A combined medicine, liposome, biomimetic liposome, preparation method thereof and application thereof in preparing anti-breast cancer lung metastasis medicine

Abstract

The application provides a combined drug, a liposome, a biomimetic liposome, a preparation method of the combined drug, the liposome and the biomimetic liposome, and application of the combined drug, the liposome and the biomimetic liposome in preparation of an anti-breast cancer lung metastasis drug, and belongs to the technical field of biological medicines. The combined drug comprises panatinib and ginsenoside Rg3. The application further provides a liposome PNT-Rg3-Lipo for simultaneously loading the two, and a biomimetic liposome MM / PNT-Rg3-Lipo coated with a macrophage membrane. Experiments show that the combination of the two can synergistically regulate chemokines CXCL1 , CXCL2 and CXCL11 , and the effect is better than the sum of the single use. The MM / PNT-Rg3-Lipo has uniform particle size, retains integrin alpha 4 / beta 1 functional proteins, can target lung metastases, significantly reduces metastatic nodules, and has good biocompatibility. The application provides an effective new strategy for resisting breast cancer lung metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a combination drug, liposomes, biomimetic liposomes, their preparation methods, and their application in the preparation of drugs for treating lung metastases of breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, with lung metastasis being a leading cause of death. Current clinical treatments for breast cancer lung metastases primarily rely on surgical resection, radiotherapy, and systemic chemotherapy, but these methods suffer from limited efficacy, significant side effects, and a high risk of drug resistance. Therefore, developing novel treatment strategies for breast cancer lung metastases is of significant clinical importance.

[0003] Ponatinib (PNT) is a third-generation tyrosine kinase inhibitor primarily used to treat chronic myeloid leukemia, and in recent years, some studies have attempted to use it for the treatment of solid tumors. However, PNT has drawbacks such as poor water solubility, short half-life in vivo, and significant non-specific distribution, which limit its full anti-tumor efficacy. Furthermore, high-dose use may cause serious adverse reactions such as cardiovascular toxicity.

[0004] Ginsenoside Rg3 is one of the main active ingredients extracted from ginseng and has been reported to have various pharmacological activities, including anti-tumor, anti-angiogenic, and immunomodulatory effects. However, Rg3 has low water solubility and oral bioavailability, and is rapidly metabolized in vivo, making it difficult to achieve effective concentrations at tumor sites, thus limiting its clinical application.

[0005] In recent years, nanomedicine delivery systems have offered new solutions to these problems. Liposomes, as classic drug carriers, offer advantages such as improved drug solubility, prolonged in vivo circulation time, and reduced toxicity. However, conventional liposomes still suffer from insufficient targeting and are easily cleared by the reticuloendothelial system. Cell membrane biomimetic technology, by coating nanoparticles with natural cell membranes, endows them with the biological functions of their source cells, such as immune escape and targeted recognition, and has become a research hotspot in the field of nanomedicine delivery. Macrophages possess chemotaxis and homing abilities in the tumor microenvironment, and their cell membrane coating strategy holds promise for enhancing the targeted delivery efficiency of nanomedicines to tumor metastases.

[0006] In summary, how to synergistically regulate tumor metastasis-related signaling pathways through rational drug combinations and delivery strategies to improve the efficacy of anti-lung metastasis treatment and reduce toxic side effects remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a combination drug, liposomes, biomimetic liposomes, their preparation method, and their application in the preparation of drugs for treating lung metastases of breast cancer.

[0008] This invention provides a combination drug containing ponatinib (PNT) and ginsenoside Rg3 for simultaneous or separate administration.

[0009] Furthermore, the mass ratio of panatinib to ginsenoside Rg3 is 1:(2~75).

[0010] Furthermore, the mass ratio of panatinib to ginsenoside Rg3 is 1:3.

[0011] The present invention also provides a liposome (PNT-Rg3-Lipo), wherein the liposome is a liposome that uses phospholipid as a carrier material and simultaneously encapsulates panatinib and ginsenoside Rg3; wherein the mass ratio of panatinib, ginsenoside Rg3 and egg yolk lecithin is 1:(1~10):(5~20).

[0012] Furthermore, the phospholipid is egg yolk lecithin; the mass ratio of panatinib, ginsenoside Rg3 and egg yolk lecithin is 1:3:10.

[0013] Furthermore, the liposomes have an average particle size of 80-150 nm, a PDI of less than 0.2, and a zeta potential of 20-40 mV.

[0014] Furthermore, the liposomes have an average particle size of 104.4 ± 1.48 nm, a PDI of 0.078 ± 0.017, and a zeta potential of 33.9 ± 0.1 mV.

[0015] Furthermore, the liposomes are prepared by thin-film dispersion method, specifically including the following steps: weigh the raw materials according to the ratio, add anhydrous ethanol to dissolve them, remove the anhydrous ethanol under reduced pressure to form a thin film; add phosphate buffer solution (PBS) and shake under a water bath at 40~50℃ to make the film completely hydrated; sonicate with a probe under ice bath conditions, filter, and the liposomes (PNT-Rg3-Lipo) are obtained.

[0016] The present invention also provides a biomimetic liposome (MM / PNT-Rg3-Lipo), wherein the biomimetic liposome is formed by mixing cell membranes derived from immune cells with the aforementioned liposomes through ultrasound.

[0017] Furthermore, the ratio of the total lipid mass in the liposomes to the membrane components derived from immune cells in the cell membrane of the immune cells is: (10~30) mg : (1~5) × 10⁻⁶ mg. 7 One immune cell.

[0018] Furthermore, the ratio of the total lipid mass in the liposomes to the membrane components derived from immune cells in the cell membrane of the immune cells is 20 mg: 3 × 10⁻⁶. 7 One immune cell.

[0019] Furthermore, the cell membrane derived from the immune cells is provided in the form of a cell membrane microcapsule solution, wherein the mixing volume ratio of the cell membrane microcapsule solution to the liposomes is (0.5~2):(0.5~2).

[0020] Furthermore, the volume ratio of the cell membrane microcapsule solution to the liposomes is 1:1.

[0021] Furthermore, the cell membrane derived from the immune cells is a macrophage membrane; the average particle size of the biomimetic liposomes is 100~160 nm, the PDI is less than 0.3, and the zeta potential is -15~0 mV.

[0022] Furthermore, the macrophage membrane is an M1 type macrophage membrane induced by lipopolysaccharide (LPS) stimulation; the average particle size of the biomimetic liposome is 130.28±3.40 nm, the PDI is 0.198±0.015, and the zeta potential is -8.1±0.2 mV.

[0023] Furthermore, the preparation method of the biomimetic liposome (MM / PNT-Rg3-Lipo) includes the following steps: (1) Extraction of macrophage membrane: Macrophages (preferably RAW264.7 cells) were cultured with lipopolysaccharide (LPS, final concentration 0.5~2 μg / mL) for 20~30 h to induce M1 type macrophages. Cells were collected, washed with pre-cooled PBS and resuspended. Membrane protein extraction reagent A containing benzyl sulfonyl fluoride (PMSF) was added to every 30 million cells and placed on ice for 10~20 min. The cell suspension was transferred to a pre-cooled glass homogenizer and homogenized 30~50 times to break most of the cells. The homogenized cell fragment suspension was centrifuged at 4℃, 600~800 g for 8~12 min and the supernatant was collected; then centrifuged at 4℃, 12000~16000 g for 25~35 min to precipitate cell membrane fragments. The cell membrane precipitate was resuspended in PBS and sonicated with a probe for 2~5 min under ice conditions to obtain macrophage membrane microcapsule solution.

[0024] (2) Preparation of biomimetic liposomes: The macrophage membrane microcapsule solution obtained in step (1) is mixed with the above liposomes (PNT-Rg3-Lipo) and sonicated under ice bath conditions for 1~10 min (preferably 5 min) to obtain biomimetic liposomes (MM / PNT-Rg3-Lipo).

[0025] The present invention also provides a method for preparing the aforementioned biomimetic liposomes, comprising the following steps: (1) Extract cell membranes from immune cells to obtain cell membrane microcapsule solutions; (2) The cell membrane microcapsule solution obtained in step (1) is mixed with the aforementioned liposomes and ultrasonically treated under ice bath conditions to obtain the final product.

