Piezoelectric nanomaterials for triggering mitochondrial fusion and preparation methods and applications thereof

Abstract

The present invention discloses a piezoelectric nanomaterial for triggering mitochondrial fusion, a preparation method and an application thereof, and belongs to the field of biotechnology. The piezoelectric material provided by the present invention can depolarize the mitochondrial membrane potential (ΔΨ) by targeted supplementation of electrons under ultrasonic stimulation. ΔΨ depolarization significantly promotes mitochondrial fusion and inhibits its fission by activating long optic atrophy 1 (OPA1) and calcium ion influx, thereby activating mitochondrial fusion proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), inhibiting the recruitment of dynamin-related protein 1 (Drp1), and reducing the expression of mitochondrial fission protein 1 (Fis1). Moreover, the JC‑1 dye evaluates ΔΨ, and the observed changes in mitochondrial dynamics are positively correlated. The use of nanomaterials to regulate mitochondrial fusion provides a new method for targeting tumor metabolism and improving cancer treatment outcomes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a piezoelectric nanomaterial for triggering mitochondrial fusion, and a preparation method and application thereof. Background Art

[0002] Mitochondria, as important intracellular organelles, participate in important roles such as energy metabolism, cell signaling, cell cycle regulation and apoptosis. The morphology and number of mitochondria are dynamically regulated by fission and fusion, which is collectively referred to as mitochondrial dynamics. Mitochondrial fission regulates the accumulation of dysfunctional mitochondria, helping cells cope with environmental stresses such as oxidative stress and energy shortage. In addition, mitochondrial fission can ensure that mitochondria are evenly distributed to daughter cells during cell division, thereby maintaining their normal function and growth potential and promoting cell proliferation. Mitochondrial fusion optimizes energy metabolism by maintaining the consistency of mitochondrial function. The process of fission and fusion maintains the dynamic balance of the mitochondrial network, maintains the quality and function of mitochondria, and thus ensures normal cell activity. Tumor cells often manipulate mitochondrial dynamics to adapt to harsh living environments, thereby gaining significant survival and proliferation advantages. Studies have found that tumor cells often show increased mitochondrial fission and reduced fusion, allowing tumor cells to better adapt to hypoxia and nutrient-deficient conditions. Mitochondrial fusion has the potential to reverse or disrupt the metabolic adaptability favored by tumor cells and affect their dependence on glycolysis and oxidative phosphorylation. This disruption makes the regulation of mitochondrial fusion a promising and feasible cancer treatment strategy with the potential to significantly affect tumor growth and survival.

[0003] At present, there are several small molecule regulators that can regulate mitochondrial fusion, such as M1 and Chimera, which promote mitochondrial fusion by enhancing the GTPase activity of Mitochondrin 1 (Mfn1) and enhancing the function of Mitochondrin 2 (Mfn2), respectively, and have shown therapeutic promise in preclinical studies. In addition, BGP-15 has been found to enhance mitochondrial inner membrane integrity by stabilizing optic atrophy 1 (OPA1), which is particularly useful in the treatment of diseases involving mitochondrial dysfunction. However, these regulators also have potential risks. For example, the nonspecific effects of M1 may inadvertently affect other GTPases, indirectly leading to cellular dysfunction and metabolic disorders. Although Chimera can effectively enhance the function of Mfn2, its biological stability is poor, and long-term use may cause concerns about cumulative toxicity. Although BGP-15 has the effect of stabilizing OPA1, it may cause unexpected side effects in different cell types due to its multifunctional nature. Given the potential negative effects, the clinical application of these regulators faces significant difficulties and obstacles. Therefore, there is an urgent need to develop new strategies and methods for regulating mitochondrial fusion with higher specificity and safety. Regulating mitochondrial membrane potential (ΔΨ) depolarization plays a key role in cellular stress response, triggering a series of molecular events and signaling pathways that directly affect key regulatory proteins of mitochondrial fission and fusion. Specifically, ΔΨ depolarization activates the production of long-form OPA1 (L-OPA1), which is beneficial to promote mitochondrial inner membrane fusion. In addition, depolarization is accompanied by the influx of calcium ions, which as important signaling molecules activate mitochondrial fusion proteins such as Mfn1 and Mfn2, thereby enhancing the fusion capacity of the mitochondrial outer membrane and forming a larger mitochondrial network. At the same time, ΔΨ depolarization inhibits the recruitment of Drp1 to the mitochondrial outer membrane, because Drp1 requires a suitable ΔΨ to effectively recruit Drp1. In addition, ΔΨ depolarization alters cellular stress response and signaling pathways, leading to the inhibition of mitochondrial fission-related proteins such as Fis1. Therefore, inducing ΔΨ depolarization may be a safer and more effective way to promote mitochondrial fusion, providing a promising strategy for effective cancer treatment.

