Glucose oxidase / simvastatin magnetic nanomotor and preparation method and application thereof

Abstract

The application discloses a preparation method of glucose oxidase / simvastatin magnetic nanomotor, and steps are as follows: FeCl3.6H2O is added into deionized water and stirred, sodium citrate, urea and PAM are added, and after stirring, reaction is carried out in a Teflon autoclave to obtain Fe3O4 nano solution; PAA and ammonia water are added and stirred, and then isopropyl alcohol is added dropwise to obtain Fe3O4-PAA nano solution; tetraethyl orthosilicate is added and stirred, and then washing is carried out to obtain Fe3O4-PAA / mSiO2 nano solution; simvastatin is mixed with the nano solution to obtain Sim-loaded Fe3O4-PAA / mSiO2 nanoparticles; EDC and NHS solution are added and stirred, and then glucose oxidase is added, and finally glucose oxidase-simvastatin magnetic nanomotor is obtained; the method utilizes the driving of the magnetic nanomotor in the magnetic field and the catalytic reaction of GOx, enhances the targeting and permeability of the drug in the tumor, and the loaded simvastatin can affect the lipid metabolism of tumor cells and inhibit glucose uptake, and compared with the use of GOx alone, the anti-tumor activity is significantly improved.

Description

Technical Field

[0001] This invention relates to a glucose oxidase / simvastatin magnetic nanomotor, its preparation method, and its application. Background Technology

[0002] If tumor cells cannot obtain sufficient oxygen and nutrients to sustain their rapid growth, they will die due to lack of energy support; this process is known as "tumor starvation therapy." Since most tumor cells have a significantly faster metabolic rate than normal cells, and glucose is the primary energy source for tumor cell growth and metabolism, tumor cells are highly sensitive to changes in intracellular glucose concentration. Glucose deficiency can activate metabolism and related signaling pathways, leading to cell death. Due to the complexity, diversity, and heterogeneity of tumors, single glucose oxidase starvation therapy is unlikely to be an effective clinical treatment strategy. Therefore, multimodal synergistic therapies based on glucose oxidase will be an important research focus in the future.

[0003] Combining glucose oxidase with hypoxia-activated prodrugs can achieve synergistic therapeutic effects of tumor starvation therapy and hypoxia-activated therapy. Researchers have innovatively developed a new tumor treatment strategy by combining glucose oxidase-based starvation therapy with AQ4N-based hypoxia-activated therapy using liposome nanocarriers. Furthermore, glucose oxidase catalyzes the production of gluconic acid and hydrogen peroxide from glucose, showing potential applications in the synergistic treatment of tumor starvation and oxidation therapies. Researchers have reported a glucose-reactive nanodrug composed of glucose oxidase nanogels, which can regulate hydrogen peroxide production and can be used for starvation and oxidation therapy in melanoma. Photothermal therapy is a method of treating tumors by converting near-infrared light energy into heat energy using photothermal agents. Its therapeutic effect is not affected by the hypoxic tumor microenvironment; therefore, researchers have used photothermal agents as nanocarriers to deliver glucose oxidase to tumor tissue to improve therapeutic efficacy. Photodynamic therapy has attracted much attention due to its ability to directly irradiate tumor sites and its relatively low invasiveness. However, glucose oxidation mediated by glucose oxidase can increase hypoxia in tumor tissue, which is detrimental to photodynamic therapy. Therefore, some researchers have combined glucose oxidase and catalase. Catalase can promote the decomposition of endogenous hydrogen peroxide to produce oxygen, thereby promoting the decomposition of glucose by glucose oxidase and accelerating the production of cytotoxic reactive oxygen species under light conditions. The combination of the two has the ability to form a cascade biological reaction and can be used for synergistic treatment of starvation therapy and photodynamic therapy.

