A cell preparation targeting bone metastatic tumor cells, its preparation method and application

Abstract

This invention discloses a cell preparation targeting bone metastatic tumor cells, its preparation method, and its application, belonging to the field of biomedical technology. The cell preparation comprises senescent neutrophils and drug-loaded cationic liposomes internalized within them. The cationic liposomes encapsulate a STING agonist and a pan-photokinase inhibitor. The preparation method includes: preparing drug-loaded liposomes using a thin-film dispersion-ultrasound method; obtaining senescent neutrophils through in vitro culture of extracted neutrophils; and preparing a senescent neutrophil preparation loaded with liposomes. This invention utilizes the natural bone marrow homing ability of senescent neutrophils to achieve precise targeting of bone metastatic tumor cells, and co-delivers two drugs through liposomes, simultaneously activating anti-tumor immunity and inducing tumor cell apoptosis / autophagy, synergistically treating bone metastases. This system has high drug loading capacity, good encapsulation efficiency, and sustained-release effect, and the preparation process is simple and controllable, providing a new strategy for the treatment of cancer bone metastases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cell preparation that targets bone metastatic tumor cells, its preparation method, and its application. Background Technology

[0002] Bone metastasis is a common complication of advanced malignant tumors, especially in cancers such as breast cancer and prostate cancer, severely impacting patients' quality of life. Bone metastasis not only increases patient suffering but also significantly reduces their quality of life. With advancements in medical technology, including recent developments in gene testing, targeted therapy, immunotherapy, and stereotactic radiotherapy, the survival time of patients with advanced malignant tumors has increased, but the risk of bone metastasis and bone-related events has significantly increased. However, due to insufficient blood supply to bone tissue and the presence of the bone marrow-blood barrier, conventional drugs struggle to effectively reach the bone marrow, limiting treatment efficacy. Therefore, developing novel drug delivery systems to improve drug concentration and efficacy in the bone marrow is a current research focus.

[0003] Current research in the therapeutic field focuses on identifying targets that may trigger or enhance anti-tumor immune responses. Vadimezan (Vad), a STING agonist, is a multi-target small molecule compound that directly binds to and activates the STING protein, promoting its transport from the endoplasmic reticulum to the Golgi apparatus. This, in turn, activates TBK1 kinase and transcription factor IRF3, ultimately inducing the secretion of type I interferon (IFN-α / β) and various pro-inflammatory cytokines. This process can initiate an innate immune response, promote the maturation of dendritic cells (DCs) and the cross-presentation of tumor antigens, thereby enhancing the infiltration and activity of CD8+ T cells and transforming the tumor microenvironment into an immune-activated state. Furthermore, Vaad exhibits anti-angiogenic effects, competitively binding to the extracellular domain of VEGFR2, blocking VEGF-mediated angiogenesis signals, disrupting tumor tissue angiogenesis, and reducing tumor blood supply. In mouse models of tumors, Vaad also demonstrates significant distal effects, promoting dendritic cell maturation and significantly enhancing the body's immune response to tumor cells by inducing tumor cell immunogenic death (ICD) and the release of damage-associated molecular patterns (DAMPs). Danusertib (Dan) is a pan-auricular kinase inhibitor that induces cell cycle arrest in tumor cells during the G2 / M phase by inhibiting auricular kinase activity. This arrest is characterized by downregulation of CDK1 / CDC2 and cyclin B1 expression, while upregulation of p21, p27, and p53 protein expression. This cell cycle arrest leads to polyploid accumulation, ultimately triggering apoptosis. The apoptosis mechanism involves activation of the mitochondrial pathway, manifested by increased expression of pro-apoptotic proteins such as Bax and Puma, decreased expression of anti-apoptotic proteins such as Bcl-2 and Bcl-xl, release of cytochrome c from mitochondria, and activation of Caspase-9 and Caspase-3. Furthermore, Dan can induce autophagy by activating the AMPK signaling pathway and inhibiting the PI3K / Akt / mTOR signaling axis, upregulating Beclin1 expression, and promoting the conversion of LC3-I to LC3-II. Cationic liposomes (Lip) prepared from DOTAP, DOPE, and cholesterol are highly efficient non-viral gene delivery systems. Composed of these three key lipid components in a specific ratio, this liposome system has significant applications in gene therapy and drug delivery. Compared to traditional cationic polymer carriers, this liposome system offers advantages such as low cytotoxicity and good biocompatibility, and has been widely used in in vitro cell transfection and in vivo gene therapy research.

