Lipid polymer complex, preparation method and application in treating tumor bone metastasis

Abstract

The present invention discloses a lipid polymer complex, a preparation method and an application in treating tumor bone metastasis, which belongs to the field of biomedicine technology, and particularly relates to a lipid polymer complex, including an oxidation-responsive polymer STING agonist (OPSA) and a GSDMB nucleic acid drug. The preparation method comprises: OPSA is complexed with a GSDMB nucleic acid drug through electrostatic interaction to form a nanocomplex of OPSA and GSDMB nucleic acid drug; a layer of liposome membrane is coated on the surface to obtain a lipid polymer complex LipoNCs. The present invention proposes to construct a lipid polymer complex LipoNCs based on an oxidation-responsive polymer STING agonist (OPSA) to achieve efficient in vivo delivery of a GSDMB nucleic acid drug for the synergistic treatment of tumor bone metastasis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and in particular relates to a lipid polymer complex and a preparation method thereof and an application thereof in treating tumor bone metastasis. Background Art

[0002] Bone is one of the common metastatic sites of malignant tumors. The occurrence of bone metastasis involves a series of bone-related events (SREs), such as the spread and proliferation of tumor cells in the bone microenvironment, osteolysis, pathological fractures, and bone cancer pain, which greatly affect the patient's prognosis and quality of life. In addition, tumor bone metastasis not only manifests as a rapid onset process, but also can use the dormant state of tumor cells to escape the killing of the immune system. These dormant tumor cells can be reactivated under certain stimuli, leading to cancer recurrence. Therefore, tumor bone metastasis is a malignant disease with high diagnostic difficulty, hidden onset, complex clinical manifestations, and extremely poor prognosis. The cell interaction in the complex metastatic microenvironment of the bone is one of the most unique and complex characteristics of tumor bone metastasis compared to primary malignant tumors, and is also one of the fundamental reasons why tumor bone metastasis is difficult to treat. In this process, while tumor cells migrate to bone tissue and proliferate, they have continuous and complex interactions with resident cells in the bone, including: tumor cells destroy bone matrix by activating osteoclasts and release a large number of growth factors from it to provide nutritional conditions for tumor proliferation; tumor cells can recruit and manipulate peripheral nerves to promote tumor metastasis and bone cancer pain.

[0003] Tumor cells invade bone tissue and settle and proliferate therein, which is the root cause of a series of subsequent interactions. How to achieve efficient tumor killing is a top priority. For patients with tumor bone metastasis, activating the innate immune system in the body, identifying tumor cells and presenting them to the acquired immune system is an emerging strategy to improve the response to tumor immunotherapy. The stimulator of interferon genes (STING) located on the endoplasmic reticulum has a strong immune activation effect. Activation of the STING pathway can induce the secretion of type I interferon (IFN-I) and the production of a variety of pro-inflammatory factors and chemokines, thereby promoting the maturation and migration of dendritic cells (DCs), providing effective antigen presentation, thereby triggering spontaneous immune responses of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK), turning "cold" tumors into "hot" tumors, thereby inhibiting tumor growth and increasing the sensitivity of tumors to immune checkpoint blockade (ICB) therapy. It is a star target in immunotherapy. However, in clinical studies, small molecule STING agonists have the defects of rapid metabolism in the body and poor tumor specificity, which are the main obstacles to their safe and effective clinical application. Developing high-molecular-weight STING agonists to efficiently target STING protein, activate the host's immune system, and achieve long-lasting immune activation effects different from small-molecule agonists is a feasible approach.

[0004] Synergistic tumor immunotherapy can enhance the immunogenicity of tumors by integrating multiple therapeutic methods, thereby promoting the immune system's attack on tumors. Pyroptosis is an efficient and rapid way of programmed cell death. It mainly mediates the activation of multiple caspases including caspase-1 through inflammasomes, causing shearing and polymerization of gasdermin (GSDM) family members, forming cell membrane perforation, accompanied by changes in osmotic pressure, loss of membrane potential and cell swelling, and ultimately leading to cell membrane rupture and release of cell contents. Compared with apoptosis and necrosis, pyroptosis occurs faster and is an active and controlled cell death method. It can directly cause tumor cell death by perforating the cell membrane until it ruptures. At the same time, in this process, in addition to the release of a large number of pro-inflammatory factors such as IL-1β and IL-18, intracellular antigens such as RNA, DNA, proteins and lipids are also released to the extracellular space, stimulating various immune cells as danger-associated pattern molecules (DAMPs) and pathogen-associated pattern molecules (PAMPs), stimulating tumor immunogenicity and triggering a strong anti-tumor immune response. It not only solves the problem of negative immune regulation of STING signaling, but also provides a means of efficient tumor killing. Experiments have shown that inducing pyroptosis in only 15% of tumor cells can eliminate the entire breast tumor (4T1) transplant in a mouse model. GSDM family proteins are like "little bombs" that positively amplify the anti-tumor immune response, leading to a significant increase in CTLs in the tumor microenvironment, showing its huge killing potential. However, the efficient killing of pyroptosis also means its hidden dangers in safety. How to accurately "detonate" these "little bombs" at the tumor site while avoiding attacks on non-target organs is a prerequisite for safe and effective synergistic treatment. There is a special protein Gasdermin B (GSDMB) in the GSDM family. Unlike other GSDM family proteins, it does not activate Caspase through non-specific inflammatory signals to perform cleavage to perform the punching function, but relies on perforin and granzyme A (GZMA) released by CTLs and NK cells after recognizing tumor cells to achieve subsequent pyroptosis. From the mechanism of action, it avoids the killing of non-target organs and is more suitable for patients with high organ load in bone metastasis. However, in most tumor cells, the expression level of GSDMB is usually low, especially 4T1 cells are found to hardly express GSDMB. Using the immune activation effect of STING to coordinate tumor cells to overexpress GSDMB to induce pyroptosis may become a new idea to enhance tumor immunity and achieve precise and efficient tumor killing. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a lipid polymer complex and a preparation method and an application in the treatment of tumor bone metastasis, so as to achieve efficient in vivo delivery of GSDMB nucleic acid drugs for the synergistic treatment of tumor bone metastasis, starting from the tumor-nerve bidirectional approach, and treating tumor bone metastasis by regulating the tumor-nerve interaction loop.

[0006] To achieve the above-mentioned object, the present invention provides a lipid polymer complex, including an oxidation-responsive polymer STING agonist OPSA and a GSDMB nucleic acid drug.

