Nanovesicles targeting metastatic cancer and methods of making and using the same

Abstract

The application belongs to the technical field of medicine, and particularly relates to a nano vesicle for targeting metastatic carcinoma and a preparation method and application thereof. The nano vesicle for targeting metastatic carcinoma is prepared by screening raw material ratio and preparation conditions. The nano vesicle is composed of bone marrow mesenchymal stem cell membranes (mNVs) and tetrahedral framework nucleic acid. After the tetrahedral framework nucleic acid is loaded with a chemotherapeutic drug, the nano vesicle has excellent storage stability and blood flow stability. When the nano vesicle is used for treating breast cancer bone metastasis and breast cancer systemic metastasis, the nano vesicle can effectively gather at the tumor site, inhibit tumor growth and invasion, effectively inhibit tumor metastasis to other important organs, and has good biological safety. In particular, tFNA and mNVs have a synergistic effect in promoting the role of DOX. After the adaptability of the chemotherapeutic drug is adjusted, the nano vesicle prepared by the application can be used for treating various metastatic carcinomas, and has a good prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a nanovesicle for targeting metastatic cancer, its preparation method, and its uses. Background Technology

[0002] Metastatic cancer refers to cancer that has spread from its original (primary) tumor site to other parts of the body. It is characterized by high invasiveness, rapid growth, and high cellular heterogeneity. If left untreated or without effective treatment, cancer cells proliferate in new sites, destroying the structure and function of normal tissues and leading to organ failure. Furthermore, metastatic cancer can cause complications such as pain, bleeding, and infection, severely impacting the patient's quality of life and survival. Triple-negative breast cancer is a highly aggressive subtype of breast cancer with a high risk of recurrence and metastasis, and it is prone to visceral and brain metastases. Effective treatment of metastatic cancer, especially triple-negative breast cancer, is of great significance.

[0003] Treatment options for triple-negative breast cancer are relatively limited, and chemotherapy remains one of the main clinical treatments, especially for surgically unresectable lesions (multiple metastases). However, due to the poor targeting and tumor tissue penetration of chemotherapy drugs, many toxic side effects occur during clinical use. Therefore, increasing the targeting and tissue penetration of chemotherapy drugs while reducing toxic side effects is crucial.

[0004] Studies have shown that biomimetic nanomaterials based on bone-marrow mesenchymal stem cells (BMSCs) and their derived extracellular vesicles (BMSC-EVs) have been widely used in the delivery of bone-targeted drugs. The BMSC cell membrane encapsulates the nanomaterial core, providing a kind of "camouflage," and its membrane proteins act as "passports" for the core nanomaterials in the immune system, thereby enabling efficient, precise targeted delivery and long-term systemic circulation of the core material. However, when using mesenchymal stem cell (MSC)-based delivery systems for systemic drug administration, the passive retention phenomenon in various tissues cannot be ignored, and there are also issues such as low drug loading levels and difficulty in ensuring effective therapeutic concentrations at tumor tissues. Furthermore, its safety requires further investigation.

[0005] Therefore, it is challenging to construct a novel nanovesicle drug that can effectively target tumor tissue and achieve high drug loading and good safety for the treatment of metastatic cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a nanovesicle delivery system for targeting metastatic cancer, aiming to target tumor tissue and safely and effectively treat triple-negative breast cancer.

[0007] This invention provides a nanovesicle for targeting metastatic cancer, wherein the nanovesicle comprises a bone marrow mesenchymal stem cell membrane and a tetrahedral framework nucleic acid loaded with chemotherapy drugs;

[0008] The molar ratio of the chemotherapy drug to the tetrahedral framework nucleic acid is 76-84:1, and the mass ratio of the bone marrow mesenchymal stem cell membrane to the tetrahedral framework nucleic acid loaded with the chemotherapy drug is 9.5-10.5:1.

[0009] In the nanovesicles, a tetrahedral framework nucleic acid containing chemotherapy drugs is encapsulated by a bone marrow mesenchymal stem cell membrane.

[0010] Preferably, the bone marrow mesenchymal stem cell membrane is prepared according to the following steps:

[0011] Step 1: Collect bone marrow mesenchymal stem cells, dissolve them in hypotonic buffer, and collect the supernatant;

[0012] Step 2: Centrifuge the supernatant from Step 1 and collect the precipitate.

[0013] Preferably, the concentration of the hypotonic buffer solution in step 1 is 25±1.25%, and the dissolution time in the hypotonic buffer solution is 10-15 minutes;

[0014] And / or, the supernatant was obtained under centrifugation conditions at a speed of 1500±75 rpm;

[0015] And / or, in step 2, the centrifugation speed is ≥150,000 rpm, the centrifugation temperature is 0-4℃, and the centrifugation time is ≥30 minutes.

[0016] Preferably, the tetrahedral framework nucleic acid is composed of four single-stranded DNA molecules through complementary base pairing; the molar ratio of the four single-stranded DNA molecules is 1:1:1:1.

[0017] Preferably, in the tetrahedral framework structure of the tetrahedral framework nucleic acid, each edge contains a double-stranded nucleic acid with 19-21 base pairs, and each vertex contains 3-6 free deoxyribonucleotides.