[0004] Currently, cell-based drug delivery systems, such as those using erythrocytes, macrophages, and stem cells, have attracted considerable attention due to their excellent biocompatibility and natural tissue tropism. However, these delivery systems still face numerous challenges, including complex cell acquisition and engineering processes, limited target specificity, and potential side effects arising from their inherent physiological functions. Previous studies have successfully utilized the natural bone marrow homing characteristics of senescent neutrophils (SNEs) for drug delivery, but this strategy has limitations in terms of the depth and complexity of therapeutic mechanisms, and it remains unresolved how to precisely intervene in the complex disease microenvironment within the bone marrow (such as the niche of tumor bone metastasis) through multi-mechanism synergy based on this targeting. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention aims to provide a cell preparation targeting bone metastatic tumor cells, its preparation method, and its application. This solves the technical problems of limited drug loading methods, low drug loading capacity, and a single therapeutic mechanism in existing neutrophil-based bone marrow targeted delivery systems.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is to provide a cell preparation that targets bone metastatic tumor cells, comprising senescent neutrophils and drug-loaded cationic liposomes internalized by the senescent neutrophils; the drug-loaded cationic liposomes are encapsulated with a STING agonist and a pan-aurora kinase inhibitor.

[0007] The cell preparation in the above technical solution is loaded with cationic liposomes containing STING agonist and pan-polar kinase inhibitor through the internalization of senescent neutrophils (such as endocytosis and macropinocytosis). This cell preparation can target the bone metastasis tumor area, release the drug while initiating an immune response and inhibiting tumor cell proliferation to induce tumor cell apoptosis or autophagy, thereby achieving targeted and synergistic treatment of cancer bone metastasis tumors. The encapsulation of the drug pair with cationic liposomes can also reduce the toxicity of the drug to cells and tissues and improve biosafety.

[0008] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the STING agonists are Vadimezan, cGAMP, ADU-S100, MSA-2, diABZI, SR-717, or MK-1454; the pan-photokinase inhibitors are Danusertib, Barasertib, Tozasertib, AMG 900, or CCT137690.

[0009] Furthermore, the mass ratio of STING agonist to pan-polar light kinase inhibitor is 8~10:1~2.

[0010] Furthermore, the cationic lipids are DOTAP (1,2-dioleoyl-3-trimethylammonium propyl ammonium chloride), DODAP (N,N-dioleoyl-N,N-dimethyl-1,2-diaminopropane), DODMA (1,2-dioleoyloxy-3-dimethylaminopropane), SM-102, or ALC-0315; the phospholipids are DOPE (1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), DSPE (1,2-distearyl-sn-glycerol-3-phosphoethanolamine), or DPPG (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine).

[0011] Furthermore, the mass ratio of cationic lipids, phospholipids, and cholesterol to cholesterol is 0.5~1.5:14~16:3~5.

[0012] This invention also discloses a method for preparing the above-mentioned cell preparation targeting bone metastasis tumor cells, which includes the following steps: S1: Preparation of drug-loaded cationic liposomes encapsulated with STING agonist and panoptic kinase inhibitor; S2: Neutrophils were extracted and isolated, and senescent neutrophils were obtained through in vitro culture; S3: Co-incubate drug-loaded cationic liposomes with senescent neutrophils to allow the senescent neutrophils to internalize the drug-loaded cationic liposomes, thus obtaining the drug-loaded cationic liposomes.

