Ultrasonic catalytic nanomodulators for reprogramming CAFs to enhance immunotherapy, their preparation methods and applications

Abstract

This invention discloses an ultrasound-catalyzed nanomodulator for reprogramming cancer cells (CAFs) to enhance immunotherapy, its preparation method, and its application, belonging to the field of biomedical technology. The nanomodulator comprises Nb₂GeTe₄ nanosheets, dasatinib loaded on the surface of the Nb₂GeTe₄ nanosheets, and a CAF cell membrane coating the Nb₂GeTe₄ nanosheets and dasatinib. The Nb₂GeTe₄ nanosheets are two-dimensional sheet structures with a lateral dimension of 100-200 nm; the ultrasound-catalyzed nanomodulator has a particle size of 150-220 nm. This invention achieves a combined application of stromal barrier removal and tumor microenvironment intervention through homologous targeted delivery to the CAF cell membrane, phenotypic reprogramming of CAFs by dasatinib, and the catalytic regulation function of Nb₂GeTe₄ under ultrasound excitation. This effectively improves the therapeutic effect of stromal-rich tumors, while the ultrasound triggering mechanism ensures the controllability and biosafety of the treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an ultrasound-catalyzed nanomodulator for reprogramming CAFs to enhance immunotherapy, its preparation method, and its application. Background Technology

[0002] Cancer-associated fibroblasts (CAFs) are the most abundant stromal cell population in the tumor microenvironment (TME), and are highly concentrated in stromal-rich tumors such as breast cancer and pancreatic cancer. CAFs continuously secrete extracellular matrix (ECM) components and release various pro-fibrotic factors and immunosuppressive cytokines, constructing a dual barrier of "physical barrier + immunosuppression" in tumor tissue. This significantly limits the infiltration of immune effector cells and the anti-tumor immune response, becoming a key factor restricting the efficacy of immunotherapy for stromal-rich tumors.

[0003] Various intervention strategies targeting cancer cell fibroblasts (CAFs) have been explored, including direct clearance, signaling pathway blocking, and phenotypic reprogramming. Dasatinib (DAS), a multi-target tyrosine kinase inhibitor, has shown potential in improving the tumor stromal barrier by blocking CAF-dependent signaling pathways such as PDGFR, thereby inducing CAF phenotypic normalization and reducing ECM deposition. However, while DAS-based CAF reprogramming can alleviate the physical barrier, it is insufficient to effectively reverse the immunosuppressive microenvironment caused by abnormal immune metabolism, resulting in limited anti-tumor function of immune cells. This indicates that a single CAF reprogramming strategy is insufficient for comprehensive regulation of the tumor microenvironment, necessitating the exploration of new approaches that can synergistically intervene in the immunosuppressive microenvironment while simultaneously alleviating the stromal barrier.

[0004] In recent years, piezoelectric catalytic nanomaterials have generated electron-hole pairs under ultrasonic (US) excitation, driving the in-situ generation of reactive oxygen species (ROS), providing new insights for the precise regulation of tumor microenvironment (TME). For example, it has been reported that two-dimensional BiFeO3 (BFO) nanosheets can efficiently generate ROS under ultrasonic excitation, and combined with glycosylation inhibitors, they can reshape the tumor immunosuppressive microenvironment. However, existing piezoelectric catalytic systems (such as BiFeO3 and MoS2) mainly rely on the generation of short-lived hydroxyl radicals (·OH) or superoxide anions (·O2). - It directly kills tumor cells or induces immunogenic cell death, and the ROS it produces has an extremely short half-life (·OH half-life is about 10). -9The piezoelectric catalytic materials mentioned above have limited action range (seconds) and lack the ability to effectively intervene in key targets of immune metabolism. In addition, they generally suffer from problems such as the need to improve catalytic efficiency, poor biocompatibility, and lack of targeted delivery capability of CAFs.

[0005] On the other hand, cell membrane-coated biomimetic nanodelivery technology has shown significant advantages in the field of tumor-targeted delivery. Cell membrane-coated nanoparticles derived from CAFs can selectively accumulate in the tumor matrix region through homologous targeting, and have been used in CAF-related drug delivery. However, combining CAF membrane coating technology with piezoelectric catalytic materials to simultaneously achieve CAF reprogramming and immune microenvironment intervention has not yet been reported.

