Nanomaterial for preventing tumor bone metastasis, preparation method therefor, and use thereof

Abstract

The present invention relates to a nanomaterial for preventing tumor bone metastasis, a preparation method therefor, and use thereof. The present invention has discovered a spatiotemporal coupling interaction between tumor cells and osteoclasts, and provides a tumor-bone initial metastasis behavior-targeting strategy which can accurately prevent tumor metastasis on the basis of the source, and can avoid drug resistance and biochemical drug resistance. On this basis, the present invention designs a physical killing nanomaterial targeting tumor-osteoclast conjugates, the nanomaterial being a bone targeting group-modified nanovesicle encapsulating a carbonate compound and a phosphate compound. The nanomaterial can be effectively concentrated in bone tissue; when tumor cells are activated, the acid secretion function of tumor-related osteoclasts in nearby tumor-osteoclast conjugates which are in contact with the tumor cells triggers the carbonate compound to generate carbon dioxide gas and promotes the release of the phosphate compound, which forms calcium phosphate crystals with calcium ions so as to kill nearby tumor cells, thus achieving a specific very-early tumor metastasis inhibition effect.

Description

A nanomaterial for preventing tumor bone metastasis and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese Application No. 202410007256.9, filed on January 3, 2024, and Chinese Application No. 202410194342.5, filed on February 21, 2024. Said application Nos. 202410007256.9 and 202410194342.5 are hereby incorporated by reference in their entirety. Technical Field

[0003] The present invention relates to the technical field of tumor bone metastasis treatment, and in particular to a nanomaterial for preventing tumor bone metastasis, and a preparation method and application thereof. Background Art

[0004] Among the many organs to which tumors metastasize, bones are the most common preferential metastatic site for many malignancies, particularly breast and prostate cancers. Furthermore, reprogramming of tumor cells within the bone microenvironment promotes secondary metastasis to other organs. Therefore, preventing bone metastasis is a crucial step in preventing systemic metastasis.

[0005] Unlike primary tumors, which are typically treated with local surgery or radiotherapy, tumor metastasis is a systemic, disseminated disease. Current treatments primarily include chemotherapy, targeted therapy, and immunotherapy, such as doxorubicin, bisphosphonates, and PD-1 monoclonal antibodies. However, tumor cells remain in a non-proliferative state for a long time during the initial phase and evade immune surveillance through the formation of the tumor microenvironment. Due to the presence of isoproteins, signaling pathways, and tumor heterogeneity, single-molecule or signaling pathway-based therapies are prone to drug resistance.

[0006] As the early tumor microenvironment continues to develop, inducing conservative behaviors in tumor stromal cells may itself represent an unappreciated new target for inhibiting metastasis at the earliest stages. Due to the unique "hard-substrate" interface of the bone matrix, "softening" the bone matrix is ​​a prerequisite for initial metastasis. Therefore, osteoclasts, the only cells with acid-secreting and osteolytic functions, are key in the initial microenvironment of tumor bone metastasis. Osteoclast activation clearly indicates the activation of bone metastases, so triggering targeted physical destruction of bone metastases through osteoclasts is a promising approach to circumvent drug resistance. Summary of the Invention

[0007] The present invention discovered the spatiotemporal coupling interaction between tumor cells and osteoclasts, and based on the spatiotemporal characteristics of tumor-osteoclast coupling in the initial metastasis process, proposed a behavioral targeting strategy for tumor-bone initial metastasis. By inducing physical killing of calcium phosphate crystals through an in situ decoupling-killing liposome system triggered by osteoclasts, it can not only accurately prevent tumor metastasis based on the source, but also avoid drug resistance and biochemical resistance.

[0008] In view of this, the present invention provides a physical killing nanomaterial targeting the tumor-osteoclast coupling, as well as its preparation method and application. To achieve the above objectives, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a nanomaterial, which is a nanovesicle modified with a bone-targeting group and encapsulating a carbonate compound and a phosphate compound.

[0010] In one embodiment, the nanovesicles comprise nanoliposomes.

[0011] In one embodiment, the nanomaterial targets the tumor-osteoclast couple.

[0012] In one embodiment, the nanomaterial is capable of physically killing tumors. Preferably, the physical killing is achieved through calcium-phosphate crystals formed by phosphate compounds and calcium ions.

[0013] In one embodiment, the nanomaterial has an average particle size of 50-1000 nanometers, more preferably, an average particle size of 50-200 nanometers, and most preferably, an average particle size of less than 100 nanometers.

[0014] In one embodiment, the bone targeting group includes at least one of tetracycline, phosphonate, calcein, and aspartic acid polypeptide sequences, and the phosphonate is, for example, alendronate sodium; preferably, the bone targeting group is tetracycline.

[0015] In the present invention, the carbonate compound can react with acid to generate gas; preferably, it is selected from carbonate salts or bicarbonate salts, more preferably, it is selected from at least one of sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, and ammonium carbonate, and most preferably, sodium bicarbonate is used.

[0016] In the present invention, the phosphate compound can form calcium phosphate crystals with calcium ions; preferably, it is selected from hydrogen phosphate or dihydrogen phosphate, more preferably, it is selected from at least one of disodium hydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and ammonium dihydrogen phosphate, and most preferably, disodium hydrogen phosphate is used.

[0017] In one embodiment, the molar ratio of the carbonate compound to the phosphate compound is 1-4:4-1, preferably 1-3:3-1 or 1-2:2-1, more preferably 1:1.

[0018] In one embodiment, the liposome membrane of the nanoliposome comprises bone-targeting phospholipid, other phospholipids other than bone-targeting phospholipid, and cholesterol.

[0019] Preferably, the bone-targeting phospholipid is obtained by covalently binding a functionalized PEGylated phospholipid to a bone-targeting group molecule. More preferably, the bone-targeting phospholipid is a bone-targeting group-PEGylated phospholipid.