[0013] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, in step S1, drug-loaded cationic liposomes are prepared using either thin-film dispersion-ultrasound or microfluidic-solvent injection.

[0014] Furthermore, the total incubation time in step S3 is 0.5~1.5h.

[0015] The present invention also discloses the application of the above-mentioned cell preparation having targeting bone metastasis tumor cells in the preparation of a drug for treating bone metastasis of cancer.

[0016] The beneficial effects of this invention are: 1. Targeting: Previous reports only focused on organ targeting, but the cell preparation of this invention can move from organ targeting to achieving specific lesions and lesion cells.

[0017] 2. The preparation process is simple and controllable. The drug-loaded cationic liposomes are prepared using a thin-film dispersion-ultrasound method, which is a mature, simple, and easily scalable process. Furthermore, this cell preparation does not require complex genetic engineering modification of the carrier cells; simply extracting neutrophils, inducing their natural senescence through in vitro culture, and then co-incubating them with the drug-loaded liposomes yields a cell preparation with targeted bone metastases and therapeutic functions.

[0018] 3. Multiple synergistic therapeutic effects: The cell preparation in this invention not only utilizes the natural bone marrow homing ability of senescent neutrophils to achieve precise targeting and further enrichment in the tumor microenvironment, but also, through liposome co-delivery of drugs, can not only activate the immune cell response but also inhibit tumor cell proliferation, induce tumor cell apoptosis and autophagy, thereby achieving targeted synergistic treatment of cancer bone metastases.

[0019] 4. The carrier system optimizes drug performance. The cationic liposomes used have a high encapsulation efficiency, which can effectively load and protect the drug, and significantly reduce the toxicity of the drug to normal cells and carrier cells. Liposomes have sustained-release properties, which can prolong the drug's action time and further improve efficacy and safety. Attached Figure Description

[0020] Figure 1 The graph shows the particle size and zeta potential of the four liposomes prepared in Example 1. Figure 2 TEM images of the four types of liposomes prepared in Example 1; Figure 3 Image of a physical liposome; Figure 4 This is a trypan blue staining image of neutrophils. Figure 5 This is a flow cytometry image showing the purity of neutrophils after double staining with Gr-1 and CLM-5 antibodies. Figure 6 Laser confocal microscope images of DiD-Lip@SNE at different time points; Figure 7 Flow cytometry analysis of CXCR4 expression in Vad&Dan-Lip@SNE; Figure 8 In vivo fluorescence images for preliminary experiments to construct a mouse bone transfer model; Figure 9 In vivo fluorescence imaging of major organs and hind limb bones 24 hours after intravenous injection of different formulation groups; Figure 10 A bar chart showing the quantitative analysis of hindlimb bone fluorescence intensity in each group; Figure 11 Immunofluorescence staining images of paraffin sections of the femur from each group of mice. Detailed Implementation

[0021] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0022] Example 1: Preparation and Characterization of Cationic Liposomes 1. Preparation of Vad & Dan-Lip: Example 1: DOTAP, DOPE, and cholesterol were dissolved separately in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed in a mass ratio of 1:15:4 for DOTAP, DOPE, and cholesterol. A Vad solution (solvent: tetrahydrofuran) with a concentration of 1 mg / mL and a Dan solution (solvent: chloroform) with a concentration of 1 mg / mL were added, with a mass ratio of Vad to Dan of 10:1. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0023] Example 2: DODAP, DOPC, and cholesterol were dissolved separately in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed in a mass ratio of DODAP, DOPC, and cholesterol of 1.5:15:3. A Vad solution (solvent: tetrahydrofuran) with a concentration of 1 mg / mL and a Dan solution (solvent: chloroform) with a concentration of 1 mg / mL were added, with a mass ratio of Vad to Dan of 8:2. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0024] Example 3: DODMA, DSPC, and cholesterol were dissolved in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed in a mass ratio of DODMA, DSPC, and cholesterol of 1:16:3. A Vad solution (solvent: tetrahydrofuran) with a concentration of 1 mg / mL and a Dan solution (solvent: chloroform) with a concentration of 1 mg / mL were added, with a mass ratio of Vad to Dan of 9:1.5. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0025] Example 4: DOTAP, DPPC, and cholesterol were dissolved separately in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed in a mass ratio of 0.5:14:5 for DOTAP, DPPC, and cholesterol. A Vad solution (solvent: tetrahydrofuran) with a concentration of 1 mg / mL and a Dan solution (solvent: chloroform) with a concentration of 1 mg / mL were added, with a mass ratio of Vad to Dan of 8:1. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0026] The Vad&Dan-Lip prepared above have similar performance. The following experiments will be conducted using the Vad&Dan-Lip prepared in the first example as an example.