[0006] In summary, current technologies lack a synergistic regulatory platform capable of actively targeting CAFs and combining drug delivery with ultrasound-responsive catalysis. Developing such a nanomodulator to enhance the treatment efficacy of stromal-rich tumors (especially breast cancer) through the synergistic effect of removing the stromal barrier and regulating the tumor microenvironment has significant scientific and clinical value. Summary of the Invention

[0007] To address the aforementioned technical problems, this application provides an ultrasound-catalyzed nanomodulator for reprogramming CAFs to enhance immunotherapy, its preparation method, and its application.

[0008] The technical solution adopted in this application is as follows: In a first aspect, this application provides an ultrasound-catalyzed nanomodulator for reprogramming CAFs to enhance immunotherapy, comprising Nb2GeTe4 nanosheets, dasatinib loaded on the surface of the Nb2GeTe4 nanosheets, and CAF cell membranes coated on the surfaces of the Nb2GeTe4 nanosheets and dasatinib. The Nb2GeTe4 nanosheets are two-dimensional sheet structures with a lateral dimension of 100~200 nm. The particle size of the ultrasonic catalytic nano-regulator is 150~220nm.

[0009] Furthermore, the mass ratio of the above-mentioned Nb2GeTe4 nanosheets, dasatinib, and CAF cell membrane is 8~12:1~2:1~1.5.

[0010] Furthermore, the aforementioned CAF cell membrane is derived from activated cancer-associated fibroblasts.

[0011] Secondly, this application provides a method for preparing the above-mentioned ultrasonic catalytic nano-regulator, which includes the following steps: (1) Millimeter-sized Nb2GeTe4 single crystals were obtained by chemical vapor transport method, and then Nb2GeTe4 nanosheets were obtained by ultrasonic exfoliation; (2) Dasatinib was loaded onto the surface of the Nb2GeTe4 nanosheets to obtain the Nb2GeTe4 / dasatinib complex; (3) Extract CAF cell membrane fragments, mix the Nb2GeTe4 / dasatinib complex with the CAF cell membrane fragments, and obtain the ultrasonic catalytic nanomodulator by extrusion.

[0012] Further, the chemical vapor transport method described in step (1) above includes: mixing niobium powder, germanium powder and tellurium powder in a stoichiometric ratio of Nb:Ge:Te=1.9~2.1:1.05~1.1:3.9~4.1, sealing the mixture under vacuum, and placing it in a dual-temperature zone tube furnace with a source zone temperature of 740~785℃ and a growth zone temperature of 640~685℃, reacting at a constant temperature for 7~8 days and then naturally cooling.

[0013] Further, the conditions for ultrasonic ablation in step (1) above are as follows: Nb2GeTe4 single crystals are dispersed in isopropanol, and ultrasonic treatment is performed under ice bath conditions with an ultrasonic power of 200~300W for 2~3 seconds, with an interval of 2~3 seconds, for a total duration of 40~60 minutes.

[0014] Further, the loading in step (2) above is as follows: Dasatinib is dissolved in the Nb2GeTe4 nanosheet dispersion, stirred at room temperature for 16-24 h, and washed 2-3 times with deionized water.

[0015] Further, the step of extracting the CAF cell membrane fragments in step (3) above is as follows: centrifuge the CAFs at 1200~1500rpm for 10~12min, collect the precipitate and resuspend it in 1~1.5mL of ultrapure water, then sonicate it at 200~300W for 15~20min, and centrifuge the sonicated suspension at 11000~13000rpm for 45~60min.

[0016] Thirdly, this application provides the application of the above-mentioned ultrasonic catalytic nanomodifier or the ultrasonic catalytic nanomodifier prepared by the above-mentioned preparation method in the preparation of antitumor drugs.

[0017] Fourthly, this application provides an antitumor drug composition comprising the above-mentioned ultrasonic catalytic nanomodulator or the ultrasonic catalytic nanomodulator prepared by the above-mentioned preparation method, and a pharmaceutically acceptable carrier.