[0004] Currently, there is still a long way to go before glucose oxidase (GOx)-based cancer therapies can be truly applied clinically and solve existing challenges in tumor treatment. First, reducing the dosage of glucose oxidase, eliminating adverse reactions caused by high-dose nanomedicines, and exploring new treatment strategies are all issues that need to be addressed. Second, while glucose oxidase is inherently biocompatible and non-toxic, and several studies have demonstrated the biosafety of currently used glucose oxidase nanomedicines, it is still a protein and therefore has several drawbacks, such as poor stability and a short half-life in vivo. Furthermore, simvastatin, a commonly used lipid-lowering drug, has been shown to have some anti-tumor potential, but the exact mechanism is still unclear, and its use alone is unlikely to achieve ideal anti-tumor effects. Therefore, designing an appropriate carrier is crucial for achieving sustained delivery and precise control of glucose oxidase and simvastatin release, while also further enhancing the response to traditional anticancer therapies. Summary of the Invention

[0005] The purpose of this invention is to provide a glucose oxidase / simvastatin magnetic nanomotor, its preparation method, and its application. On the one hand, glucose oxidase is loaded onto magnetic nanoparticles through a condensation reaction, preserving the bioactivity of GOx. On the other hand, the magnetic nanomotor is driven in a magnetic field, and GOx provides driving force to the motor through catalytic reactions in the tumor microenvironment, promoting drug penetration into tumor tissue and enhancing drug targeting. Simultaneously, the loaded simvastatin can not only affect the lipid metabolism of tumor cells but also inhibit the uptake of glucose by tumor cells. This dual intervention of GOx and tumor cell energy metabolism significantly enhances the anti-tumor activity of GOx compared to glucose oxidase starvation therapy alone.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A method for preparing a glucose oxidase / simvastatin magnetic nanomotor includes the following steps:

[0008] 1) Add FeCl3·6H2O to deionized water and stir magnetically for 5-15 minutes. Then mix the suspension with sodium citrate, urea and PAM, and stir for another 20-40 minutes. Transfer the mixture to a Teflon autoclave and seal it. Keep it at 190-210℃ for 22-26 hours, and then let it cool naturally at room temperature. Wash it several times with deionized water and ethanol. After centrifugation, disperse the precipitate in deionized water to obtain a hollow mesoporous Fe3O4 nano solution.

[0009] 2) Add PAA solution and ammonia water to Fe3O4 nano solution respectively, and stir mechanically for 20-40 minutes; then slowly add isopropanol dropwise using a separatory funnel, and continue stirring for 20-40 minutes to obtain Fe3O4-PAA nano solution for the next step of the experiment;

[0010] 3) Add tetraethyl orthosilicate to the Fe3O4-PAA nano solution and continue stirring for 22-26 hours; wash with deionized water 2-4 times, centrifuge and resuspend in deionized water to obtain Fe3O4-PAA / mSiO2 nano solution;

[0011] 4) The lipid-soluble drug simvastatin was dissolved in an organic solvent to form a simvastatin solution, which was then mixed with the nanoparticle solution obtained in step 3). The mixture was stirred evenly in a metal bath, and the precipitate was collected after centrifugation to obtain Sim-loaded Fe3O4-PAA / mSiO2 nanoparticles.

[0012] 5) Resuspend the precipitate obtained in step 4) in buffer solution, add EDC and NHS solutions respectively, and stir for 20-40 minutes;

[0013] 6) Add glucose oxidase to deionized water and vortex to dissolve it to obtain an enzyme solution;

[0014] 7) Add the enzyme solution obtained in step 6) to the mixture in step 5), continue stirring for 3-5 hours, centrifuge, and resuspend the precipitate in deionized water to obtain glucose oxidase-simvastatin magnetic nanomotor.

[0015] Preferably, in step 1), the concentrations of FeCl3·6H2O, sodium citrate, and urea are 0.05M, 0.10M, and 0.15M, respectively; the centrifugation speed is 8000-10000 rpm, and the time is 10-20 minutes; magnetic separation can also be used.

[0016] Preferably, in step 2), the mass concentration of the Fe3O4 nanosolution is 0.5 mg / mL, and the volume ratio of isopropanol to nanoparticle suspension is 29:1.

[0017] Preferably, in step 3), the centrifugation speed is 8000-10000 rpm and the time is 10-20 minutes, or magnetic separation can also be used.

