth, with the GE-GC group showing the most significant tumor-suppressing effect. Due to the lung-targeting enhancement effect of GE, the tumor volume was reduced by about 96.2% compared with the control group.

[0080] (5) At the end of the experiment, lungs from each group were dissected and observed. The results are as follows: Figure 21 As shown, multiple metastatic lesions were observed in the lungs of the control group and the GE-GR group; only isolated tumor nodules were observed in the free siRNA and GC groups; while the tumors in the GE-GC group had essentially regressed. Further, lung sections were prepared by fixing in 10% neutral buffered formaldehyde, embedding in paraffin, and then subjected to H&E staining to assess histopathological changes. Results are as follows... Figure 22 As shown, the control group showed large areas of necrosis in the lung tissue, and the free siRNA group still had obvious tumors; the GE-GC group showed significant tumor cell apoptosis, which not only effectively inhibited the growth of the primary tumor, but also significantly prevented the spread of the tumor.

[0081] (6) To assess the system's biosafety, blood samples were collected via cardiac puncture on days 7, 15, and 23 for complete blood count and biochemical analysis. The results are shown in Table 1:

[0082] Table 1. Changes in blood biochemical parameters in mice of different treatment groups

[0083]

[0084] Table 1 shows that liver and kidney function indicators in all treatment groups were within the normal range, and serum IL-6 and TNF-α levels were significantly elevated in the GE-GC group. Figure 23 This indicates that it can effectively activate the immune response.

Claims

1. A ginseng exosome-coated gold nanocluster complex, characterized in that, The complex has a core-shell structure, wherein the core is a gold nanocluster loaded with siCD47, and the shell is a ginseng exosome encapsulating the core.

2. The complex according to claim 1, characterized in that, The siCD47 is a small interfering RNA sequence that targets the CD47 gene.

3. A method for preparing the ginseng exosome-coated gold nanocluster complex according to claim 1 or 2, characterized in that, Includes the following steps: S1. Provide gold nanocluster solution and SiCD47 solution; S2. The gold nanocluster solution and the siCD47 solution are mixed through the first stage of a microfluidic chip, so that the gold nanoclusters and siCD47 self-assemble to form a gold nanocluster composite loaded with siCD47. S3. The ginseng exosome solution and the complex solution obtained in step S2 are mixed in the second stage of the microfluidic chip, so that the ginseng exosomes encapsulate the gold nanocluster complex loaded with siCD47, to obtain the ginseng exosome-coated gold nanocluster complex.

4. The method according to claim 5, characterized in that, The first stage of the microfluidic chip includes a three-inlet DC reaction channel; the second stage includes a symmetrical spiral structure mixing unit.

5. The use of the ginseng exosome-coated gold nanocluster complex according to claim 1 or 2 in the preparation of a drug for treating lung cancer.

Images