[0018] In summary, this application has the following beneficial effects: (1) This application introduces Nb2GeTe4 material into the biomedical field for the first time, providing a novel ultrasound-responsive piezoelectric catalytic nanoplatform. Existing piezoelectric catalytic materials (such as BiFeO3, MoS2, etc.) have limitations in terms of catalytic efficiency and biocompatibility. This invention selects Nb2GeTe4 as the core material for ultrasound catalysis. Its two-dimensional sheet structure (lateral dimension 100~200 nm) can effectively generate reactive oxygen species under ultrasound excitation. Ultrathin Nb2GeTe4 nanosheets generate ONOO under ultrasound. - (half-life 10) -2 Its strong oxidizing ability can persistently destroy IDO-1, far exceeding the effect of traditional short-lived ROS (such as ·OH, ·O2). - It also possesses good biocompatibility, providing a new material option for regulating the tumor microenvironment.

[0019] (2) This application achieves synergistic delivery of CAF reprogramming and ultrasound-catalyzed intervention. Dasatinib is loaded onto the surface of Nb2GeTe4 nanosheets. Through a single nanomodulator, dasatinib simultaneously exerts its phenotypic reprogramming effect on CAFs (reducing extracellular matrix deposition and alleviating the interstitial physical barrier) and the catalytic regulation function of Nb2GeTe4 under ultrasound excitation, achieving dual intervention on the matrix barrier and immunosuppression in the tumor microenvironment, which has a better anti-tumor effect than a single strategy.

[0020] (3) This application uses CAF-derived cell membrane-coated nanomodulators to achieve selective enrichment of the matrix-rich tumor region by utilizing homologous targeting, effectively improving the drug delivery efficiency in the tumor matrix and reducing non-specific adsorption to normal tissues. At the same time, natural cell membrane coating helps to prolong the in vivo circulation time and reduce immune clearance.

[0021] (4) The nano-modulator of this application generates reactive oxygen species and triggers the release of dasatinib only under ultrasonic excitation, thereby achieving external control and targeting of the treatment process, avoiding off-target effects of drugs and catalytic active ingredients in vivo, and showing no significant systemic toxicity in in vivo experiments. It has both high efficiency and biocompatibility. Attached Figure Description

[0022] Figure 1 This is a TEM image of the Nb2GeTe4 two-dimensional nanosheets obtained in Example 1 of the present invention; Figure 2 This is a particle size distribution diagram of the NGT / DAS@C nano-regulator obtained in Example 2 of the present invention; Figure 3 XPS image of Nb2GeTe4 two-dimensional nanosheets obtained in Example 3 of this invention; Figure 4 The image shows the PFM diagram of the Nb2GeTe4 two-dimensional nanosheets obtained in Example 1. Figure 5 The graph shows the DPBF degradation capacity of the NGT / DAS@C nanomodifier obtained in Example 2. Figure 6 The image shows the ultrasonic catalytic oxygen production from water using the NGT / DAS@C nano-modifier obtained in Example 3. Figure 7 This is a schematic diagram of the tumor inhibition rate of the NGT / DAS@C nanomodulator obtained in Example 3 in tumor-bearing mice. Detailed Implementation

[0023] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] The inventive concept of this application is as follows: using CAF cell membrane coating as a targeted delivery method, the nanomodulator is selectively enriched in the stromal-rich tumor region by utilizing homologous targeting; based on targeted delivery, dasatinib loaded on the surface of Nb2GeTe4 nanosheets exerts a phenotypic reprogramming effect on CAFs, reducing extracellular matrix deposition and alleviating the stromal physical barrier; simultaneously, the piezoelectric catalysis of Nb2GeTe4 nanosheets under ultrasonic excitation generates reactive oxygen species, thereby achieving catalytic regulation of the tumor microenvironment. The synergistic effect of these three factors achieves a dual effect of "stromal barrier relief + immune microenvironment regulation," enhancing the anti-tumor therapeutic effect of stromal-rich tumors (especially breast cancer).

[0025] The technical solution of this invention is as follows: An ultrasound-catalyzed nanomodulator for reprogramming CAFs to enhance immunotherapy comprises Nb2GeTe4 nanosheets, dasatinib loaded on the surface of the Nb2GeTe4 nanosheets, and CAF cell membranes coated on the surfaces of the Nb2GeTe4 nanosheets and dasatinib.