[0018] And / or, the nucleotide sequences of the four single-stranded DNAs are as shown in SEQ ID NO.1-4.

[0019] Preferably, the chemotherapy drug is selected from at least one of doxorubicin hydrochloride, epirubicin, paclitaxel, cisplatin, cyclophosphamide, fluorouracil, gemcitabine, oxaliplatin, and tegafur.

[0020] This invention provides a method for preparing nanovesicles targeting metastatic cancer as described in any one of the above claims, the method comprising:

[0021] Step A: Mix tetrahedral framework nucleic acids with chemotherapy drugs, incubate, centrifuge, and collect the precipitate;

[0022] Step B involves pressing the precipitate from Step A and the bone marrow mesenchymal stem cell membrane through a filter membrane to obtain the final product.

[0023] Preferably, the incubation conditions in step A are: incubation at 0-4℃ for 24±1.2 hours; and the centrifugation is performed using a 30±3kDa ultrafiltration tube.

[0024] And / or, the filter membrane in step B comprises one or both of filter membranes with pore sizes of 400±40 nm and 200±20 nm; the extrusion is repeated in a micro-liposome extruder.

[0025] The present invention provides the use of the nanovesicles that target metastatic cancer as described in any of the above claims in the preparation of cancer treatment drugs.

[0026] Preferably, the cancer includes triple-negative breast cancer and bone metastases.

[0027] This invention, through screening raw material ratios and preparation conditions, prepares nanovesicles targeting metastatic cancer. These nanovesicles consist of tetrahedral framework nucleic acids encapsulated within a bone marrow mesenchymal stem cell membrane. The tetrahedral framework nucleic acids, when loaded with chemotherapeutic drugs, exhibit excellent stability. When used to treat bone metastases and systemic metastases of breast cancer, they effectively accumulate at the tumor site, inhibiting tumor growth and invasion, effectively suppressing metastasis to other vital organs, and demonstrating good biocompatibility. In particular, tFNA and mNVs have a synergistic effect in promoting the efficacy of DOX. After adaptive adjustments, the chemotherapeutic drugs of this invention can be used to treat various metastatic cancers, showing promising potential.

[0028] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0029] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0030] Figure 1 A schematic diagram illustrating the synthesis of DOX-tFNA@mNVs, a nanovesicle delivery system for targeting metastatic cancer.

[0031] Figure 2 a is the polyacrylamide (PAGE) electrophoresis diagram synthesized by tFNA; Figure 2 b is a graph showing the hydration particle size results for tFNA and DOX-tFNA.

[0032] Figure 3 This is a transmission electron microscope image of the BMSCs cell membrane.

[0033] Figure 4 Transmission electron microscopy images of DOX-tFNA and DOX-tFNA@mNVs.

[0034] Figure 5 This is a standard curve graph for DOX.

[0035] Figure 6 The storage stability results of DOX@mNVs and DOX-tFNA@mNVs are shown in Figure 1; a is a photograph taken after different days of storage, and b is a graph showing the proportion of DOX in the supernatant after centrifugation.

[0036] Figure 7 The following are the results of blood flow stability of DOX@mNVs and DOX-tFNA@mNVs: a) Schematic diagram of in vitro blood flow stability experiment, b) Photograph after shaking for 24 hours, c) DOX content in the supernatant after centrifugation.

[0037] Figure 8 Figures show the DOX release results of DOX@mNVs and DOX-tFNA@mNVs at different pH values; a is a schematic diagram of drug release at pH 7.4, b is a drug release curve at pH 7.4, c is a schematic diagram of drug release at pH 5.5, and d is a drug release curve at pH 5.5.

[0038] Figure 9 Figure showing the cytotoxic effects of free DOX, DOX-tFNA, DOX@mNVs, and DOX-tFNA@mNVs on 4T1 cells.

[0039] Figure 10 The inhibitory effects of free DOX, DOX-tFNA, DOX@mNVs and DOX-tFNA@mNVs on bone metastasis of triple-negative breast cancer are shown in Figure a. Treatment process diagram, Figure b. Leg circumference change of mice with bone metastasis, Figure c. Photograph of excised leg, Figure d. Spleen, Figure e. Tumor weight result of excised leg, and Figure f. Spleen weight.

[0040] Figure 11 This is a bioimaging image of a mouse with bone metastasis from triple-negative breast cancer.

[0041] Figure 12Image a shows the micro-CT analysis results of the detached leg; Figure 12 b is the HE and Ki67 staining diagram; Figure 12 c is a graph showing the results of bone mineral density (BMD) analysis of isolated bone; Figure 12 d represents the analysis results of Ki67.

[0042] Figure 13 The aggregation of Cy5-labeled DOX-tFNA@mNVs, DOX-tFNA@rNVs, and DOX-tFNA in a breast cancer bone metastasis model; a is a fluorescence image, b is fluorescence intensity analysis.

[0043] Figure 14 Distribution of Cy5-labeled DOX-tFNA@mNVs, DOX-tFNA@rNVs, and DOX-tFNA in the heart, liver, spleen, lung, kidney, and brain of a mouse model of breast cancer bone metastasis.

