Composite silicon-based nanomotor and preparation method and application thereof

Abstract

The application discloses a composite silicon-based nanomotor and a preparation method and application thereof, and belongs to the field of nanomedicines. The composite silicon-based nanomotor comprises a hollow silica nanobottle and calcium peroxide nanoparticles and manganese oxide nanoparticles loaded in the bottle. The outer wall of the hollow silica nanobottle is hydrophobic, and the inner wall is hydrophilic. The synthesized nanomotor is a high-permeability gas-driven composite silicon-based nanomotor, has good permeability in tumors, has small treatment side effects, and can generate gas to be imaged under ultrasonic waves, so that diagnosis and treatment are integrated. In addition, the preparation method is relatively simple, and the drug efficacy is good.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, and in particular relates to a composite silicon-based nanomotor, its preparation method and application. Background Technology

[0002] Breast cancer has become the most common malignant tumor among women, with approximately 400,000 to 420,000 new cases annually, and its incidence rate is increasing by 2% to 4%. Currently, the most commonly used clinical treatments for tumors are surgery, radiotherapy, and chemotherapy. These treatments have serious side effects and limited efficacy. Developing novel tumor treatments with high efficacy and fewer side effects is crucial for improving the quality of life for cancer patients. With the development of nanobiomedicine, researchers have developed various cancer treatment methods based on biofunctional nanomaterials. Among them, amplifying oxidative stress within tumor cells has been proven to be an effective way to inhibit tumor growth. Based on oxidative stress-related phenomena, various tumor treatment methods using nanomedicines have emerged, including photodynamic therapy (PDT), sonodynamic therapy (SDT), and chemodynamic therapy (CDT).

[0003] Compared to photodynamic therapy and sonodynamic therapy, chemokinetic therapy has advantages such as less invasiveness, higher tumor targeting, and independence from external conditions like light and sound. Hydrogen peroxide within tumors can be converted into toxic hydroxyl radicals (·OH) with the help of transition metal catalysts, generating higher levels of oxidative stress than hydrogen peroxide to kill tumor cells. However, traditional chemokinetic therapy mainly focuses on the Fenton reaction mediated by ferrous ions, which has a relatively low reaction rate. Furthermore, the relatively insufficient level of hydrogen peroxide in tumor cells and the high levels of reduced glutathione (GSH) can disrupt the formation of intracellular reactive oxygen species (ROS), limiting the therapeutic potential of chemokinetic therapy. In addition, the poor penetration of small molecule drugs into tumor tissue due to their lack of active delivery capability is also a significant factor limiting their effectiveness.

[0004] To address the difficulty of drug penetration in solid tumors, motile nanomotors have been increasingly recognized by researchers in recent years for their unique mobility, which can significantly facilitate drug penetration into tissues. In recent years, micro- and nanomotors have shown great potential in biomedical applications due to their unique mobility. Through ingenious design, micro- and nanomotors can also identify and sense changes in pH, temperature, and chemical composition at lesion sites, and responsively release drugs or other carriers at the lesion site, accelerating local response and achieving efficient, precise, and targeted therapy. However, existing micro- and nanomotors are costly, have complex fabrication methods, and offer relatively limited functionality. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a composite silicon-based nanomotor, its preparation method, and its application. The composite silicon-based nanomotor prepared by this invention is a highly permeable gas-driven composite silicon-based nanomotor, which has good permeability in tumors, few therapeutic side effects, and the generated gas can be visualized under ultrasound, achieving integrated diagnosis and treatment.

[0006] To achieve the above objectives, the present invention provides a composite silicon-based nanomotor, comprising a hollow silicon dioxide nanobottle (silicon-based nanobottle) and calcium peroxide nanoparticles and manganese oxide nanoparticles loaded inside the bottle; The outer wall of the silica hollow nanobottle is hydrophobic, while the inner wall is hydrophilic.

[0007] Furthermore, the silica hollow nanobottle includes a bottle body with a hollow chamber and a narrow neck; The average diameter of the bottle body is 450±100nm, and the diameter of the bottleneck is 100~200nm.

[0008] Furthermore, based on mass parts, one part of the bottle contains 2 to 4 parts of calcium peroxide nanoparticles and 2 to 4 parts of manganese oxide nanoparticles, preferably one part of the bottle contains 3 parts of calcium peroxide nanoparticles and 3 parts of manganese oxide nanoparticles.