[0004] Under the stimulation of ultrasound, piezoelectric materials can precisely generate electrons and holes, which can be used to regulate the membrane depolarization process. This ability makes them uniquely potential for biophysical modulation. However, despite the extensive application and research of nanomaterials in biomedicine, whether they can effectively manipulate mitochondrial dynamics remains to be explored. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a piezoelectric nanomaterial for triggering mitochondrial fusion and a preparation method and application thereof.

[0006] The present invention is achieved by a method for preparing a piezoelectric nanomaterial that triggers mitochondrial fusion, comprising the following steps:

[0007] 1) Preparation of Ba x Ca 1-x Ti y Ce 1-y O3 nanoparticles (BCT:Ce NPs): BCT:Ce NPs were prepared by hydrothermal method using titanium tetrachloride, barium chloride, calcium chloride and cerium trichloride as raw materials, where 0.8≤x<1, 0.8≤y<1;

[0008] 2) Preparation of graphene oxide / Ba x Ca 1-x Ti y Ce 1-y O3 nanoparticles (GO / BCT: Ce NPs): GO / BCT: Ce NPs were prepared by a hydrothermal method using the BCT: Ce NPs prepared in step 1), graphene and polyvinyl pyrrolidone as raw materials.

[0009] The diameter of the BCT:Ce NPs is 100 to 200 nanometers; the diameter of the GO / BCT:Ce NPs is 200 to 300 nanometers.

[0010] Step 1) Ba x Ca 1-x Ti y Ce 1-y Preparation method of O3 nanoparticles (BCT:Ce NPs):

[0011] 1) BaCl2·2H2O and CaCl2 solid powders were weighed and dissolved in deionized water according to the molar ratio of Ba:Ca=x:1-x to prepare a mixed solution, and TiCl4 and CeCl3·7H2O solid powders were dissolved in anhydrous ethanol and deionized water according to the molar ratio of Ti:Ce=y:1-y respectively;

[0012] 2) Take the above solution according to the molar ratio of the product molecular formula, mix and stir for 30 minutes, add NaOH and polyvinyl pyrrolidone, continue stirring for 30 minutes, transfer the suspension to an autoclave, and react at 100-200°C for 1-20 hours; in terms of molar number, the amount of NaOH added is 7x mmol, the amount of polyvinyl pyrrolidone added is 0.012x mmol, the average molecular weight of polyvinyl pyrrolidone is 8000, and x is the molar number of Ba;

[0013] 3) After the reaction is completed, the obtained precipitate is repeatedly washed with deionized water and anhydrous ethanol until the pH value is 7, and then dried in an oven at 80°C for 4 to 12 hours.

[0014] Step 2) Graphene oxide / Ba x Ca 1-x Ti y Ce 1-y Preparation method of O3 nanoparticles (GO / BCT: Ce NPs):

[0015] 1) Weigh the BCT:Ce NPs, graphene oxide (GO) and polyvinyl pyrrolidone (PVP) prepared in step 1), dissolve them in deionized water and stir them evenly; wherein the mass ratio of BCT:Ce NPs, GO, PVP and deionized water is (800-1100): (90-120): (400-600): (20-35);

[0016] 2) The suspension was transferred to an autoclave and reacted at 100-200°C for 1-20 hours. After the reaction, the obtained precipitate was repeatedly washed with deionized water and anhydrous ethanol to pH = 7, and then dried in an oven at 80°C for 4-12 hours to obtain GO / BCT: Ce nanoparticles.

[0017] The autoclave is a stainless steel autoclave lined with tetrafluoroethylene.

[0018] The piezoelectric nanomaterial for triggering mitochondrial fusion is prepared by the above preparation method.

[0019] Preferably, the Ba x Ca 1-x Ti y Ce 1-y O3 is Ba 0.85 Ca 0.15 Ti 0.9 Ce 0.1 O3.