[0044] Figure 15 Statistical distribution of Cy5-labeled DOX-tFNA@mNVs, DOX-tFNA@rNVs, and DOX-tFNA in the heart, liver, spleen, lung, kidney, and brain of a mouse model of breast cancer bone metastasis.

[0045] Figure 16 Image of spleen stained with HE.

[0046] Figure 17 This figure shows the statistical results of organ metastasis in each group of mice after treatment with free DOX, DOX-tFNA, DOX@mNVs, and DOX-tFNA@mNVs in a mouse model of breast cancer bone metastasis.

[0047] Figure 18 Ki67 staining images of metastatic lesions in the brain, heart, liver, lungs, and kidneys.

[0048] Figure 19 The image shows the results of Ki67 staining analysis of metastatic lesions in the bone, brain, heart, liver, lungs, and kidneys.

[0049] Figure 20 HE staining images of the brain, heart, liver, lungs, and kidneys, with red stars marking metastatic lesions.

[0050] Figure 21 The graph shows the changes in mouse body weight during treatment with free DOX, DOX-tFNA, DOX@mNVs, and DOX-tFNA@mNVs.

[0051] Figure 22The inhibitory effects of free DOX, DOX-tFNA, and DOX-tFNA@mNVs on a systemic breast cancer metastasis model are shown in figure a. Treatment diagram; figure b. Survival results for each group.

[0052] Figure 23 for 18 The micro-PET / CT detection of FDG@mNVs prepared from F-labeled glucose (FDG) shows the distribution of FDG@mNVs in normal mice and mice with systemic breast cancer metastasis.

[0053] Figure 24 This is a bioluminescence detection image.

[0054] Figure 25 This is a statistical chart showing the metastasis of breast cancer to various organs throughout the body.

[0055] Figure 26 Ki67 staining images of metastatic lesions in the brain, heart, liver, lungs, and kidneys.

[0056] Figure 27 The image shows the results of Ki67 staining analysis of the brain, heart, liver, lungs, kidneys, and bones.

[0057] Figure 28 HE staining images of the brain, heart, liver, spleen, lungs, and kidneys of mice with systemic metastasis, with red stars marking metastatic lesions.

[0058] Figure 29 The image shows the results of complete blood count (white blood cells, WBC; red blood cells, RBC; platelets, PLT; hemoglobin, Hb) and biochemical tests (ALB; LDH; CREA; ALT; AST) after three injections of the drug.

[0059] Figure 30 The image shows the results of complete blood count (white blood cells, WBC; red blood cells, RBC; platelets, PLT; hemoglobin, Hb) and biochemical tests (ALB; LDH; CREA; ALT; AST) after 7 injections of the drug. Detailed Implementation

[0060] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0061] Example 1: A nanovesicle delivery system for targeting metastatic cancer, DOX-tFNA@mNVs

[0062] I. Preparation of DOX-tFNA@mNVs

[0063] DOX-tFNA@mNVs follows these steps ( Figure 1 The following was prepared:

[0064] 1. Synthesis of tFNA

[0065] Four single-stranded DNA molecules (S1, S2, S3, S4, Table 1) were dissolved in TM Buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0), with a final concentration of 1 μM for each molecule. The mixture was thoroughly mixed, rapidly heated to 95°C and held for 10 minutes, then rapidly cooled to 4°C and held for at least 20 minutes to obtain the DNA tetrahedron. Polyacrylamide gel electrophoresis (PAGE) confirmed the successful synthesis of the tetrahedral framework nucleic acid tFNA. Figure 2 a).

[0066] Table 1

[0067]

[0068] 2. Preparation of DOX-tFNA

[0069] 80 μM DOX (doxorubicin hydrochloride) was mixed with 1 μM tFNA and incubated at 4 °C for 24 h. The mixture was then centrifuged using a 30 kDa ultrafiltration tube, and the precipitate was collected as DOX-tFNA. Dynamic light scattering experiments confirmed the successful preparation of DOX-tFNA. Figure 2 b).

[0070] 3. Extraction and synthesis of bone marrow mesenchymal stem cell membranes (mNVs)

[0071] BMSCs were extracted and cultured using the classic whole bone marrow adherent method. First, C57BL / 6 mice (female, 4-5 weeks old) were euthanized, and the femur and tibia were rapidly separated. The femurs and tibias were washed with PBS buffer to remove adhering connective tissue and other non-culture-required tissues. After washing, the femurs and tibias were sequentially transferred to buffer solutions containing different concentrations of penicillin. The ends of the femurs and tibias were cut off, and cells were flushed from the bone marrow cavity. The cells were repeatedly pipetted until the bone marrow tissue fragments separated into cell clusters or dispersed single cells, facilitating BMSC adherent culture. After 3-5 days of culture, primary BMSCs adhered to the flask walls and grew, at which point the culture medium was partially replaced. After another 2-3 days of culture, the culture medium was completely replaced. Once the BMSCs had confluently grown to the flask walls (P0 generation), they were passaged.