[0026] Nb2GeTe4 nanosheets are two-dimensional sheet structures with a lateral dimension of 100~200 nm. The particle size of the ultrasonic catalytic nano-regulator is 150~220nm.

[0027] The CAF cell membrane in this application may be derived from the following pathways: (1) Primary CAFs: Primary cancer-associated fibroblasts isolated from tumor tissues of patients with breast or pancreatic cancer.

[0028] (2) Cell line activation: The mouse fibroblast cell line NIH / 3T3 or the human fibroblast cell line MRC-5 was treated with TGF-β1 (transforming growth factor-β1) at a concentration of 5~10 ng / mL for 48~72 h to induce activation into CAFs-like cells for cell membrane extraction.

[0029] The extraction method for CAF cell membranes is as follows: CAFs are centrifuged at 1200-1500 rpm for 10-12 min to collect the precipitate. The precipitate is resuspended in 1-1.5 mL of ultrapure water and then sonicated at 200-300 W for 15-20 min under ice bath conditions. The sonicated suspension is then centrifuged at 11000-13000 rpm for 45-60 min to collect the supernatant, which yields the CAF cell membrane fragments. The extracted cell membrane protein concentration can be determined using the BCA protein quantification method, and the vesicle morphology can be observed using transmission electron microscopy to confirm successful extraction.

[0030] The reason why this application uses CAF-derived cell membranes instead of other types of cell membranes (such as tumor cell membranes, erythrocyte membranes, etc.) is that the surface of CAF cell membranes retains adhesion molecules and chemokine receptors unique to CAF cells, which can produce specific affinity for homologous tumor stroma regions, thereby achieving selective enrichment of nanomodulators in tumor stroma regions enriched by CAFs.

[0031] In this embodiment, dasatinib (DAS), with the molecular formula C... 22 H 26 ClN7O2S·H2O, CAS No. 302962-49-8, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of ≥98%.

[0032] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0033] Example 1:

[0034] This embodiment provides an NGT / DAS@C nanomodifier, the preparation method of which includes: (1) High-purity niobium powder (99.95%, 200 mesh), germanium powder (99.999%, 200 mesh), and tellurium powder (99.999%) are mixed in a stoichiometric ratio of Nb:Ge:Te = 1.9:1.05~1.1:3.9, with germanium powder in excess of 5~10% to suppress the formation of NbTe2 impurity phase. The mixed raw materials are loaded into quartz ampoules (15~20 mm in diameter and 15~20 cm in length) cleaned with aqua regia, and vacuumed to 100°C. -5Torr and then seal. The ampoule was placed in a horizontal dual-temperature zone tube furnace, with the source temperature set at 740~780℃ and the growth temperature at 640~680℃. The temperature was increased to 300℃ at 2℃ / min and held at that temperature for 2 hours for pre-reaction. Then the temperature was increased to the target temperature at 3℃ / min and held at that temperature for 7 days before natural cooling to obtain millimeter-sized Nb2GeTe4 single crystals.

[0035] (2) The obtained millimeter-sized Nb2GeTe4 single crystals were placed in a glass bottle, and an appropriate amount of isopropanol (concentration of about 1 mg / mL) was added as a dispersion solvent. The sample bottle was placed in an ice-water bath, and intermittent sonication was performed using an ultrasonic cell disruptor (power 300W, frequency 20kHz) (sonication for 2 seconds, interval 3 seconds), with a total duration of 60 min. After sonication, the suspension was centrifuged at 1000 rpm for 5 min to remove the unpeeled thick sheets, and the supernatant was then centrifuged at 8000 rpm for 15 min to collect the Nb2GeTe4 nanosheet precipitate.

[0036] (3) Weigh 1.5 mg of dasatinib (DAS) and dissolve it in 25 mL of the above Nb2GeTe4 nanosheet dispersion. Stir at room temperature for 24 h and wash three times with deionized water to obtain NGT / DAS complex dispersion. Store at 4 °C for later use.

[0037] (4) Aliquot the CAFs into centrifuge tubes, centrifuge at 1500 rpm for 10 min, collect the precipitate, resuspend in 1 mL of ultrapure water, and then sonicate for 20 min using a 300 W cell disruptor. Centrifuge the sonicated suspension at 12000 rpm for 45 min to obtain CAF cell membrane fragments. Mix the above NGT / DAS complex dispersion with the CAF cell membrane fragments, and extrude the mixture through a 200 nm liposome extruder to obtain the sample NGT / DAS@C.