[0007] Preferably, the lipid polymer complex LipoNCs is prepared by encapsulating a liposome membrane with a nanocomplex of OPSA and GSDMB nucleic acid drugs.

[0008] The present invention also provides a method for preparing the lipid polymer complex, comprising the following steps:

[0009] (1) OPSA complexes with GSDMB nucleic acid drugs through electrostatic interaction to form a nanocomplex of OPSA and GSDMB nucleic acid drugs;

[0010] (2) The surface of the nanocomplex of OPSA and GSDMB nucleic acid drugs obtained in step (1) is coated with a layer of liposome membrane to obtain lipid polymer complex LipoNCs.

[0011] Preferably, the OPSA in step (1) is represented by the following formula I:

[0012] ;

[0013] In formula I, n is an integer from 10 to 200; X is NH or O; and R1 is a methyl group or a hydrogen atom.

[0014] Preferably, the mass ratio of the nanocomplex of OPSA and GSDMB nucleic acid drugs to the liposome membrane in step (2) is 1:240, and the liposome membrane in step (2) is composed of 1,2-dioleoylphosphatidylethanolamine DOPE, cholesterol succinate CHEMS and distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 DSPE-PEG.

[0015] Preferably, the liposome membrane in step (2) is composed of DOPE, CHEMS and DSPE-PEG in a molar ratio of 7.65:2:1.35.

[0016] The present invention also provides the use of the lipid polymer complex or the lipid polymer complex prepared by the preparation method in preparing a drug for treating tumor bone metastasis.

[0017] Preferably, the lipid polymer complex uses tumor-specific highly expressed ROS to trigger dissociation to release a high-molecular-weight STING agonist and a GSDMB nucleic acid drug. On the one hand, the high-molecular-weight STING agonist activates tumor innate immunity, promotes the maturation of DC cells, and then induces CTLs to kill tumor cells. The GZMA secreted by CTLs, with the auxiliary perforation of perforin and LipoNCs, effectively enters tumor cells to cut GSDMB and induce cell pyroptosis. The inflammatory factors, chemokines and DAMPs produced thereby further enhance the immunogenicity of the tumor and promote anti-tumor immune response. On the other hand, the high-molecular-weight STING agonist activates the IFN-I signaling pathway to act on the sensory nerves around the tumor, inhibits the pathological changes of the nerves, and treats tumor bone metastasis by regulating the tumor-nerve interaction loop.

[0018] The present invention also provides a medicine for treating tumor bone metastasis, comprising the lipid polymer complex or the lipid polymer complex prepared by the preparation method.

[0019] The present invention also provides a medicine for treating tumor bone metastasis, comprising the lipid polymer complex prepared by the preparation method.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention proposes to construct a lipid polymer complex LipoNCs based on an oxidation-responsive polymer STING agonist (OPSA) to achieve efficient in vivo delivery of GSDMB nucleic acid drugs for the synergistic treatment of tumor bone metastasis. Nerves are an important "accomplice" of tumor growth. The interaction between nerves and tumor cells is an important cause of promoting the growth and invasion of tumor cells in bone tissue and causing bone cancer pain. Studies have found that tumor cells stimulate and sensitize peripheral nerve endings by secreting inflammatory factors (such as TNF-α, IL-1β, IL-6), nerve growth factors (such as NGF) and neurotransmitters (such as glutamate), promoting pathological growth of nerve axons, thereby producing irreversible pathological changes. Even if the tumor is controlled or completely removed, the patient will still suffer from persistent pain; at the same time, peripheral neurons release neuropeptides (such as substance P) and growth factors (such as brain-derived neurotrophic factor, BDNF), which in turn promote tumor growth and invasion, providing conditions for subsequent tumor recurrence, forming a mutually reinforcing closed loop. Therefore, neural intervention is another indispensable element for regulating tumor-nerve interaction and comprehensive treatment of tumor bone metastasis and bone cancer pain. Activation of the STING pathway can effectively inhibit bone cancer pain by inducing IFN-I production and inhibiting the excitability of peripheral sensory neurons. In addition, cutting off tumor-nerve interaction can have a significant negative regulatory effect on tumor proliferation, providing a way to activate the STING pathway to solve the problem of tumor-nerve interaction.

[0022] The system described in the present invention can deliver drugs to tumor sites through blood circulation, and utilizes tumor-specific highly expressed ROS to trigger LipoNCs dissociation, releasing high-molecular-weight STING agonists and GSDMB nucleic acid drugs to express GSDMB; at the same time, the high-molecular-weight STING agonists, on the one hand, activate tumor inherent immunity, reshape immunosuppressive "cold" tumors into "hot" tumors infiltrated with immune effector cells, and promote CTLs to kill tumor cells. The GZMA secreted by them effectively enters tumor cells and cuts GSDMB to induce cell pyroptosis under the auxiliary perforation of perforin and LipoNCs, and the inflammatory factors, chemokines and DAMPs produced thereby further enhance the immunogenicity of the tumor and promote anti-tumor immune response; on the other hand, the high-molecular-weight STING agonists activate the IFN-I signaling pathway to act on the sensory nerves around the tumor, inhibit the pathological changes of the nerves, and treat tumor bone metastasis by regulating the tumor-nerve interaction loop from the perspective of tumor-nerve bidirectionality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 is the OPSA in Example 1 1 H-NMR spectrum, where a~k represent H at different positions;

[0025] Figure 2 is the OPSA under oxidative conditions in Example 1 1 H-NMR spectrum, where a~b and a'~b' represent H at different positions;

[0026] Figure 3 The particle size distribution and Zeta potential diagram and gel retardation electrophoresis diagram of OPSA NCs with different N / P ratios in Example 1, wherein a is the particle size distribution and Zeta potential diagram, and b is the gel retardation electrophoresis diagram;

[0027] Figure 4 Figures 1 and 2 are transmission electron microscopy images, particle size changes, and gel retardation electrophoresis images of OPSA NCs under oxidative conditions in Example 1, wherein a is a transmission electron microscopy image of OPSA NCs, with a scale of 100 nm, b is a transmission electron microscopy image of OPSA NCs after H2O2 treatment, with a scale of 100 nm, c is a particle size change image, and d is a gel retardation electrophoresis image;

[0028] Figure 5 The transfection effect, cytotoxicity and effect of oxidation conditions on transfection efficiency of OPSA NCs in 4T1 cells in Example 1, wherein a is the transfection effect, b is the cytotoxicity, and c is the effect of oxidation conditions on transfection efficiency;