[0072] Well-grown P3 generation BMSCs were trypsin-digested and collected in centrifuge tubes. After centrifugation to remove the cell culture medium, the cells were washed three times with PBS buffer. The cells were resuspended in 25% hypotonic PBS buffer for 15 minutes to allow the BMSCs to swell and lyse. The liquid was centrifuged at low speed (1500 rpm), and the bottom precipitate was removed. The supernatant was aliquoted into conical centrifuge tubes. Centrifugation was performed at ultra-high speed (150,000 rpm) for at least 30 minutes to obtain cell membrane fragments. These fragments were resuspended in PBS buffer and centrifuged repeatedly at least three times to obtain cell membrane fragments. Transmission electron microscopy confirmed the successful extraction and receipt of BMSC cell membranes. Figure 3 ).

[0073] 4. Synthesis of DOX-tFNA@mNVs

[0074] Synthesized DOX-tFNA (1 mg) and extracted BMSCs cell membrane mNVs (10 mg) were repeatedly extruded through a micro-liposome extruder (Avanti) using filters with pore sizes of 400 nm and 200 nm to prepare DOX-tFNA@mNVs. Transmission electron microscopy images confirmed the successful preparation of DOX-tFNA@mNVs. Figure 4 ).

[0075] Comparative Example 1: DOX@mNVs

[0076] 1. Bone marrow mesenchymal stem cell membranes (mNVs) were extracted and synthesized according to the method in Example 1;

[0077] 2. 80 μM DOX was mixed with 10 mg / ml mNVs and placed in a liposome extruder, then passed through 400 nm and 200 nm filter membranes to prepare the liposome.

[0078] Comparative example 2DOX-tFNA

[0079] DOX-tFNA was prepared according to the method in Example 1.

[0080] Comparative example 3DOX-tFNA@rNVs

[0081] DOX-tFNA@rNVs were prepared according to the method in Example 1, except that BMSCs cell membrane mNVs were replaced with erythrocyte membrane rNVs.

[0082] The technical solution of the present invention will be further explained through experiments below. The sample DOX-tFNA@mNVs in the following experimental examples was prepared by the method of Example 1, and DOX@mNVs, DOX-tFNA, and DOX-tFNA@rNVs were prepared by the methods of Comparative Examples 1-3.

[0083] Experiment Example 1: Screening of Raw Material Proportions

[0084] I. Experimental Methods

[0085] First, the OD values ​​of different concentrations of DOX were measured, and a standard curve was plotted. Next, 10, 20, 40, 60, 80, and 100 μM DOX were mixed with 1 μM tFNA and incubated. Then, using a 30 kDa ultrafiltration tube, after centrifugation, the supernatant was collected, the concentration of free DOX was measured, and the encapsulation efficiency was calculated. The formula for the encapsulation efficiency is as follows:

[0086] Encapsulation efficiency (%) = (Initial DOX concentration - Free DOX concentration) / Initial DOX concentration × 100% II. Experimental Results

[0087] DOX standard curve as follows Figure 5 As shown, the highest encapsulation efficiency is achieved when DOX is used at a concentration of 80 μM. Therefore, the preferred concentration of DOX in this invention is 80 μM.

[0088] Experiment Example 2: Stability Experiment

[0089] I. Experimental Methods

[0090] 1. Storage stability

[0091] DOX, DOX@mNVs, and DOX-tFNA@mNVs were dissolved in TM buffer and incubated at 25°C for 1, 3, 5, 7, and 10 days. After centrifugation, the storage stability was evaluated by measuring the percentage of DOX in the supernatant.

[0092] 2. Blood flow stability.

[0093] DOX, DOX@mNVs, and DOX-tFNA@mNVs were dissolved in blood, shaken in a shaker at 200 rpm for 24 hours, and centrifuged. Blood flow stability was evaluated by measuring the percentage of DOX in the supernatant.

[0094] II. Experimental Results

[0095] 1. Storage stability

[0096] Storage stability results are as follows Figure 6 As shown, the amount of DOX released by DOX@mNVs gradually increased with prolonged storage time. Compared with DOX@mNVs, DOX-tFNA@mNVs released significantly less DOX, indicating that DOX-tFNA@mNVs have better storage stability, with no obvious drug leakage after 10 days of storage.

[0097] 2. Blood flow stability

[0098] Blood flow stability results as follows Figure 7 As shown, compared with DOX@mNVs, DOX-tFNA@mNVs released significantly less DOX, indicating that DOX-tFNA@mNVs have excellent blood flow stability, with almost no significant drug leakage even after 24 hours in a flowing blood environment.

[0099] Experimental Example 3: pH-responsive drug release

[0100] I. Experimental Methods

[0101] DOX@mNVs (Comparative Example 1) and DOX-tFNA@mNVs (prepared by the method in Example 1) were incubated in PBS buffer at pH 7.4 and 5.5 for different times, respectively. After centrifugation, the DOX content in the supernatant was measured.

[0102] II. Experimental Results

[0103] Drug release results as follows Figure 8 As shown in the figure, in a buffer solution at pH 7.4, DOX-tFNA@mNVs showed no significant drug release compared to DOX@mNVs; however, in a buffer solution at pH 5.5, DOX-tFNA@mNVs exhibited rapid drug release with increasing incubation time, with a release curve similar to that of DOX@mNVs, reaching equilibrium after 12 hours of incubation. These results indicate that DOX-tFNA@mNVs can release the drug in a pH-responsive manner, rapidly releasing DOX in a pH 5.5 environment. The tumor microenvironment is weakly acidic, demonstrating its responsive drug release within the tumor microenvironment.