[0027] 2. Preparation of Lip: Dissolve DOTAP, DOPE and cholesterol separately in chloroform to obtain a solution with a concentration of 1 mg / mL; then mix the three solutions according to the mass ratio of DOTAP, DOPE and cholesterol of 1:15:4, remove the solvent by vacuum water bath rotary evaporation at room temperature and 50 rpm to obtain the adherent lipid layer, add ultrapure water, and sonicate in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds and pause for 5 seconds).

[0028] 3. Preparation of Dan-Lip: DOTAP, DOPE, and cholesterol were dissolved in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed at a mass ratio of 1:15:4 for DOTAP, DOPE, and cholesterol. A Dan solution with a concentration of 1 mg / mL (chloroform as solvent) was added. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0029] 4. Preparation of Vad-Lip: DOTAP, DOPE, and cholesterol were dissolved in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed at a mass ratio of 1:15:4 for DOTAP, DOPE, and cholesterol. Vad solution with a concentration of 1 mg / mL (solvent: tetrahydrofuran) was added. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0030] 5. Characterization Figure 1The above four types of liposomes are shown in the particle size and zeta potential statistics. The measurements were taken using a Malvern particle size analyzer. The PDI in the bar chart was less than 0.3 at the time of measurement. Figure 2 These are TEM scan images of four types of liposomes. The encapsulation efficiency of Vad & Dan-Lip was determined to be 99.02% after ultrafiltration using HPLC. Figure 3 This is a real photo of a Lip sample.

[0031] 6. Preparation of cationic liposomes loaded with other STING agonists and panoptic kinase inhibitors Example 1: DOTAP, DOPE, and cholesterol were dissolved separately in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed in a mass ratio of 1:15:4 for DOTAP, DOPE, and cholesterol. A Vad solution (solvent: tetrahydrofuran) with a concentration of 1 mg / mL and a Barasertib solution (solvent: dimethyl sulfoxide) with a concentration of 1 mg / mL were added, with a mass ratio of Vad to Barasertib of 10:2. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0032] Example 2: DOTAP, DOPE, and cholesterol were dissolved in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed in a mass ratio of 1.2:15:3 for DOTAP, DOPE, and cholesterol. A 1 mg / mL diABZI solution (solvent: dimethyl sulfoxide) and a 1 mg / mL Tozasertib solution (solvent: dimethyl sulfoxide) were added, with a mass ratio of diABZI to Tozasertib of 10:1. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0033] Example 3: DOTAP, DOPE, and cholesterol were dissolved separately in chloroform to obtain a solution with a concentration of 1 mg / mL. Then, the three solutions were mixed in a mass ratio of 1:15:4 for DOTAP, DOPE, and cholesterol. SR-717 solution (solvent: dimethyl sulfoxide) with a concentration of 1 mg / mL and AMG 900 solution (solvent: dimethyl sulfoxide) with a concentration of 1 mg / mL were added, with a mass ratio of SR-717 to AMG 900 of 8:1.5. The solvent was removed by rotary evaporation in a vacuum water bath at room temperature and 50 rpm to obtain an adherent lipid layer. Ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min to obtain the final product (ultrasonic power 350 W, sonication for 5 seconds, pause for 5 seconds).