[0029] Figure 6 is the activation effect of OPSA NCs on the STING pathway of 4T1 cells in Example 1, wherein a is the immunoblotting result of STING pathway-related proteins, b is the expression of CXCL10 gene, c is the expression of IFNB1 gene, and d is the content of IFN-β in the supernatant of cell culture medium;

[0030] Figure 7 The results of OPSA NCs inducing cell pyroptosis in Example 1, wherein a is the immunoblotting result of PEI NCs and OPSA NCs in 4T1 cells transfected with GSDMB, b is the AnnxinV / PI staining result of 4T1 cells electroporated and delivered with GZMA induced pyroptosis after untreated, OPSA, NCs@pVector, and NCs@pGSDMB treatment, the scale is 10 μm, c is the release ratio of LDH in the supernatant of the culture medium of each group of 4T1 cells after induced pyroptosis, d is the concentration of ATP in the supernatant of the culture medium of each group of 4T1 cells after induced pyroptosis, and e is the immunoblotting result of the full-length GSDMB protein and its cleavage fragments in each group of 4T1 cells after induced pyroptosis;

[0031] Figure 8 The particle size distribution and cryo-EM image of Lipo NCs in Example 1, wherein a is the particle size distribution and b is the cryo-EM image;

[0032] Fig. 9 In Example 1, the phenomenon and mechanism of LipoNCs inducing cell perforation by reducing cell membrane fluidity, wherein a is untreated, treated PEI LipoNCs, OPSA Scanning electron microscopy results of the 4T1 cell surface after LipoNCs treatment, the scale is 500nm, b is the statistical result of the number of pores on the surface of 4T1 cells in each group, c is the statistical result of the diameter of the pores on the surface of 4T1 cells in each group, d is the statistical result of the diameter of the pores on the surface of 4T1 cells in each group, and PEI LipoNCs, OPSA Fluorescence images of 4T1 cells treated with LipoNCs and FITC dye, the scale bar is 150 μm, e is untreated, PEI LipoNCs, OPSA Flow cytometry analysis results of 4T1 cells treated with LipoNCs and then added with FITC dye. f is the statistical results of the fluorescence intensity of 4T1 cells in each group, g is the statistical results of the fluorescence intensity of 4T1 cells without treatment and after treatment. PEILipoNCs, OPSA The images of fluorescence bleaching of 4T1 cells incubated with LipoNCs. The circled area represents the fluorescence bleaching site. The scale bar is 10 μm. h is the statistical result of the normalized fluorescence intensity during the fluorescence bleaching experiment of 4T1 cells in each group.

[0033] Fig.10 is the result of LipoNCs inducing cell pyroptosis by perforation in Example 1, wherein a is 4T1 cells overexpressing GSDMB without treatment or by PEI LipoNCs, OPSA a The bright field image of GZMA delivered after LipoNCs treatment, the scale bar is 100 μm, and the scale bar of the magnified image is 40 μm, b is the immunoblotting results of the full-length GSDMB protein and its cleavage fragments in the above groups;

[0034] Fig.11 The in vivo distribution of LipoNCs labeled with DiR in mice with bone tumors in Example 1, wherein a is the in vivo imaging results of various organs and tumor tissues of mice 8h and 48h after injection, and b is the statistical results of the fluorescence intensity of various organs and tumor tissues 8h and 48h after injection;

[0035] Fig.12 The immune activation effect of LipoNCs on mice with tumor bone metastasis in Example 1, wherein a is the number of CD4 + T cell ratio, b is the CD8 + The proportion of T cells, c is the proportion of mature DC cells in the tumor-draining lymph nodes of mice in each group, d is the proportion of effective memory T cells (TEM) in the spleen of mice in each group, e is the proportion of central memory T cells (TCM) in the spleen of mice in each group, f is the proportion of regulatory T cells (Tregs) in the tumor tissue of mice in each group, and g is the proportion of cytotoxic T cells (CTLs) in the tumor tissue of mice in each group;

[0036] Fig.13 The anti-tumor effect of LipoNCs on mice with bone metastasis in Example 1, wherein a is the in vivo imaging image results of mice in each group injected with PBS, LipoNCs@pVector, and LipoNCs@pGSDMB at different time points, b is the in vivo imaging fluorescence statistical results of mice in each group at different time points, c is the tumor size of the tumor-bearing legs of mice in each group, and d is the weight of the tumor tissue of mice in each group;

[0037] Fig.14The protective effect of LipoNCs on bone metastasis of mice in Example 1, wherein a is the Micro-CT result of the bone tissue on the tumor-bearing side of the mice injected with PBS, LipoNCs@pVector, and LipoNCs@pGSDMB, b is the statistical result of the bone volume fraction, c is the statistical result of the bone density, d is the statistical result of the number of trabeculae, and e is the statistical result of the spacing between trabeculae;

[0038] Fig.15 The results of LipoNCs in Example 1 are as follows: antagonizing the abnormality of peripheral nerves in tumors of mice with bone metastasis; wherein, a is the fluorescence image of nerve tracing in tumors of mice injected with PBS, LipoNCs@pVector, and LipoNCs@pGSDMB; b is the Tuj1 fluorescence staining image of dorsal root ganglion cells of innervated parts derived from mice in each group in vitro; c is the statistical result of the axon length of L1-L4 dorsal root ganglion cells derived from mice in each group; and d is the statistical result of the number of branches of L1-L4 dorsal root ganglion cells derived from mice in each group;

[0039] Fig.16 The results are as follows: Figure 1 shows the effect of LipoNCs in antagonizing the abnormal excitability of tumor peripheral nerves in mice with bone metastasis and inhibiting bone cancer pain, wherein a is the calcium imaging fluorescence image of L1-L4 dorsal root ganglion cells from the sham operation group and the mice injected with PBS, LipoNCs@pVector, and LipoNCs@pGSDMB under potassium chloride stimulation, b is the statistical result of the normalized calcium imaging fluorescence, c is the statistical result of the acetone cold pain reaction time of mice in each group, d is the statistical result of the pain threshold of the Von Frey acupuncture experiment of mice in each group, e is the statistical result of the gait behavior of the tumor-bearing legs of mice in each group, f is the statistical result of the ground contact strength of the tumor-bearing legs of mice in each group, and g is the statistical result of the bottom contact time of the tumor-bearing legs of mice in each group. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1