[0026] Furthermore, the conditions for ultrasonic treatment in step (2) are: ultrasonic treatment at 0~5℃ for 1~10 minutes.

[0027] The present invention also provides the use of the aforementioned combination drugs, the aforementioned liposomes, or the aforementioned biomimetic liposomes in the preparation of drugs for the prevention and / or treatment of breast cancer.

[0028] Furthermore, the drug is an anti-breast cancer lung metastasis drug.

[0029] Furthermore, the drug is a drug that inhibits the p38 MAPK / STAT1 signaling pathway, inhibits chemokines CXCL1 or CXCL2, or activates chemokine CXCL11.

[0030] The present invention has achieved the following beneficial effects: (1) Significant synergistic effect: When PNT and Rg3 are used in combination, they reduce chemokines. CXCL1 and CXCL2 mRNA levels and elevated CXCL11 In terms of mRNA levels, the effect was significantly better than the sum of the effects of the two drugs used alone, showing an unexpected synergistic effect (Tables 4 and 5).

[0031] (2) Excellent formulation characteristics and tumor microenvironment responsiveness: PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo have uniform particle size, good dispersibility, and high stability (no significant change in particle size within 72 h). PNT-Rg3-Lipo can rapidly release the drug under weakly acidic conditions (pH 4.8, 5.8), with complete cumulative release within 24 h, which is beneficial for responsive drug release in the tumor microenvironment. Figures 1-2 , Figure 5 (Tables 1-2)

[0032] (3) Functional proteins are preserved intact and cellular uptake is selective: MM / PNT-Rg3-Lipo successfully preserves integrin α4 and β1 functional proteins on the macrophage membrane surface. This biomimetic liposome can enhance the uptake by breast cancer 4T1 cells while inhibiting the phagocytosis of macrophages by RAW264.7, which is beneficial to reducing the clearance of the reticuloendothelial system in vivo. Figures 6-11 ).

[0033] (4) Strong in vitro and in vivo antitumor activity with good safety: MM / PNT-Rg3-Lipo can significantly downregulate p38 MAPK / STAT1 signaling pathway-related proteins and metastasis-promoting chemokines ( CCL2 , CXCL1 , CXCL2 The expression of ) upregulates antitumor immune-related chemokines. CXCL11 The expression of [the drug / formula]. In vivo experiments confirmed that this formulation can target and accumulate in lung metastases, significantly reduce the number of lung metastatic nodules, improve the pathological state of lung tissue, and has a hemolysis rate of less than 5% and no significant decrease in mouse body weight, demonstrating good biocompatibility and low systemic toxicity. Figures 12-27 (Tables 3-7)

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0036] Figure 1 Particle size distribution diagrams of PNT-Lipo (A), PNT-Rg3-Lipo (B), and MM / PNT-Rg3-Lipo (C) and their particle size distribution comparison diagram (D).

[0037] Figure 2 The figure shows the stability test results for PNT-Lipo (A), PNT-Rg3-Lipo (B), and MM / PNT-Rg3-Lip (C).

[0038] Figure 3 TEM images of PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo.

[0039] Figure 4 The standard curves for PNT (A) and Rg3 (B) are shown.

[0040] Figure 5 The graph shows the in vitro cumulative drug release of PNT(A) and Rg3(B) in release media at pH 4.8, pH 5.8 and pH 7.4.

[0041] Figure 6 This is an SDS-PAGE full-protein map of membrane proteins from M1 macrophages, MM, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo.

[0042] Figure 7This image shows the protein detection of integrin α4 and β1 on M1 macrophages, MM, and MM / PNT-Rg3-Lipo.

[0043] Figure 8 The results of qualitative detection of drug uptake by 4T1 breast cancer cells using laser confocal microscopy (scale bar = 25 μm).

[0044] Figure 9 The graph shows the cellular uptake of each drug group by breast cancer 4T1 cells by flow cytometry (A) and the quantitative analysis of fluorescence intensity (B).

[0045] Figure 10 The results of qualitative detection of drug uptake by RAW264.7 macrophages for each group using laser confocal microscopy (scale bar = 25 μm).

[0046] Figure 11 The graph shows the cellular uptake of each drug group by RAW264.7 macrophages by flow cytometry (A) and the quantitative analysis of fluorescence intensity (B).

[0047] Figure 12 The figure shows the effects of free PNT, PNT-Lipo, PNT-Rg3-Lipo, MM / PNT-Rg3-Lipo, and Rg3-Lipo on the viability of the 4T1 cell line.

[0048] Figure 13 The effect of free PNT, PNT-Lipo, PNT-Rg3-Lipo, MM / PNT-Rg3-Lipo and Rg3-Lipo on the cell viability of the MDA-MB-231 cell line is shown in the figure.

[0049] Figure 14 This diagram illustrates the regulatory effects of DMSO, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo on p-STAT1, STAT1, p-p38, p-38, and c-Jun proteins in 4T1 cells.

[0050] Figure 15 The figure shows the quantitative results of the effects of DMSO, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo on the expression of p-STAT1, STAT1, p-p38, p-38, and c-Jun proteins in 4T1 cells.

[0051] Figure 16 The combined use of PNT and Rg3 to treat 4T1 breast cancer cells CCL2 , CXCL1 , CXCL2 as well as CXCL11 The regulatory effect diagram.

[0052] Figure 17 The effects of DMSO, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo on 4T1 breast cancer cells. CCL2 , CXCL1 , CXCL2 as well as CXCL11 The diagram illustrates the regulatory effects of [the substance / mechanism].

[0053] Figure 18 The graph shows the hemolysis of each group of drugs detected by the in vitro direct contact method, including a hemolysis experiment photograph (A) and a bar chart of hemolysis rate (B).

[0054] Figure 19 This is a map showing the in vivo distribution of each drug group in a mouse model of lung metastasis in breast cancer.

[0055] Figure 20 This is a distribution map of each drug group in the tissues and organs of a mouse model of lung metastasis in breast cancer.

[0056] Figure 21 A schematic diagram of an animal experiment design scheme.

[0057] Figure 22 The results of the observation of the number of lung metastatic nodules in the lung tissue of mice after drug treatment in each group and their quantitative statistical graphs.

[0058] Figure 23 Organ index of the lungs in each group treated with drugs (A) and statistical graph of mouse body weight change during the experiment (B).

[0059] Figure 24 The images show the pathological results of lung tissue after drug treatment in various tissues.

[0060] Figure 25 This diagram illustrates the regulatory effects of PBS, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo on p-STAT1, STAT1, p-p38, p-38, and c-Jun proteins in lung metastatic nodules of mice with breast cancer lung metastases.

[0061] Figure 26 Statistical graph showing the quantitative effects of PBS, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo on p-STAT1, STAT1, p-p38, p-38, and c-Jun proteins in lung metastatic nodules of mice with breast cancer lung metastases.

[0062] Figure 27The effects of PBS, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo on lung metastatic nodules in mice with breast cancer lung metastases. CCL2 , CXCL1 , CXCL2 as well as CXCL11 Its regulatory role. Detailed Implementation

[0063] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0064] In this invention, "room temperature" refers to 15~35℃.

[0065] Example 1: Preparation of biomimetic liposomes coated with macrophage membranes This embodiment provides a method for preparing biomimetic liposomes coated with macrophage membranes using a thin-film dispersion method, specifically including: 1. Preparation of conventional PNT liposomes (PNT-Lipo) 2 mg of PNT (Ponatinib), 8 mg of cholesterol, and 40 mg of egg yolk lecithin (mass ratio 1:4:20, w / w) were weighed into a 100 mL round-bottom flask. 5 mL of anhydrous ethanol was added to completely dissolve the drug and carrier material. The round-bottom flask was placed on a rotary evaporator to remove the anhydrous ethanol until a uniform thin film formed at the bottom of the flask. 5 mL of phosphate buffer solution (PBS) was added, and the film was shaken at 45°C to ensure complete hydration. Subsequently, the film was sonicated for 5 min at 4°C on an ice bath and filtered through a 0.8 μm microporous membrane to obtain PNT-Lipo (the total lipid mass of PNT-Lipo is the total amount of cholesterol and egg yolk lecithin, i.e., 48 mg).