[0009] Furthermore, the calcium peroxide nanoparticles have a particle size of 10-20 nm, and the manganese oxide nanoparticles have a particle size of 10-30 nm.

[0010] A method for preparing the composite silicon-based nanomotor includes the following steps: Polyvinylpyrrolidone (PVP) was dissolved in n-pentanol, and ammonia water containing ethanol and trisodium citrate was added and mixed evenly. Tetrabutyl orthosilicate (TBOS) and 3-chloropropyltrimethoxysilane (CPTMS) were added and mixed evenly. The mixture was reacted at 20-25°C. After the reaction was completed, the mixture was centrifuged and washed to obtain silica hollow nanobottles. The silica hollow nanobottle, calcium peroxide nanoparticles, and manganese oxide nanoparticles were added to a solution, mixed, sonicated, centrifuged, and washed to obtain the composite silicon-based nanomotor.

[0011] Furthermore, the mass ratio of polyvinylpyrrolidone to trisodium citrate is 3000:86.7.

[0012] Furthermore, the volume ratio of tetrabutyl orthosilicate to 3-chloropropyltrimethoxysilane is 10:1.

[0013] Furthermore, the ultrasound duration is 20-60 minutes.

[0014] Furthermore, the method for preparing the composite silicon-based nanomotor includes the following steps: (1) Synthesis of silica hollow nanobottles (SiNBs): 3.0 g PVP (mv=40000) was dissolved in 30.0 mL n-pentanol, and ammonia water containing 3.0 mL ethanol and 98 mg trisodium citrate was added. The mixture was vortexed for three minutes until it was homogeneous. Then 300 μL TBOS and 30 μL CPTMS were added, and the mixture was shaken for 5 minutes. The mixture was then placed on a rotator and reacted for 5 hours at a temperature of 20~25℃ and a rotation speed of 40 rpm. The mixture was then centrifuged and washed with ethanol to obtain SiNBs. (2) Synthesis of composite silicon-based nanomotors (CaO2 / MnO@SiNBs): Mix 2-4 parts of calcium peroxide nanoparticles, 2-4 parts of manganese oxide nanoparticles and 1 part of silica hollow nanobottle evenly by mass and sonicate for 20-60 min. Separate by centrifugation and wash the precipitate with pure water to obtain CaO2 / MnO@SiNBs.

[0015] In this invention, CPTMS molecules hydrolyze when they encounter water droplets under alkaline conditions. Since the hydrolyzed CPTMS molecules are amphiphilic, their hydrophilic Si(O)3 component inserts into the water droplet, while the hydrophobic chloropropyl tail remains in the pentanol, thus forming a thin silica film on the droplet surface. When TBOS migrates from the oil phase to the aqueous phase, it hydrolyzes and condenses on the aforementioned silica film to form a "bowl-shaped" silica with a thicker shell. Under the co-condensation of hydrolyzed CPTMS and TBOS, the bowl-shaped silica is drawn into the hollow silica shell. The hydrolyzed TBOS continuously deposits in the shell from the water side, squeezing out water and creating a new water-oil interface. The co-condensation at the newly formed water-oil interface causes the silica to form a flask-shaped structure with an opening. Therefore, the composite silicon-based nanomotor of this invention has a flask-shaped structure.

[0016] An ultrasound contrast agent is prepared from the above-mentioned composite silicon-based nanomotors.

[0017] The application of the composite silicon-based nanomotor in the preparation of drugs for the treatment or prevention of breast cancer.