[0046] (1) Synthesis of OPSA carrier

[0047] N-(2-Hydroxyethyl)hexamethylenediamine (2.00 g, 13.96 mmol) and triethylamine (2.12 g, 20.94 mmol) were dissolved in anhydrous dichloroethane (50 mL), placed in a reaction flask and cooled in an ice bath. Methacryloyl chloride (1.75 g, 16.75 mmol) dissolved in dichloroethane (20 mL) was added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was gradually warmed to room temperature and stirred overnight to ensure complete reaction. The reaction mixture was washed with water, saturated sodium bicarbonate solution and saturated brine in sequence. The organic layer was separated and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary evaporator. The crude product was purified by vacuum distillation to obtain N-(2-hydroxyethyl)hexamethylenediamine methacrylate with a yield of 76%. N-(2-hydroxyethyl)hexamethylenediamine acrylate can be prepared by replacing methacryloyl chloride with acryloyl chloride. N-(2-aminoethyl)hexamethylenediamine can be substituted for N-(2-hydroxyethyl)hexamethylenediamine to obtain N-(2-aminoethyl)hexamethylenediamine (meth)acrylamide.

[0048] 4-Cyano-4-(phenylthiocarbonylthio)pentanoic acid (9.45 mg, 0.034 mmol), N-(2-hydroxyethyl)hexamethylenediamine methacrylate (0.50 g, 2.37 mmol) and azobisisobutyronitrile (1.67 mg, 0.010 mmol) were dissolved in dioxane (2 mL). The mixture was degassed by three freeze-pump-thaw cycles and sealed under argon atmosphere. The sealed tube was immersed in an oil bath preheated to 75 °C and stirred for 12 h to complete the polymerization reaction. The polymerization reaction was quenched in liquid nitrogen, and the product was precipitated in hexane three times, centrifuged and dried under vacuum to obtain poly(N-(2-hydroxyethyl)hexamethylenediamine methacrylate) with a yield of 70%. Poly(N-(2-hydroxyethyl)hexamethylenediamine acrylate) and poly(N-(2-aminoethyl)hexamethylenediamine (meth)acrylamide) were obtained using the same method.

[0049] Poly(N-(2-hydroxyethyl)hexamethylenediamine methacrylate) (0.2 g, 0.95 mmol tertiary amine) was dissolved in DMF (10 mL), and 4-(bromomethyl)phenylboronic acid (0.41 g, 1.89 mmol) was then added to the solution. The reaction mixture was stirred at room temperature for 24 h. The resulting solution was poured into tetrahydrofuran to precipitate the product. The precipitate was collected, washed with tetrahydrofuran, and dried under vacuum. The crude product was dissolved in water and dialyzed overnight in deionized water using a dialysis membrane with a molecular weight cutoff of 3500 KDa. The purified product was freeze-dried to obtain OPSA as a white solid (yield: 68%). The same method can be used to obtain quaternized poly(N-(2-hydroxyethyl)hexamethylenediamine acrylate) and poly(N-(2-aminoethyl)hexamethylenediamine (meth)acrylamide).

[0050] The reaction formula is as follows:

[0051] .

[0052] In order to preliminarily verify that the oxidation-responsive polymer STING agonist can be used as an efficient delivery carrier for nucleic acid drugs, an oxidation-responsive cationic polymer was first synthesized. 1 H-NMR Figure 1 As shown. Boric acid is oxidized by H2O2, followed by a 1,4-elimination reaction, and the quaternary ammonium salt quickly turns into a tertiary amine, while the generated quinone quickly turns into p-hydroxybenzyl alcohol in water. 1 H-NMR characterizes the structural changes of OPSA under oxidative conditions. Figure 2 As shown, the peaks at a and b disappear and become a′ and b′, ​​proving that an oxidation response occurs and the quaternary ammonium salt becomes a tertiary amine.

[0053] (2) Preparation of OPSA NCs

[0054] Positively charged OPSA can form stable nanocomplexes OPSA NCs with negatively charged GSDMB nucleic acid drugs through electrostatic self-assembly. First, 8.1 mg of OPSA was dissolved in 500 μL of DMSO solution to prepare OPSA stock solution. Then, HEPES buffer solution (pH 7.4, 10 mM) was used to dilute OPSA solutions with concentrations of 270, 540, 810, 1080, 1350, and 1620 μg / mL. GSDMB plasmid (pGSDMB) was selected as the nucleic acid drug, and pGSDMB was diluted to a concentration of 40 μg / mL with HEPES buffer solution (pH 7.4, 10 mM). Then, OPSA solution and nucleic acid drug solution were mixed in a volume ratio of 1:1, vortexed for 15 s, and allowed to stand at room temperature for 30 min. OPSA and nucleic acid drugs were electrostatically self-assembled to obtain a series of stable nanocomplexes OPSA NCs with N / P ratios (5, 10, 15, 20, 25, and 30). The particle size of OPSA NCs can be compressed to 40~60nm, and the Zeta potential is distributed between 10~16mV (e.g. Figure 3 (a). Gel retardation electrophoresis results (such as Figure 3 (b) shows that the nanocomposite OPSA NCs can effectively block the migration of GSDMB nucleic acid drugs, proving that GSDMB nucleic acid drugs can be stably encapsulated in the nanocomposite. TEM observation (such as Figure 4 In (a), OPSA NCs with N / P=15 appear as spherical nanoparticles with regular and uniform morphology and a particle size of 40 nm, which is basically consistent with the results measured by DLS.

[0055] In order to further verify the oxidation response characteristics of the system, the nanocomposite OPSA NCs with N / P = 15 were incubated at 37 ° C for 1 h under the action of 0.1 mM H2O2. The looseness of the nanocomposite OPSA NCs was different in morphology (e.g. Figure 4 b) and particle size changes (e.g. Figure 4This is fully reflected in (c), where OPSA NCs are dispersed and irregular, and the particle size is significantly larger, proving that OPSA NCs have undergone significant dissociation. In order to prove the successful release of nucleic acid drugs from OPSA NCs, subsequent electrophoresis gel experiments were performed. 10 μL of untreated pGSDMB and 10 μL of pGSDMB incubated at 37°C for 1 h under the action of 200 μM H2O2 were added to the agarose gel as controls, and then 20 μL of OPSA NCs incubated at 37°C for 1 h under the action of 0, 5, 10, 20, 40, 100, and 200 μM H2O2 were added. Gel electrophoresis was performed at a voltage of 140 V for 30 minutes, and the agarose gel after electrophoresis was imaged in a UV gel imaging analyzer. The results of gel retardation electrophoresis showed that the nanocomplex can quickly and effectively release GSDMB nucleic acid drugs (such as Figure 4 (d)