[0104] Experimental Example 4: Inhibitory effect on bone metastasis of triple-negative breast cancer

[0105] The materials DOX@mNVs and DOX-tFNA used in this experiment were prepared using the methods of Comparative Examples 1 and 2, respectively, while DOX-tFNA@mNVs were prepared using the method of Example 1. The molar concentrations of DOX-tFNA and DOX-tFNA@mNVs were calculated based on the molar concentration of tFNA, both being 1 μM, while the molar concentration of DOX@mNVs was calculated based on the molar concentration of DOX, being 80 μM.

[0106] I. Experimental Methods

[0107] 1. The killing effect of DOX-tFNA@mNVs on breast cancer cells

[0108] After culturing 4T1 cells (triple-negative breast cancer cells) in 96-well plates for 24 hours, 80 μM DOX, 1 μM DOX-tFNA, DOX@mNVs containing 80 μM DOX, and 1 μM DOX-tFNA@mNVs were co-cultured with 4T1 cells for 24 hours. CCK8 solution was then added and the OD values ​​of each group were measured at 450 nm to detect the inhibitory effect on cell viability.

[0109] 2. Establishment and index detection of a triple-negative breast cancer bone metastasis model

[0110] The treatment diagram is shown in Figure 10a. 4T1-Luc cells were injected into the right tibia of BalBc mice. Five days later, the modeling process was observed using a small animal in vivo imaging system. Mice were randomly divided into five groups, treated with saline (physiological saline), DOX (80 μM), DOX-tFNA (containing 1 μM tFNA), DOX@mNVs (containing 80 μM DOX), and DOX-tFNA@mNVs (containing 1 μM tFNA), respectively. The dosage was 100 μL per mouse, administered every two days for seven treatments. Healthy mice were designated as the sham group. Leg circumference and body weight changes were measured concurrently in each group during treatment. After treatment, organs (heart, liver, spleen, lungs, kidneys, brain, femur-tibia, etc.) were collected for micro-CT, HE staining, and immunohistochemical staining.

[0111] 3. Tumor targeting

[0112] To verify tumor targeting, small animal optical imaging systems and tissue section staining techniques were used. Cy5-labeled DOX-tFNA@mNVs, DOX-tFNA@rNVs, and DOX-tFNA were injected intravenously into mice with breast cancer bone metastases. After 12, 24, and 48 hours, the mice were euthanized, and the organs (heart, liver, spleen, lung, kidney, brain, femur-tibia, etc.) were harvested and fixed in paraformaldehyde at 4°C for at least 24 hours. The in vivo metabolism and distribution of different drugs were detected using a small animal optical imaging system. Tissue section staining was used to detect the fluorescence intensity of the aforementioned materials in various tissues, comparing the distribution and aggregation effects of the materials on important tissues and organ toxicity, thus clarifying the targeting of DOX-tFNA@mNVs to tumor tissue and its distribution and metabolism in important organs.

[0113] 4. Bioluminescence detection experiment

[0114] This experiment used 4T1-luc cells transfected with luciferase, which can be used for bioimaging to observe the location and dynamic changes of tumor metastases.

[0115] 5. Transfer rate experiment

[0116] HE staining was performed on tissues from the heart, liver, spleen, lungs, kidneys, brain, and femoral-tibial region. The HE-stained sections were observed under an optical microscope. Metastatic lesions were identified and counted under the microscope, and the metastasis rate was calculated. The metastasis rate formula is as follows:

[0117]

[0118] II. Experimental Results

[0119] 1. Killing effect on triple-negative breast cancer cells

[0120] Experimental results are as follows Figure 9 As shown, DOX, DOX-tFNA, DOX@mNVs, and DOX-tFNA@mNVs all exhibited significant cytotoxic effects on 4T1 cells.

[0121] 2. Effects on the legs

[0122] Leg circumference results as follows Figure 10 As shown in b and c. Compared with other control groups, DOX-tFNA@mNVs significantly inhibited the growth of leg circumference, with a stronger inhibitory effect than DOX-tFNA or DOX@mNVs; its inhibitory effect was comparable to that of healthy mice, indicating that tFNA and mNVs have a significant synergistic effect in promoting the inhibitory effect of DOX on leg circumference growth. The tumor weight of the excised leg also showed the same results as the leg circumference. Figure 10 e) indicates that DOX-tFNA@mNVs can effectively inhibit the growth of bone metastatic tumors. Bioimaging results of metastatic cancer ( Figure 11 Further evidence indicates that DOX-tFNA@mNVs can effectively accumulate in bone metastases of breast cancer in mice, effectively inhibiting the growth of bone metastases. Micro-CT and bone mineral density (BMD) analysis results of the ex vivo leg are shown below. Figure 12 As shown in a and c, DOX-tFNA@mNVs can effectively reduce osteolytic destruction. HE and Ki67 staining results are as follows... Figure 12 As shown in b and d, DOX-tFNA@mNVs can effectively inhibit the growth of metastatic tumors.