[0018] Preferably, the organic solvent mentioned in step 4) is one or a combination of two or more of acetonitrile, ethanol, methanol, acetone, and dimethyl sulfoxide; the mass ratio of simvastatin to nanoparticles is 1:4 to 1:5; the metal bath temperature is 37°C, the rotation speed is 800 to 1000 rpm, and the time is 2 to 12 hours; the centrifugation speed is 8000 to 10000 rpm, and the time is 10 to 20 minutes.

[0019] Preferably, the EDC and NHS concentrations in step 5) are 5 mg / mL, and the mass ratio to the nanoparticles is 1:1 to 1:1.5. Other carboxylic acid activators can also be used. The buffer solution is phosphate buffer or 2-morpholine ethanesulfonic acid buffer with a pH of 6.0 to 6.2. The metal bath temperature is 37°C, the rotation speed is 800 to 1000 rpm, and the time is 0.5 to 2 hours.

[0020] Preferably, in step 6), the concentration of the enzyme solution is 0.1–10 mg / mL.

[0021] Preferably, in step 7), the mass ratio of glucose oxidase to nanoparticles is 1:0.2 to 1:0.5; the metal bath temperature is 37°C, the rotation speed is 800 to 1000 rpm, and the time is 2 to 4 hours; the centrifugation speed is 8000 to 10000 rpm, and the time is 10 to 20 minutes.

[0022] Another technical problem to be solved by the present invention is to provide a glucose oxidase / simvastatin magnetic nanomotor prepared by the method described in any of the above-mentioned methods, wherein the simvastatin loading rate in the glucose oxidase / simvastatin magnetic nanomotor is 15% to 25%, the glucose oxidase content is 20% to 35%, and the glucose oxidase / simvastatin magnetic nanomotor can reach a speed of 20 to 25 μm / s when the magnetic field frequency is 25 Hz.

[0023] Another technical problem to be solved by the present invention is to provide a glucose oxidase / simvastatin magnetic nanomotor prepared by the method described in any of the above-mentioned methods, or the application of the above-mentioned glucose oxidase / simvastatin magnetic nanomotor in the preparation of a tumor treatment drug, wherein the tumor cells are 4T1 breast cancer cells.

[0024] The beneficial effects of this invention are:

[0025] The nanomotor prepared by this method has good driving force in a magnetic field. At a magnetic field frequency of 25 Hz, the speed can reach 20-25 μm / s and has good directionality. At the same time, glucose oxidase can provide driving force for the nanomotor through catalytic reaction in the tumor microenvironment, enhancing self-diffusion motion. This dual-driven nanomotor, as a carrier, can enhance its ability to target lesion sites and promote drug penetration in tumor tissue.

[0026] Ferric oxide nanoparticles with a hollow mesoporous structure can serve as carriers for the drug simvastatin. In addition to affecting the energy supply of tumor cells by reducing cholesterol production, simvastatin can also inhibit glucose uptake by tumor cells by blocking glucose transport and glucose metabolism. At the same time, simvastatin exerts anti-tumor effects by inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and inhibiting tumor cell invasion and migration.

[0027] Glucose oxidase modified on the outer surface of nanocarriers can consume glucose in tumor tissue, further blocking the energy supply of tumor cells and exhibiting a highly efficient tumor-suppressing effect.

[0028] Through a relatively simple preparation process, a nanomotor with excellent targeting and multiple combined tumor treatment effects was obtained, minimizing the shortcomings of single treatment and ultimately achieving its highly efficient synergistic treatment of tumors. Attached Figure Description

[0029] Figure 1 This is a transmission electron microscope image of hollow mesoporous Fe3O4NPs;

[0030] Figure 2 This is a scanning electron microscope image of the FPS-Sim-GOx magnetic nanomotor;

[0031] Figure 3 It shows the motion trajectory of the FPS-Sim-GOx magnetic nanomotor at different magnetic field frequencies;

[0032] Figure 4 The movement speed of the FPS-Sim-GOx magnetic nanomotor at different magnetic field frequencies;

[0033] Figure 5 It is the mean square displacement (MSD) of the FPS-Sim-GOx magnetic nanomotor at different magnetic field frequencies;

[0034] Figure 6 It is the enzyme chemotaxis of the FPS-Sim-GOx magnetic nanomotor;

[0035] Figure 7 This is a graph showing the cytotoxicity of CCK-8 cells treated with different drug administration groups.