[0020] The above-mentioned piezoelectric nanomaterials that trigger mitochondrial fusion are used in the preparation of drugs that promote mitochondrial fusion in tumor cells.

[0021] The advantages and technical effects of the present invention are as follows:

[0022] 1) Polyvinyl pyrrolidone was used as a binder to combine BCT: Ce NPs with graphene oxide to make the composite material more stable. GO / BCT: Ce with a size of 260.69±17.27 nm was prepared, and its interplanar spacing was 1.99 Å, which was aligned with the crystal plane of BTO. 2) The doping of Ca and Ce and the change of their electronic structure improved the conductivity and covalent bond strength of GO / BCT: Ce nanoparticles. 3) The piezoelectric coefficient d33 reflects the piezoelectric properties of nanoparticles. The d33 of GO / BTO is only 2.04 mV / V, while the d33 of GO / BCT: Ce AEM is as high as 11.25 mV / V, indicating that the doping of Ca and Ce elements improves its piezoelectric properties by about 5.51 times, further confirming that the synthesized GO / BCT: Ce AEM has excellent piezoelectric properties. 4) In GO / BCT: Ce piezoelectric nanomaterials regulate mitochondrial fusion and inhibit its fission by promoting the depolarization of mitochondrial membrane potential, significantly increase the levels of activated mitochondrial fusion proteins Mfn1, Mfn2 and Opa1, inhibit the recruitment of dynamin Drp1 and the expression of mitochondrial fission protein Fis1, and thus affect the metabolism and growth of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is GO / BCT: Ce nanoparticle TEM.

[0024] Figure 2 This is a high magnification TEM of GO / BCT: Ce nanoparticles.

[0025] Figure 3 is a high-angle annular dark-field scanning electron microscope image.

[0026] Figure 4 TEM-mapping images of GO / BCT: Ce (merged and individual elements: Ba, Ti, O, Ca, Ce).

[0027] Figure 5 is the GO / BCT: Ce X-ray diffraction pattern.

[0028] Figure 6 This is the GO / BCT: Ce X-ray photoelectron spectrum.

[0029] Figure 7 This is the GO / BCT: Ce XPS pattern.

[0030] Figure 8 This is the infrared spectrum of GO / BCT: Ce.

[0031] Fig. 9 This is the Raman spectrum of GO / BCT: Ce.

[0032] Fig.10 It is the thermogravimetric curve of GO / BCT: Ce.

[0033] Fig.11 This is a high-intensity structured illumination microscopy image of the mitochondrial morphological changes after ultrasound treatment.

[0034] Fig.12 These are immunofluorescence images of Mfn-1, Mfn-2, and Opa-1 proteins.

[0035] Fig.13 are immunofluorescence images of dynamin-related protein 1 (Drp-1) and mitochondrial fission protein 1 (Fis1) proteins.

[0036] Fig.14 These are flow cytometry and fluorescence images of changes in mitochondrial membrane potential after ultrasound treatment.

[0037] Fig.15 These are images of tumor volume changes in B16-F10 tumor-bearing mice during GO / BCT: Ce ultrasound treatment.

[0038] Fig.16 These are hematoxylin and eosin (H&E) stained images of tumor tissue sections after GO / BCT: Ce ultrasound treatment. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The method for preparing the piezoelectric nanomaterial for triggering mitochondrial fusion of the present invention comprises the following steps:

[0041] 1) Preparation of Ba x Ca 1-x Ti y Ce 1-y O3 nanoparticles (BCT:Ce NPs): BCT:Ce NPs were prepared by hydrothermal method using titanium tetrachloride, barium chloride, calcium chloride and cerium trichloride as raw materials, where 0.8≤x<1, 0.8≤y<1;

[0042] 2) Preparation of graphene oxide / Ba x Ca 1-x Ti y Ce 1-y O3 nanoparticles (GO / BCT: Ce NPs): GO / BCT: Ce NPs were prepared by a hydrothermal method using the BCT: Ce NPs prepared in step 1), graphene and polyvinyl pyrrolidone as raw materials.

[0043] The diameter of the BCT:Ce NPs is 100 to 200 nanometers; the diameter of the GO / BCT:Ce NPs is 200 to 300 nanometers.