[0056] (3) Study on the behavior and mechanism of OPSA NCs in activating STING and inducing cell pyroptosis

[0057] i. Gene transfection efficiency of OPSA NCs: The transfection effect of OPSA NCs with different nitrogen-phosphorus ratios in 4T1 cells was studied by transfection with luciferase plasmid (pLuc). First, 4T1 cells were plated at 1.5×10 4 4T1 cells were inoculated in 96-well plates and incubated for 24 hours in a 37°C, 5% CO2 incubator environment with DMEM medium containing 10% fetal bovine serum (FBS). After 24 hours, the serum-containing medium in the 96-well plate was discarded and replaced with 200 μL serum-free DMEM medium, and OPSA NCs with a series of N / P ratios (5, 10, 15, 20, 25, 30) containing 0.5 μg pLuc were added to the medium and incubated for 4 hours. After 4 hours, the medium containing OPSA NCs was discarded and replaced with DMEM medium containing 10% FBS, and the culture was continued for 44 hours. After 44 hours, the cell lysis buffer was added to lyse the 4T1 cells, and the chemiluminescence intensity was detected by a chemiluminometer. The protein concentration of the lysate was determined using a BCA detection kit. The chemiluminescence intensity was normalized by the protein concentration, and its unit was the luminescence intensity per milligram protein RLU / mgprotein.

[0058] Luciferase reporter assay results Figure 5 As shown in Figure a, compared with the positive control group PEI, the transfection efficiency of OPSA NCs at N / P = 15 can be increased by 4 orders of magnitude, while having lower cytotoxicity (e.g. Figure 5 b), so the nanocomposite with N / P=15 was selected for subsequent research.

[0059] To further demonstrate the effect of intracellular ROS on OPSA NCs transfection, different concentrations of H2O2 were added to the culture medium to increase the intracellular ROS concentration. 4T1 cells were cultured at 1.5×10 4 The cells were inoculated in 96-well plates and incubated for 24 hours in a 37°C, 5% CO2 incubator environment with DMEM medium containing 10% FBS. After 24 hours, the serum-containing medium in the 96-well plate was discarded and replaced with 200 μL serum-free DMEM medium, and OPSA NCs with N / P=15 containing 0.1 μg pLuc were added to the serum-free DMEM medium and incubated for 4 hours. After 4 hours, the medium containing OPSA NCs was discarded and replaced with DMEM medium containing 10% FBS and 0, 10, 50, 100, 200 μM H2O2, and the culture was continued for 44 hours. After 44 hours, the cell lysis solution was added to lyse the 4T1 cells cultured under different concentrations of H2O2, and the chemiluminescence intensity was detected by a chemiluminometer. The protein concentration of the lysate was determined using a BCA detection kit. The chemiluminescence intensity was normalized by the protein concentration, and its unit was the luminescence intensity per milligram of protein RLU / mg protein.

[0060] The results showed that the luciferase gene transfection efficiency of cells treated with H2O2 increased by 1 order of magnitude without affecting cell growth (100 μM H2O2). Figure 5 (c) shows that the highly reactive oxygen environment of tumor cells will promote the release of GSDMB nucleic acid drugs and achieve more efficient transfection.

[0061] ii. Study on the activation of STING by OPSA NCs: OPSA NCs will undergo oxidative response in the highly reactive oxygen environment of tumor cells, releasing polymer-type PC7A that binds to the STING protein on the endoplasmic reticulum and undergoes conformational changes. 4T1 cells were plated at 3×10 54T1 cells were inoculated in 6-well plates and incubated for 24 hours in a 37°C, 5% CO2 incubator environment with DMEM medium containing 10% FBS. Then, OPSA NCs with N / P=15 were obtained in the same manner as above. After 4T1 cells were incubated for 24 hours, the serum-containing DMEM medium in the 6-well plate was discarded, and the medium in the 6-well plate was replaced with 2 mL of serum-free DMEM. The following reagents were added: OPSA NCs with N / P=15 containing 4 μg of blank plasmid (pVector), OPSA NCs with N / P=15 containing 4 μg of GSDMB plasmid (pGSDMB), and OPSA solution without plasmid but with the same polymer content, and incubated for 4 hours. After 4 hours, the medium was discarded and replaced with DMEM medium containing 10% FBS. After 44 hours, the cell culture supernatant was collected for subsequent ELISA detection. The cells were washed three times with phosphate buffered saline (PBS), and then lysed with RIPA lysis buffer or Trizol lysis buffer to extract the corresponding cell proteins / RNA. The above collected protein / RNA / medium supernatant samples were detected by WB, qPCR, and ELISA to detect STING pathway-related proteins (STING, TBK1, IRF3 and its phosphorylated proteins pSTING, pTBK1, pIRF3) (such as Figure 6 a), genes (IFNB1, CXCL10), and cytokines (IFN-β) (e.g. Figure 6 In b, such as Figure 6 In c and Figure 6 The expression of OPSA NCs in 4T1 cells was demonstrated to activate the STING pathway.

[0062] iii. Study on OPSA NCs-induced cell pyroptosis: 4T1 cells hardly express GSDMB. In order to further explore whether DNA transfection can induce cell pyroptosis, WB was used to detect the expression of GSDMB after OPSA@pGSDMB transfection. First, 4T1 cells were plated at 3×10 54T1 cells were inoculated in 6-well plates and incubated for 24 hours in a 37°C, 5% CO2 incubator environment with DMEM medium containing 10% FBS. Then, OPSA NCs with N / P=15 were obtained in the same way as above. After 4T1 cells were incubated for 24 hours, the serum-containing DMEM medium in the 6-well plates was discarded, and the medium in the 6-well plates was replaced with 2 mL of serum-free DMEM, and then divided into a control group and a treatment group. No other reagents were added to the control group. The following reagents were added to the treatment groups: PEI NCs with N / P=7 containing 4 μg of GSDMB plasmid (pGSDMB), and OPSA NCs with N / P=15 containing 4 μg of GSDMB plasmid (pGSDMB), and incubated for 4 hours. After 4 hours, the medium containing NCs was discarded and replaced with DMEM medium containing 10% FBS. After 44 hours, the cells in each group were digested with 0.25% trypsin, centrifuged after FBS digestion, and resuspended in PBS. After resuspending, 10 mg of GZMA protein was added to every 1 million cells of 4T1 cells, and electroporation was performed by an electroporator under the conditions of 300 V and 20 ms pulses. The electroporated 4T1 cells were resuspended in DMEM medium containing 10% FBS and inoculated in a 6-well plate. After incubation for 6 hours, each group of 4T1 cells was photographed under a bright field microscope, and the cell supernatant was collected. RIPA lysis buffer was subsequently added to collect the 4T1 cell proteins of each group, and the protein expression was detected by WB experiment.