[0020] Preferably, the functionalized PEGylated phospholipid is a PEGylated phospholipid modified with a reactive functional group. The reactive functional group may be a hydroxyl group, a carboxyl group, an amino group, a maleimide group, etc. The molecular weight of the polyethylene glycol may be 500-50,000, preferably 800-6,000.

[0021] Preferably, the functionalized PEGylated phospholipid is functionalized DSPE-PEG, and more preferably, the functionalized PEGylated phospholipid is DSPE-PEG-NHS.

[0022] Preferably, the functionalized PEGylated phospholipids are DSPE-PEG2000-NHS and DSPE-PEG5000-NHS.

[0023] Therefore, preferably, the bone-targeting phospholipid is DSPE-PEG2000-TC (DSPE-PEG2000-tetracycline) or DSPE-PEG5000-TC (DSPE-PEG5000-tetracycline).

[0024] Preferably, the phospholipid other than the bone-targeted phospholipid is at least one of natural phospholipids, semi-synthetic phospholipids and fully synthetic phospholipids; preferably, the phospholipid other than the bone-targeted phospholipid is at least one of lecithin, hydrogenated lecithin and cephalin, and more preferably, the phospholipid is lecithin.

[0025] Therefore, preferably, the liposome membrane comprises bone-targeting phospholipids, phosphatidylcholine and cholesterol.

[0026] Preferably, the mass ratio of bone-targeted phospholipid, phospholipid other than bone-targeted phospholipid, and cholesterol is 5-40:100:10-25, preferably 15-25:100:12-20, and more preferably 20:100:16.

[0027] Preferably, the mass proportion of the bone-targeting phospholipids is 10-40%, preferably 14-28% of the liposome membrane.

[0028] In a second aspect, the present invention further provides a method for preparing the nanomaterial, comprising the following steps:

[0029] Bone-targeted phospholipids, phospholipids other than bone-targeted phospholipids, and cholesterol are placed in an organic solvent. After removing the solvent, solutions of carbonate compounds and phosphate compounds are added and ultrasonically hydrated. The liposomes are placed in a dialysis bag for dialysis and extruded to obtain bone-targeted group-modified nanoliposomes encapsulating carbonate compounds and phosphate compounds.

[0030] In one embodiment, the organic solvent may be at least one of an alcohol solvent, an ester solvent, a halogenated hydrocarbon, a nitrile solvent, and an ether solvent; preferably, the organic solvent may be at least one of methanol, ethanol, methyl acetate, ethyl acetate, dichloromethane, chloroform, acetonitrile, and diethyl ether.

[0031] In one embodiment, the molar ratio of the carbonate compound to the phosphate compound is 1-4:4-1, preferably 1-3:3-1 or 1-2:2-1, more preferably 1:1.

[0032] In one embodiment, in the solution of carbonate compounds and phosphate compounds, the concentration of the carbonate compound is 30-120 mM, and the concentration of the phosphate compound is 20-80 mM; more preferably, the concentration of the carbonate compound is 50-70 mM, and the concentration of the phosphate compound is 50-70 mM.

[0033] In one embodiment, the bone-targeted phospholipid can be prepared by the following method: covalently binding a functionalized PEGylated phospholipid to a bone-targeting group molecule, and purifying to obtain the bone-targeted phospholipid.

[0034] Preferably, the reaction conditions for covalently binding the functionalized PEGylated phospholipid and the bone-targeting group molecule are as follows: placing the functionalized PEGylated phospholipid and the bone-targeting group molecule in an organic solvent, adjusting the pH to 7.5-9.0 with an organic base, and stirring the reaction at room temperature for 6-48 hours. The organic solvent may be at least one of an alcohol solvent, an ester solvent, a halogenated hydrocarbon, a nitrile solvent, and an ether solvent; preferably, the organic solvent may be at least one of methanol, ethanol, methyl acetate, ethyl acetate, dichloromethane, chloroform, acetonitrile, and diethyl ether.

[0035] Preferably, after stirring the reaction, the solvent is replaced with water, and then purified to obtain the bone-targeted phospholipid.

[0036] Preferably, the molar ratio of the functionalized PEGylated phospholipid to the bone targeting group molecule is 1:1-4, preferably 1:1-3, and more preferably 1:1-2.5.

[0037] Preferably, the pH is adjusted to 8.0-8.4; and the mixture is stirred for 18-30 hours.

[0038] Preferably, the purification is selected from at least one of dialysis and chromatography.

[0039] Preferably, the nanoliposomes obtained by extrusion have an average particle size of 50-1000 nm, more preferably, an average particle size of 50-200 nm, and most preferably, an average particle size of less than 100 nm.

[0040] In a third aspect, the present invention also provides an application of the nanomaterial. The nanomaterial is injected intravenously and is found to accumulate in bone tissue. Upon tumor activation and induction of tumor-osteoclast coupling, it is released to form calcium-phosphorus crystals, which are used to physically destroy tumor-osteoclast couplings in bone metastases.

[0041] Therefore, the present invention provides a use of the nanomaterial in the preparation of a medicament for killing tumor-osteoclast couples in bone metastases. Furthermore, the present invention provides a method for killing tumor-osteoclast couples in bone metastases, comprising providing the nanomaterial to an individual in need.

[0042] In one embodiment, the killing is physical killing. More preferably, the killing is physical killing caused by calcium phosphate crystals.

[0043] In one embodiment, the source of bone metastases includes at least one of breast tumors, prostate tumors, bone tumors (including osteoporosis), lung tumors, liver tumors, kidney tumors, gastrointestinal tumors (including gastric tumors and intestinal tumors), and pancreatic tumors.

[0044] In addition, the present invention also provides a use of the nanomaterial in preparing a drug for preventing tumor bone metastasis. In addition, the present invention also provides a method for preventing tumor bone metastasis, which comprises providing the nanomaterial to an individual in need.