[0018] The aforementioned composite silicon-based nanomotor is used as an ultrasound contrast agent.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects: Based on the unique physicochemical properties of the tumor microenvironment, this invention constructs a composite silicon-based nanomotor, which is an oxygen-driven silicon-based bottle-shaped nanomotor (Si-motor). When loaded with MnO and CaO2, the silicon-based nanomotor reacts with excess hydrogen peroxide and acidic ions in the tumor to generate oxygen, autonomously driving the nanoparticles and improving the system's penetration into the tumor. Simultaneously, calcium peroxide induces intracellular Ca...2+ Mitochondrial damage caused by overload, Mn 2+ GSH-mediated depletion and MnO-mediated chemokinetic therapy (CDT) synergistically amplify oxidative stress within tumors, leading to significant cell death. Ultrasound can increase intracellular calcium ion influx, further sensitizing calcium overload and amplifying the oxidative stress effect within tumor cells. This silicon-based nanomotor is stable under physiologically neutral or alkaline conditions and exhibits good biocompatibility. Furthermore, the oxygen microbubbles driving its movement can be visualized under ultrasound and used as an ultrasound contrast agent to monitor the movement of the silicon-based nanomotor within the tumor in real time, achieving integrated diagnosis and therapy. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The image shows the effect of the composite silicon-based nanomotor obtained in Example 1. In the image, A is the particle size distribution curve of the hollow silicon dioxide nanobottle; B is the infrared spectrum of the hollow silicon dioxide nanobottle; C and D are transmission electron microscope images of the composite silicon-based nanomotor loaded with calcium peroxide nanoparticles and manganese oxide nanoparticles, where ① is the nanoparticle loaded inside the hollow silicon dioxide nanobottle, and ② is the nanoparticle outside the hollow silicon dioxide nanobottle. Figure 2 The image shows the effect of the composite silicon-based nanomotor obtained by the method of the present invention. Figure 3 Dissolved oxygen curves in acidic hydrogen peroxide solution for different groups (10 mM calcium peroxide group (CaO2), 5 mM manganese oxide group (MnO), physical mixture of calcium peroxide and manganese oxide group (MnO+CaO2) and silicon nanomotor group containing 10 mM calcium peroxide and 5 mM manganese oxide (i.e., the composite silicon-based motor group of Example 1, Si-motor). Figure 4 The composite silicon-based nanomotor obtained in Example 1 has the motion trajectory and instantaneous velocity in H2O2 solutions of different concentrations. AE represents the motion trajectory under hydrogen peroxide concentrations of 0, 0.01, 0.1, 1, and 10 mM, respectively; FG represents the motion velocity and cumulative motion distance under different hydrogen peroxide concentrations; and H represents the mean square displacement calculated from the motion distance. Figure 5 The permeation effect of the composite silicon-based nanomotor obtained in Example 1 in 3D tumor spheres (200 μm). Figure 6The in vitro antitumor effect of the composite silicon-based nanomotors obtained in Example 1 is shown in Figure A, which represents the survival rate of Mcf-7 cells after incubation with samples of different concentrations under neutral and acidic conditions; Figure B represents the survival rate of Mcf-7 cells after incubation with different samples under pH 6.5 conditions; and Figure C represents the survival rate of normal mouse physiological cells 3T3 cells after co-incubation with composite silicon nanomotors of different concentrations under neutral physiological conditions. Figure 7 The ability of manganese oxide to deplete glutathione; Figure 8 The graph shows the results of the tumor-suppressing experiment of the composite silicon-based nanomotor obtained in Example 1 in tumor-bearing nude mice. Figure 9 The image shows an in vitro ultrasound image of the composite silicon-based nanomotor obtained in Example 1 after injection into a tumor. Figure 10 The image shows an in vivo ultrasound image of the composite silicon-based nanomotor obtained in Example 1 after injection into a tumor. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] In the embodiments of this invention, the number of “parts” refers to the number of parts by mass.

[0027] All raw materials used in this invention were obtained through purchase.

[0028] In this embodiment of the invention, 3T3 refers to the cell line obtained by GJ Todaro et al. from Swiss mouse fetuses, used to identify transformed cells; MCF-7 is human breast cancer cell, and both of the above cell lines were obtained through purchase.

[0029] The effect image of the composite silicon-based nanomotor obtained by the method of this invention is shown in the figure. Figure 2 .

[0030] Example 1 I. Fabrication of Oxygen-Driven Silicon-Based Bottle-Shaped Nanomotors (Si-motors) (1) Synthesis of SiNBs: 3.0 g PVP (mv≈40000) was dissolved in 30.0 mL n-pentanol, and ammonia water (1 mM) containing 3.0 mL ethanol and 86.7 mg trisodium citrate was added. The mixture was vortexed for 3 minutes until it was homogeneous. Then 300 μL TBOS and 30 μL CPTMS were added, and the mixture was shaken for 5 minutes. The mixture was then placed on a rotator and reacted for 5 hours at 25 °C and 40 rpm. The mixture was then centrifuged and washed with ethanol to obtain SiNBs. (2) Synthesis of CaO2 / MnO@SiNBs: According to the mass parts, 3 parts of calcium peroxide nanoparticles (particle size of 15nm), 3 parts of manganese oxide nanoparticles (particle size of 15nm) and 1 part of SiNBs were mixed evenly and sonicated for 40min. After centrifugation, the precipitate was washed with pure water to obtain the composite silicon-based nanomotor CaO2 / MnO@SiNBs, which is the oxygen-driven silicon-based bottle-shaped nanomotor (Si-motor).