[0036] Figure 8 It is the permeation effect of FPS-Sim-GOx magnetic nanomotors on 3D tumor cell spheres in different glucose concentrations;

[0037] Figure 9 It is the permeation effect of FPS-Sim-GOx magnetic nanomotors on 3D tumor cell spheres in different magnetic field frequencies. Specific implementation methods

[0038] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. Example

[0040] A method for fabricating an FPS-Sim-GOx magnetic nanomotor includes the following steps:

[0041] (1) Add 0.4055g FeCl3·6H2O to 30mL (0.05M) deionized water and stir magnetically for 10 minutes. Then add 0.8823g sodium citrate (0.10M), 0.2702g urea (0.15M) and 0.3000g PAM in sequence and stir vigorously for another 30 minutes. Transfer the mixture to a 50mL Teflon autoclave and seal it. Keep it at 200℃ for 24 hours. Then cool it naturally at room temperature. Centrifuge the solution at high speed (10000rpm, 10 minutes) to collect the precipitate. Wash it with ethanol and deionized water in sequence. Finally disperse it in deionized water to obtain a hollow mesoporous Fe3O4NPs solution (0.5mg / mL).

[0042] (2) 80 μL of PAA solution (0.2 g / mL) and 100 μL of ammonia (5 mol / L) were added to 10 mL of the above Fe3O4 nanoparticle solution, and mechanically stirred at room temperature for 30 minutes. Then, 290 mL of isopropanol (1:29) was slowly added dropwise using a separatory funnel, and stirring was continued for 30 minutes to obtain Fe3O4-PAA nanoparticles. 450 μL of tetraethyl orthosilicate was added to the above system, and stirring was continued for 24 h. The solution was centrifuged (10000 rpm, 10 min), the precipitate was collected, washed three times with deionized water, and then resuspended in deionized water to obtain FPS nanoparticle solution (4 mg / mL).

[0043] (3) Dissolve 8 mg of simvastatin in 1 mL of acetonitrile, mix with 500 μL of the above Fe3O4-PAA / mSiO2 nanoparticle solution, stir in a metal bath for 4 hours (1000 rpm, 37 °C), centrifuge (10000 rpm, 10 min) to collect the precipitate, resuspend in 2 mL of deionized water to obtain FPS-Sim nanoparticle solution (1 mg / mL);

[0044] (4) Add 1 mg of glucose oxidase to 1 mL of deionized water and vortex to dissolve to obtain an enzyme solution of 1 mg / mL; weigh 5 mg of EDC and NHS and add them to 1 mL of deionized water to obtain EDC and NHS solutions of 5 mg / mL; take 1 mL of FPS-Sim nanoparticle solution, centrifuge (10000 rpm, 10 min) and resuspend in 1 mL of phosphate buffer (pH = 6.0), add 200 μL of EDC and NHS solutions respectively, stir in a metal bath for 30 min (1000 rpm, 37 °C), add 400 μL of glucose oxidase solution, and continue stirring for 4 hours to obtain FPS-Sim-GOx magnetic nanomotors.

[0045] Example 2

[0046] A method for fabricating an FPS-Sim-GOx magnetic nanomotor includes the following steps:

[0047] (1) Add 0.3500g FeCl3·6H2O to 35mL of 0.04M deionized water and stir magnetically for 15 minutes. Then add 0.8000g sodium citrate (0.08M), 0.2500g urea (0.12M) and 0.2500g PAM in sequence and stir vigorously for 40 minutes. Transfer the mixture to a 50mL Teflon autoclave and seal it. Keep it at 180℃ for 20 hours and then let it cool naturally at room temperature. Collect the precipitate by high-speed centrifugation (10000rpm, 12 minutes), wash it with ethanol and deionized water in sequence, and finally disperse it in deionized water to obtain a hollow mesoporous Fe3O4NPs solution (0.6mg / mL).