[0063] The results showed that OPSA@pGSDMB could efficiently express GSDMB in 4T1 cells, which was much higher than that of PEI group (e.g. Figure 7 After GZMA was delivered by electroporation simulating CTLs, 4T1 cells transfected with GSDMB showed obvious cell swelling and expansion, forming a large number of vesicles (such as Figure 7 In b), the release of LDH and ATP in the cell culture supernatant increased significantly (e.g. Figure 7 Medium c and Figure 7 d), GSDMB was activated and cleaved, indicating that it induced cell pyroptosis (e.g. Figure 7 (e).

[0064] (4) Construction of LipoNCs and study of their in vivo and in vitro behavior

[0065] Construction of LipoNCs and evaluation of assisted pyroptosis effect and tumor targeting: First, liposomes were prepared according to the thin film hydration method. DOPE (11.06 mg), CHEMS (1.88 mg) and DSPE-PEG2000 (7.35 mg) were dissolved in 2 mL of chloroform, and then the organic solvent was removed by reduced pressure evaporation to form a uniform lipid film. The lipid film was hydrated with 1 mL of HEPES buffer (10 mM, pH 7.4) at room temperature overnight with slight stirring. To obtain a uniform suspension, the hydrated liposome solution was sonicated for 10 min under ice bath conditions. Subsequently, the obtained liposome solution was filtered through a 0.22 μm filter membrane to obtain the liposomes used in subsequent experiments. Then, OPSA NCs with N / P=15 were obtained by the same method as above. The obtained liposomes and OPSANCs were mixed in a volume ratio of 1:1, vortexed for 15 s, and assembled at room temperature overnight. When the mass ratio of liposomes to OPSA NCs was 240:1, surface PEGylated LipoNCs were successfully constructed with a particle size of approximately 110 nm and a Zeta potential of -3 mV (e.g. Figure 8 Cryo-TEM images show that LipoNCs are spherical nanoparticles, and the particle size is basically consistent with the results measured by DLS (e.g. Figure 8 (b)

[0066] Next, the perforation ability of LipoNCs and its corresponding assisted pyroptosis effect were investigated. FITC is a hydrophilic dye that cannot penetrate the cell membrane. It is usually difficult to internalize into cells and will only enter cells when holes are formed on the cell surface. 4T1 cells were plated at 1.5×10 5 4T1 cells were inoculated on a 6-well plate and incubated for 24 hours in a 37°C, 5% CO2 incubator with DMEM medium containing 10% FBS. After 4T1 cells were incubated for 24 hours, the serum-containing DMEM medium in the 6-well plate was discarded, and the medium in the 6-well plate was replaced with 2 mL of serum-free DMEM. The cells were divided into a control group and a treatment group. No other reagents were added to the control group. The following reagents were added to the treatment groups: N / P=7 containing 4 μg of GSDMB plasmid (pGSDMB) PEI LipoNCs, containing 4 μg of GSDMB plasmid (pGSDMB) with N / P=15 OPSA LipoNCs, cells were incubated at 37°C, 5% CO2 incubator for 7 h. 4T1 cells treated as above were subsequently subjected to transmission electron microscopy, flow cytometry or fluorescence photobleaching assay (FRAP).

[0067] For transmission electron microscopy, the 4T1 cells in each group were not digested. After washing with PBS three times, they were fixed with 2.5% pentanediol (pH 7.4) at 4°C overnight in a flat state on a cell slide. The fixed cells were washed with PBS three times, dehydrated with gradient ethanol, dried in a Hitachi HCP-2 dryer, and sprayed with gold. Finally, the prepared samples were imaged with a scanning electron microscope to observe the pore size on the cell surface. The results showed that 4T1 cells were exposed to LipoNCs containing OPSA NCs ( OPSA After LipoNCs treatment, a large number of pores with a diameter of about 50 nm will be generated on the cell membrane surface (such as Fig. 9 Middle a, Fig. 9 Medium b and Fig. 9 c). For flow cytometry experiments, after incubation for 7 hours, 1 μL of FITC solution (1 mg / mL) was added to the cell culture medium and incubated for another 1 hour. After 1 hour, the cells were digested with 0.25% trypsin and digested with FBS. After resuspending in PBS, the fluorescence intensity was detected by flow cytometry to determine whether FITC in the culture medium entered the cells through the holes on the cell surface.

[0068] The results showed that compared with LipoNCs containing PEI NCs ( PEI Compared with cells with LipoNCs and cells without LipoNCs, OPSA LipoNCs have significantly enhanced fluorescence signals (e.g. Fig. 9 Middle d, Fig. 9 Zhongehe Fig. 9 f), confirmed OPSA The LipoNCs delivery system has the ability to penetrate the pores. OPSA The mechanism of perforation of LipoNCs was preliminarily studied. For the FRAP experiment, after the cells were incubated for 8 hours, the cell culture medium was discarded, and the cell membrane was marked by staining with the cell membrane dye NBD-C6-HPC at room temperature for 15 minutes, and then the culture medium was replaced with 10% FBS DMEM. The edge of the stained 4T1 cell membrane was selected and continuously irradiated with a 488nm laser at 100% intensity (25mW) through a confocal microscope to quench the fluorescence of the cell membrane in the selected area. The cell membrane has fluidity, and the fluorescent dyes in other areas will restore the fluorescence of the area due to the flow. The membrane fluidity of each group of cells was calculated by observing and recording the fluorescence recovery of the selected area, and normalizing the change of fluorescence intensity over time. The results show that compared with LipoNCs containing PEI NCs ( PEI Compared with cells with and without LipoNCs, OPSA LipoNCs significantly inhibited membrane fluidity (e.g. Fig. 9 Medium g and Fig. 9 h). OPSA The positive charge and hydrophobicity of OPSA encapsulated in LipoNCs suggest that it may bind to the cell membrane through charge attraction, reduce membrane fluidity and integrity, cause membrane stability to decrease, and ultimately lead to membrane rupture. The occurrence of pyroptosis depends on the effective delivery of GZMA into the cell by CTLs through perforin. OPSA Can LipoNCs assist GZMA to enter cells and promote pyroptosis by perforating? 5 4T1 cells overexpressing GSDMB were seeded in 6-well plates and incubated for 24 hours in a 37°C, 5% CO2 incubator with DMEM medium containing 10% FBS. After the 4T1 cells were incubated for 24 hours, the serum-containing DMEM medium in the 6-well plates was discarded, and the medium in the 6-well plates was replaced with 2 mL of serum-free DMEM, and then divided into a control group and a treatment group. Only 1 μg of GZMA protein was added to the control group. In addition to adding 1 μg of GZMA protein to the treatment group, the following reagents were added: N / P=7 containing 4 μg of GSDMB plasmid (pGSDMB) PEI LipoNCs, containing 4 μg of GSDMB plasmid (pGSDMB) with N / P=15 OPSA LipoNCs, cells were incubated at 37°C, 5% CO2 incubator for 8 h and then observed under bright field microscope, and then protein samples were collected for WB pyroptosis detection. The results showed that the addition of GZMA and OPSA LipoNCs can successfully induce the generation of pyroptotic bodies (e.g. Fig.10 (a) WB results also confirmed the cleavage of GSDMB protein by GZMA (e.g. Fig.10 (b) shows that the LipoNCs lipid polymer complex constructed by the present invention can induce pyroptosis by assisting perforation.