[0066] 2. Preparation of PNT-Rg3 combined liposomes (PNT-Rg3-Lipo) 2 mg of PNT, 6 mg of Rg3 (ginsenoside Rg3), and 20 mg of egg yolk lecithin (1:3:10, w / w) were weighed into 100 mL round-bottom flasks. PNT-Rg3-Lipo was prepared using the same method as PNT-Lipo (the total lipid mass of PNT-Rg3-Lipo was the total amount of egg yolk lecithin, i.e., 20 mg).

[0067] 3. Preparation of biomimetic nanoliposomes MM / PNT-Rg3-Lipo (1) Extraction of macrophage membrane On the second day after passage of RAW264.7 macrophages, diluted lipopolysaccharide (LPS) was added to the cell culture dish to a final concentration of 1 μg / mL. After stimulating the cells with LPS for 24 h, the LPS-containing medium was discarded, and the cells were washed twice with PBS to induce M1 macrophages. The cells were gently scraped off with a cell scraper and collected in centrifuge tubes. The tubes were centrifuged at 1000 rpm for 3 min at room temperature, the supernatant was discarded, and the cell pellet was kept for later use. The cell pellet was gently resuspended with a suitable amount of pre-chilled PBS. A small number of cells were counted, and the remaining cells were centrifuged at 4°C, 600 g for 5 min. After discarding the supernatant, the cells were centrifuged again at 4°C, 600 g for 1 min to precipitate the residual liquid on the centrifuge tube wall and further precipitate the cells. The residual liquid was discarded. Before use, 1 mL of membrane protein extraction reagent A containing benzyl sulfonyl fluoride (PMSF) was added per 30 million cells, the cells were gently resuspended by pipetting, and the cells were incubated on ice for 15 min. Transfer the cell suspension to a pre-chilled glass homogenizer and homogenize approximately 30-50 times until at least 70% of the cells are lysed. Transfer the homogenized cell fragment suspension to a centrifuge tube and centrifuge at 700 g for 10 min at 4°C. Collect the supernatant into a new centrifuge tube and centrifuge again at 14000 g for 30 min at 4°C to precipitate cell membrane fragments. Resuspend the cell membrane pellet in 1 mL of PBS and sonicate for 3 min at 4°C on ice to obtain a macrophage membrane microcapsule solution (each mL of solution contains cells derived from 3 × 10⁻⁶ cells). 7 (Membranes of macrophages), used for the subsequent preparation of biomimetic liposomes.

[0068] (2) Preparation of MM / PNT-Rg3-Lipo The biomimetic liposome MM / PNT-Rg3-Lipo was prepared by coating macrophage membranes onto the surface of PNT-Rg3-Lipo using an ultrasonic method. The macrophage membrane microcapsule solution and PNT-Rg3-Lipo were thoroughly mixed at a volume ratio of 1:1 and ultrasonicated for 5 min in an ice bath (4℃) to obtain MM / PNT-Rg3-Lipo.

[0069] The following experimental examples demonstrate the beneficial effects of the present invention.

[0070] Example 1: Characterization of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo 1. Particle size, PDI, and zeta potential of PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo (1) Experimental method: The particle size, PDI and ζ potential of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo were determined by dynamic light scattering nanoparticle size analyzer.

[0071] (2) Experimental results: The particle size distributions of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo are as follows: Figure 1 As shown in (A)-(D), the three formulations formed nanoparticles with uniform particle size in aqueous solution. The average particle size of PNT-Lipo was 162.02±2.37 nm, with a PDI of 0.262±0.003 and a zeta potential of -1.8±0.2. The average particle size of PNT-Rg3-Lipo was 104.4±1.48 nm, with a PDI of 0.078±0.017 and a zeta potential of 33.9±0.1. The average particle size of MM / PNT-Rg3-Lipo was 130.28±3.40 nm, with a PDI of 0.198±0.015 and a zeta potential of -8.1±0.2. PDI was used to evaluate the distribution of nanoparticles. The low PDI of all three formulations indicates that they are well dispersible and have a uniform particle size distribution in aqueous solution. After being coated with the macrophage membrane, the particle size of MM / PNT-Rg3-Lipo increased by 25~26 nm. In addition, the zeta potential of MM / PNT-Rg3-Lipo was much lower than that of PNT-Rg3-Lipo, suggesting that the macrophage membrane was successfully coated on the surface of PNT-Rg3-Lipo.

[0072] 2. Stability of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo (1) Experimental methods: The particle size of the prepared PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo was measured again at 24, 48 and 72 h using a Malvern nanoparticle size potentiometry instrument to evaluate the stability of the prepared liposomes.

[0073] (2) Experimental results: The dispersion stability of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo in PBS is as follows: Figure 2 As shown in (A)-(C) and Table 1, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo showed no significant change in particle size in PBS within 72 h, demonstrating their good stability.

[0074] Table 1. Particle size of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo within 72 h 3. Morphology of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo (1) Experimental method: The morphological characteristics of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo were detected by a visual transmission electron microscope (TEM).

[0075] (2) Experimental results: such as Figure 3 As shown, TEM results indicate that both PNT-Lipo and PNT-Rg3-Lipo are nanoscale spherical particles. The MM / PNT-Rg3-Lipo core liposomes (PNT-Rg3-Lipo) are spherical and relatively uniformly distributed, with a 25-26 nm thick "shell" structure on their surface. TEM clearly reveals the core-shell spherical structure of MM / PNT-Rg3-Lipo. Combined with its particle size being 25-26 nm larger than PNT-Rg3-Lipo and its zeta potential being negative compared to PNT-Rg3-Lipo, this collectively demonstrates that the macrophage membrane successfully coats the surface of PNT-Rg3-Lipo.

[0076] 4. Encapsulation efficiency (EE) and drug loading (LE) of PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo (1) Experimental method: The peak areas of PNT at 254 nm were determined by high performance liquid chromatography (HPLC) for standard concentrations of 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, 7.81 μg / mL, 3.91 μg / mL, and 1.95 μg / mL, respectively. A standard curve was obtained by fitting the PNT concentration (X) and peak area (Y) to the curve, as shown below. Figure 4 As shown in (A), the linear equation of the standard curve is given by formula (3): R 2 =1 Formula (3) Similarly, high-performance liquid chromatography (HPLC) was used to determine the peak areas of Rg3 at 203 nm for standard concentrations of 750 μg / mL, 375 μg / mL, 187.5 μg / mL, 93.75 μg / mL, 46.88 μg / mL, 23.44 μg / mL, and 11.72 μg / mL. A standard curve was obtained by fitting the concentration (X) and peak area (Y) of Rg3 to the curve, as shown below. Figure 4 As shown in (B), the linear equation of the standard curve is given by formula (4): R 2 =0.9995 Formula (4) The prepared PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo were mixed with twice the volume of methanol, respectively, and ultrasonically demulsified. The peak area was detected by HPLC, and the content was calculated by formula (3) and formula (4). The EE and LE of each formulation were calculated according to formula (1) and formula (2).

[0077] Formula (1) Formula (2) (2) Experimental results: As shown in Table 2, the EE% of PNT in PNT-Lipo was 90.58±0.70, and the LE% was 3.96±0.11. In PNT-Rg3-Lipo, the EE% of PNT was 96.16±0.59, the EE% of Rg3 was 80.65±3.29, the LE% of PNT was 7.00±0.09, and the LE% of Rg3 was 14.76±0.55. In MM / PNT-Rg3-Lipo, the EE% of PNT was 96.00±0.72, and the EE% of Rg3 was 79.63±1.26. This indicates that PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo have good drug loading capacity for PNT and Rg3.

[0078] Table 2. Particle size, PDI, zeta potential, EE, and LE of PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo 5. In vitro drug release characteristics of PNT-Rg3-Lipo (1) Experimental method: The in vivo environment was simulated by three different pH values ​​(pH4.8, pH5.8 and pH7.4) to investigate the in vitro release behavior of PNT-Rg3-Lipo. Appropriate amounts of release medium were collected at 0, 1, 2, 4, 8, 12, 24, 48 and 72 h after release. The drug concentration in the release medium at each time point was determined by HPLC, and the release of the drug at different pH values ​​was statistically analyzed.