[0034] Example 2: Extraction and identification of senescent neutrophils (SNE) 6-8 week old BALB / c female mice were acclimatized for 3 days, then euthanized by cervical dislocation. Femurs and tibias were collected, disinfected with 75% alcohol, and placed in RPMI-1640 complete culture medium. The bone marrow was repeatedly flushed with an insulin syringe to obtain cell suspension. After removing impurities through a cell filter, the suspension was centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 1.5 mL of PBS. 55%, 65%, and 80% Percoll solutions were prepared in a density gradient, and the cell suspension was added to the top. The suspension was centrifuged at 1200g for 30 min. Mature neutrophils located at the 65% and 80% boundary were collected, washed with PBS, treated with 4°C pre-chilled erythrocyte lysis buffer, and washed twice with PBS. Finally, the cells were resuspended in complete culture medium and incubated in culture dishes.

[0035] The viability of the extracted neutrophils was determined using trypan blue staining, and the staining pattern is shown below. Figure 4 As shown, the cell viability was >99% after counting with a hemocytometer. Figure 5 The cell purity was determined by staining the collected NEs with Gr1 and CLM-5 antibodies and then detecting the neutrophil purity by flow cytometry. The result showed a neutrophil purity of 94.2%.

[0036] Example 3: Preparation and Intracellular Behavior of Cellular Preparation (Vad & Dan-Lip@SNE) Four hours after neutrophil extraction, the complete culture medium for neutrophil culture was replaced with serum-free medium, and the cells were starved for 1 hour. The serum-free medium was then replaced with 5% glucose isotonic solution. Vad&Dan-Lip 5% glucose solution was added and incubated for 1 hour. The cell preparation incubation was completed 6 hours after neutrophil extraction, yielding Vad&Dan-Lip@SNE.

[0037] After repeated freeze-thaw cycles to lyse Vad&Dan-Lip@SNE cells and adding anhydrous ethanol to break the liposomes, the drug loading of 1000w neutrophils was determined by HPLC to be 38.02μg Vad and 3.8ug Dan.

[0038] For tracing, 4 hours after neutrophil extraction, the complete culture medium for neutrophil culture was replaced with serum-free culture medium, and the cells were starved for 1 hour. The serum-free culture medium was then replaced with 5% glucose isotonic solution. DiD-Lip (prepared in the same way as in Example 1) 5% glucose solution was added and incubated for 1 hour to obtain DiD-Lip@SNE.

[0039] Figure 6These are CLSM images of DiD-Lip@SNE at different time points. It can be seen that at 2 h, liposomes and lysosomes co-localize; at 4 h, liposomes begin to escape from lysosomes; and at 8 h, cell apoptosis begins, and liposomes are released. Simultaneously, intracellular tracking data also confirms that DiD-Lip is taken up into the cell by SNE. Figure 7 The results are from flow cytometry analysis of Vad&Dan-Lip@SNE after CXCR4 staining. The results show that the cell preparation has a high degree of senescence, which can help prove that Vad&Dan-Lip@SNE is highly targeted to bone marrow.

[0040] Example 4: Verification of the in vivo targeting and anti-bone metastasis effects of cell preparations ① Establishment of bone metastasis model A quiescent model of microscopic bone metastasis was established by injecting 4T1 cells with IIA. The model was established using 6- to 8-week-old female BALB / c mice injected with 5 × 10⁻⁶ cells of IIA. 5 4T1 / GFP cells. Figure 8 This is a live fluorescence image from a pre-modeling experiment. The left side shows the fluorescent GFP of tumor cells, and the right side shows the DiD fluorescence of DiD-Lip@SNE. The main organs from top to bottom are the heart, liver, spleen, lung, and kidney. Next to them are the femur (highlighted by the tumor), tibia, and fibula.