[0045] In one embodiment, the tumor comprises at least one of a breast tumor, a prostate tumor, a bone tumor (including a skeletal tumor), a lung tumor, a liver tumor, a kidney tumor, a gastrointestinal tumor (including a stomach tumor and a bowel tumor), or a pancreatic tumor.

[0046] In this invention, upon tumor cell activation, the nanomaterial triggers the production of carbon dioxide gas from carbonate compounds through the acid secretion of tumor-associated osteoclasts in close contact with the tumor-osteoclast complex. This creates pores in the liposome membrane, promoting the release of phosphate compounds. These phosphate compounds then form calcium-phosphate crystals with calcium ions to kill nearby tumor cells, achieving a specific, very early stage tumor metastasis inhibition effect. This behavior-targeted physical killing method offers a new approach for targeting early bone metastases and avoiding pharmacological and biochemical resistance.

[0047] The present invention can also use other bone-targeting groups and other liposome component ratios to achieve similar technical effects.

[0048] Therefore, in a fourth aspect, the present invention further provides a method for preventing tumor bone metastasis, wherein the method targets tumor cells to induce the behavior of osteoclasts.

[0049] In one embodiment, the method uncouples and kills the tumor-osteoclast couple.

[0050] In one embodiment, the method is triggered by tumor-associated osteoclasts.

[0051] In one embodiment, the method produces chemical or physical killing of tumor cells.

[0052] In one embodiment, the method is performed by bone-targeting the drug.

[0053] Preferably, the bone-targeted drug is triggered by tumor-associated osteoclasts.

[0054] Preferably, the bone-targeted drug has the ability to chemically or physically kill tumor cells.

[0055] Preferably, the bone-targeted drug is the nanomaterial described in the present invention.

[0056] In another aspect, the present invention studies the spatiotemporal characteristics of tumor-induced osteoclast maturation. Since the development of classical osteoclasts undergoes continuous stimulation of M-CSF and RANKL, in order to explore the critical period of tumor-induced osteoclast formation, we divided the osteoclast growth stage into three key periods: osteoclast precursor (OCP), RANKL 1-day stimulation of osteoclast precursor (R1-OCP) and RANKL 5-day stimulation of osteoclast precursor (R5-OCP). By co-culturing with tumor cells, it was found that R1-OCP can be induced into osteoclasts. By separating and culturing with tumor cells through Transwell chambers, it was found that R1-OCP can only be induced into osteoclasts under spatial conditions of contact with tumor cells.

[0057] Therefore, in a fifth aspect, the present invention further provides a method for culturing tumor-associated osteoclasts, the method comprising culturing osteoclast precursors pre-stimulated by RANKL together with tumor cells, thereby obtaining tumor-associated osteoclasts.

[0058] Preferably, the RANKL pre-stimulated osteoclast precursors are osteoclast precursors stimulated with RANKL for 1-3 days, preferably osteoclast precursors stimulated with RANKL for 1 day.

[0059] Preferably, the tumor includes at least one of breast tumor, prostate tumor, bone tumor (including bone and muscle tumor), lung tumor, liver tumor, kidney tumor, gastrointestinal tumor (including gastric tumor and intestinal tumor), and pancreatic tumor.

[0060] In the present invention, the calcium phosphate crystal refers to a crystal formed by the reaction of a phosphate compound with calcium ions, and the main component is calcium carbonate.

[0061] In addition, the present invention also provides the following solutions:

[0062] The invention provides a physical killing nano material targeting a tumor-osteoclast coupling. The nano material is a nano liposome modified with a bone targeting group and encapsulating sodium bicarbonate and sodium hydrogen phosphate.

[0063] In one embodiment, the particle size of the nanoliposome is 50-1000 nanometers. Preferably, the particle size of the nanoliposome is 100 nanometers.

[0064] In one embodiment, the bone targeting group comprises tetracycline, phosphonate, or calcein.

[0065] The present invention provides a method for preparing a physical killing nanomaterial of a tumor-osteoclast coupling, the method comprising the following steps:

[0066] Step 1, preparation of bone-targeted phospholipids: covalently binding functionalized PEGylated phospholipids to bone-targeted group molecules, and obtaining bone-targeted phospholipids by dialysis and chromatography;

[0067] Step 2. Preparation of bone-targeting group-modified nanoliposomes encapsulating sodium bicarbonate and sodium hydrogen phosphate: Bone-targeting phospholipids, lecithin, and cholesterol are dissolved in chloroform and spin-dried by suspension evaporation. A mixed solution of sodium bicarbonate and sodium hydrogen phosphate is added, followed by ultrasonic hydration and extrusion to obtain bone-targeting group-modified nanoliposomes encapsulating sodium bicarbonate and sodium hydrogen phosphate.

[0068] In one embodiment, the functionalized PEGylated phospholipid is functionalized DSPE-PEG.

[0069] In one embodiment, the functionalized PEGylated phospholipid is a functionalized PEGylated phospholipid using DSPE-PEG2000-NHS to react with tetracycline. The reaction conditions are: using triethylamine in chloroform to adjust the pH to 8.0-8.4, and stirring at room temperature for 24 hours.

[0070] In one embodiment, the mass ratio of the bone-targeted phospholipid is 10-30% of the liposome membrane, and the liposome membrane comprises bone-targeted phospholipid, phosphatidylcholine, and cholesterol; more preferably, the mass ratio of bone-targeted phospholipid, phosphatidylcholine, and cholesterol is 5:20:4.

[0071] In one embodiment, the concentration of sodium bicarbonate is 30-120 mM, and the concentration of sodium hydrogen phosphate is 20-80 mM. More preferably, 60 mM sodium bicarbonate and 60 mM sodium hydrogen phosphate are used.

[0072] The present invention also provides an application of a physical killing nano material targeting a tumor-osteoclast coupling in the physical killing of the tumor-osteoclast coupling in bone metastases.

[0073] In one embodiment, the sources of bone metastases include breast tumors, prostate tumors, bone tumors, lung tumors, liver tumors, kidney tumors, and gastrointestinal tumors.