[0123] 3. Drug distribution

[0124] The aggregation of Cy5 fluorescently labeled materials in the legs is as follows: Figure 13As shown in the results, compared with other groups, DOX-tFNA@mNVs significantly accumulated in bone metastases 12 hours after administration; at 24 hours after administration, DOX-tFNA@mNVs reached its maximum accumulation in bone metastases, with significantly higher accumulation than in other groups; after more than 24 hours after administration, DOX-tFNA@mNVs were effectively metabolized in bone metastases, reaching low levels after 48 hours. This indicates that DOX-tFNA@mNVs can effectively accumulate in bone metastases and then be effectively metabolized.

[0125] The aggregation of Cy5 fluorescently labeled materials in various organs, as shown in... Figure 14 , 15 As shown in the figure. The results showed that, compared with other groups, DOX-tFNA@mNVs showed significant accumulation in the liver, lungs, kidneys, and brain 12 hours after administration, but less distribution in the heart and spleen; 12 hours after administration, there was still a high level of accumulation in the liver and kidneys, which was gradually metabolized over time. This indicates that DOX-tFNA@mNVs are widely distributed in organs and tissues and can be effectively metabolized.

[0126] 4. Effects on the spleen

[0127] The experimental results of spleen size and weight are as follows: Figure 10 As shown in d and f, the spleen of the mouse model of breast cancer bone metastasis became enlarged and significantly heavier, indicating that the spleen, as the largest immune organ, showed activation of the immune system. Treatment with DOX, DOX-tFNA, DOX@mNVs, and DOX-tFNA@mNVs resulted in spleen shrinkage and lighter weight; in particular, DOX-tFNA@mNVs treatment significantly reduced spleen size and weight. HE staining results of the spleen are shown below. Figure 16 As shown in the figure. The results indicate that DOX-tFNA@mNVs are beneficial for the restoration of normal spleen function and improve the overall health of mice with breast cancer bone metastases.

[0128] 5. Status of organ metastasis

[0129] (1) Transfer rate

[0130] Results of organ metastasis as follows Figure 17 As shown in the figure, compared with other control groups, the metastasis rate of tumors to the heart, liver, and lungs was significantly reduced in the DOX-tFNA@mNVs group; the metastasis rate to the kidneys and brain was comparable to that of DOX@mNVs, and was also significantly reduced. The results indicate that DOX-tFNA@mNVs can effectively inhibit the tumor metastasis rate to other vital organs.

[0131] (2) Tumor invasiveness and growth activity

[0132] Ki67 staining and analysis results of various organs are as follows: Figure 18 , 19As shown, DOX-tFNA@mNVs can effectively reduce the invasiveness of tumors in various organs and significantly reduce tumor proliferation activity. Its inhibitory effect is stronger than that of DOX-tFNA or DOX@mNVs, indicating that tFNA and mNVs have a synergistic effect in promoting DOX's inhibition of tumor proliferation and tumor invasion.

[0133] Results of HE staining experiments on various organs are as follows Figure 20 As shown, the results indicate that the number of metastatic lesions in various organs of mice in the DOX-tFNA@mNVs group was significantly reduced.

[0134] 6. Biosafety

[0135] Changes in mouse body weight during treatment, as follows Figure 21 As shown, the results indicate that only the mice in the DOX-tFNA@mNVs group showed almost no decrease in body weight, and their biocompatibility was significantly better than that of DOX, DOX-tFNA, and DOX@mNVs.

[0136] The above results indicate that DOX-tFNA@mNVs can effectively accumulate in bone metastases of breast cancer in mice, reducing osteolytic destruction; and can effectively inhibit the growth and invasion of metastatic tumors; in addition, it can also effectively inhibit the tumor metastasis rate, number and size of metastatic lesions in other important organs; its inhibitory effect is stronger than that of DOX-tFNA or DOX@mNVs, and tFNA and mNVs have a synergistic effect in promoting the function of DOX.

[0137] Inhibitory effect of Experiment Example 5 on a systemic metastasis model of triple-negative breast cancer

[0138] The DOX-tFNA materials used in this experiment were prepared using the method described in Comparative Example 2, and the DOX-tFNA@mNVs were prepared using the method described in Example 1. The molar concentration of DOX-tFNA was calculated based on the molar concentration of tFNA and was 1 μM.

[0139] I. Experimental Methods

[0140] 1. Establishment of a systemic metastasis model of triple-negative breast cancer

[0141] The treatment diagram is shown in Figure 22a. 4T1-Luc cells were injected into the left ventricle of BalBc mice. Five days later, the modeling process was observed using a small animal in vivo imaging system. Mice were randomly divided into four groups, treated with saline (physiological saline), DOX (80 μM), DOX-tFNA (1 μM), and DOX-tFNA@mNVs (1 μM), respectively, at 100 μL / mouse, administered every two days for seven treatments. Healthy mice were designated as the sham group. Leg circumference and body weight changes were measured simultaneously in each group during treatment. After treatment, organs (heart, liver, spleen, lungs, kidneys, brain, femur-tibia, etc.) were collected for HE staining and immunohistochemical staining. In this experimental example, the molar concentrations of DOX-tFNA and DOX-tFNA@mNVs are calculated based on the molar concentration of tFNA.