[0048] (2) Add 90 μL of PAA solution (0.18 g / mL) and 120 μL of ammonia (4 mol / L) to 10 mL of Fe3O4 nanoparticle solution and stir at room temperature for 40 minutes; slowly add 250 mL of isopropanol (1:25) dropwise using a separatory funnel and continue stirring for 40 minutes to obtain Fe3O4-PAA nanoparticles; add 350 μL of tetraethyl orthosilicate to the above system and continue stirring for 22 hours; collect the precipitate after centrifugation (10000 rpm, 12 minutes), wash three times with deionized water, and then resuspend in deionized water to obtain FPS nanoparticle solution (5 mg / mL);

[0049] (3) Dissolve 10 mg simvastatin in 1.2 mL acetonitrile, mix with 600 μL of the above Fe3O4-PAA / mSiO2 nanoparticle solution, and place in a metal bath and stir for 5 hours (1100 rpm, 40 °C); centrifuge (10000 rpm, 12 min) to collect the precipitate, and resuspend in 2.5 mL of deionized water to obtain FPS-Sim nanoparticle solution (1.2 mg / mL);

[0050] (4) Dissolve 1.2 mg glucose oxidase in 1.5 mL of deionized water and vortex to obtain an enzyme solution of 1 mg / mL; weigh 6 mg EDC and 6 mg NHS and add them to 1.2 mL of deionized water to obtain EDC and NHS solutions of 6 mg / mL; take 1.2 mL of FPS-Sim nanoparticle solution, centrifuge (10000 rpm, 12 min) and resuspend in 1.2 mL of phosphate buffer (pH = 6.5), add 250 μL of EDC and NHS solutions respectively, stir in a metal bath for 40 min (1100 rpm, 40 °C); add 500 μL of glucose oxidase solution and continue stirring for 5 hours to finally obtain FPS-Sim-GOx magnetic nanomotors.

[0051] Example 3

[0052] A method for fabricating an FPS-Sim-GOx magnetic nanomotor includes the following steps:

[0053] (1) Add 0.3000g FeCl3·6H2O to 40mL of 0.06M deionized water and stir magnetically for 20 minutes. Then add 1.0000g sodium citrate (0.12M), 0.3000g urea (0.18M) and 0.3500g PAM in sequence and stir vigorously for 45 minutes. Transfer the mixture to a 50mL Teflon autoclave and seal it. Keep it at 190℃ for 22 hours and then let it cool naturally at room temperature. Collect the precipitate by high-speed centrifugation (10000rpm, 12 minutes), wash it with ethanol and deionized water in sequence, and finally disperse it in deionized water to obtain a hollow mesoporous Fe3O4NPs solution (0.7mg / mL).

[0054] (2) Add 100 μL of PAA solution (0.22 g / mL) and 150 μL of ammonia water (4.5 mol / L) to 10 mL of Fe3O4 nanoparticle solution and stir at room temperature for 50 minutes; slowly add 280 mL of isopropanol (1:27) dropwise using a separatory funnel and continue stirring for 50 minutes to obtain Fe3O4-PAA nanoparticles; add 400 μL of tetraethyl orthosilicate to the above system and continue stirring for 20 hours; collect the precipitate after centrifugation (10000 rpm, 12 minutes), wash three times with deionized water, and then resuspend in deionized water to obtain FPS nanoparticle solution (6 mg / mL);

[0055] (3) Dissolve 12 mg simvastatin in 1.5 mL acetonitrile, mix with 700 μL of the above Fe3O4-PAA / mSiO2 nanoparticle solution, and place in a metal bath and stir for 6 hours (1200 rpm, 42 °C); centrifuge (10000 rpm, 12 min) to collect the precipitate, and resuspend in 3 mL of deionized water to obtain FPS-Sim nanoparticle solution (1.5 mg / mL);

[0056] (4) Dissolve 1.5 mg glucose oxidase in 1.5 mL deionized water and vortex to obtain an enzyme solution of 1 mg / mL; weigh 7 mg EDC and 7 mg NHS and add them to 1.5 mL deionized water to obtain EDC and NHS solutions of 7 mg / mL; take 1.5 mL of FPS-Sim nanoparticle solution, centrifuge (10000 rpm, 12 min) and resuspend in 1.5 mL of phosphate buffer (pH = 6.2), add 300 μL of EDC and NHS solutions respectively, stir in a metal bath for 50 min (1200 rpm, 42 °C); add 600 μL of glucose oxidase solution and continue stirring for 6 hours to finally obtain FPS-Sim-GOx magnetic nanomotors.