[0038] Example 2:

[0039] This embodiment provides an NGT / DAS@C nanomodifier, the preparation method of which includes: (1) High-purity niobium powder (≥99.95%, 200 mesh), germanium powder (≥99.999%, 200 mesh), and tellurium powder (≥99.999%) are mixed in a stoichiometric ratio of Nb:Ge:Te = 2.1:1.05~1.1:4.1, with germanium powder in excess of 5~10% to suppress the formation of by-products. The mixed raw materials are loaded into high-purity quartz ampoules (φ15~20mm, L 15~20cm) treated with aqua regia, and vacuumed to 100°C. -5After sealing, the Torr was placed in a horizontal dual-temperature zone tube furnace, with the source region set at 745~785℃ and the growth region at 645~685℃. The temperature was increased to 300℃ at a rate of 1~2℃ / min and held at that temperature for 2 hours for pre-reaction. Then, the temperature was increased to the target temperature at a rate of 3℃ / min and held at that temperature for 7 days before natural cooling to obtain millimeter-sized single crystals.

[0040] (2) The obtained single crystal was placed in a glass bottle containing isopropanol (1 mg / mL) and subjected to intermittent sonication (2s on / 3s off, 30~60 min) using an ultrasonic cell disruptor (300W, 20kHz) under ice-water bath conditions. Then, it was centrifuged at 1000 rpm / 5 min and 8000 rpm / 15 min to obtain Nb2GeTe4 nanosheets with uniform thickness.

[0041] (3) Weigh 1.5 mg of dasatinib (DAS) and dissolve it in 25 mL of the above Nb2GeTe4 nanosheet dispersion. Stir at room temperature for 24 h and wash three times with deionized water to obtain NGT / DAS complex dispersion. Store at 4 °C for later use.

[0042] (4) Aliquot the CAFs into centrifuge tubes, centrifuge at 1500 rpm for 10 min, collect the precipitate, resuspend in 1 mL of ultrapure water, and then sonicate for 20 min using a 300 W cell disruptor. Centrifuge the sonicated suspension at 12000 rpm for 45 min to obtain CAF cell membrane fragments. Mix the above NGT / DAS complex dispersion with the CAF cell membrane fragments, and extrude the mixture through a 200 nm liposome extruder to obtain the sample NGT / DAS@C.

[0043] Example 3:

[0044] This embodiment provides a method for preparing an NGT / DAS@C nanomodulator and evaluating its in vivo antitumor effect.

[0045] (1) High-purity niobium powder (≥99.95%, 200 mesh), germanium powder (≥99.999%, 200 mesh), and tellurium powder (≥99.999%) are mixed in a stoichiometric ratio of Nb:Ge:Te = 2:1.05~1.1:4, with germanium powder in excess of 5~10% to suppress the formation of by-products. The mixed raw materials are loaded into high-purity quartz ampoules (φ15~20mm, L 15~20cm) treated with aqua regia, and vacuumed to 100°C. -5 After sealing, the Torr was placed in a horizontal dual-temperature zone tube furnace, with the source region set at 745~785℃ and the growth region at 645~685℃. The temperature was increased to 300℃ at a rate of 1~2℃ / min and held at that temperature for 2 hours for pre-reaction. Then, the temperature was increased to the target temperature at a rate of 3℃ / min and held at that temperature for 7 days before natural cooling to obtain millimeter-sized single crystals.

[0046] (2) The obtained single crystal was placed in a glass bottle containing isopropanol (1 mg / mL) and subjected to intermittent sonication (2s on / 3s off, 30~60min) using an ultrasonic cell disruptor (300W, 20kHz) under ice-water bath conditions. Then, it was centrifuged at 1000rpm / 5min and 8000rpm / 15min in sequence to obtain Nb2GeTe4 nanosheets with uniform thickness.

[0047] (3) Weigh 2.5 mg of dasatinib (DAS) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and dissolve it in 35 mL of the above Nb2GeTe4 nanosheet dispersion. Stir at room temperature for 24 h and wash three times with deionized water to obtain NGT / DAS complex dispersion. Store at 4 °C for later use.