[0142] also, 18 F-labeled glucose (FDG) was prepared into FDG@mNVs. FDG@mNVs were injected into healthy mice and mouse models of systemic breast cancer metastasis, respectively, to clarify the location of tumor metastasis and the tumor targeting of mNVs-based nanomedicines.

[0143] 2. Bioluminescence detection experiment

[0144] The experiment was conducted according to the method described in Experiment Example 4, Experiment Method 4.

[0145] 3. Biosafety testing

[0146] Saline, free DOX, DOX-tFNA, mNVs, DOX@mNVs, and DOX-tFNA@mNVs were injected into healthy BalBc mice 3 or 7 times, respectively, and then blood routine tests were performed.

[0147] II. Experimental Results

[0148] 1. Survival rate

[0149] Survival results as follows Figure 22 As shown in b, mice in the saline, free DOX, and DOX-tFNA groups had extremely low survival rates during or at the end of treatment; while mice in the DOX-tFNA@mNVs group had high survival rates, indicating that DOX-tFNA@mNVs can effectively prolong the survival rate of mice with systemic breast cancer metastasis.

[0150] 2. Tumor targeting of mNVs

[0151] The micro-PET / CT results of FDG@mNVs are as follows Figure 23As shown, the distribution of FDG@mNVs differed between normal mice and mice with metastatic breast cancer. In mice with metastatic breast cancer, FDG@mNVs were mainly distributed in the tumor region, demonstrating that mNVs can provide tumor targeting, enabling mNV-based materials to accumulate in mouse breast cancer metastases.

[0152] 3. Status of organ metastasis

[0153] (1) Results of bioluminescence experiments

[0154] Bioluminescence detection results as follows Figure 24 As shown, in mice treated with Saline, DOX, and DOX-tFNA, tumors spread throughout the body after 7 days, resulting in a high mortality rate. However, all mice in the DOX-tFNA@mNVs group survived the experiment, and tumor distribution was significantly inhibited with prolonged treatment. This indicates that DOX-tFNA@mNVs can effectively inhibit tumor metastasis.

[0155] (2) Transfer rate

[0156] Results of organ metastasis as follows Figure 25 As shown in the figure, compared with other control groups, the DOX-tFNA@mNVs group showed a significant reduction in tumor metastasis rates to the heart, liver, lungs, kidneys, brain, and bones. The results indicate that DOX-tFNA@mNVs can effectively inhibit tumor metastasis to other vital organs.

[0157] (3) Tumor invasiveness and growth activity

[0158] Ki67 staining and analysis results of various organs are as follows: Figure 26 , 27 As shown in the figure, compared with the control group, DOX-tFNA@mNVs can effectively reduce the invasiveness of tumors in various organs and significantly reduce tumor proliferation activity, indicating that DOX-tFNA@mNVs can effectively inhibit metastatic lesions and tumor proliferation in other important organs.

[0159] HE tissue staining results are as follows: Figure 28 As shown, the number of metastatic lesions in various organs of mice in the DOX-tFNA@mNVs group was significantly reduced.

[0160] 4. Biosafety

[0161] The blood routine and biochemical test results after 3 injections of the drug are as follows: Figure 29As shown in the results, DOX significantly decreased white blood cell count and platelet count, and significantly increased ALT (alanine aminotransferase), AST (aspartate aminotransferase), CREA (creatinine), and LDH (lactate dehydrogenase), indicating that DOX causes adverse reactions and has toxic effects on important organs and tissues such as the bone marrow hematopoietic system, liver, and kidneys. The DOX-tFNA group showed decreased white blood cell count and abnormally elevated AST, CREA, and LDH, indicating that DOX still has significant toxicity after being loaded with tFNA. The DOX@mNVs group also showed decreased white blood cell count and significantly elevated LDH, indicating that DOX also has significant toxicity after being loaded with mNVs. However, the indicators in the DOX-tFNA@mNVs group were comparable to those in the Saline control group, indicating that DOX-tFNA@mNVs has good biocompatibility.

[0162] The blood routine and biochemical test results after 7 injections of the drug are as follows: Figure 30 As shown, with increasing injection frequency, the toxicity of DOX intensified, particularly with a significant decrease in white blood cell count and platelet count, and a significant increase in ALT, AST, and LDH. In the DOX-tFNA group, the white blood cell count further decreased, while AST and LDH significantly increased, indicating that DOX, even after being loaded with mNVs, still exhibits significant toxicity with increasing injection frequency. The white blood cell count in the DOX@mNVs group also further decreased, indicating that DOX, even after being loaded with mNVs, still exhibits significant toxicity with increasing injection frequency. However, the indicators in the DOX-tFNA@mNVs group were comparable to those in the Saline control group, indicating that DOX-tFNA@mNVs has good biocompatibility.