[0069] In order to verify the intrabone tumor targeting of LipoNCs, mice on the 7th day of intrabone tumor modeling were injected with fluorescently labeled LipoNCs through the tail vein. At 8h and 48h after injection, the mice were killed by carbon dioxide, and the heart, liver, spleen, lung, kidney, and tumor-bearing leg of the mice were isolated for in vitro imaging. The IVIS imaging results showed that the fluorescence signal of LipoNCs was significantly enriched in the tumor-bearing bone compared with the heart, liver, spleen, lung, and kidney (e.g. Fig.11 ), demonstrating that LipoNCs have the property of targeting intrabone tumors.

[0070] In order to demonstrate the in vivo immune activation effect of LipoNCs, a mouse intrabone tumor model was established using the 4T1-Luc tumor cell line. After the mice were anesthetized, the skin at the right knee of the mice was incised, the surface fascia and muscles were bluntly separated, the patellar ligament was exposed, and the position of the patellar ligament was moved to expose the medial and lateral femoral condyles. 5×10 4 4T1-Luc tumor cells were injected into the growth plate between the medial and lateral condyles of the femur, and then the patellar ligament was restored to its original position, and the muscle and skin were sutured layer by layer. Seven days after modeling, the mice with successful modeling were randomly divided into a control group (Control), a LipoNCs group carrying an empty plasmid (LipoNCs@pVector), and a LipoNCs group carrying pGSDMB (LipoNCs@pGSDMB). The DNA dose was 1 mg / kg, and the tail vein was administered every 1 day for a total of 5 times. The mice were euthanized on the 28th day, and then the spleen, inguinal lymph nodes on the tumor-bearing side, and tumor tissues of the mice were removed. The cells were fixed after grinding, and the various immune indicators were stained and marked by flow cytometry antibodies with fluorescent labels. Flow analysis was performed to evaluate the immune activation of mice in different treatment groups. The experimental results showed that LipoNCs@pVector can significantly increase the CD in the spleen of mice by activating the STING pathway. 4+ and CD 8+ The proportion of T cells, and GSDMB-mediated cell pyroptosis further enhanced the immune regulatory effect (such as Fig.12 A and Fig.12 In addition, it was demonstrated that LipoNCs@pGSDMB effectively promoted the maturation of dendritic cells (DCs) in the draining lymph nodes of the bone tumor site, creating conditions for the effective presentation of antigens (e.g. Fig.12 (c), LipoNCs@pGSDMB also promoted the formation of central memory T cells (TCM) and effector memory T cells (TEM) in the spleen of mice, providing long-lasting immune protection against tumor bone metastasis (e.g. Fig.12 Medium D and Fig.12 Furthermore, immune activation strategies were used to successfully reduce the infiltration of regulatory T cells (Treg) in tumor tissues (e.g. Fig.12 f), and promoted the accumulation of cytotoxic T cells (CTLs) to tumor sites (e.g. Fig.12 Middle g).

[0071] (5) Effect and safety study of LipoNCs in regulating the closed loop of tumor-nerve interaction

[0072] i. Anti-tumor effect and anti-bone destruction ability of LipoNCs: In order to explore the anti-tumor effect of LipoNCs, the intrabone tumor model of mice was established and grouped in the above manner. The mice in the Control group, LipoNCs@pVector group and LipoNCs@pGSDMB group were imaged every week. 15 mg / mL of luciferase substrate was intraperitoneally injected into each mouse at a dose of 150 mg / kg body weight, and fluorescence imaging was performed within 15 minutes. The luciferase signal intensity of the tumor site in the mouse was calculated. The mice were euthanized on day 28, and the complete tumor-bearing femur and tibia (including tumor and adhesion tissue) were taken, the surrounding skin and muscle tissue were peeled off, the tumor-bearing femur and tibia were exposed, and the tumor tissue was photographed and weighed, and the size of the intrabone tumor in each group was compared. The experimental results showed that the LipoNCs@pGSDMB group could significantly inhibit the growth of intrabone tumors, with a tumor inhibition rate of up to 94%, which was significantly higher than that of the LipoNCs@pVector treatment group (51%) (such as Fig.13 ). Tumor bone metastasis will seriously damage the bone in the corresponding part. Effective tumor suppression can reduce bone loss. After removing the tumor tissue from the tumor-bearing bone, the bone defect area of ​​the tumor-bearing part of the mouse was scanned and three-dimensionally reconstructed by Micro-CT (such as Fig.14 In a), the results showed that the bone volume fraction (BV / TV) of the LipoNCs@pGSDMB-treated group (e.g. Fig.14 b), bone mineral density (BMD) (such as Fig.14 c) and trabecular bone number (TB.N) (e.g. Fig.14 d) were significantly increased, and the trabecular separation (Th.Sp) (e.g. Fig.14 e) is reduced, and has significant anti-bone destruction ability.