[0074] Beneficial effects of the present invention:

[0075] 1) The present invention triggers tumor-associated osteoclasts to "behavioral target" tumor cells, avoiding the nonspecific killing of traditional targeting methods.

[0076] 2) The present invention releases high concentrations of hydrogen phosphate, which rapidly forms calcium-phosphate crystals with in-situ calcium ions, thereby physically killing tumor cells and avoiding biochemical drug resistance of tumors.

[0077] 3) The sodium bicarbonate and disodium hydrogen phosphate used in the present invention are both physiological environment substances and have high biosafety in drug metabolism.

[0078] 4) The method for preventing tumor bone metastasis provided by the present invention targets tumor cells to induce the behavior of osteoclasts, has a definite effect in the early prevention of bone metastasis, provides more ideas for the development of tumor bone metastasis prevention drugs, and has broad significance.

[0079] 5) The method of culturing osteoclasts in vitro of the present invention can more effectively obtain tumor-induced osteoclasts, which provides a more powerful means for studying the spatiotemporal coupling between tumor and osteoclasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0081] Figure 1 shows fluorescence-TRAP images of tumor-osteoclast complexes in bone metastases of GFP-labeled tumor cells in vivo. GFP fluorescence is used to locate tumor cells, and TRAP signals are used to locate osteoclasts.

[0082] Figure 2 shows TRAP staining of tumor-osteoclast conjugates obtained by contact culture. The difference in osteoclast induction between OCP and R1-OCP after co-culture of 4T1 tumor cells suggests that RANKL pre-stimulation is a necessary temporal condition for tumor-associated osteoclastogenesis.

[0083] Figure 3 shows TRAP staining of tumor-osteoclast complexes obtained by Transwell culture. No osteoclasts were observed after separating R1-OCP from 4T1 tumor cells, suggesting that close contact is a necessary spatial condition for tumor-associated osteoclasts.

[0084] Figure 4 shows a schematic diagram of the regional spatial culture and TRAP staining. R1-OCPs that were only in contact with tumor cells were induced to become osteoclasts, while R1-OCPs outside the tumor cell range were not induced to become osteoclasts, confirming that close contact with tumor cells is a necessary spatial location for tumor-associated osteoclastogenesis.

[0085] Figure 5 is a schematic diagram of the structure of tetracycline-modified sodium bicarbonate & disodium hydrogen phosphate nanoliposomes (HC&HP@TNL), a physical killing nanomaterial targeting the tumor-osteoclast couple obtained according to Example 2.1 of the present invention. In the corresponding bar graph for each organ in the right figure, the left side is ICG@NL and the right side is ICG@TNL.

[0086] FIG6 is a cryo-electron microscopy image of calcium phosphate crystals before and after the acid-responsive release of HC&HP@TNL obtained in Example 2.1 of the present invention (scale bar: 100 nm).

[0087] FIG7 is an elemental energy spectrum of calcium-phosphorus crystals after acid-responsive release of HC&HP@TNL obtained according to Example 2.1 of the present invention.

[0088] FIG8 is an in vivo imaging diagram and quantitative statistics of the bone targeting ability of HC&HP@TNL obtained according to Example 2.1 of the present invention.

[0089] FIG9 is a quantitative analysis of the fluorescence intensity of HC&HP@TNL released at different cell-bone slice interfaces according to Example 2.1 of the present invention.

[0090] FIG10 is a diagram showing the targeted release of HC&HP@TNL in bone metastases in vivo according to Example 2.1 of the present invention.

[0091] FIG11 is a TRAP staining diagram showing the decoupling-killing ability of HC&HP@TNL obtained according to Example 2.1 of the present invention on the coupling of tumor cells from different sources with tumor-associated osteoclasts.

[0092] FIG12 shows the tumor cell killing effect of HC&HP@TNL obtained according to Example 2.1 of the present invention, as represented by the GFP expression signal.

[0093] FIG13 is an electron micrograph of the tumor cells of HC&HP@TNL obtained according to Example 2.1 of the present invention being physically killed by calcium phosphate crystal mineralization.

[0094] Figure 14 shows the inhibitory effect of HC&HP@TNL, obtained according to Example 2.1 of the present invention, on bone metastasis in an in vitro model compared to the classic anti-tumor drug DOX. In Figure d, the bar graphs in each group are, from left to right, Control, DOX, Cl@TNL, HC@TNL, and HC&HP@TNL.

[0095] FIG15 shows the in vivo imaging statistics of the bone metastasis inhibition effect of HC&HP@TNL obtained according to Example 2.1 of the present invention.

[0096] Figure 16 shows the Micro-CT statistics of the metaphyseal bone loss inhibition effect of HC&HP@TNL obtained according to Example 2.1 of the present invention. In each group of histograms in the right figure, the left side is Cl@TNL and the right side is HC&HP@TNL. DETAILED DESCRIPTION

[0097] The following describes in detail a physical killing nanomaterial targeting tumor-osteoclast coupling, its preparation method and application provided by the present invention in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0098] The following examples are further given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description herein, and are not necessarily limited to the specific numerical values ​​exemplified below.

[0099] Example 1: Study on the spatiotemporal coupling relationship between tumor and osteoclasts

[0100] First, we observed the relative relationship between tumor metastases and osteoclasts in vivo in a mouse bone metastasis model using the following steps:

[0101] (1) Select 8-10 week old female Balb / c mice and anesthetize them.

[0102] (2) Make a 1.5 cm incision between the 4th and 5th nipples in the right lower quadrant. Separate the muscles to expose the iliac artery.

[0103] (3) 5x10 6 GFP-expressing 4T1 breast tumor cells (0.1 mL) were injected into the iliac artery through a 31G needle.

[0104] (4) Use a cotton tip to press the arterial incision area to stop bleeding and suture the incision.