[0163] The above experimental results demonstrate that the DOX-tFNA@mNVs prepared in this invention exhibit excellent storage and blood flow stability; it can achieve responsive drug release in the tumor microenvironment (acidic); in mouse models of breast cancer bone metastasis, DOX-tFNA@mNVs can effectively accumulate in the bone metastases of mouse breast cancer, reducing osteolytic destruction; and effectively inhibit the growth and invasion of metastatic tumors; in addition, it can also effectively inhibit the tumor metastasis rate, number, and size of metastatic lesions in other important organs; its inhibitory effect is stronger than DOX-tFNA or DOX@mNVs, and tFNA and mNVs have a synergistic effect in promoting the action of DOX; in mouse models of systemic breast cancer metastasis, DOX-tFNA@mNVs can effectively prolong the survival rate of mice with systemic breast cancer metastasis, can effectively accumulate in the metastatic lesions of mouse breast cancer; it can also effectively inhibit the tumor metastasis rate, number, and size of metastatic lesions in other important organs, and has good biocompatibility.

[0164] As can be seen from the above embodiments and experimental examples, this invention, through screening the raw material ratios and preparation conditions, prepares nanovesicles targeting metastatic cancer. These nanovesicles are composed of tetrahedral framework nucleic acids encapsulated by bone marrow mesenchymal stem cell membranes. After loading chemotherapeutic drugs, the tetrahedral framework nucleic acids exhibit excellent stability. When used to treat bone metastases and systemic metastases of breast cancer, they can effectively accumulate at the tumor site, inhibiting tumor growth and invasion, effectively inhibiting tumor metastasis to other vital organs, and demonstrating good biocompatibility. In particular, tFNA and mNVs have a synergistic effect in promoting the function of DOX. After adaptive adjustments, the chemotherapeutic drugs of this invention can be used to treat various metastatic cancers, showing promising prospects.

Claims

1. A nanovesicle targeting metastatic cancer, characterized in that: The nanovesicles include a bone marrow mesenchymal stem cell membrane and a tetrahedral framework nucleic acid loaded with chemotherapy drugs; The molar ratio of the chemotherapy drug to the tetrahedral framework nucleic acid is 76-84:1, and the mass ratio of the bone marrow mesenchymal stem cell membrane to the tetrahedral framework nucleic acid loaded with the chemotherapy drug is 9.5-10.5:

1. In the nanovesicles, a tetrahedral framework nucleic acid containing chemotherapy drugs is encapsulated by a bone marrow mesenchymal stem cell membrane.

2. The nanovesicles for targeted cancer metastasis according to claim 1, characterized in that, The bone marrow mesenchymal stem cell membrane is prepared according to the following steps: Step 1: Collect bone marrow mesenchymal stem cells, dissolve them in hypotonic buffer, and collect the supernatant; Step 2: Centrifuge the supernatant from Step 1 and collect the precipitate.

3. The nanovesicles for targeted cancer metastasis according to claim 2, characterized in that: The concentration of the hypotonic buffer solution mentioned in step 1 is 25±1.25%, and the dissolution time in the hypotonic buffer solution is 10-15 minutes; And / or, the supernatant was obtained under centrifugation conditions at a speed of 1500±75 rpm; And / or, in step 2, the centrifugation speed is ≥150,000 rpm, the centrifugation temperature is 0-4℃, and the centrifugation time is ≥30 minutes.

4. The nanovesicles for targeted cancer metastasis according to claim 1, characterized in that: The tetrahedral framework nucleic acid is composed of four single-stranded DNA molecules that are paired by complementary bases; the molar ratio of the four single-stranded DNA molecules is 1:1:1:

1.

5. The nanovesicles for targeted cancer metastasis according to claim 4, characterized in that: In the tetrahedral framework structure of the aforementioned tetrahedral framework nucleic acid, each edge contains a double-stranded nucleic acid with 19-21 base pairs, and each vertex contains 3-6 free deoxyribonucleotides. And / or, the nucleotide sequences of the four single-stranded DNAs are as shown in SEQ ID NO.1-4.

6. The nanovesicles for targeted cancer metastasis according to claim 1, characterized in that: The chemotherapy drug is selected from at least one of doxorubicin hydrochloride, epirubicin, paclitaxel, cisplatin, cyclophosphamide, fluorouracil, gemcitabine, oxaliplatin, and tegafur.

7. The method for preparing targeted metastatic cancer nanovesicles according to any one of claims 1-6, characterized in that, The preparation method includes: Step A: Mix tetrahedral framework nucleic acids with chemotherapy drugs, incubate, centrifuge, and collect the precipitate; Step B involves pressing the precipitate from Step A and the bone marrow mesenchymal stem cell membrane through a filter membrane to obtain the final product.

8. The preparation method according to claim 7, characterized in that: The incubation conditions described in step A are: incubation at 0-4℃ for 24±1.2 hours; the centrifugation is performed using a 30±3kDa ultrafiltration tube. And / or, the filter membrane in step B comprises one or both of filter membranes with pore sizes of 400±40 nm and 200±20 nm; the extrusion is repeated in a micro-liposome extruder.

9. Use of the nanovesicles targeting metastatic cancer according to any one of claims 1-6 in the preparation of a cancer treatment drug.

10. The use according to claim 9, characterized in that: The cancers mentioned include triple-negative breast cancer and bone metastases.

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