[0031] II. Identification of Oxygen-Driven Silicon-Based Bottle-Shaped Nanomotors (Si-motor) The empty silicon-based nanobottles (SiNBs) obtained in Example 1 were diluted, and the particle size was measured using a dynamic light scattering (DLS) instrument. DLS analysis showed that the particle size distribution (PSD) of the silicon-based nanobottles was 400–500 nm. Figure 1 (A); Fourier transform infrared (FTIR) spectrum of -CH2- (1400 cm⁻¹) -1 and 1425 cm -1 The symmetrical bending vibrations of NBs demonstrate that the outer surface of NBs is covered with organic 3-chloropropyl groups. Figure 1 (B). The morphology was characterized using transmission electron microscopy (TEM), which showed that the synthesized silica nanoparticles resembled a flask shape, with an average bottom diameter of 450 ± 100 nm and a neck diameter of 100–200 nm. Figure 1 (C). Due to the hydrophobic outer surface and hydrophilic inner surface of the silicon-based nanobottle, hydrophilic manganese oxide nanoparticles and calcium peroxide nanoparticles can be successfully loaded into the interior of the silicon-based nanobottle under ultrasound via amphiphilic interactions. The lattice of the manganese oxide nanoparticles located on the outside of the silicon-based nanobottle can be clearly distinguished, while the lattice of the manganese oxide nanoparticles loaded inside the silicon-based nanobottle is less distinct. Figure 1 (D), this is mainly due to the electron blocking effect of the silicon shell of the silicon-based nanobottle.

[0032] Example 2 I. Fabrication of Oxygen-Driven Composite Silicon-Based Bottle-Shaped Nanomotors (Si-motors) (1) Synthesis of SiNBs: Weigh 3.0g PVP (mv≈40000) and dissolve it in 30.0mL n-pentanol. Add ammonia water (1mM) solution containing 3.0mL ethanol and 86.7mg trisodium citrate. Vortex the mixture for three minutes until it is homogeneous. Then add 300μL TBOS and 30μL CPTMS. After shaking for 5 minutes, place it on a rotator and react for 5 hours at 20℃ and 40rpm. Centrifuge and wash with ethanol to obtain SiNBs.

[0033] (2) Synthesis of CaO2 / MnO@SiNBs: By mass, 2 parts of calcium peroxide nanoparticles (particle size of 10nm), 2 parts of manganese oxide nanoparticles (particle size of 10nm) and 1 part of SiNBs were mixed evenly and sonicated for 30min. After centrifugation, the precipitate was washed with pure water to obtain the composite silicon-based nanomotor system CaO2 / MnO@SiNBs, which is the oxygen-driven silicon-based bottle-shaped nanomotor (Si-motor).

[0034] II. Identification of Oxygen-Driven Silicon-Based Nanomotors (Si-motors) The empty silicon-based nanobottles (i.e., SiNBs) obtained in Example 2 were diluted, and the particle size was measured using a dynamic light scattering (DLS) instrument. DLS analysis showed that the particle size distribution (PSD) of the silicon nanobottles was 400–500 nm; the Fourier transform infrared (FTIR) spectrum showed that -CH2- (1400 cm⁻¹) -1 and 1425 cm -1The symmetrical bending vibrations of the nanoparticles (NBs) confirmed that the outer surface of the NBs was covered with organic 3-chloropropyl groups. The morphology was characterized using transmission electron microscopy (TEM). The synthesized silica nanoparticles resembled flasks, with an average bottom diameter of 450 ± 100 nm and a neck diameter of approximately 100–200 nm. The amount of hydrophilic manganese oxide and calcium peroxide nanoparticles loaded into the silica nanoflasks was relatively small.