[0048] (4) Aliquot the CAFs into centrifuge tubes, centrifuge at 1500 rpm for 10 min, collect the precipitate, resuspend in 1 mL of ultrapure water, and then sonicate for 10 min using a 200 W cell disruptor. Centrifuge the sonicated suspension at 12000 rpm for 45 min to obtain CAF cell membrane fragments. Mix the above NGT / DAS complex dispersion with the CAF cell membrane fragments, and extrude the mixture through a 200 nm liposome extruder to obtain the sample NGT / DAS@C.

[0049] Example 4:

[0050] This embodiment provides a method for preparing NGT / DAS@C nanomodifiers.

[0051] (1) The preparation steps of Nb2GeTe4 nanosheets in this embodiment are the same as in Example 1, except that: niobium powder, germanium powder and tellurium powder are mixed in a stoichiometric ratio of Nb:Ge:Te=2.0:1.07:4.0. The temperature of the source zone of the dual-temperature zone tubular furnace is set to 760℃ and the temperature of the growth zone is set to 660℃. The temperature is increased to 300℃ at 1.5℃ / min and held at that temperature for 2 hours for pre-reaction. Then, the temperature is increased to the target temperature at 2.5℃ / min and held at that temperature for 7.5 days before natural cooling. The ultrasonic exfoliation conditions are: ultrasonic power of 250W and frequency of 20kHz under ice-water bath conditions, intermittent ultrasonication (ultrasonic 2s, interval 2s), total duration of 50min. Then, the nanosheets are obtained by centrifugation at 1050rpm / 4min and 8500rpm / 18min respectively.

[0052] (2) Weigh 2.0 mg of dasatinib (DAS) and dissolve it in 30 mL of the above Nb2GeTe4 nanosheet dispersion. Stir at room temperature for 20 h and wash three times with deionized water to obtain NGT / DAS complex dispersion. Store at 4 °C for later use.

[0053] (3) The extraction of CAF cell membrane is the same as in Example 1, except that: CAFs are centrifuged at 1300 rpm for 11 min to collect the precipitate, resuspended in 1.2 mL of ultrapure water, and then sonicated for 18 min using a 250 W cell disruptor. The sonicated suspension is centrifuged at 12000 rpm for 50 min to obtain CAF cell membrane fragments.

[0054] (4) The above NGT / DAS complex dispersion was mixed with CAF cell membrane fragments and repeatedly extruded 25 times through a liposome extruder with a specification of 200 nm to obtain the ultrasonic catalytic nano-regulator NGT / DAS@C.

[0055] Example 5:

[0056] This embodiment provides a method for preparing NGT / DAS@C nanomodifiers.

[0057] Preparation of Nb₂GeTe₄ nanosheets: Same as in Example 2, except that niobium powder, germanium powder, and tellurium powder were mixed in a stoichiometric ratio of Nb:Ge:Te = 2.1:1.1:4.1, the source region temperature was set to 785℃, and the growth region temperature was set to 685℃. The ultrasonic exfoliation conditions were: ultrasonic power of 300W and frequency of 20kHz under ice-water bath conditions, intermittent ultrasonication (3s ultrasonication, 2s interval), for a total duration of 40min, followed by centrifugation at 1100rpm / 3min and 9000rpm / 15min respectively to obtain Nb₂GeTe₄ nanosheets.

[0058] (2) Weigh 2.0 mg of dasatinib (DAS) and dissolve it in 30 mL of the above Nb2GeTe4 nanosheet dispersion. Stir at room temperature for 16 h, wash twice with deionized water to obtain NGT / DAS complex dispersion, and store at 4 °C for later use.

[0059] (3) The extraction of CAF cell membrane is the same as in Example 2, except that: CAFs are centrifuged at 1200 rpm for 12 min to collect the precipitate, resuspended in 1.5 mL of ultrapure water, and then sonicated for 15 min using a 200 W cell disruptor. The sonicated suspension is centrifuged at 11000 rpm for 60 min to obtain CAF cell membrane fragments.