[0079] (2) Experimental results: such as Figure 5As shown in (A)-(B), in a neutral solution at pH 7.4, PNT and Rg3 were slowly released from the PNT-Rg3-Lipo nanocarrier. At 12 h, the cumulative release percentage of PNT from PNT-Rg3-Lipo was 21%, and the cumulative release percentage of Rg3 was 24%. At 72 h, the cumulative release percentage of PNT was 55%, and the cumulative release percentage of Rg3 was 49%. However, in acidic environments at pH 4.8 and 5.8, the release rates of PNT and Rg3 were faster. In a release medium at pH 4.8, at 12 h, the cumulative release percentage of PNT from PNT-Rg3-Lipo was 63%, and the cumulative release percentage of Rg3 was 91%. Complete drug release from PNT-Rg3-Lipo was achieved at 24 h. In a release medium at pH 5.8, after 12 h, the cumulative release percentage of PNT-Rg3-Lipo was 52% for PNT and 86% for Rg3; similarly, complete drug release from PNT-Rg3-Lipo was achieved after 24 h. This demonstrates that PNT-Rg3-Lipo rapidly releases the encapsulated drug in an acidic environment. Given the weakly acidic nature of the tumor microenvironment, we hypothesize that PNT-Rg3-Lipo readily interacts with it. The weakly acidic environment induces a phase transition in PNT-Rg3-Lipo, leading to lipid membrane instability and thus facilitating rapid drug release.

[0080] 6. Characterization of the complete MM / PNT-Rg3-Lipo protein (1) Experimental Methods: In this invention, the total protein of LPS-stimulated RAW264.7 cells was detected by SDS-PAGE protein gel electrophoresis. Proteins from macrophage membranes and MM / PNT-Rg3-Lipo were extracted, and PNT-Rg3-Lipo was used as a control. The specific method was as follows: First, RIPA lysis buffer containing broad-spectrum protease and phosphatase inhibitors was used to lyse LPS-stimulated RAW264.7 cells, macrophage membranes, and centrifuged PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo precipitates for 30 min on ice. After centrifuging each group of samples at 4℃ and 12000 rpm for 15 min, the supernatant was collected, and SDS-PAGE protein loading buffer was added at a volume ratio of 5:1. The samples were then heated at 100℃ for 5 min to fully denature the proteins. Subsequently, the processed protein samples and protein markers were added to the wells of a polyacrylamide gel and electrophoresis was performed in a Tris-Glycine-SDS buffer system (80 V for the stacking gel stage, adjusted to 100 V after entering the separating gel). After electrophoresis, the gels were removed and stained in Coomassie Brilliant Blue at room temperature for 1 h, followed by destaining in destaining solution on a shaker at room temperature for 5 h until the background was clear and the protein bands were distinct. Finally, the gels were photographed and analyzed using a gel imaging system.

[0081] (2) Experimental results: The total protein maps of each group of samples are as follows Figure 6 As shown, the number of protein types in the extracted macrophage membrane was reduced compared to the total protein in RAW264.7 cells. The protein bands in the prepared MM / PNT-Rg3-Lipo were located almost identically to those in the extracted cell membrane, and no protein was detected in the PNT-Rg3-Lipo group. Therefore, this invention further confirms that the corresponding membrane proteins of RAW264.7 macrophages were successfully retained on the surface of the liposome drug, providing a reliable guarantee for the biofunctionalization of MM / PNT-Rg3-Lipo.

[0082] 7. Detection of characteristic proteins in MM / PNT-Rg3-Lipo (1) Experimental method: The present invention uses Western blotting to detect integrin α4 and β1 proteins on M1 macrophages, MM and MM / PNT-Rg3-Lipo, and uses β-actin as a reference to show the purity of the extracted cell membrane.

[0083] (2) Experimental results: such as Figure 7As shown, integrin α4 and β1 proteins were detected in M1 macrophages, MM cells, and MM / PNT-Rg3-Lipo cells, confirming that characteristic functional proteins are retained on MM / PNT-Rg3-Lipo cells. β-actin is only present in the cytoplasm of cells and is easily detected in M1 macrophage samples, but almost undetectable in MM and MM / PNT-Rg3-Lipo cells, demonstrating the high purity of the isolated macrophage membranes. These results indicate that MM / PNT-Rg3-Lipo cells can retain the characteristic functional proteins integrin α4 and β1, providing a possibility for targeting lung metastases from breast cancer.

[0084] Experimental Example 2: In vitro uptake of MM / PNT-Rg3-Lipo by macrophages and breast cancer 4T1 cells 1. Qualitative experiment on the uptake of MM / PNT-Rg3-Lipo by 4T1 breast cancer cells (1) Experimental methods: The uptake of MM / PNT-Rg3-Lipo by breast cancer 4T1 cells was qualitatively assessed using laser confocal microscopy. To facilitate fluorescence observation and subsequent quantitative determination, fluorescent probe C6 was used instead of PNT in this invention, and various fluorescent liposomes, namely C6-Lipo, C6-Rg3-Lipo, and MM / C6-Rg3-Lipo, were prepared using the same preparation method as in Example 1. The dosage of C6 in each group was 100 ng / mL. Hoechst 33342 was used to stain the nuclei of live cells, which appeared blue, while C6, which replaced PNT, showed green fluorescence.

[0085] (2) Experimental results: such as Figure 8 As shown, after 4 h of co-incubation with each group of fluorescent liposomes, 4T1 cells in all groups exhibited a green fluorescent signal of C6. Compared with the free C6 group, the fluorescence intensity of the C6-Lipo group was significantly enhanced; the fluorescence intensity of the C6-Rg3-Lipo group, which used Rg3 to replace cholesterol, was significantly enhanced compared with the conventional liposome group C6-Lipo; when C6-Rg3-Lipo was coated with a macrophage membrane, the uptake of MM / C6-Rg3-Lipo by 4T1 cells was further enhanced, and the fluorescence signal was enhanced compared with the C6-Rg3-Lipo group. This indicates that compared with the C6-Lipo group, replacing cholesterol with Rg3 increased the uptake by 4T1 cells; the macrophage membrane-modified MM / C6-Rg3-Lipo group further increased the uptake by 4T1 cells.

[0086] 2. Quantitative experiment on the uptake of MM / PNT-Rg3-Lipo by 4T1 breast cancer cells (1) Experimental method: The present invention uses flow cytometry to quantitatively detect the uptake of liposomes by 4T1 cells in each group.

[0087] (2) Experimental results: such as Figure 9 As shown in (A)-(B), consistent with the characterization results of laser confocal microscopy, after 4 h of co-incubation with the drug, the cellular uptake signal of C6-Rg3-Lipo in 4T1 cells was 1.68 times higher than that of C6-Lipo, indicating that C6-Rg3-Lipo was more readily taken up by 4T1 cells. The cellular uptake signal of MM / C6-Rg3-Lipo was 1.25 times higher than that of C6-Rg3-Lipo. These results suggest that macrophage membrane decoration in MM / C6-Rg3-Lipo enhances cellular uptake.

[0088] 3. Qualitative experiment on the uptake of MM / PNT-Rg3-Lipo by RAW264.7 macrophages (1) Experimental method: The operation steps are the same as those described above in "Qualitative experiment on uptake of MM / PNT-Rg3-Lipo by breast cancer 4T1 cells".

[0089] (2) Experimental results: such as Figure 10 As shown, after RAW264.7 cells were co-incubated with each group of fluorescent liposomes for 4 h, the fluorescence intensity of the C6-Lipo group was enhanced compared with the free C6 group; the fluorescence intensity of the C6-Rg3-Lipo group, which used Rg3 to replace cholesterol, was enhanced compared with the conventional liposome group C6-Lipo; however, when C6-Rg3-Lipo was modified with a macrophage membrane, the green fluorescence signal in RAW264.7 cells was weakened. This indicates that, compared with C6-Rg3-Lipo, macrophage membrane-coated liposomes MM / C6-Rg3-Lipo can inhibit macrophage phagocytosis.

[0090] 4. Quantitative experiment on the uptake of MM / PNT-Rg3-Lipo by RAW264.7 macrophages (1) Experimental method: The operation steps are the same as those described above in "Quantitative experiment on uptake of MM / PNT-Rg3-Lipo by 4T1 breast cancer cells".