[0035] Example 3 I. Fabrication of Oxygen-Driven Composite Silicon-Based Bottle-Shaped Nanomotors (Si-motors) (1) Synthesis of SiNBs: Weigh 3.0g PVP (mv≈40000) and dissolve it in 30.0mL n-pentanol. Add it to ammonia water (1mM) solution containing 3.0mL ethanol and 86.7mg trisodium citrate. Shake the mixture vigorously until it is homogeneous. Then add 300μL TBOS and 30μL CPTMS. Shake for 5 minutes and place it on a rotator. React for 5 hours at 25℃ and 40rpm. Centrifuge and wash with ethanol to obtain SiNBs.

[0036] (2) Synthesis of CaO2 / MnO@SiNBs: According to the mass parts, 4 parts of calcium peroxide nanoparticles (particle size of 20nm), 4 parts of manganese oxide nanoparticles (particle size of 30nm) and 1 part of SiNBs were mixed evenly and sonicated for 30min. After centrifugation, the precipitate was washed with pure water to obtain the composite silicon-based nanomotor system CaO2 / MnO@SiNBs, which is the oxygen-driven silicon-based bottle-shaped nanomotor (Si-motor).

[0037] II. Identification of Oxygen-Driven Silicon-Based Nanomotors (Si-motors) The empty silicon-based nanobottles (i.e., SiNBs) obtained in Example 3 were diluted, and the particle size was measured using a dynamic light scattering (DLS) instrument. DLS analysis showed that the particle size distribution (PSD) of the silicon nanobottles was 400–500 nm; the Fourier transform infrared (FTIR) spectrum showed that -CH2- (1400 cm⁻¹) -1 and 1425 cm -1 The symmetrical bending vibrations of the nanoparticles (NBs) confirmed that the outer surface of the NBs was covered with organic 3-chloropropyl groups. Their morphology was characterized using transmission electron microscopy (TEM). The synthesized silica nanoparticles resembled a flask shape, with an average bottom diameter of 450 ± 100 nm and a neck diameter of approximately 100–200 nm. The number of hydrophilic manganese oxide and calcium peroxide nanoparticles loaded inside the silica nanoflasks was not significantly different from that in Example 1.

[0038] The following section uses the composite silicon-based nanomotor obtained in Example 1 as an example to conduct performance tests: Application Example 1: Detection of Enhanced Tumor Penetration Capacity through Autonomous Motion of In Vivo and In Vitro Composite Silicon-Based Nanomotors I. Samples and Reference Standards The sample was the composite silicon-based nanomotor of Example 1 (i.e., CaO2 / MnO@SiNBs); the control was an empty silicon-based nanobottle (i.e., SiNBs).

[0039] II. In vitro gas production experiment The synergistic effect of calcium peroxide and manganese oxide nanoparticles reacting with hydrogen peroxide to generate oxygen was studied by comparing the dissolved oxygen values ​​of calcium peroxide nanoparticles alone, manganese oxide nanoparticles, and a silicon-based bottle motor loaded with both nanoparticles in a hydrogen peroxide environment (100 μM). Different groups (10 mM calcium peroxide (CaO2), 5 mM manganese oxide (MnO), a physical mixture of calcium peroxide and manganese oxide (MnO+CaO2), and a silicon nanomotor group containing 10 mM calcium peroxide and 5 mM manganese oxide (i.e., the composite silicon-based motor group of Example 1, Si-motor)) were thoroughly mixed with hydrogen peroxide. After sealing the liquid surface with oil, the oxygen content of the reaction solution was measured using a dissolved oxygen meter for 15 minutes. Figure 3 As shown, compared with the individual manganese oxide nanoparticle groups and the individual calcium peroxide nanoparticle groups, the composite silicon-based motor group exhibits a significantly higher dissolved oxygen content and demonstrates rapid and sustained oxygen release behavior. Furthermore, the oxygen generation efficiency of the physical mixture of manganese oxide and calcium peroxide nanoparticles is lower, indicating that under hydrogen peroxide conditions, the release of oxygen by the Janus-structured composite silicon nanomotor can drive its autonomous movement, leading to enhanced diffusion.