[0060] (4) The above NGT / DAS complex dispersion was mixed with CAF cell membrane fragments and repeatedly extruded 20-30 times through a liposome extruder with a specification of 200 nm to obtain the ultrasonic catalytic nano-regulator NGT / DAS@C.

[0061] Example 6:

[0062] This embodiment provides a method for preparing NGT / DAS@C nanomodifiers.

[0063] The preparation method of the NGT / DAS@C nano-regulator in this embodiment is the same as in Example 3. The only difference is that: niobium powder, germanium powder and tellurium powder are mixed in a stoichiometric ratio of Nb:Ge:Te=1.9:1.05:3.9; the source region temperature is set to 740℃ and the growth region temperature is set to 640℃; the ultrasonic exfoliation conditions are: ultrasonic power 200W, frequency 20kHz, intermittent ultrasonication (ultrasonication for 2s, interval for 3s), total duration 60min; the DAS dosage is 1.5mg, and the dispersion volume is 25mL; the CAF cell membrane extraction conditions are: centrifugation at 1500rpm for 10min, resuspension in 1mL of ultrapure water, and ultrasonic disruption at 300W for 20min.

[0064] Structural characterization and performance determination: 1. The morphology of the Nb2GeTe4 nanosheets obtained in Example 1 was characterized using transmission electron microscopy, and the results are as follows: Figure 1 As shown, Nb2GeTe4 exhibits a typical two-dimensional sheet-like structure with an average lateral dimension of approximately 150 nm.

[0065] 2. The piezoelectric properties of the Nb2GeTe4 nanosheets obtained in Example 1 were further characterized using piezoelectric force microscopy (PFM), and the results are as follows: Figure 4 As shown, Nb2GeTe4 nanosheets exhibit good piezoelectric response characteristics.

[0066] 3. The particle size distribution of NGT / DAS@C in Example 2 was characterized using a laser particle size analyzer, and the results are as follows: Figure 2 As shown, the particle size distribution of NGT / DAS@C is around 180 nm.

[0067] 4. The reactive oxygen species (ROS) generation capacity of NGT / DAS@C under ultrasonic catalysis in Example 2 was detected using a 1,3-diphenylisobenzofuran (DPBF) probe. The DPBF probe was added to the NGT / DAS@C dispersion, and the absorbance change of DPBF at 410 nm was measured at different time points under ultrasonic irradiation. The results are as follows: Figure 5 As shown, with the extension of ultrasonic irradiation time, the characteristic absorbance of DPBF at 410 nm decreased significantly, reflecting the presence of superoxide anion (·O2). - (Continuously generated.)

[0068] 5. The chemical composition of NGT / DAS@C provided in Example 3 was analyzed by X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 3 As shown, the successful loading of elements such as Nb, Ge, and Te is confirmed.

[0069] 6. Evaluate the piezoelectric catalytic oxygen production performance of NGT / DAS@C provided in Example 3 under ultrasonic (US) stimulation. The NGT / DAS@C dispersion was placed in a closed system, and the change in O2 content in the system was detected by gas chromatography (GC) under ultrasonic irradiation. The results are as follows: Figure 6 As shown, the O2 signal in the system continued to increase with the extension of US irradiation time, verifying that NGT / DAS@C has a high oxygen production capacity under ultrasonic stimulation.

[0070] 7. Verify the in vivo antitumor effect of the NGT / DAS@C nanomodulator provided in Example 3. Using a breast cancer-bearing mouse model, the mice were randomly divided into different treatment groups, including the NGT / DAS@C group (no ultrasound irradiation) and the NGT / DAS@C+US group (ultrasound irradiation after administration). Tumor volume was measured periodically and growth curves were plotted. The results are as follows: Figure 7 As shown, the tumor volume in the NGT / DAS@C+US group was significantly lower than that in the NGT / DAS@C group, indicating that NGT / DAS@C can enhance the anti-tumor therapeutic effect by reprogramming CAFs and synergistically enhancing the ultrasound catalytic effect.