[0105] (5) After 8 days, the right femur of the mice was serially sectioned and subjected to fluorescence scanning and TRAP staining scanning, respectively, to observe the localization of GFP-positive tumor cells and TRAP-positive osteoclasts. It was found that tumor-associated osteoclasts and metastatic lesions had obvious co-localization and "surrounding phenomenon" (Figure 1).

[0106] Secondly, we established a cell culture model to explore the spatiotemporal relationship between tumor cells and osteoclasts, which was carried out in the following steps:

[0107] (1) Mouse femurs were removed and washed with culture medium to obtain bone marrow cells, which were then cultured in α-MEM medium containing 20 ng / mL M-CSF for 5 days.

[0108] (2) Digest the cells and plate them, setting it as day 0. The cells obtained at this time are osteoclast precursors (OCP). Add 50 ng / mL RANKL to the culture system and culture for 1 day. Then remove RANKL. On the first day, osteoclast precursors (R1-OCP) pre-stimulated with RANKL for 1 day are obtained. If RANKL is not removed and RANKL is continued to be used for 4 days, osteoclast precursors (R5-OCP) stimulated with RANKL for 5 days are obtained on the fifth day. These are also classic osteoclasts (OC) in the conventional culture system.

[0109] (3) On day 1, 4T1 tumor cells were added to the OCP, R1-OCP, and R5-OCP groups, respectively, and cultured until day 5. It was found that 4T1 cells could induce R1-OCP into osteoclasts, while OCP did not have the ability to be induced into osteoclasts by 4T1 cells (Figure 2). Therefore, RANKL pre-stimulation is a necessary time series condition for tumor-associated osteoclasts.

[0110] (4) Keeping other conditions unchanged, the above-mentioned 4T1 tumor cells were cultured on a Transwell chamber separated from osteoclast precursors. It was found that under non-contact conditions, R1-OCP could not be induced into osteoclasts (Figure 3). To further verify, we inoculated 4T1 in the middle area of ​​R1-OCP cells and continued to culture for 4 days. It was found that only R1-OCP in contact with tumor cells was induced into osteoclasts, while R1-OCP outside the tumor cell range was not induced into osteoclasts (Figure 4). Therefore, close contact with tumor cells is a necessary spatial position condition for tumor-associated osteoclasts.

[0111] The above results indicate that there is a strict spatiotemporal coupling relationship between tumor cells and tumor-associated osteoclasts, especially a strict spatial co-localization. Because tumor cells are difficult to target, and the acid-producing characteristics of osteoclasts make them an ideal target, local and precise killing of tumor bone metastases can be achieved by targeting tumor-associated osteoclasts.

[0112] On this basis, we designed a physical killing nanomaterial that targets the tumor-osteoclast couple.

[0113] Example 2: Synthesis of Physical Killing Nanomaterials Targeting Tumor-Osteoclast Couples

[0114] 2.1. Tetracycline-modified sodium bicarbonate & disodium hydrogen phosphate nanoliposomes

[0115] DSPE-PEG2000-NHS and tetracycline were dissolved in 10 mL of chloroform at a ratio of 20 mg:6.8 mg. 100 μL of triethylamine was added to adjust the pH to 8.4. The mixture was stirred at room temperature for 24 hours and then dissolved in water after rotary evaporation. Excess tetracycline was removed by dialysis to obtain tetracycline-modified PEG-2000 phospholipids. Tetracycline-modified PEG-2000 phospholipids, phosphatidylcholine, and cholesterol were dissolved in 10 mL of chloroform at a ratio of 20 mg:100 mg:16 mg. The mixture was then dried by suspension evaporation at 37°C and ultrasonically hydrated with 10 mL of a 60 mM sodium bicarbonate and 60 mM sodium hydrogen phosphate solution. The mixture was then dialyzed in a dialysis bag and extruded to obtain tetracycline-modified sodium bicarbonate and sodium hydrogen phosphate nanoliposomes.

[0116] 2.2. Alendronate modified sodium bicarbonate & disodium hydrogen phosphate nanoliposomes

[0117] DSPE-PEG5000-NHS and alendronate sodium were dissolved in 10 mL of chloroform at a ratio of 20 mg:4.3 mg. 100 μL of triethylamine was added to adjust the pH to 8.4. The mixture was stirred at room temperature for 24 hours and then dissolved in water after rotary evaporation. Excess alendronate sodium was removed by dialysis to obtain alendronate-modified PEG 5000 phospholipid. Alendronate-modified PEG 5000 phospholipid, lecithin, and cholesterol were dissolved in 10 mL of chloroform at a ratio of 30 mg:100 mg:16 mg. The mixture was then dried by suspension evaporation at 37°C. 10 mL of a mixed solution of 30 mM sodium bicarbonate and 80 mM sodium hydrogen phosphate was added and ultrasonically hydrated. The mixture was dialyzed in a dialysis bag and extruded to obtain alendronate-modified sodium bicarbonate and sodium hydrogen phosphate nanoliposomes.

[0118] 2.3. Tetracycline-modified sodium bicarbonate & disodium hydrogen phosphate nanoliposomes

[0119] DSPE-PEG2000-NHS and tetracycline were dissolved in 10 mL of chloroform at a ratio of 20 mg:6.8 mg. 100 μL of triethylamine was added to adjust the pH to 8.0. The mixture was stirred at room temperature for 24 hours and then dissolved in water after rotary evaporation. Excess tetracycline was removed by dialysis to obtain tetracycline-modified PEG-2000 phospholipids. Tetracycline-modified PEG-2000 phospholipids, phosphatidylcholine, and cholesterol were dissolved in 10 mL of chloroform at a ratio of 45 mg:100 mg:16 mg. The mixture was then dried by suspension evaporation at 37°C and ultrasonically hydrated with 10 mL of a mixed solution of 50 mM sodium bicarbonate and 20 mM sodium hydrogen phosphate. The mixture was then dialyzed in a dialysis bag and extruded to obtain tetracycline-modified sodium bicarbonate and sodium hydrogen phosphate nanoliposomes.