[0091] (2) Experimental results: The results are as follows Figure 11 As shown in (A)-(B), consistent with the characterization results of laser confocal microscopy, after 4 h of co-incubation with the drug, the cellular fluorescence signal of C6-Rg3-Lipo in RAW264.7 cells was 1.75 times higher than that of C6-Lipo; compared with the C6-Rg3-Lipo group, macrophage uptake of MM / C6-Rg3-Lipo was reduced by approximately 53%. The results indicate that MM / C6-Rg3-Lipo can significantly inhibit macrophage phagocytosis.

[0092] Experimental Example 3: The regulatory effects of MM / PNT-Rg3-Lipo on the proliferation of breast cancer 4T1 cells, the p38 MAPK / STAT1 pathway, and its downstream chemokines. 1. CCK-8 assay for the cytotoxicity of MM / PNT-Rg3-Lipo to breast cancer cells (1) Experimental methods: The cytotoxicity of MM / PNT-Rg3-Lipo against breast cancer 4T1 cells and MDA-MB-231 cells was evaluated using the CCK-8 assay. Each group of drugs was co-incubated with 4T1 cells for 24 h. Specifically, 4T1 and MDA-MB-231 cells in logarithmic growth phase were seeded at a density of 8000 cells / well in 96-well plates and cultured in an incubator for 24 h. Then, the original culture medium was aspirated, and 100 μL of drug-containing culture medium containing different concentration gradients was added, including free PNT (0.001~8 μg / mL), Rg3-Lipo (1~150 μg / mL), and PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo (0.0001~12 μg / mL). Ten concentration gradients were set for each drug group, with five replicates for each concentration. Drug-free cell control wells and cell-free blank wells were also set up. After co-incubation for 24 h, the drug-containing culture medium was aspirated, and 100 μL of CCK-8 working solution (90 μL of LMEM complete culture medium mixed with 10 μL of CCK-8 reagent) was added to each well. The wells were then incubated in the dark for 1 h. Finally, the absorbance of each well at 450 nm was measured using a microplate reader, and the cell viability was calculated according to formula (5).

[0093] ×100% formula (5) (2) Experimental results: The cytotoxic effects of each group of drugs are shown in the figure. Figure 12 The half-maximal inhibitory concentration (IC50) of free PNTs. 50 The concentration of PNT-Lipo was 1.207 μg / mL. 50 The IC50 of PNT-Rg3-Lipo was 0.52 μg / mL. 50 The IC50 of MM / PNT-Rg3-Lipo was 0.45 μg / mL. 50 The concentration was 0.37 μg / mL. The results indicated that the combined administration of PNT and Rg3 enhanced cytotoxicity against 4T1 cells. Modification with macrophage membranes further enhanced cytotoxicity against 4T1 cells.

[0094] The cytotoxicity of each drug group against metastatic breast cancer cells MDA-MB-231 was comparable to that against the 4T1 cell line, as shown in the results. Figure 13As shown. In MDA-MB-231 cells, the IC50 of free PNTs... 50 The IC50 of PNT-Lipo was 2.48 μg / ml. 50 The IC50 of PNT-Rg3-Lipo was 0.92 μg / ml. 50 IC50 of 0.66 μg / ml, MM / PNT-Rg3-Lipo 50 The concentration was 0.51 μg / ml. Simultaneously, this invention also tested the cytotoxicity of Rg3-Lipo against 4T1 and MDA-MB-231 cells, and its IC50 values ​​in both cell lines were [not specified]. 50 The values ​​were 72.89 μg / ml and 74.93 μg / ml, respectively.

[0095] 2. Regulatory effects of MM / PNT-Rg3-Lipo on p-STAT1, p-p38, and c-Jun proteins in breast cancer 4T1 cells. (1) Experimental methods: Free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo were co-incubated with breast cancer 4T1 cells for 24 h. The PNT content in each group was 0.36 μg / mL, and the Rg3 content in the Rg3-Lipo group and the PNT-Rg3-Lipo group was the same. Total protein was extracted from each group of cells, and the protein expression of p-STAT1, p-p38, and c-Jun in the 4T1 cell line after drug treatment was analyzed by Western blotting.

[0096] (2) Experimental results: Table 3. Regulatory effects of each drug group on p-STAT1, STAT1, p-p38, p-38 and c-Jun proteins in 4T1 cells. (n=3) Note: Compared with the DMSO group, # P<0.05; ## P<0.01 Compared with the model control group, P<0.05； P<0.01 .

[0097] from Figure 14 , Figure 15 As shown in Table 3, in 4T1 cells, compared with the DMSO group, the expression level of p-STAT1 / STAT1 decreased after treatment with free PNTs. P<0.05Compared with DMSO, the expression of p-STAT1 / STAT1 in the Rg3-Lipo group showed no significant change; compared with free PNT, the p-STAT1 / STAT1 ratio in the PNT-Lipo and PNT-Rg3-Lipo groups showed a decreasing trend, but there was no statistically significant difference; the expression level of p-STAT1 / STAT1 protein was decreased in MM / PNT-Rg3-Lipo compared with free PNT. P<0.05 ).

[0098] Compared to the DMSO group, the expression level of c-Jun protein was significantly reduced after treatment with free PNT, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo groups. P<0.01 Compared with free PNT, the expression levels of c-Jun protein in the PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo groups showed a decreasing trend, but the difference was not statistically significant. Rg3-Lipo had almost no effect on the expression level of c-Jun protein.

[0099] Compared to the DMSO group, the expression level of p-p38 / p38 protein showed a decreasing trend after administration of free PNT; compared to DMSO, the expression level of p-p38 / p38 in the Rg3-Lipo group did not change significantly; compared to free PNT, the expression level of p-p38 / p38 protein in the PNT-Lipo group showed a decreasing trend, but there was no statistically significant difference; the PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo groups significantly reduced the expression level of p-p38 / p38 protein compared to free PNT. P<0.01 ).

[0100] This indicates that, at the same PNT content, MM / PNT-Rg3-Lipo has a better effect on downregulating p-STAT1 and p-p38 compared to the free PNT group.

[0101] 3. The effects of PNT and Rg3 alone and in combination on 4T1 breast cancer cells. CCL2 , CXCL1 , CXCL2 as well as CXCL11 Regulatory role (1) Experimental methods: Real-time quantitative PCR was used to detect the downstream chemokines of the p38 MAPK-STAT1 signaling pathway in 4T1 cells after PNT and Rg3 were administered alone and in combination. CXCL1 and CXCL2 And two other chemokines CXCL11 , CCL2 The mRNA expression level was determined. The specific method was as follows: 4T1 cells in logarithmic growth phase were harvested and subjected to a 2×10⁻⁶ mRNA expression assay.5 Cells were seeded at a density of PNTs / mL in six-well plates and cultured for 24 h. Then, they were treated with free PNTs, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo (the final PNT concentration in each group was 0.36 μg / mL). 24 h after drug administration, total RNA was extracted from each group using the Trizol reagent method, and its purity (A260 / A280 between 1.8 and 2.1) and concentration were determined spectrophotometrically. Subsequently, 1000 ng of total RNA was used, and after removing genomic DNA at 42℃ for 2 min, cDNA was synthesized by reverse transcription at 50℃ for 15 min and 85℃ for 5 sec. The diluted cDNA was used as a template for amplification using AceQ qPCR SYBR Green Master Mix on a real-time quantitative PCR instrument. The reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 sec, 60℃ annealing for 10 sec, and 72℃ extension for 20 sec, for a total of 40 cycles. Finally, using GAPDH as an internal reference gene, the measured Ct value was used to analyze the reaction mixture. -ΔΔCt Method for calculating target genes CXCL1 , CXCL2 , CXCL1 1 and CCL2 The relative mRNA expression level.

[0102] (2) Experimental results: Table 4. Effects of combined PNT and Rg3 therapy on 4T1 breast cancer cells. CCL2 , CXCL1 , CXCL2 as well as CXCL11 Regulatory role Note: Compared with the DMSO group, # P<0.05; ## P<0.01 Compared with the model control group, P<0.05； P<0.01 .

[0103] from Figure 16 As shown in Table 4, compared with the DMSO group, after administration of 1 μM PNT alone, CCL2 Decreased mRNA levels ( P<0.05 ); 50 μM Rg3 alone CCL2 The mRNA level was significantly reduced ( P<0.01 When PNT is used in combination with Rg3, compared to PNT alone, CCL2 Decreased mRNA levels ( P<0.05 ).