[0071] In summary, this invention achieves a combined application of targeted delivery, matrix reprogramming, and ultrasound-catalyzed intervention through a three-layer structure design of Nb2GeTe4 nanosheets, dasatinib, and CAF cell membrane. CAF cell membrane encapsulation endows the nanomodulator with homologous targeting capabilities to stromal-rich tumor regions, improving delivery efficiency; dasatinib loading enables phenotypic reprogramming of CAFs, reducing extracellular matrix deposition; and Nb2GeTe4 generates reactive oxygen species under ultrasound excitation, exerting catalytic regulatory functions. The combined action of these three components on the tumor microenvironment achieves the dual effects of stromal barrier removal and immune microenvironment regulation, effectively improving the anti-tumor treatment efficiency of stromal-rich tumors. Simultaneously, the ultrasound triggering mechanism ensures the controllability and biosafety of the treatment process.

[0072] The specific embodiments described above are merely explanations of this application and are not intended to limit it. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An ultrasound-catalyzed nanomodulator for reprogramming CAFs to enhance immunotherapy, characterized in that, It includes Nb2GeTe4 nanosheets, dasatinib loaded on the surface of the Nb2GeTe4 nanosheets, and CAF cell membranes coated on the surface of the Nb2GeTe4 nanosheets and dasatinib; The Nb2GeTe4 nanosheets are two-dimensional sheet structures with a lateral dimension of 100~200 nm. The particle size of the ultrasonic catalytic nano-regulator is 150~220nm.

2. The ultrasonic catalytic nano-regulator according to claim 1, characterized in that, The mass ratio of the Nb2GeTe4 nanosheets, dasatinib, and CAF cell membrane is 8~12:1~2:1~1.

5.

3. The ultrasonic catalytic nano-regulator according to claim 1, characterized in that, The CAF cell membrane is derived from activated cancer-associated fibroblasts.

4. A method for preparing the ultrasonic catalytic nano-regulator according to any one of claims 1 to 3, characterized in that, It includes the following steps: (1) Millimeter-sized Nb2GeTe4 single crystals were obtained by chemical vapor transport method, and then Nb2GeTe4 nanosheets were obtained by ultrasonic exfoliation; (2) Dasatinib was loaded onto the surface of the Nb2GeTe4 nanosheets to obtain the Nb2GeTe4 / dasatinib complex; (3) Extract CAF cell membrane fragments, mix the Nb2GeTe4 / dasatinib complex with the CAF cell membrane fragments, and obtain the ultrasonic catalytic nanomodulator by extrusion.

5. The preparation method according to claim 4, characterized in that, The chemical vapor transport method described in step (1) includes: mixing niobium powder, germanium powder and tellurium powder in a stoichiometric ratio of Nb:Ge:Te=1.9~2.1:1.05~1.1:3.9~4.1, sealing the mixture under vacuum, and placing it in a dual-temperature zone tube furnace with a source zone temperature of 740~785℃ and a growth zone temperature of 640~685℃. The mixture is kept at a constant temperature for 7~8 days and then naturally cooled.

6. The preparation method according to claim 4, characterized in that, The conditions for ultrasonic ablation in step (1) are as follows: Nb2GeTe4 single crystals are dispersed in isopropanol, and ultrasonic power of 200~300W is used for treatment under ice bath conditions, with ultrasonic treatment for 2~3 seconds and intervals of 2~3 seconds, for a total duration of 40~60 minutes.

7. The preparation method according to claim 4, characterized in that, The loading in step (2) is as follows: Dasatinib is dissolved in the Nb2GeTe4 nanosheet dispersion, stirred at room temperature for 16-24 hours, and washed 2-3 times with deionized water.

8. The preparation method according to claim 4, characterized in that, The steps for extracting the CAF cell membrane fragments in step (3) are as follows: centrifuge the CAFs at 1200~1500rpm for 10~12min, collect the precipitate and resuspend it in 1~1.5mL of ultrapure water, then sonicate it at 200~300W for 10~20min, and centrifuge the sonicated suspension at 11000~13000rpm for 45~60min.

9. The use of an ultrasonic catalytic nanomodulator as described in any one of claims 1 to 3 or an ultrasonic catalytic nanomodulator prepared by the preparation method described in any one of claims 4 to 8 in the preparation of antitumor drugs.

10. An antitumor drug composition, characterized in that, It comprises the ultrasonic catalytic nanomodifier according to any one of claims 1 to 3 or the ultrasonic catalytic nanomodifier prepared by the preparation method according to any one of claims 4 to 8, and a pharmaceutically acceptable carrier.

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