[0044] Step 1) Ba x Ca 1-x Ti y Ce 1-y Preparation method of O3 nanoparticles (BCT:Ce NPs):

[0045] 1) BaCl2·2H2O and CaCl2 solid powders were weighed and dissolved in deionized water according to the molar ratio of Ba:Ca=x:1-x to prepare a mixed solution, and TiCl4 and CeCl3·7H2O solid powders were dissolved in anhydrous ethanol and deionized water according to the molar ratio of Ti:Ce=y:1-y respectively;

[0046] 2) Take the above solution according to the molar ratio of the product molecular formula, mix and stir for 30 minutes, add NaOH and polyvinyl pyrrolidone, continue stirring for 30 minutes, transfer the suspension to an autoclave, and react at 100-200°C for 1-20 hours; in terms of molar number, the amount of NaOH added is 7x mmol, the amount of polyvinyl pyrrolidone added is 0.012x mmol, the average molecular weight of polyvinyl pyrrolidone is 8000, and x is the molar number of Ba;

[0047] 3) After the reaction is completed, the obtained precipitate is repeatedly washed with deionized water and anhydrous ethanol until the pH value is 7, and then dried in an oven at 80°C for 4 to 12 hours.

[0048] Step 2) Graphene oxide / Ba x Ca 1-x Ti y Ce 1-y Preparation method of O3 nanoparticles (GO / BCT: Ce NPs):

[0049] 1) Weigh the BCT:Ce NPs, graphene oxide (GO) and polyvinyl pyrrolidone (PVP) prepared in step 1), dissolve them in deionized water and stir them evenly; wherein the mass ratio of BCT:Ce NPs, GO, PVP and deionized water is (800-1100): (90-120): (400-600): (20-35);

[0050] 2) The suspension was transferred to an autoclave and reacted at 100-200°C for 1-20 hours. After the reaction, the obtained precipitate was repeatedly washed with deionized water and anhydrous ethanol to pH = 7, and then dried in an oven at 80°C for 4-12 hours to obtain GO / BCT: Ce nanoparticles.

[0051] The autoclave is a stainless steel autoclave lined with tetrafluoroethylene.

[0052] The piezoelectric nanomaterial for triggering mitochondrial fusion is prepared by the above preparation method.

[0053] Preferably, the Ba x Ca 1-x Ti y Ce 1-y O3 is Ba 0.85 Ca 0.15 Ti 0.9 Ce 0.1 O3.

[0054] The above-mentioned piezoelectric nanomaterials that trigger mitochondrial fusion are used in the preparation of drugs that promote mitochondrial fusion in tumor cells.

[0055] Specifically, the step 1) Ba x Ca 1-x Ti y Ce 1-y The preparation method of O3 nanoparticles (BCT:Ce NPs) is as follows:

[0056] 1) Synthesize Ba with a diameter of 100-200 nanometers by hydrothermal reaction x Ca 1-x Ti y Ce 1-y O3 nanoparticles. In the synthesis process, BaCl2·2H2O and CaCl2 solid powders were weighed and dissolved in deionized water according to the molar ratio of Ba:Ca=x:1-x (0.8≤x<1, two decimal places are retained) to prepare a mixed solution. Then, TiCl4 and CeCl3·7H2O solid powders were dissolved in anhydrous ethanol and deionized water respectively according to the molar ratio of Ti: Ce=y:1-y (0.8≤y<1, two decimal places are retained);

[0057] 2) After equal volumes of the clarified solutions were mixed, they were magnetically stirred in a round-bottom flask for 30 minutes to mix evenly, and 80-110 mmol NaOH and 1-1.4 g polyvinyl pyrrolidone (PVP) were added, and stirring was continued for 30 minutes. Finally, the suspension was transferred to a 50 ml stainless steel autoclave lined with tetrafluoroethylene and reacted at 100-200°C for 1-20 hours;

[0058] 3) After the reaction is completed, the obtained precipitate is repeatedly washed with deionized water and anhydrous ethanol until the pH value is 7, and then dried in an oven at 80°C for 4 to 12 hours.