[0104] Compared with the DMSO group, after administration of 1 μM PNT alone, CXCL1 The mRNA level was significantly reduced ( P<0.01 ); 50 μMRg3 alone after administration CXCL1 The mRNA level did not change significantly; when PNT was used in combination with Rg3, compared with PNT alone, CXCL1 Decreased mRNA levels ( P<0.05 Among them, the combined use of PNT and Rg3 can reduce... CXCL1 At the level of expression, the effect was greater than the sum of the effects of using PNT alone and Rg3 alone, demonstrating a synergistic effect and achieving unexpected technical results.

[0105] Compared with the DMSO group, after administration of 1 μM PNT alone, CXCL2 The mRNA level was significantly reduced ( P<0.01 ); 50 μMRg3 alone after administration CXCL2 The mRNA level was significantly reduced ( P<0.01 When PNT is used in combination with Rg3, compared to PNT alone, CXCL2 Decreased mRNA levels ( P<0.05 ).

[0106] Compared with the DMSO group, after administration of 1 μM PNT alone, CXCL11 The mRNA level was significantly increased ( P<0.01 ); 50 μM Rg3 alone CXCL11 The mRNA level did not change significantly; when PNT was used in combination with Rg3, compared with PNT alone, CXCL11 The mRNA level was significantly increased ( P<0.01 Similarly, the combined use of PNT and Rg3 in raising... CXCL11 At the expression level, its effect is also better than the sum of the effects of the two drugs used alone, further confirming the synergistic effect of the two and the unexpected technical effect.

[0107] The results showed that Rg3 and PNT synergistically reduced chemokine levels. CCL2 , CXCL1 and CXCL2 mRNA expression levels, increased chemokine levels CXCL11 The mRNA expression level is increased, thereby promoting tumor immunity.

[0108] 4. MM / PNT-Rg3-Lipo's effect on 4T1 breast cancer cells CCL2 , CXCL1 , CXCL2 as well as CXCL11 Regulatory role (1) Experimental methods: Free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo were co-incubated with breast cancer 4T1 cells for 24 h. The PNT content in each group was 0.36 μg / mL, and the Rg3 content in the Rg3-Lipo group and the PNT-Rg3-Lipo group was the same. RNA was extracted from the cells of each group, and the effects of each group of drugs on the 4T1 cell line were detected by real-time quantitative PCR. CCL2 , CXCL1 , CXCL2 as well as CXCL11 The effect of mRNA expression levels.

[0109] (2) Experimental results: Table 5. Effects of each drug group on 4T1 breast cancer cells CCL2 , CXCL1 , CXCL2 as well as CXCL11 The regulatory effect ( (n=3) Note: Compared with the DMSO group, # P<0.05; ## P<0.01 Compared with the model control group, P<0.05； P<0.01 .

[0110] from Figure 17 As shown in Table 5, compared with DMSO, free PNTs and Rg3-Lipo groups significantly reduced the concentration of PNTs in 4T1 cells. CCL2 mRNA levels; compared with the free PNT group, the PNT-Lipo group showed mRNA levels after drug administration. CCL2 The mRNA levels in the PNT-Rg3-Lipo group showed a decreasing trend, but the difference was not statistically significant. CCL2 Decreased mRNA levels ( P<0.05 ), MM / PNT-Rg3-Lipo group CCL2 The mRNA level was significantly reduced ( P<0.01 ).

[0111] Compared with DMSO, free PNTs significantly reduced the number of 4T1 cells. CXCL1 The mRNA levels in the Rg3-Lipo group were almost unaffected. CXCL1 mRNA expression levels; compared with the free PNT group, the PNT-Lipo group showed mRNA expression levels after drug administration. CXCL1 Decreased mRNA levels ( P<0.05 PNT-Rg3-Lipo group and MM / PNT-Rg3-Lipo group CXCL1The mRNA level was significantly reduced ( P<0.01 Among them, PNT-Rg3-Lipo makes CXCL1 The mRNA expression level was reduced to 0.17 (a decrease of 0.83), which was better than the combined effects of PNT-Lipo alone (expression level 0.20, decrease of 0.80) and Rg3-Lipo alone (expression level 1.06, decrease of -0.06). This indicates that PNT and Rg3 co-loaded in the same liposome produced a synergistic effect.

[0112] Compared with DMSO, free PNTs significantly reduced the number of 4T1 cells. CXCL2 mRNA levels ( P<0.01 ), Rg3-Lipo group CXCL2 The mRNA expression level of the PNT group did not change significantly; compared with the free PNT group, the PNT-Lipo group showed no significant change after drug administration. CXCL2 The mRNA levels showed a decreasing trend, but there was no statistically significant difference between the PNT-Rg3-Lipo group and the MM / PNT-Rg3-Lipo group. CXCL1 Decreased mRNA levels ( P<0.05 Similarly, PNT-Rg3-Lipo makes CXCL2 The mRNA expression level was reduced to 0.44 (a reduction of 0.56), which was better than the combined effect of PNT-Lipo alone (expression level 0.62, reduction of 0.38) and Rg3-Lipo alone (expression level 0.87, reduction of 0.13), thus confirming the synergistic effect.

[0113] Compared with DMSO, free PNTs significantly increased the concentration of PNTs in 4T1 cells. CXCL11 mRNA levels in the Rg3-Lipo group CXCL11 The mRNA expression level of the PNT group did not change significantly; compared with the free PNT group, the PNT-Lipo group showed no significant change after drug administration. CXCL11 The mRNA levels in the PNT-Rg3-Lipo group showed an increasing trend, but there was no statistically significant difference. CXCL11 Increased mRNA levels ( P<0.05 ), MM / PNT-Rg3-Lipo group CXCL11 The mRNA level was significantly increased ( P<0.01 ).

[0114] Therefore, it can be concluded that the PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo groups can significantly downregulate chemokines compared with the free PNT group. CCL2 , CXCL1 and CXCL2 mRNA expression levels, increased chemokine levels CXCL11The mRNA expression level of PNT-Rg3-Lipo was reduced. This result is attributed to the combined use of PNT and Rg3, and may also be due to the enhanced uptake of PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo by 4T1 cells. In summary, PNT-Rg3-Lipo reduces... CXCL1 , CXCL2 The effect of the MM / PNT-Rg3-Lipo group was greater than that of PNT-Lipo + Rg3-Lipo (the sum of their individual effects), demonstrating a clear synergistic effect. This result is something that could not be achieved by combining the two liposomes alone, and is an unexpected technical effect. Meanwhile, the MM / PNT-Rg3-Lipo group showed... CXCL1 (0.15) and CXCL2 (0.41) also showed a very significant decrease, further supporting the robustness of this synergistic effect.

[0115] Experimental Example 4: Biocompatibility and In vivo distribution of MM / PNT-Rg3-Lipo 1. In vitro direct contact method for detecting hemolysis of MM / PNT-Rg3-Lipo (1) Experimental methods: The hemolysis of free PNT, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo was detected by in vitro direct contact method. The specific method was as follows: Blood from the marginal ear vein of rabbits was collected in heparin sodium blood collection tubes, washed and centrifuged (2000 rpm, 15 min) multiple times with fibrinogen-free saline, and the basal red blood cells were collected and prepared into a 2% red blood cell suspension with saline. The experiment was divided into a negative control group (saline), a positive control group (ultrapure water), and an experimental group (free PNT, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo, with a final PNT concentration of 10 μg / mL in each group). 0.75 mL of the control solvent or the sample from each experimental group was mixed with an equal volume of 0.75 mL of the 2% red blood cell suspension. After incubating the mixture in a 37°C water bath for 3 h, centrifuge at 1500 rpm for 15 min, take the supernatant into a 96-well plate, use an ELISA reader to measure the absorbance of each well at a wavelength of 576 nm, and calculate the hemolysis rate of each group of samples according to formula (6).