[0073] ii. Neurointervention effect of LipoNCs: Bone cancer pain is one of the most obvious symptoms of tumor bone metastasis. Tumor cells metastasized to the bone will interact with the nerves around the tumor, leading to pathological occurrence and abnormal excitability of the nerves around the tumor. In order to explore the neurointervention effect of LipoNCs, the sensory nerves in the tumor were traced anterogradely. 21 days after modeling, the dorsal root ganglia of mice that innervated the corresponding segments of the tumor site were injected with nerve tracing dyes. Specifically, after the anesthetized mice separated the back skin with a scalpel, the muscles and fascia were bluntly separated to expose the dorsal root ganglion swellings at L1-L4. 10μL of 1mM concentration of DiI lipid dye was injected into the dorsal root ganglion swellings at the corresponding sites through a microsyringe. The injected lipid dye will flow to the innervated site, making the nerve tissue in the terminal tumor fluorescent. The mice were euthanized on day 28, and the tumor tissues were frozen and sectioned, and the degree of pathological nerve growth in the tumor was evaluated by fluorescence scanning. The experimental results show that LipoNCs@pGSDMB can significantly inhibit the growth of nerves into the tumor (such as Fig.15 In a), the dorsal root ganglia of the corresponding parts were cultured in vitro and then stained. The dorsal root ganglia of L1-L4 were separated after exposure in the above manner, and then digested with 2% type II collagenase for 1 hour. After FBS termination, they were ground with a 1mL syringe, filtered through a 40μm filter, and then cultured in vitro for 48 hours at 37°C and 5% CO2 incubator. After 48 hours, they were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and confocal photography was performed after Tuj1 antibody labeling and staining to observe the in vitro growth of dorsal root ganglion cells from each group of mice.

[0074] The results showed that the dorsal root ganglion cells cultured in vitro had a significant growth inhibition, and the length and branching of the axons were reduced (e.g. Fig.15 In b, such as Fig.15 Medium c and Fig.15 d), is the basis for preventing subsequent bone cancer pain. Bone cancer pain is not only due to abnormal growth of nerves toward the tumor, but also related to abnormal excitability of nerves. To this end, we separated the dorsal root ganglia of the corresponding segments for in vitro culture in the above manner. After 48 hours of in vitro culture, the dorsal root ganglion cells were labeled and stained with Fluo-4AM calcium ion probe, and then the excitability of the sensory nerves of mice was evaluated by calcium imaging technology. The fluorescence intensity of dorsal root ganglion cells was recorded every 5 seconds, and a 1mM high-concentration KCl solution was added at 10 seconds to stimulate neuronal excitation, and the recording was continued for a total of 600 seconds. The changes in the calcium ion fluorescence intensity of the dorsal root ganglion cells in each group were statistically analyzed by comparing the instantaneous fluorescence intensity with the initial fluorescence intensity to determine the excitability of the dorsal root ganglion cells in each group of mice.

[0075] The experimental results showed that under KCl stimulation, the calcium flow of mice in the LipoNCs@pGSDMB group increased less, and the time required to recover to the resting state was relatively faster, with lower excitability (such as Fig.16 A and Fig.16 b). Nerve excitability is specifically manifested as pain in animal experiments. Cold pain, mechanical pain, and Catwalk behavioral experiments were further conducted. Each group of mice was placed in a special animal experimental room. After 30 minutes of adaptation, acetone solution was evenly dripped onto the sole of the tumor-bearing side of the mouse. The acetone solution evaporated quickly, causing the temperature to drop. The mice licked their feet. The duration of the reaction to acetone cold pain in each group of mice was recorded to determine the sensitivity of the mice to cold pain. On the next day, after repeating the above adaptation process, the Vonfrey acupuncture experiment was performed on the sole of the tumor-bearing side of the mouse. When the mouse felt pain and had a foot retraction reaction, the instrument automatically recorded the peak pressure at that time as the stimulation threshold of the mouse Vonfrey experiment. The average value was taken after three repetitions to record the foot retraction threshold of each group of mice. On the next day, the mice were placed in a specific Catwalk gait analyzer to record the walking pattern. The instrument automatically measured and recorded the time, pressure, and other data of the tumor-bearing side of the mouse foot contacting the ground and performed subsequent analysis. Cold pain, mechanical pain, and Catwalk behavioral experiments further supported the prevention of bone cancer pain. Fig.16 Middle c, Fig.16 Middle d, Fig.16 Middle e, Fig.16 Medium f and Fig.16 As shown in middle g, the treated mice showed reduced abnormal sensitivity to cold pain and mechanical pain, and had a walking pattern closer to that of normal mice. They were able to walk with greater ground contact intensity and longer ground contact time, which significantly inhibited bone cancer pain.

[0076] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A lipid polymer complex, characterized in that Including OPSA and GSDMB nucleic acid drugs based on the oxidation-responsive polymer STING agonist; The OPSA is shown in the following formula I: ； In formula I, n is an integer from 10 to 200; X is O; R1 is a methyl group or a hydrogen atom; The GSDMB nucleic acid drug is a GSDMB plasmid.

2. The lipid polymer complex according to claim 1, characterized in that The lipid polymer complex is prepared by encapsulating a liposome membrane with a nanocomposite of OPSA and GSDMB nucleic acid drugs.

3. The method for preparing a lipid polymer complex according to claim 1 or 2, characterized in that: The following steps are involved: (1) OPSA complexes with GSDMB nucleic acid drugs through electrostatic interaction to form a nanocomplex of OPSA and GSDMB nucleic acid drugs; (2) The surface of the nanocomplex of OPSA and GSDMB nucleic acid drugs obtained in step (1) is coated with a layer of liposome membrane to obtain a lipid polymer complex.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the nanocomplex of OPSA and GSDMB nucleic acid drugs to the liposome membrane in step (2) is 1:240, and the liposome membrane in step (2) is composed of 1,2-dioleoylphosphatidylethanolamine DOPE, cholesterol succinate CHEMS and distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000DSPE-PEG.

5. The preparation method according to claim 4, characterized in that: The liposome membrane in step (2) is composed of DOPE, CHEMS and DSPE-PEG in a molar ratio of 7.65:2:1.

35.

6. Use of the lipid polymer complex according to claim 1 or 2 or the lipid polymer complex prepared by the preparation method according to any one of claims 3 to 5 in the preparation of a drug for treating tumor bone metastasis.

7. A drug for treating tumor bone metastasis, characterized in that: Comprising the lipid polymer complex as claimed in claim 1 or 2.

8. A drug for treating tumor bone metastasis, characterized in that: The method comprises preparing the lipid polymer complex according to any one of claims 3 to 5.

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