[0057] exist Figure 1 The image shows a transmission electron microscope (TEM) image of hollow mesoporous Fe3O4 nanoparticles (Fe3O4NPs). This image demonstrates that Fe3O4NPs possess a hollow structure and excellent mesoporous properties, providing a suitable material for the subsequent construction of nanomotors.

[0058] The Fe3O4NPs prepared by the above method were further processed to obtain FPS-Sim-GOx magnetic nanomotors. Figure 2 These are scanning electron microscope (SEM) images of FPS-Sim-GOx magnetic nanomotors, showing the morphology and surface structure of these magnetic nanomotors and verifying the morphology and distribution of nanoparticles during the preparation process.

[0059] Experimental Example 1

[0060] Self-driving behavior of an FPS-Sim-GOx magnetic nanomotor in a magnetic field:

[0061] The FPS-Sim-GOx magnetic nanomotor was resuspended in PBS buffer. A custom-designed five-coil magnetic field generator was used as the magnetocontrol system. The motor's motion was observed under an inverted fluorescence microscope at magnetic field frequencies of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 Hz. The recorded motion video of the motor was analyzed using ImageJ and Chemotaxis software to track the motor's trajectory and calculate the mean square displacement (MSD).

[0062] Figure 3 The figure shows the motion trajectory of the FPS-Sim-GOx magnetic nanomotor under different magnetic field frequencies. This demonstrates that the FPS-Sim-GOx magnetic nanomotor exhibits different motion modes under the influence of magnetic fields of different frequencies, enabling it to achieve a certain degree of self-driven motion. Figure 4 The study further demonstrated the motion speed of the FPS-Sim-GOx magnetic nanomotor under different magnetic field frequencies. It can be seen that changes in magnetic field frequency significantly affect the motion speed of the nanomotor; as the frequency increases, the motor speed changes, providing a better basis for motion control. Figure 5 This demonstrates the mean square displacement (MSD) of the FPS-Sim-GOx magnetic nanomotor under different magnetic field frequencies. By analyzing the motion trajectory, the MSD variation of the nanomotor at different frequencies is obtained, further proving the influence of magnetic field frequency on the motor's motion characteristics.

[0063] Experimental Example 2

[0064] Enzymatic chemotaxis of an FPS-Sim-GOx magnetic nanomotor:

[0065] A three-inlet, one-outlet microfluidic device was used. The three inlet channels, from top to bottom, contained 5mM glucose solution, FPS-Sim-GOx magnetic nanomotor solution, and PBS buffer solution. The solution was delivered by an injection pump to simulate blood flow in capillaries in vivo. The middle channel was observed using an inverted microscope, and the chemotactic displacement of the nanomotor was determined by the change in fluorescence intensity perpendicular to the flow direction compared with the control group.

[0066] Figure 6 The enzymatic chemotaxis of the FPS-Sim-GOx magnetic nanomotor was demonstrated. The chemotactic behavior of the nanomotor in glucose solution was observed using a microfluidic device simulating blood flow. The changes in fluorescence intensity clearly show that the FPS-Sim-GOx magnetic nanomotor can chemotactically move towards the target site under the influence of glucose solution, demonstrating its enzyme-catalyzed chemotactic properties.

[0067] Experimental Example 3

[0068] Detection of tumor-killing effects of FPS-Sim-GOx magnetic nanomotors at the 2D cellular level:

[0069] 4T1 breast cancer cells were used as model cells. After resuscitation, the cells were passaged three times and then digested. A cell suspension at a density of 1 × 10⁵ cells / mL was added to 96-well plates and cultured for 24 hours in a 5% CO₂ incubator at 37°C. Cells were treated according to the following regimens: Group 1 was the control group; Group 2 received different concentrations of Simvastatin; Group 3 received FPS-Sim nanoparticles; Group 4 received FPS-Sim-GOx magnetic nanomotors; and Group 5 received FPS-Sim-GOx magnetic nanomotors followed by magnetic field treatment (25 Hz). Four hours after adding CCK-8 solution, the absorbance was measured at 450 nm using a microplate reader, and the relative cell viability of each well was calculated. Relative viability = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group).