[0120] 2.4. Tetracycline-modified sodium bicarbonate & disodium hydrogen phosphate nanoliposomes

[0121] DSPE-PEG2000-NHS and tetracycline were dissolved in 10 mL of chloroform at a ratio of 20 mg:6.8 mg. 100 μL of triethylamine was added to adjust the pH to 8.2. The mixture was stirred at room temperature for 24 hours and then dissolved in water after rotary evaporation. Excess tetracycline was removed by dialysis to obtain tetracycline-modified PEG-2000 phospholipids. Tetracycline-modified PEG-2000 phospholipids, phosphatidylcholine, and cholesterol were dissolved in 10 mL of chloroform at a ratio of 30 mg:100 mg:16 mg. The mixture was then dried by suspension evaporation at 37°C and ultrasonically hydrated with 10 mL of a mixed solution of 120 mM sodium bicarbonate and 50 mM sodium hydrogen phosphate. The mixture was then dialyzed in a dialysis bag and extruded to obtain tetracycline-modified sodium bicarbonate and sodium hydrogen phosphate nanoliposomes.

[0122] Example 3: Application of Tetracycline-Modified Sodium Bicarbonate & Disodium Hydrogen Phosphate Nanoliposomes for Physical Killing of Tumor-Osteoclast Couple in Breast Cancer Bone Metastasis

[0123] Tetracycline-modified sodium bicarbonate & disodium hydrogen phosphate nanoliposomes HC&HP@TNL prepared in Example 2.1 with an average particle size of about 100 nm were selected. The typical structure thereof is shown in FIG5 . The physicochemical properties thereof for targeting tumor-osteoclast coupling were first tested:

[0124] (1) Cryo-TEM:

[0125] 1) The copper grid was treated with glow discharge for 120 s to increase its hydrophilicity, and 2.5 μL of HC&HP@TNL (control or adding hydrochloric acid solution and calcium chloride solution to trigger its release) was added to the copper grid.

[0126] 2) Set the conditions to pressurize for 5 seconds, relax for 3 seconds to form a thin liquid layer, and immerse in liquid nitrogen for vitrification.

[0127] 3) The sample was observed under a 200 kV cryo-transmission electron microscope.

[0128] 4) It was observed that the originally intact liposomes ruptured upon triggering, and the released sodium hydrogen phosphate combined with calcium ions to produce a large number of crystals (Figure 6).

[0129] (2) Crystallization element analysis:

[0130] 1) The copper mesh was treated with glow discharge for 120 s to increase its hydrophilicity, and 2.5 μL of HC&HP@TNL, which was released by adding hydrochloric acid solution and calcium chloride solution, was added to the copper mesh.

[0131] 2) Place the sample under a transmission electron microscope and perform EDS spectrum measurement in scanning mode to determine the ratio of elements such as Ca, P, and O.

[0132] 3) The formed crystals were verified by measurement and calculation to be calcium phosphate crystals (Figure 7).

[0133] (3) In vivo imaging tracing:

[0134] 1) 0.5 mg / mL indocyanine green (ICG) was added during the ultrasonic hydration of HC&HP@TNL for in vivo tracing.

[0135] 2) Tetracycline-modified ICG@TNL and tetracycline-free ICG@NL were injected into Balb / c mice via the tail vein at a dose of 10 mL / kg.

[0136] 3) The distribution of liposomes in mice was observed by IVIS in vivo imaging at 5 minutes, 3 hours, and 6 hours.

[0137] 4) In vivo imaging data analysis revealed that tetracycline-modified liposomes can be effectively enriched in bone tissue, with a bone / liver signal ratio of approximately 1 (Figure 8).

[0138] (4) Cell-specific release experiment:

[0139] 1) A pH probe, Lysotracker, was added during the ultrasonic hydration process of HC&HP@TNL for liposome release characterization.

[0140] 2) Tumor-associated osteoclasts (TAOC), 4T1 tumor cells, osteoblasts, and mesenchymal stem cells were cultured on bone slices using α-MEM medium in a 37°C incubator for 1 day.

[0141] 3) Liposomes were added to the culture medium at a volume ratio of 1:10, and the cell membrane was stained with WGA-Rhod.

[0142] 4) The bone slices were inverted and placed on a glass slide, and the Lysotracker release signal at the interface between cells and bone slices was observed using a confocal microscope.

[0143] 5) Analysis of the image data revealed that liposome release was only observed at the interface between TAOC and the bone slice, indicating that TAOC can effectively trigger the release of HC&HP@TNL for killing the tumor-osteoclast couple (Figure 9).

[0144] (4) In vivo release experiment:

[0145] 1) Select 8-10 week old female Balb / c mice and anesthetize them.

[0146] 2) Make a 1.5 cm incision in the right lower quadrant between the 4th and 5th nipples. Split the muscles to expose the iliac arteries.

[0147] 3) 5x10 6 GFP-expressing 4T1 breast tumor cells (0.1 mL) were injected into the iliac artery through a 31G needle.

[0148] 4) Use a cotton tip to press the arteriotomy area to stop bleeding and suture the incision.

[0149] 5) TRITC fluorescent molecules were added during the ultrasonic hydration process of HC&HP@TNL for fluorescence localization of the released area.

[0150] 6) Liposomes were injected into mice via the tail vein at a dose of 10 mL / kg.

[0151] 7) Take the mouse femur for tissue sectioning and perform fluorescence scanning.

[0152] 8) TRITC signals were found in tumor metastases, indicating that tumor cells and the area affected by liposome release were highly co-localized, indicating that HC&HP@TNL can achieve precise release of tumor metastases through TAOC triggering (Figure 10).

[0153] Secondly, we verified the decoupling and killing effects of HC&HP@TNL on the tumor-osteoclast couple:

[0154] (1) Tumor-associated osteoclast induction and uncoupling-killing effects in tumors of different origins:

[0155] 1) Mouse femurs were removed and washed with culture medium to obtain bone marrow cells, which were then cultured in α-MEM medium containing 20 ng / mL M-CSF for 5 days.