[0059] Step 2) GO / Ba x Ca 1-x Tiy Ce 1-y The preparation method of O3 nanoparticles (GO / BCT: Ce NPs) is as follows:

[0060] 1) Synthesis of GO / Ba with a diameter of 200-300 nanometers by hydrothermal reaction x Ca 1-x Ti y Ce 1-y O3 nanoparticles. During the synthesis process, 800–1100 mg of BCT:Ce nanoparticles, 90–120 mg of GO, and 400–600 mg of PVP were weighed and dissolved in 20–35 mL of deionized water and stirred evenly in a 50 mL round-bottom flask;

[0061] 2) The suspension was transferred to a 50 ml stainless steel autoclave lined with tetrafluoroethylene and reacted at 100-200°C for 1-20 hours. After the reaction, the precipitate was repeatedly washed with deionized water and anhydrous ethanol to pH = 7, and then dried in an oven at 80°C for 4-12 hours to obtain GO / BCT: Ce nanoparticles.

[0062] The experimental steps of GO / BCT:Ce NPs ultrasound treatment to change mitochondrial morphology are as follows:

[0063] 1) B16-F10 cells were incubated in a 35 mm confocal culture dish for 24 h, and then incubated with PBS, BTO, GO / BTO, GO / BCT, and GO / BCT: Ce for 12 h. After incubation, ultrasound (1.5 W / cm 2 , 1 MHz, 50% duty cycle) for 2 minutes. Ultrasonic stimulation changed the mitochondrial morphology. The average area and length of mitochondria increased. Subsequently, high-intensity structural microscopy (HIS-SIM) was used to observe the changes in mitochondrial morphology, confirming that piezoelectric materials promoted mitochondrial fusion in tumor cells under ultrasound stimulation;

[0064] 2) B16-F10 cells were incubated in a 35 mm confocal culture dish for 24 h, and then incubated with PBS, BTO, GO / BTO, GO / BCT, and GO / BCT: Ce for 12 h. After incubation, ultrasound (1.5 W / cm 2, 1 MHz, 50% duty cycle) for 2 minutes, let stand for 4 minutes, add 0.1 μM immunofluorescence dye, incubate at 37°C for 30 minutes, and observe the changes in different fluorescence intensities using a confocal fluorescence microscope. In the GO / BCT: Ce ultrasound group, compared with other treatment groups, the levels of fusion-related proteins Mitofusins ​​(Mfn1 and Mfn2) and Opa1 increased significantly, while the expression of proteins related to mitochondrial fission, including dynamin-related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1), decreased significantly. The results show that GO / BCT: Ce combined with ultrasound stimulation can effectively promote mitochondrial fusion while inhibiting mitochondrial fission.

[0065] The experimental steps of GO / BCT:Ce NPs ultrasound therapy-induced changes in mitochondrial dynamic homeostasis are as follows:

[0066] The JC-1 probe was used to detect the mitochondrial membrane potential (ΔΨ) of B16-F10 cells. B16-F10 cells were seeded in 6-well plates and 35 mm confocal microplates and cultured overnight. PBS (pH 7.4), BTO, GO / BTO, GO / BCT, and GO / BCT: Ce were added and incubated for 12 h. Each well was sonicated (1.5 W / cm 2 , 1 MHz, 50% duty cycle) for 2 min and allowed to stand for 4 h. JC-1 fluorescent dye was added and incubated at 37°C for 20 min. After washing with cold PBS, the fluorescence intensity of each group was analyzed using a fluorescence confocal laser scanning microscope and flow cytometer.

[0067] The present invention will be further described in the following examples, but the present invention is not limited thereto.

[0068] Example 1

[0069] Ba 0.85 Ca 0.15 Ti 0.9 Ce 0.1 Preparation of O3 nanoparticles (BCT:Ce NPs) was performed as follows:

[0070] 1) Synthesis of Ba by hydrothermal reaction 0.85 Ca 0.15 Ti 0.9 Ce 0.1O3 nanoparticles. During the synthesis process, 1.707 g (9 mmol) of TiCl4 powder was weighed and dissolved in 10 ml of anhydrous ethanol to prepare a TiCl4 anhydrous ethanol solution. Then 3.114 g (12.75 mmol) of BaCl2·2H2O, 0.166 g (2.25 mmol) of CaCl2 and 0.373 g (1 mmol) of CeCl3·7H2O solid powder were weighed and dissolved in 30 ml of deionized water to prepare a mixed solution.

[0071] 2) After the two clear solutions were mixed evenly in a 100 mL round-bottom flask under magnetic stirring for 10 minutes, 3.6 g NaOH and 1.2 g polyvinyl pyrrolidone (PVP) were added and stirred for another 30 minutes. Finally, the suspension was transferred to a 50 mL stainless steel autoclave lined with tetrafluoroethylene and reacted at 200°C for 20 hours.