[0070] Figure 7 This is a graph showing the cytotoxicity of CCK-8 cells treated with different drug groups. The graph displays the cell viability of different groups. By comparing the results of the FPS-Sim-GOx magnetic nanomotor treatment group with the control group, it was found that the FPS-Sim-GOx magnetic nanomotor can significantly reduce cell viability at a certain concentration, exhibiting strong cytotoxicity.

[0071] Experiment Example 4

[0072] Tumor penetration capability of FPS-Sim-GOx magnetic nanomotors detected at the 3D cellular level:

[0073] (1) 3D tumor cell spheres were prepared by selecting 4T1 breast cancer cells and 3T3 fibroblasts at a ratio of 1:5, with 1200 cells per well in each well of a low-adsorption U-shaped 96-well plate, and centrifuged at 3500 rpm for 5 minutes. The morphology and size were observed under a microscope from the second to the fifth day. When the diameter of the tumor spheres was about 250 mm, they could be used as models.

[0074] (2) The culture medium of the tumor cell sphere model was replaced with a solution containing the same concentration of fluorescently labeled FPS-Sim-GOx magnetic nanomotors and glucose concentrations of 0, 50, 100 and 200 μg / mL. After incubation for 4 h, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde solution. The cells were then observed under a confocal laser scanning microscope, and fluorescence scanning was performed on the cell spheres from different layers to evaluate the permeation effect of FPS-Sim-GOx magnetic nanomotors in the tumor spheres. The above operation was repeated to evaluate the permeation effect of FPS-Sim-GOx magnetic nanomotors in the tumor spheres at magnetic field frequencies of 0, 5, 15 and 25 Hz.

[0075] Figure 8 This study demonstrates the penetration of FPS-Sim-GOx magnetic nanomotors into 3D tumor cell spheres at different glucose concentrations. The results in the figure show that the FPS-Sim-GOx magnetic nanomotors can effectively penetrate into the interior of tumor cell spheres at different glucose concentrations, and the penetration effect is enhanced with increasing glucose concentration.

[0076] Figure 9 This demonstrates the penetration of FPS-Sim-GOx magnetic nanomotors into 3D tumor cell spheres under different magnetic field frequencies. The figure shows that the penetration ability of the FPS-Sim-GOx magnetic nanomotors varies with different magnetic field frequencies. Increasing the magnetic field frequency effectively promotes the penetration of the nanomotors, demonstrating the significant impact of magnetic field modulation on its penetration effect.

[0077] The above embodiments of the present invention are not intended to limit the scope of protection of the present invention. The implementation of the present invention is not limited thereto. All other modifications, substitutions or alterations made to the above structure of the present invention based on the above content of the present invention, in accordance with ordinary technical knowledge and common practice in the field, without departing from the basic technical idea of ​​the present invention, shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing glucose oxidase / simvastatin magnetic nanomotor, characterized in that, Comprising the following steps: 1) FeCl3·6H2O is added to deionized water and stirred magnetically for 5-15 minutes, then the suspension is mixed with sodium citrate, urea and PAM, and further stirred for 20-40 minutes; the mixture is transferred to a Teflon autoclave and sealed, kept at 190-210℃ for 22-26 hours, and then naturally cooled at room temperature; washed several times with deionized water and ethanol, and after centrifugation, the precipitate is dispersed in deionized water to obtain a hollow mesoporous Fe3O4 nano solution; 2) PAA solution and ammonia are added to the Fe3O4 nano solution respectively, and mechanically stirred for 20-40 minutes; Then isopropanol is slowly added dropwise with a separatory funnel, and stirring is continued for 20-40 minutes to obtain a Fe3O4-PAA nano solution for the next step experiment; 3) Tetraethyl orthosilicate is added to the Fe3O4-PAA nano solution, and stirring is continued for 22-26 hours; washed 2-4 times with deionized water, and after centrifugation, resuspended in deionized water to obtain a Fe3O4-PAA / mSiO2 nano solution; 4) Liposoluble drug simvastatin is dissolved in an organic solvent to form a simvastatin solution, mixed with the nanoparticle solution obtained in step 3), and stirred uniformly in a metal bath; after centrifugation, the precipitate is collected to obtain Sim-loaded Fe3O4-PAA / mSiO2 nanoparticles; 5) The precipitate obtained in step 4) is resuspended in a buffer solution, and EDC and NHS solutions are added respectively, and stirred for 20-40 minutes; 6) Glucose oxidase is added to deionized water, and vortexed to dissolve to obtain an enzyme solution; 7) The enzyme solution obtained in step 6) is added to the mixture in step 5), and stirring is continued for 3-5 hours; after centrifugation, the precipitate is resuspended in deionized water to obtain a glucose oxidase-simvastatin magnetic nanomotor.