[0116] Formula (6) (2) Experimental results: such as Figure 18As shown in (A), the PNT content in free PNT, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo was 10 μg / mL. After incubation with blood, each group was centrifuged. The supernatant in the negative control group was clear after centrifugation; the solution in the positive control group was blood-red, indicating erythrocyte destruction; the supernatant of free PNT showed slight hemolysis; the supernatants in the PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo groups were clear and showed no significant difference compared to the negative control, suggesting that erythrocytes were not destroyed.

[0117] The OD values ​​of the supernatants in each group were measured at 576 nm using a multi-functional microplate reader, and the hemolysis rate was calculated. The results are as follows: Figure 18 As shown in (B), the calculated hemolysis percentages of PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo groups were all less than 5%, indicating that when the PNT content is the same, compared with free drugs, PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo do not cause damage to red blood cells and all have good blood compatibility.

[0118] 2. In vivo distribution of MM / PNT-Rg3-Lipo in a mouse model of breast cancer lung metastasis. (1) Experimental Methods: The targeting ability of MM / PNT-Rg3-Lipo in an established mouse model of lung metastasis was investigated. Fourteen days after intravenous injection of 4T1-luc cells into the tail vein of mice, D-luciferin potassium salt was injected intraperitoneally to activate luciferase in vivo. Bioluminescence detection was then performed. Figure 19 The results show that there are bioluminescent signals in the lungs of mice, proving that the lung metastasis model of breast cancer was successfully established.

[0119] Subsequently, mice were injected via tail vein with the following drugs: Free DIR, DIR-Lipo, DIR-Rg3-Lipo, and MM / DIR-Rg3-Lipo. The amount of DIR in each group was the same, 1 mg / kg. Chemiluminescence imaging of the drugs in mice was performed at 1, 2, 4, 8, 12, and 24 hours post-administration.

[0120] (2) Experimental results: From Figure 19 As can be seen, the drugs in each group were concentrated in the liver, which has a rich blood flow, before 2 hours. As time progressed, DIR-Rg3-Lipo and MM / DIR-Rg3-Lipo began to accumulate in the lungs. Free DIR and DIR-Lipo showed no significant accumulation in the lungs 24 hours after administration. At 24 hours, MM / DIR-Rg3-Lipo showed significant accumulation in lung metastases.

[0121] 3. Distribution of MM / PNT-Rg3-Lipo in tissues and organs of a mouse model of breast cancer lung metastasis (1) Experimental method: In order to determine the targeting activity of MM / PNT-Rg3-Lipo, mice were euthanized by dislocation 24 h after administration, and the heart, liver, spleen, lung and kidney of the mice were removed for chemiluminescence imaging. The drug signal was displayed as red fluorescence. Bioluminescence imaging was performed on the lung tissue, and the metastatic lesions were displayed as blue-green fluorescence to determine the location of metastatic nodules.

[0122] (2) Experimental results: From Figure 20 As can be seen, 4T1-luc cells colonized and formed pulmonary nodules in lung tissue. No significant chemiluminescent signals were observed in heart and kidney tissues for any of the drug groups; MM / DIR-Rg3-Lipo showed significantly higher enrichment in pulmonary nodules than the other three groups. This result indicates that MM / DIR-Rg3-Lipo has targeting ability for metastatic pulmonary nodules.

[0123] Experimental Example 5: In vivo evaluation of MM / PNT-Rg3-Lipo's inhibition of lung metastasis in breast cancer. 1. MM / PNT-Rg3-Lipo effectively reduces the number of lung metastatic nodules in mice with breast cancer lung metastases. (1) Experimental methods: On day 0 of the experiment, 4T1-luc cells were injected into the tail vein of mice to establish a lung metastasis model of breast cancer. On day 4 after modeling, the mice were randomly divided into 6 groups: PBS group, free PNT group, Rg3-Lipo group, PNT-Lipo group, PNT-Rg3-Lipo group, and MM / PNT-Rg3-Lipo group. The drugs were administered via tail vein injection once every 2 days for a total of 11 times. The PNT dosage in each group was 10 mg / kg. The Rg3 content in the Rg3-Lipo group and the PNT-Rg3-Lipo group was the same. The experimental and administration diagram is shown below. Figure 21 As shown.

[0124] (2) Experimental Results: After the last administration, lung tissue from each group of mice was removed, weighed, photographed, and the number of lung nodules was recorded. Figure 22The results show that the PBS group had an average of 16.5±8.4 visible lung metastatic nodules, the free PNT group had an average of 10.3±2.2 obvious lung metastatic lesions, and the Rg3-Lipo group had an average of 10.8±2.5 visible lung metastatic nodules. The number of obvious lung metastatic nodules in the PNT-Lipo, PNT-Rg3-Lipo, and MM / PNT-Rg3-Lipo groups were 5.9±1.8, 2.6±1.4, and 1.5±1.5, respectively. Quantitative results indicate that compared with the PBS group, the number of lung metastatic nodules was reduced in both the free PNT and Rg3-Lipo groups; compared with free PNT, the number of lung metastatic nodules in the PNT-Lipo group showed a decreasing trend, but there was no statistically significant difference; compared with the free PNT group, the number of lung metastases in the PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo groups was significantly reduced. P <0.01). Weigh the lung tissue of the mouse and calculate the organ index of the lung. Figure 23 As can be seen in (A), compared with free PNT, the organ indices of the PNT-Rg3-Lip and MM / PNT-Rg3-Lipo groups were significantly reduced. This result indicates that MM / PNT-Rg3-Lipo has a good inhibitory effect on lung metastasis of breast cancer.

[0125] 2. Effects of MM / PNT-Rg3-Lipo on body weight changes in mice (1) Experimental method: Monitor the weight changes of mice during the experiment.

[0126] (2) Experimental results: From Figure 23 As shown in (B), the body weight of mice in the PBS group decreased in the later stage of the experiment, while the body weight of mice in the PNT-Lipo, PNT-Rg3-Lip, and MM / PNT-Rg3-Lipo groups did not change significantly before and after the experiment. This indicates that PNT-Lipo, PNT-Rg3-Lip, and MM / PNT-Rg3-Lipo can alleviate the body weight loss in mice caused by the breast cancer lung metastasis model. On the other hand, it also shows that PNT-Lipo, PNT-Rg3-Lip, and MM / PNT-Rg3-Lipo have no obvious systemic toxicity.

[0127] 3. Lung tissue pathology results (1) Experimental method: H&E staining was used.

[0128] (2) Experimental results: The H&E staining results of lung tissue sections are as follows Figure 24As shown, obvious micrometastatic lesions were observed in the PBS group, free PNT group, Rg3-Lipo group, PNT-Lipo group, and PNT-Rg3-Lipo group, while no obvious metastatic lesions were observed in the MM / PNT-Rg3-Lipo group, which is consistent with the results of macroscopic observation, indicating that MM / PNT-Rg3-Lipo has a significant effect on inhibiting lung metastasis of breast cancer.

[0129] 4. Regulatory effects of MM / PNT-Rg3-Lipo on p-STAT1, p-p38, and c-Jun proteins in lung metastatic nodules of mice with breast cancer lung metastases. (1) Experimental method: In order to further reveal the mechanism of action of MM / PNT-Rg3-Lipo at the in vivo level, lung tissues of mice were taken out after being continuously administered PBS, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo 11 times. Total protein was extracted from the lung metastatic nodules of each group of mice. The protein expression of p-STAT1, p-p38 and c-Jun in the lung metastatic nodules of each group of mice after drug treatment was analyzed by Western blotting.

[0130] (2) Experimental results: Table 6. Regulatory effects of each drug group on p-STAT1, STAT1, p-p38, p-38 and c-Jun proteins in lung metastatic nodules of breast cancer mice. ±s, n=3) Note: Compared with the DMSO group, # P<0.05 ; ## P<0.01 Compared with the model control group, P<0.05 ; P<0.01 .

[0131] from Figure 25 , Figure 26 As shown in Table 6, compared with the PBS group, the expression level of p-STAT1 / STAT1 decreased after treatment with free PNTs in the lung metastatic nodule tissues of each group. P <0.05); Compared with the PBS group, there was no significant change in p-STAT1 / STAT1 expression in the Rg3-Lipo group; Compared with free PNT, the expression of p-STAT1 / STAT1 in the PNT-Lipo and PNT-Rg3-Lipo groups showed a decreasing trend, but there was no statistical difference; The expression level of p-STAT1 / STAT1 in MM / PNT-Rg3-Lipo was lower than that in free PNT ( P<0.05).