[0156] 2) Digest the cells and plate them on day 0. Add 50 ng / mL RANKL to the culture system and culture for 1 day. Then remove RANKL to obtain R1-OCP. Add tumor cells from different sources (4T1, 4T1.2, EMT-6: breast cancer cells; RM-1: prostate cancer cells; K7M2: osteosarcoma cells; Hepa1-6: liver cancer cells; LLC: lung cancer cells; Panc02: pancreatic cancer cells; MFC: gastric cancer cells; Renca: renal cancer cells) to the R1-OCP for co-culture.

[0157] 3) Treatment groups were set up, and tetracycline-modified nanoliposomes loaded with sodium chloride (Cl@TNL), tetracycline-modified nanoliposomes loaded with sodium bicarbonate (HC@TNL), and tetracycline-modified nanoliposomes loaded with sodium bicarbonate and disodium hydrogen phosphate (HC&HP@TNL) were added to the culture medium at a volume ratio of 1:10.

[0158] 4) Continue culturing until the fifth day, fix with 4% paraformaldehyde, and perform TRAP staining.

[0159] 5) We found that the phenomenon of tumor-associated osteoclast production is universal across tumor cells of various origins. Furthermore, after adding HC&HP@TNL, we found that it had a decoupling and killing effect on tumor-osteoclast couples of various tumors (Figure 11).

[0160] (2) Tumor cell killing verification:

[0161] 1) GFP-expressing 4T1 tumor cells were plated at 1,000 cells / well in a 96-well cell culture plate containing 10,000 R1-OCP cells / well.

[0162] 2) Tetracycline-modified nanoliposomes loaded with sodium chloride (Cl@TNL), tetracycline-modified nanoliposomes loaded with sodium bicarbonate (HC@TNL), and tetracycline-modified nanoliposomes loaded with sodium bicarbonate and disodium hydrogen phosphate (HC&HP@TNL) were added to the culture medium at a volume ratio of 1:10, respectively.

[0163] 3) One day later, the 96-well cell culture plate was placed on a multifunctional microplate reader for quantification of GFP signal.

[0164] 4) The fluorescence intensity of GFP verified that HC&HP@TNL caused tumor cell killing and growth inhibition under the triggering of tumor-associated osteoclasts ( Figure 12 ).

[0165] 5) Fix the injured cells with glutaraldehyde and prepare samples for transmission electron microscopy.

[0166] 6) After resin fixation, the cell samples were ultrathinly sectioned.

[0167] 7) Observation under a transmission electron microscope revealed the presence of a large number of calcium-phosphorus crystals on the surface and inside the TAOC and 4T1 cells, which destroyed the integrity of the tumor cells and achieved direct physical killing of the tumor cells ( FIG13 ).

[0168] (3) In vitro evaluation of tumor suppression effects:

[0169] 1) 8-10 week old Balb / c female mice were selected, their femurs were isolated, and the femurs were cultured in vitro in cell culture plates. We constructed a bone-in-culture array (BICA).

[0170] 2) On day 0, 4T1 tumor cells expressing Luciferase were injected into the femur to establish a bone metastasis model.

[0171] 3) On days 1, 3, 5, and 7, tetracycline-modified nanoliposomes loaded with sodium chloride (Cl@TNL), tetracycline-modified nanoliposomes loaded with sodium bicarbonate (HC@TNL), tetracycline-modified nanoliposomes loaded with sodium bicarbonate and disodium hydrogen phosphate (HC&HP@TNL), and the classic anti-tumor drug DOX were perfused into the femur at a low rate of 10 μL / min using a microinjector.

[0172] 4) On day 14, 1.5 mg / mL of luciferin potassium salt substrate was added to each sample, and the effects were compared by IVIS imaging ( FIG. 14 a ).

[0173] 5) Comparing the effects of HC&HP@TNL and the traditional chemotherapy drug doxorubicin (DOX) in BICA, HC&HP@TNL showed a more pronounced tumor inhibitory effect and less side effects on other cells compared with DOX (Figure 14b-d).

[0174] (4) Mouse bone metastasis inhibition experiment:

[0175] 1) Select 8-10 week old female Balb / c mice and anesthetize them.

[0176] 2) Make a 1.5 cm incision in the right lower quadrant between the 4th and 5th nipples. Split the muscles to expose the iliac arteries.

[0177] 3) 5x10 6 GFP-expressing or Luciferase-expressing 4T1 breast tumor cells (0.1 mL / mL) were injected into the iliac artery through a 31G needle.

[0178] 4) Use a cotton tip to press the arterial incision area to stop bleeding, suture the incision, and consider it as Day 0.

[0179] 5) Tetracycline-modified nanoliposomes loaded with sodium chloride (Cl@TNL), tetracycline-modified nanoliposomes loaded with sodium bicarbonate (HC@TNL), and tetracycline-modified nanoliposomes loaded with sodium bicarbonate and disodium hydrogen phosphate (HC&HP@TNL) were injected via the tail vein at a dose of 10 ml / kg on days 1, 3, 5, and 7, respectively.

[0180] 6) On day 8, the femurs of mice inoculated with GFP-4T1 cells were taken for Micro-CT scanning, and their metaphyseal bone parameters were measured.

[0181] 7) On day 28, each group of mice inoculated with Luciferase-4T1 cells was injected with 150 mg / kg of luciferin potassium salt substrate via the tail vein, and the growth of tumor metastases was observed using IVIS imaging.