[0040] Among them, the 10mM calcium peroxide group refers to the hydrogen peroxide solution containing 10mM calcium peroxide obtained by directly adding calcium peroxide nanoparticles to hydrogen peroxide. The 5mM manganese oxide group refers to the hydrogen peroxide solution containing 5mM manganese oxide obtained by directly adding manganese oxide nanoparticles to hydrogen peroxide. The physical mixture of calcium peroxide and manganese oxide refers to the direct addition of manganese oxide nanoparticles and calcium peroxide nanoparticles to hydrogen peroxide to obtain a hydrogen peroxide solution containing 5 mM manganese oxide and 10 mM calcium peroxide.

[0041] III. External Motion Capacity Testing After confirming the successful synthesis of the composite silicon-based nanomotors (i.e., CaO2 / MnO@SiNBs of Example 1), the motion trajectories of the composite silicon-based nanomotors were observed and photographed under a Nikon Ti2-A inverted optical microscope equipped with a high-speed camera (sCMOS) and NIS Elements AR3.2 software. Motion behavior was recorded for 10 s, and at least 40 nanomotors were analyzed under each hydrogen peroxide concentration condition. The motion trajectories and motion characteristics of the composite silicon nanomotors in the video were manually tracked and analyzed using the ImageJ plugin, including average velocity and cumulative motion distance. The mean square displacement (MSD) was calculated by Equation (1), where Δt is the time interval (in s), x is the lateral distance of particle movement in the ImageJ trajectory analysis (in μm), and y is the longitudinal distance of movement (in μm). Without the addition of hydrogen peroxide, the composite silicon-based nanomotors exhibited significant Brownian motion, while with the gradual increase of hydrogen peroxide substrate concentration (0, 0.01, 0.1, 1, and 10 mM), the diffusion of the composite silicon-based nanomotors was significantly enhanced (e.g., ...). Figure 4 (Middle AE). When the hydrogen peroxide substrate concentration increased to 10 mM, the average velocity of the nanomotor reached 3.8 μm / s, which was 1.7 times higher than that without hydrogen peroxide. Figure 4 (Middle F), and the distance traveled is also longer ( Figure 4 (G). Meanwhile, with increasing hydrogen peroxide substrate concentration, the MSD of the composite silicon-based nanomotors increases (G). Figure 4 (H), demonstrating that its autonomous motility can greatly enhance diffusion efficiency in the hydrogen peroxide-rich tumor microenvironment, thereby improving its penetration into the deep tumor.

[0042] (1) IV. 3D Tumor Sphere Permeability Test Two cell lines, 3T3 and MCF-7 (3T3:MCF-7 = 5:1, cell ratio), were mixed at a density of 1200 cells / 100 μL and seeded into U-shaped 96-well plates. After centrifugation at 3500 rpm for 5 min, the plates were incubated. After 4-5 days of incubation, tumor spheres with a diameter of 400 µm were selected and co-incubated with Nile Red-labeled composite silicon motors and empty silicon nanoflasks at pH 6.5 and pH 7.4, respectively, using 100 μM hydrogen peroxide. After 24 h, the cells were washed twice with PBS, and the penetration of different groups of materials was observed using confocal fluorescence microscopy. Figure 5As shown, under the condition of hydrogen peroxide (H2O2) at pH=6.5, the fluorescence spectra of MTSs at the 0-100 μm cross section after being penetrated by empty silicon-based nanobottles and composite silicon-based nanomotors show that the empty silicon-based nanobottles have a weak fluorescence signal on the cross section, while the composite silicon-based nanomotors can be observed to have a significant fluorescence intensity at the same depth. This is because the H2O2 substrate in the acidic environment drives the autonomous movement of the nanomotors, which can improve the penetration ability of the composite silicon-based nanomotors into three-dimensional tumor spheres.

[0043] Application Example 2: Detection of in vitro tumor suppression effect I. Sample Synthesis The sample is the oxygen-driven silicon-based bottle motor of Example 1.