[0132] Compared to the PBS group, the expression level of c-Jun protein decreased after treatment with free PNT, but the difference was not statistically significant; Rg3-Lipo had almost no effect on the expression level of c-Jun protein; compared with free PNT, the expression of c-Jun in the PNT-Lipo and PNT-Rg3-Lipo groups showed a decreasing trend, but the difference was not statistically significant; compared with free PNT, the expression of c-Jun protein was decreased in the MM / PNT-Rg3-Lipo group. P <0.05).

[0133] Compared with the PBS group, the expression level of p-p38 / p38 protein decreased after administration of free PNT, but there was no statistically significant difference. The expression levels of p-p38 / p38 protein in the PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo groups all showed a decreasing trend compared with free PNT.

[0134] The results of this experiment show that the MM / PNT-Rg3-Lipo group has a better effect on downregulating p-STAT1 and p-p38 in vivo than the free PNT group, which is consistent with the in vitro results.

[0135] 5. MM / PNT-Rg3-Lipo's effect on lung metastatic nodules in mice with breast cancer lung metastases CCL2 , CXCL1 , CXCL2 as well as CXCL11 Regulatory role (1) Experimental methods: In order to further verify the regulatory effect of MM / PNT-Rg3-Lipo on downstream chemokines of the p38 MAPK / STAT1 signaling pathway at the in vivo level, in a mouse model of breast cancer lung metastasis, PBS, free PNT, Rg3-Lipo, PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo were administered 11 times consecutively. Lung tissues of mice in each group were taken out, and RNA was extracted from lung metastasis nodules of each group of mice. The effects of each drug on lung metastasis nodules of breast cancer lung metastasis mice after drug treatment were detected by real-time quantitative PCR. CCL2 , CXCL1 , CXCL2 as well as CXCL11 The effect of mRNA expression levels.

[0136] (2) Experimental results: Table 7. Effects of each drug group on lung metastatic nodules in mice with breast cancer lung metastases. CCL2 , CXCL1 , CXCL2 as well as CXCL11 The regulatory effect ( ±s, n=3) Note: Compared with the DMSO group, # P<0.05 ; ## P<0.01 Compared with the model control group, P<0.05 ; P<0.01 .

[0137] from Figure 27 As shown in Table 7, compared with the PBS group, the free PNT and Rg3-Lipo groups had significantly lower levels of lung metastatic nodules. CCL2 The mRNA level showed a decreasing trend, but the difference was not statistically significant; compared with the free PNT group, the PNT-Lipo group showed a decrease after drug administration. CCL2 There was no significant difference in mRNA levels in the PNT-Rg3-Lipo group. CCL2 The mRNA level showed a decreasing trend in the MM / PNT-Rg3-Lipo group. CCL2 Decreased mRNA levels ( P <0.05).

[0138] Compared with the PBS group, the free PNT group significantly reduced the number of lung metastatic nodules. CXCL1 mRNA levels ( P <0.01); the Rg3-Lipo group had almost no effect. CXCL1 mRNA expression levels; compared with the free PNT group, the PNT-Lipo group showed mRNA expression levels after drug administration. CXCL1 The mRNA expression levels of the PNT-Rg3-Lipo group and the MM / PNT-Rg3-Lipo group were not significantly different. CXCL1 The mRNA levels showed a decreasing trend, but there were no significant differences.

[0139] Compared with the PBS group, the free PNT group significantly reduced the number of lung metastatic nodules. CXCL2 mRNA expression level ( P <0.01), in the Rg3-Lipo group CXCL2 The mRNA expression of PNT showed a decreasing trend, but this was not statistically significant; compared with the free PNT group, the expression of PNT-Lipo, PNT-Rg3-Lipo and MM / PNT-Rg3-Lipo groups decreased after drug administration. CXCL1 The mRNA levels showed a decreasing trend, but this was not statistically significant.

[0140] Compared with the PBS group, the free PNT group and the Rg3-Lipo group had lower concentrations of PNTs. CXCL11The mRNA expression level showed an increasing trend; compared with the free PNT group, the PNT-Lipo group and the PNT-Rg3-Lipo group showed increased expression levels after drug administration. CXCL11 The mRNA levels of all groups showed an increasing trend, but there was no statistically significant difference. In the MM / PNT-Rg3-Lipo group, after administration... CXCL11 Increased mRNA levels ( P <0.05).

[0141] The results of this experiment show that the MM / PNT-Rg3-Lipo group can downregulate chemokines better than the free PNT group. CCL2 , CXCL1 and CXCL2 mRNA expression levels, increased chemokine levels CXCL11 The mRNA expression level was consistent with the in vitro results.

[0142] In summary, this invention provides a combination drug, liposomes, biomimetic liposomes, their preparation methods, and their application in the preparation of drugs for treating lung metastases in breast cancer. Specifically, this invention provides a combination drug comprising panatinib (PNT) and ginsenoside Rg3, as well as conventional liposomes PNT-Rg3-Lipo and macrophage membrane-coated biomimetic liposomes MM / PNT-Rg3-Lipo constructed based on this combination drug. Experiments have demonstrated that PNT and Rg3 regulate chemokines. CXCL1 , CXCL2 and CXCL11 This combination exhibits a significant synergistic effect, with its combined effect exceeding the sum of the effects of the two drugs used alone. The liposomes are uniform in size and exhibit good stability. MM / PNT-Rg3-Lipo successfully retains the integrin α4 / β1 functional protein on the macrophage membrane surface and presents a typical core-shell structure. In vitro cell experiments show that this biomimetic liposome can enhance the uptake of 4T1 breast cancer cells, inhibit macrophage phagocytosis, and downregulate p38 MAPK / STAT1 signaling pathway-related proteins. CCL2 , CXCL1 , CXCL2 The expression, uplift CXCL11 The expression of [the drug / pNT-Rg3-Lipo] was further confirmed by in vivo pharmacodynamic experiments. MM / PNT-Rg3-Lipo can target and accumulate in lung metastases, significantly reducing the number of lung metastatic nodules, improving the pathological state of lung tissue, and exhibiting good biocompatibility. This invention provides an innovative biomimetic nanodelivery strategy for combating breast cancer lung metastases, with clear clinical application prospects.

Claims

1. A combination drug, characterized in that: It contains panatinib and ginsenoside Rg3 for simultaneous or separate administration.

2. The combined medication according to claim 1, characterized in that: The mass ratio of panatinib to ginsenoside Rg3 is 1:(2~75).

3. The combined medication according to claim 2, characterized in that: The mass ratio of panatinib to ginsenoside Rg3 is 1:

3.

4. A liposome, characterized in that: The liposomes are liposomes that use phospholipids as carrier materials and simultaneously encapsulate panatinib and ginsenoside Rg3; the mass ratio of panatinib, ginsenoside Rg3 and egg yolk lecithin is 1:(1~10):(5~20).

5. The liposomes according to claim 4, characterized in that: The phospholipid is egg yolk lecithin; the mass ratio of panatinib, ginsenoside Rg3 and egg yolk lecithin is 1:3:

10.

6. A biomimetic liposome, characterized in that: The biomimetic liposome is formed by mixing cell membranes derived from immune cells with the liposomes described in any one of claims 4 to 5 via ultrasound.

7. The biomimetic liposome according to claim 6, characterized in that: The ratio of the total lipid mass in the liposomes to the membrane components derived from immune cells in the cell membranes of the immune cells is: (10~30) mg : (1~5) × 10⁻⁶ mg. 7 One immune cell.

8. A method for preparing the biomimetic liposomes of claim 6 or 7, characterized in that: Includes the following steps: (1) Extract cell membranes from immune cells to obtain cell membrane microcapsule solutions; (2) The cell membrane microcapsule solution obtained in step (1) is mixed with the liposomes described in any one of claims 4 to 5 and ultrasonically treated under ice bath conditions to obtain the final product.

9. Use of the combination drug of claim 1 or 2, the liposome of any one of claims 4 to 5, or the biomimetic liposome of any one of claims 6 to 7 in the preparation of a drug for the prevention and / or treatment of breast cancer.

10. The use according to claim 9, characterized in that: The drug in question is an anti-breast cancer lung metastasis drug.

Images