[0072] 3) After the reaction, the obtained precipitate was repeatedly washed with deionized water and anhydrous ethanol until pH = 7, and then dried in an oven at 80 °C for 12 h.

[0073] Example 2

[0074] GO / Ba 0.85 Ca 0.15 Ti 0.9 Ce 0.1 The preparation method of O3 nanoparticles (GO / BCT: Ce NPs) is as follows:

[0075] 1) Synthesis of GO / Ba by hydrothermal reaction 0.85 Ca 0.15 TiO3 nanoparticles. During the synthesis, 900 mg of BCT:Ce nanoparticles, 100 mg of GO, and 500 mg of PVP were weighed and dissolved in 30 mL of deionized water and stirred well in a 50 mL round-bottom flask.

[0076] 2) The suspension was transferred to a 50 ml stainless steel autoclave lined with tetrafluoroethylene and reacted at 200°C for 12 to 20 h. After the reaction, the precipitate was repeatedly washed with deionized water and anhydrous ethanol to pH = 7, and then dried in an oven at 80°C for 12 h.

[0077] 3) Polyvinylpyrrolidone (PVP) was used as a binder to combine BCT: Ce with graphene oxide to make the composite material more stable. GO / BCT: Ce with a size of 260.69±17.27 nm was prepared. Figure 1-2 GO / BCT: Ce AEM high angle circular dark field (HAADF) imaging is shown in Figure 3As shown, element mapping shows that Ba, Ca, Ti, Ce and O are evenly distributed in the composite structure. Figure 4 The X-ray diffraction pattern clearly shows the crystal structure of the prepared nanoparticles. The positions of the different peaks are consistent with the standard chart of tetragonal BTO. Figure 5 The chemical composition and oxidation state of graphene oxide / BCT: Ce AEM were investigated by X-ray photoelectron spectroscopy. Figure 6 The peaks of Ba 3d, Ca 2p, Ti 2p, Ce 3d, O1s and C1s can be seen from the measured spectrum and detailed XPS spectrum as shown in Figure 7 Fourier transform infrared spectroscopy further proved the binding between BCT: Ce and GO. Figure 8 As shown. In the Raman spectrum, BTO has a -1 and 713 cm -1 The characteristic peak at is tetragonal BTO. Fig. 9 Thermogravimetric analysis further confirmed that the composition of hydrothermally synthesized GO / BCT: Ce is as follows Fig.10 shown.

[0078] Example 3

[0079] The experimental steps of GO / BCT:Ce NPs ultrasound treatment to change mitochondrial morphology are as follows:

[0080] 1) BTO nanoparticles were synthesized by hydrothermal method. During the synthesis, 1.897 g (10 mmol) TiCl4 was dissolved in 10 mL of anhydrous ethanol. Then, 3.664 g (15 mmol) BaCl2·2H2O was dissolved in 30 mL of deionized water. After the two clear solutions were mixed, 3.6 g (90 mmol) NaOH and 1.2 g polyvinylpyrrolidone (PVP) were added. Finally, the suspension was transferred to a 50 mL stainless steel autoclave lined with tetrafluoroethylene and reacted at 200°C for 20 h. After the reaction, the obtained precipitate was repeatedly washed with deionized water and anhydrous ethanol to pH = 7, and then dried in an oven at 80°C for 12 h.

[0081] 2) B16-F10 cells were incubated in a 35 mm confocal culture dish for 24 h, and then incubated with PBS, BTO, GO / BTO, GO / BCT, and GO / BCT: Ce (0.05 mg / mL) for 12 h. After incubation, ultrasound (1.5 W / cm 2, 1 MHz, 50% duty cycle) for 2 minutes. Ultrasonic stimulation changed the mitochondrial morphology. The average area and length of mitochondria increased. Subsequently, high-intensity structural microscopy (HIS-SIM) was used to observe the changes in mitochondrial morphology, confirming that piezoelectric materials promote tumor cell mitochondrial fusion under ultrasound stimulation. Fig.11 shown.