[0044] II. CCK-8 assay for cytotoxicity The cytotoxicity of samples with different concentrations (calcium peroxide at concentrations of 0, 40, 80, 120, and 160 μg / mL, and manganese oxide at concentrations of 0, 20, 40, 60, and 8 μg / mL, including calcium peroxide-only treatment, manganese oxide-only treatment, and composite silicon nanomotors) under acidic or neutral pH conditions was evaluated using the CCK-8 assay. A density of 1×10⁻⁶ samples was used. 4 100 μL of MCF-7 cells were seeded in 96-well plates (n=3) and co-incubated with different samples for 24 hours. Figure 6 As shown in Figure A, no cytotoxicity was observed in any group at pH 7.4. However, at pH 6.5, the MCF-7 cell viability of the group co-incubated with calcium peroxide nanoparticles for 24 h was significantly affected, indicating that calcium peroxide nanoparticles exhibit pH responsiveness. The composite silicon-based nanomotor loaded with calcium peroxide and manganese oxide nanoparticles showed the best ability to inhibit cell viability, which is attributed to the synergistic effect of calcium peroxide and manganese oxide amplifying the oxidative stress effect within tumor cells. Furthermore, the effects of different treatment groups at pH 6.5 on cell viability were investigated. Figure 6 As shown in Figure B, the physical mixture of calcium peroxide and manganese oxide had no significant inhibitory effect on cell viability. The composite silicon-based nanomotors with motility significantly increased cytotoxicity, while the composite silicon-based nanomotor incubation combined with ultrasound irradiation showed the strongest cell-killing effect. Normal human cells, under the same treatment, were able to tolerate the adverse effects of the composite silicon-based nanomotors, and their cell survival rate was much higher than that of tumor cells. Figure 6 (C)

[0045] III. Detection of Glutathione Depletion Capacity of Manganese Oxide by DTNB Method The Ellman method was used to determine the GSH-consuming capacity of MnO and the conventional Fenton reaction catalyst FeCl2. MnO (34 mM, 1 mL) and FeCl2 (34 mM, 1 mL) were separately dispersed in PBS containing 2 mM GSH and shaken at 37°C for 30 min. After centrifugation to remove the supernatant, 10 μL of DTNB (DMSO, 100 mM) was added, and the absorbance of 5-thio-2-nitrobenzoic acid (the reaction product of DTNB and GSH) at 410 nm was measured using UV-Vis absorption spectroscopy. Tubes containing only GSH (2 mM) served as the negative control group. Figure 7 As shown, MnO nanoparticles consume GSH more significantly than FeCl2, indicating that MnO has higher catalytic performance as a Fenton reaction catalyst than traditional ferrous ions.

[0046] Application Example 3: In vivo tumor suppression effect detection I. Sample Preparation The sample is the composite silicon-based nanomotor (i.e., CaO2 / MnO@SiNBs) of Example 1.

[0047] II. Establishment of Tumor-Bearing Animal Models The experimental animals used were female BALB / c nude mice. MCF-7 cells (5 × 10⁻⁶) were suspended in 50 μL PBS. 6 MCF-7 tumor model was established by subcutaneous injection into the second mammary fat pad of BALB / c nude mice. The tumor was allowed to grow to 150 mm. 3 Treatment was subsequently administered.

[0048] III. Experimental Grouping and Processing Methods 1. Experimental Grouping The sample was the composite silicon-based nanomotor from Example 1. Mice were randomly assigned to the following groups: (I) saline group (PBS); (II) saline combined with ultrasound irradiation group (US); (III) calcium peroxide group (CaO2); (IV) manganese oxide group (MnO); (V) physical mixture of calcium peroxide and manganese oxide group (MnO+CaO2); (VI) composite silicon-based nanomotor group (Si-motor); (VII) composite silicon-based nanomotor combined with ultrasound irradiation group (Si-motor+US). n=5.

[0049] 2. Ultrasonic irradiation method 24 hours after the drug was injected via the tail vein, an appropriate amount of coupling agent was applied to the tumor site of the tumor-bearing nude mouse to eliminate the air between the ultrasound therapy probe and the skin of the nude mouse. The probe was then fixed to the tumor of the nude mouse and continuously irradiated with ultrasound for 10 minutes.

[0050] IV. Tumor Suppression Experiment Each experimental group was treated with an equal amount of calcium peroxide or manganese oxide (20 mg / kg Ca, 10 mg / kg Mn per nude mouse body weight), or 200 μL of physiological saline per tumor-bearing nude mouse, administered every two days. Tumor volume was measured every two days during treatment. The inhibitory effect of each treatment group on mammary tumors in nude mice was statistically analyzed. Results showed that the composite silicon-based nanomotor combined with ultrasound irradiation group exhibited the best tumor-inhibiting effect, with tumor volume shrinking and growth inhibited after treatment. The synergistic anti-tumor effect was significantly superior to the non-nanomotor group and the calcium peroxide and manganese oxide single treatment groups. Results are as follows: Figure 8 As shown.