2. The method for preparing the glucose oxidase / simvastatin magnetic nanomotor as described in claim 1, characterized in that, In step 1), the concentrations of FeCl3·6H2O, sodium citrate and urea are 0.05M, 0.10M and 0.15M respectively; the centrifugal speed is 8000-10000 rpm, and the time is 10-20 minutes, which can also be separated by magnetic force.

3. The method for preparing the glucose oxidase / simvastatin magnetic nanomotor as described in claim 1, characterized in that, In step 2), the mass concentration of the Fe3O4 nano solution is 0.5 mg / mL, and the volume ratio of isopropanol to nanoparticle suspension is 29:

1.

4. The method for preparing the glucose oxidase / simvastatin magnetic nanomotor as described in claim 1, characterized in that, In step 3), the centrifugal speed is 8000-10000 rpm, and the time is 10-20 minutes, which can also be separated by magnetic force.

5. The method for preparing the glucose oxidase / simvastatin magnetic nanomotor as described in claim 1, characterized in that, In step 4), the organic solvent is one or a combination of two or more of acetonitrile, ethanol, methanol, acetone and dimethyl sulfoxide; the mass ratio of simvastatin to nanoparticles is 1:4-1:5; the temperature condition of the metal bath is 37℃, the speed is 800-1000 rpm, and the time is 2-12 hours; the centrifugal speed is 8000-10000 rpm, and the time is 10-20 minutes.

6. The method for preparing the glucose oxidase / simvastatin magnetic nanomotor as described in claim 1, characterized in that, The EDC and NHS mass concentration in step 5) is 5 mg / mL, the mass ratio of the nanoparticles is 1:1-1:1.5, and other carboxylic acid activators can also be used; the buffer is phosphate buffer or 2-morpholinoethanesulfonic acid buffer, the pH value is 6.0-6.2; the metal bath temperature condition is 37℃, the rotation speed is 800-1000 rpm, and the time is 0.5-2 hours.

7. The method for preparing the glucose oxidase / simvastatin magnetic nanomotor as described in claim 1, characterized in that, In step 6), the concentration of the enzyme solution is 0.1-10 mg / mL.

8. The method for preparing the glucose oxidase / simvastatin magnetic nanomotor as described in claim 1, characterized in that, In step 7), the mass ratio of the glucose oxidase and the nanoparticles is 1:0.2-1:0.5; the metal bath temperature condition is 37℃, the rotation speed is 800-1000 rpm, and the time is 2-4 hours; the centrifugal rotation speed is 8000-10000 rpm, and the time is 10-20 minutes.

9. Glucose oxidase / simvastatin magnetic nanomotor prepared by the method according to any one of claims 1-8, characterized in that, The drug loading rate of simvastatin in the glucose oxidase / simvastatin magnetic nanomotor is 15%-25%, the content of glucose oxidase is 20%-35%, and the speed of the glucose oxidase / simvastatin magnetic nanomotor can reach 20-25 μm / s at a magnetic field frequency of 25 Hz.

10. Use of the glucose oxidase / simvastatin magnetic nanomotor prepared by the method of any one of claims 1-8 or the glucose oxidase / simvastatin magnetic nanomotor of claim 9 in the preparation of a medicament for treating tumors, characterized in that, The tumor cells are 4T1 breast cancer cells.

Images