[0082] 3) B16-F10 cells were incubated in a 35 mm confocal culture dish for 24 h, and then incubated with PBS, BTO, GO / BTO, GO / BCT, and GO / BCT: Ce (0.05 mg / mL) for 12 h. After incubation, ultrasound (1.5 W / cm 2 , 1 MHz, 50% duty cycle) for 2 minutes, let stand for 4 minutes, add 0.1 μM immunofluorescence dye, incubate at 37°C for 30 minutes, and observe the changes of different fluorescence intensities under confocal fluorescence microscopy. In the GO / BCT: Ce+ultrasound group, the levels of fusion-related proteins Mitofusins ​​(Mfn1 and Mfn2) and Opa1 were significantly increased compared with the other treatment groups, such as Fig.12 The expression of proteins related to mitochondrial fission, including dynamin-related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1), was significantly reduced, as shown in Figure 2. Fig.13 shown.

[0083] 4) JC-1 probe was used to detect mitochondrial membrane potential (ΔΨ) of B16-F10 cells. B16-F10 cells were seeded in 6-well plates and 35 mm confocal microplates and cultured overnight. PBS (pH 7.4), BTO, GO / BTO, GO / BCT, and GO / BCT: Ce (0.05 mg / mL) were added and incubated for 12 h. Each well was sonicated (1.5 W / cm 2 , 1 MHz, 50% duty cycle) for 2 min and allowed to stand for 4 h. JC-1 fluorescent dye was added and incubated at 37°C for 20 min. After washing with cold PBS, the fluorescence intensity of each group was analyzed by flow cytometry and fluorescence confocal laser scanning microscopy. Fig.14 shown.

[0084] Example 4

[0085] The experimental steps for evaluating the effect of ultrasound treatment of GO / BCT: Ce NPs are as follows:

[0086] 1) Animal experiments used BI6-F10 mouse melanoma cells to construct melanoma as an example. 2×10 6B16-F10 cancer cells were used to successfully establish a mouse B16-F10 tumor model. After 7 days, the tumor-bearing mice were randomly divided into 6 groups, each of which received different ultrasound (under ultrasonic, US) treatment schemes: (i) control group (PBS solution), (ii) BTO+US group (US treatment only), (iii) GO / BTO+US group, (iv) GO / BCT+US group, (v) GO / BCT: Ce group, and (vi) GO / BCT: Ce+US group. Except for group (v), the remaining groups received 1.5 W, 1.0 MHz, 50% duty cycle ultrasound treatment 12 hours after the first intravenous injection of nanoparticles. The size of the tumor volume in each group during the 15-day treatment process was statistically analyzed as follows: Fig.15 shown.

[0087] 2) In order to further illustrate the effect of ultrasound treatment of GO / BCT: Ce NPs, the tumors of each group of mice were collected after treatment and cut into 10 μm tissue sections. The sections were stained with eosin and hematoxylin. It was observed that the tumor tissue was obviously damaged after ultrasound treatment of GO / BCT: Ce NPs. Fig.16 shown.

[0088] The piezoelectric nanomaterial graphene oxide (GO) / Ba 0.85 Ca 0.15 Ti 0.9 Ce 0.1 O3 (BCT: Ce), which can regulate mitochondrial fusion (ΔΨ) by promoting the depolarization of mitochondrial membrane potential. This regulation has an important impact on the metabolism and growth of cancer cells, and it can regulate ΔΨ depolarization. Under ultrasonic (Under ultrasonic, ultrasound) stimulation, the material generates electron holes, in which highly reducing electrons promote H + Evolution to H2, high oxidation holes oxidize NADH to NAD + . H + and NADH consumption lead to ΔΨ depolarization. This depolarization induces changes in mitochondrial dynamics, shifting the trend of mitochondrial fission to that of mitochondrial fusion. This imbalance in mitochondrial fusion and fission also shifts tumor cell respiration from aerobic glycolysis to oxidative phosphorylation. Tumor tissues exhibit pyruvate accumulation and reduced lactate production, which effectively disrupts the metabolic process of tumor cells and inhibits their growth. The piezoelectric catalysis of graphene oxide / BCT nanomaterials, combining mitochondrial dynamics regulation with piezoelectric catalysts, can inhibit tumor cell growth and metabolism, activate apoptosis pathways, and have a high therapeutic effect on melanoma, providing a new paradigm for the development of advanced biocatalytic tumor treatments with optimal efficacy, precision, and safety.