[0041] ② Distribution within the body On day 5 of modeling, the modeling mice were randomly divided into 4 groups of 3 mice each. They were intravenously injected with PBS, DiD, DiD-Lip, and DiD-Lip@SNE (DiD dose 1 mg / kg). 24 hours later, the modeling mice were euthanized, and images of major organs and hind limb bones were acquired and analyzed using small animal in vivo imaging to observe the distribution of DiD-Lip@SNE and GFP-carrying tumor cells in bone and other organs. In vivo imaging was performed 24 hours later. Figure 9 , Figure 10 The results showed that only the DiD-Lip@SNE group exhibited strong DiD fluorescence signals in the hind limb bones (femur / tibia) of mice, while the non-targeted uptake in organs such as the liver and spleen was significantly lower than that in other groups, demonstrating its excellent bone marrow targeting specificity.

[0042] Validating the targeting ability of cell preparations using tissue immunofluorescence sections Following in vivo fluorescence detection, the major organs of each group of model mice were sectioned in paraffin, and cell nuclei were stained with DAPI. Femurs were decalcified for one week in the dark using a special method; paraffin sections of the distal femoral epiphysis and metaphysis were then sectioned and stained with IF (immunoassay), with cell nuclei stained with DAPI. All slides were scanned using SLIDEVIEW VS200 ST, and images and data were analyzed using OlyVIA software. Figure 11The images show that the DiD fluorescence in the DiD-Lip@SNE group was the highest in the femoral sections, and the trend was similar to that of the DiD-Lip@SNE group. Figure 10 Consistent. Furthermore, the DiD fluorescence and GFP fluorescence of all cell preparation groups highly overlapped, while most other groups did not, indicating the targeting ability of the cell preparations designed in this invention to lesions and lesion cells.

[0043] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A cell preparation that targets bone metastatic tumor cells, characterized in that: The formulation includes senescent neutrophils and drug-loaded cationic liposomes internalized by the senescent neutrophils; the drug-loaded cationic liposomes are encapsulated with a STING agonist and a pan-aurora kinase inhibitor.

2. The cell preparation targeting bone metastasis tumor cells according to claim 1, characterized in that: The STING agonist is Vadimezan, cGAMP, ADU-S100, MSA-2, diABZI, SR-717, or MK-1454; the pan-photokinase inhibitor is Danusertib, Barasertib, Tozasertib, AMG 900, or CCT137690.

3. The cell preparation targeting bone metastasis tumor cells according to claim 1, characterized in that: The mass ratio of the STING agonist to the pan-polar light kinase inhibitor is 8-10:1-2.

4. The cell preparation targeting bone metastatic tumor cells according to claim 1, characterized in that: The drug-loaded cationic liposomes include cationic lipids, phospholipids, and cholesterol; the cationic lipids are DOTAP, DODAP, DODMA, SM-102, or ALC-0315; the phospholipids are DOPE, DOPC, DPPC, DSPC, DSPE, or DPPG.

5. The cell preparation for targeting bone metastatic tumor cells according to claim 4, characterized in that: The mass ratio of cationic lipids, phospholipids, and cholesterol to cholesterol is 0.5~1.5:14~16:3~5.

6. A method for preparing a cell preparation with targeting bone metastasis tumor cells as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1: Preparation of drug-loaded cationic liposomes encapsulated with STING agonist and panoptic kinase inhibitor; S2: Neutrophils were extracted and isolated, and senescent neutrophils were obtained through in vitro culture; S3: Co-incubate the drug-loaded cationic liposomes with the senescent neutrophils to allow the senescent neutrophils to internalize the drug-loaded cationic liposomes, thus obtaining the final product.

7. The preparation method according to claim 6, characterized in that: In step S1, the drug-loaded cationic liposomes are prepared using either thin-film dispersion-ultrasound or microfluidic-solvent injection.

8. The preparation method according to claim 6, characterized in that: The total incubation time in step S3 is 0.5~1.5h.

9. The use of the cell preparation having targeted bone metastasis tumor cells as described in any one of claims 1 to 5 in the preparation of a medicament for treating bone metastasis of cancer.

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