[0182] 8) Analysis of Micro-CT and IVIS luminescence intensity data confirmed that HC&HP@TNL physically killed the tumor-osteoclast couple, effectively inhibited bone metastasis (Figure 15) and effectively suppressed metaphyseal bone loss (BMD: 0.24±0.04 g / cm 3 vs 0.17±0.02g / cm 3 ,p=0.0007; BV / TV: 15.44±4.44% vs 8.96±1.81%, P<0.0019; Tb.N: 2.43±0.58mm -1 vs 1.25±0.14mm -1, P < 0.0001; Tb.Sp: 0.23 ± 0.03 mm vs 0.38 ± 0.05 mm, P < 0.0001) ( FIG16 ). This indicates that the nanomaterial for preventing tumor bone metastasis of the present invention can effectively inhibit tumor bone metastasis.

[0183] The above is only a preferred embodiment of the present invention. It should be pointed out that although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention and do not deviate from the scope defined by the claims of the present invention.

Claims

1. A nanomaterial, which is a nanovesicle modified with a bone-targeting group and encapsulating carbonate compounds and phosphate compounds; preferably, the nanovesicle includes a nanoliposome.

2. The nanomaterial according to claim 1, wherein The average particle size of the nanomaterial is 50 - 1000 nanometers, preferably 50 - 200 nanometers, and more preferably less than 100 nanometers.

3. The nanomaterial according to claim 1, wherein The bone-targeting group includes at least one of tetracycline, phosphonate, calcein, and aspartic acid polypeptide sequence, and the phosphonate is, for example, alendronate sodium; preferably, the bone-targeting group uses tetracycline.

4. The nanomaterial according to claim 1, characterized in that, The carbonate compound is selected from carbonate salts or bicarbonate salts, preferably at least one of sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, and ammonium carbonate, and more preferably sodium bicarbonate; the phosphate compound is selected from hydrogen phosphate salts or dihydrogen phosphate salts, preferably at least one of disodium hydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and ammonium dihydrogen phosphate, and more preferably disodium hydrogen phosphate; preferably, the molar ratio of the carbonate compound to the phosphate compound is 1 - 4:4 - 1, preferably 1 - 3:3 - 1 or 1 - 2:2 - 1, and more preferably 1:

1.

5. The nanomaterial according to claim 1, characterized in that, The lipid membrane of the nanoliposome contains bone-targeting phospholipids, other phospholipids outside the bone-targeting phospholipids, and cholesterol.

6. The nanomaterial according to claim 1, characterized in that, The bone-targeting phospholipid is obtained by covalently binding a functionalized polyethylene glycolated phospholipid with a bone-targeting group molecule; preferably, the functionalized polyethylene glycolated phospholipid refers to a polyethylene glycolated phospholipid modified with a reactive functional group, and the reactive functional group can be a hydroxyl group, a carboxyl group, an amino group, or a maleimide group, and the molecular weight of polyethylene glycol can be 500 - 50000, preferably 800 - 6000; more preferably, the functionalized polyethylene glycolated phospholipid uses functionalized DSPE-PEG; more preferably, the functionalized polyethylene glycolated phospholipid uses DSPE-PEG-NHS. The phospholipid outside the bone-targeting phospholipid is at least one of natural phospholipids, semi-synthetic phospholipids, and fully synthetic phospholipids; preferably, the phospholipid outside the bone-targeting phospholipid is at least one of lecithin, hydrogenated lecithin, and cephalin, and more preferably, the phospholipid is lecithin. Preferably, the mass ratio of the bone-targeting phospholipid, the phospholipid outside the bone-targeting phospholipid, and cholesterol is 5 - 40:100:10 - 25, preferably 15 - 25:100:12 - 20, and more preferably 20:100:

16.

7. A method for preparing the nanomaterial according to any one of claims 1 - 6, comprising the following steps: Placing the bone-targeting phospholipid, the phospholipid outside the bone-targeting phospholipid, and cholesterol in an organic solvent, removing the solvent, adding a solution of carbonate compounds and phosphate compounds, followed by ultrasonic hydration, placing it in a dialysis bag for dialysis, and performing extrusion and sizing to obtain a nanoliposome modified with a bone-targeting group and encapsulating carbonate compounds and phosphate compounds.

8. The method according to claim 7, characterized in that, The bone-targeting phospholipid is prepared by the following method: covalently binding a functionalized polyethylene glycolated phospholipid with a bone-targeting group molecule, and purifying to obtain the bone-targeting phospholipid; preferably, the reaction conditions for covalently binding the functionalized polyethylene glycolated phospholipid with the bone-targeting group molecule are: placing the functionalized polyethylene glycolated phospholipid and the bone-targeting group molecule in an organic solvent, adjusting the pH to 7.5-9.0 with an organic base, and stirring at room temperature for 6-48 hours.

9. Use of a nanomaterial according to any one of claims 1-6 in the preparation of a drug for killing tumor-osteoclast conjugates in bone metastases or for preventing tumor bone metastasis.

10. The application according to claim 9, characterized in that, The killing is physical killing, and more preferably, the killing is physical killing caused by calcium phosphate crystals.

11. The application according to claim 9, characterized in that, The sources of the bone metastases include at least one of breast tumors, prostate tumors, bone tumors (including osteosarcoma), lung tumors, liver tumors, kidney tumors, gastrointestinal tumors (including gastric tumors and intestinal tumors), and pancreatic tumors; the tumors in the tumor bone metastasis include at least one of breast tumors, prostate tumors, bone tumors (including osteosarcoma), lung tumors, liver tumors, kidney tumors, gastrointestinal tumors (including gastric tumors and intestinal tumors), and pancreatic tumors.

12. A method for culturing tumor-related osteoclasts, the method comprising co-culturing RANKL-prestimulated osteoclast precursors with tumor cells to obtain tumor-related osteoclasts.

13. The cultivation method according to claim 12, characterized in that, The RANKL-prestimulated osteoclast precursors are osteoclast precursors stimulated by RANKL for 1-3 days, preferably osteoclast precursors stimulated by RANKL for 1 day; the tumors include at least one of breast tumors, prostate tumors, bone tumors (including osteosarcoma), lung tumors, liver tumors, kidney tumors, gastrointestinal tumors (including gastric tumors and intestinal tumors), and pancreatic tumors.

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