[0089] Those skilled in the art can achieve the final preparation technology by appropriately changing the conditions and routes by referring to the contents of this article. Although the method and preparation technology of the present invention have been described by preferred embodiments, relevant technicians can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention.

Claims

1. A method for preparing a piezoelectric nanomaterial that triggers mitochondrial fusion, characterized in that: The following steps are involved: 1) Preparation of Ba x Ca 1-x Ti y Ce 1-y O3 nanoparticles: BaCl2, TiCl2, CrCl2 and CeCl3 were prepared by hydrothermal method. x Ca 1-x Ti y Ce 1-y O3 nanoparticles, where 0.8≤x<1, 0.8≤y<1; 2) Preparation of graphene oxide / Ba x Ca 1-x Ti y Ce 1-y O3 nanoparticles: Ba prepared in step 1) x Ca 1-x Ti y Ce 1-y O3 nanoparticles, graphene and polyvinyl pyrrolidone were used as raw materials to prepare graphene oxide / Ba by hydrothermal method. x Ca 1-x Ti y Ce 1-y O3 nanoparticles, including Ba x Ca 1-x Ti y Ce 1-y The mass ratio of O3 nanoparticles, graphene oxide, polyvinyl pyrrolidone and deionized water is (800-1100): (90-120): (400-600): (20-35).

2. The method for preparing the piezoelectric nanomaterial that triggers mitochondrial fusion according to claim 1, characterized in that: The Ba x Ca 1-x Ti y Ce 1-y The diameter of O3 nanoparticles is 100-200 nanometers; graphene oxide / Ba x Ca 1-x Ti y Ce 1-y The diameter of O3 nanoparticles is 200 to 300 nanometers.

3. The method for preparing the piezoelectric nanomaterial that triggers mitochondrial fusion according to claim 1, characterized in that: Step 1) Ba x Ca 1-x Ti y Ce 1-y Preparation method of O3 nanoparticles: 1) BaCl2·2H2O and CaCl2 solid powders were weighed and dissolved in deionized water according to the molar ratio of Ba:Ca=x:1-x to prepare a mixed solution, and TiCl4 and CeCl3·7H2O solid powders were dissolved in anhydrous ethanol and deionized water according to the molar ratio of Ti:Ce=y:1-y respectively; 2) Take the above solution according to the molar ratio of the product molecular formula, mix and stir for 30 minutes, add NaOH and polyvinyl pyrrolidone, continue stirring for 30 minutes, transfer the suspension to an autoclave, and react at 100-200°C for 1-20 hours; the average molecular weight of polyvinyl pyrrolidone is 8000; 3) After the reaction is completed, the obtained precipitate is repeatedly washed with deionized water and anhydrous ethanol until the pH value is 7, and then dried in an oven at 80°C for 4 to 12 hours.

4. The method for preparing the piezoelectric nanomaterial that triggers mitochondrial fusion according to claim 3, characterized in that: Step 2) Graphene oxide / Ba x Ca 1-x Ti y Ce 1-y Preparation method of O3 nanoparticles: 1) Weigh the Ba prepared in step 1) x Ca 1-x Ti y Ce 1-y O3 nanoparticles, graphene oxide, and polyvinyl pyrrolidone were dissolved in deionized water and stirred evenly; 2) The suspension was transferred to an autoclave and reacted at 100-200°C for 1-20 hours. After the reaction, the precipitate was repeatedly washed with deionized water and anhydrous ethanol until pH = 7, and then dried in an oven at 80°C for 4-12 hours to obtain graphene oxide / BaO x Ca 1-x Ti y Ce 1-y O3 nanoparticles.

5. The method for preparing the piezoelectric nanomaterial that triggers mitochondrial fusion according to claim 3 or 4, characterized in that: The autoclave is a stainless steel autoclave lined with tetrafluoroethylene.

6. The piezoelectric nanomaterial for triggering mitochondrial fusion obtained by the preparation method according to any one of claims 1 to 5.

7. The piezoelectric nanomaterial for triggering mitochondrial fusion according to claim 6, characterized in that: Ba x That 1- x IT y What 1-y O3 is Ba 0.85 That 0.15 IT 0.9 What 0.1 O3.

8. Use of the piezoelectric nanomaterial that triggers mitochondrial fusion as claimed in claim 6 in the preparation of drugs that promote mitochondrial fusion in tumor cells.

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