[0051] Application Example 4: Detection of In Vivo and In Vitro Ultrasound Imaging Effects I. Sample Preparation The sample is the composite silicon-based nanomotor of Example 1.

[0052] II. External Ultrasound Imaging In vitro ultrasound imaging was performed in B-mode using an 18 MHz probe. A water-filled balloon model was used to simulate the acidic tumor microenvironment (pH=6.5, hydrogen peroxide levels of 0 mM, 0.1 mM, 1 mM, and 10 mM). A composite silicon-based nanomotor was injected into the balloon via a three-way valve, and changes in the ultrasound echo signal were then recorded. Figure 9 It can be seen that the higher the hydrogen peroxide concentration, the more oxygen is generated, and the stronger the echo signal. Even in a 0.1 mM hydrogen peroxide solution, the echo signal of oxygen bubbles can be clearly detected. In summary, the oxygen generation of composite silicon-based nanomotors in the tumor microenvironment is detectable under ultrasound, and it holds promise for real-time monitoring of motor delivery within tumors.

[0053] III. In vivo ultrasound imaging A balb / c nude mouse MCF-7 breast tumor model was established (same as application example 4), and the tumor was allowed to grow to 80 mm. 3 Ultrasound imaging was then performed. 50 μL of PBS and 50 μL of a composite silicon-based nanomotor were injected into the tumor, respectively. Figure 10 As can be seen, no obvious echo signal was observed within the tumor after PBS injection, while a significant hyperechoic signal was observed within the tumor after injection of the composite silicon-based nanomotor. Subsequently, the hyperechoic signal diffused throughout the entire tumor. This also demonstrates the potential of the composite silicon-based nanomotor as an ultrasound contrast agent.

[0054] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A composite silicon-based nanomotor, characterized in that, This includes hollow silica nanobottles and calcium peroxide nanoparticles and manganese oxide nanoparticles loaded inside the bottles; The outer wall of the silica hollow nanobottle is hydrophobic, and the inner wall is hydrophilic. Based on mass fractions, one bottle contains 2-4 parts of calcium peroxide nanoparticles and 2-4 parts of manganese oxide nanoparticles.

2. The composite silicon-based nanomotor according to claim 1, characterized in that, The silica hollow nanobottle includes a bottle body with a hollow chamber and a narrow neck. The average diameter of the bottle body is 450±100nm, and the diameter of the bottleneck is 100~200nm.

3. The composite silicon-based nanomotor according to claim 1, characterized in that, The calcium peroxide nanoparticles have a particle size of 10-20 nm, and the manganese oxide nanoparticles have a particle size of 10-30 nm.

4. A method for preparing a composite silicon-based nanomotor according to any one of claims 1 to 3, characterized in that, Includes the following steps: Polyvinylpyrrolidone was dissolved in n-pentanol, and ammonia water containing ethanol and trisodium citrate was added and mixed evenly. Tetrabutyl orthosilicate and 3-chloropropyltrimethoxysilane were added and mixed evenly. The mixture was reacted at 20~25℃. After the reaction was completed, the mixture was centrifuged and washed to obtain hollow silica nanobottles. The silica hollow nanobottle, calcium peroxide nanoparticles, and manganese oxide nanoparticles were added to a solution, mixed, sonicated, centrifuged, and washed to obtain the composite silicon-based nanomotor.

5. The method for preparing the composite silicon-based nanomotor according to claim 4, characterized in that, The mass ratio of polyvinylpyrrolidone to trisodium citrate is 3000:86.

7.

6. The method for preparing the composite silicon-based nanomotor according to claim 4, characterized in that, The volume ratio of tetrabutyl orthosilicate to 3-chloropropyltrimethoxysilane is 10:

1.

7. An ultrasound contrast agent, characterized in that, It is prepared from the composite silicon-based nanomotor described in any one of claims 1 to 3.

8. The use of the composite silicon-based nanomotor according to any one of claims 1 to 3 in the preparation of drugs for treating breast cancer.

9. The use of the composite silicon-based nanomotor according to any one of claims 1 to 3 in the preparation of ultrasound contrast agents.

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