Low-frequency magnetic field triggered magnetic nanoscale enzyme and preparation method and application thereof
Patent Information
- Application Number
- CN202611183807.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-05
- Publication Date
- 2026-09-15
Smart Images
Figure CN122745286A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of nanozyme technology, and in particular to a low-frequency magnetic field triggered magnetic nanozyme (denoted as Ti / Fe-CN@CM nanozyme, or TFC@CM nanozyme or TFC@CM) and its preparation method and application. Background Technology
[0002] Central nervous system (CNS) tumors are among the deadliest types of malignant tumors. Glioblastoma (GBM) is the most common and aggressive primary brain tumor, often exhibiting highly metabolically adaptive characteristics, significant redox homeostasis regulation, and a highly immunosuppressive tumor microenvironment (TME). Although various treatments such as surgery, radiotherapy, and chemotherapy are available, the indistinct borders and diffuse, infiltrative nature of GBM limit the effectiveness of traditional treatments. The 5-year survival rate for GBM patients remains below 10%, with a median survival of approximately 14.6 months.
[0003] Currently, exogenous stimulation-based treatment strategies for GBM mainly include magnetothermal therapy (MHT), sonodynamic therapy (SDT), and photodynamic therapy (PDT). While MHT can overcome the limitations of tissue penetration depth and compensate for the shortcomings of PDT and SDT, it relies on heat conduction to kill tumor cells, often lacking precision and easily causing damage to surrounding normal tissues. In recent years, regulatory nanomedicine has developed into a controllable, precise, and highly efficient anti-cancer strategy by intervening in tumor-specific metabolic pathways or reshaping the pathological microenvironment to regulate cellular homeostasis. Among them, nanozymes, as a class of artificial catalysts that combine the mimicry activity of natural enzymes with the multifunctional properties of nanomaterials, have significant advantages such as high stability, low preparation cost, and diverse catalytic functions. Nanozymes can be specifically activated at the tumor site, catalyzing the generation of toxic reactive oxygen species (ROS) from endogenous substrates, thereby achieving targeted killing of tumor cells.
[0004] However, despite the numerous advantages of nanozymes, they still face three major obstacles in the treatment of GBM: First, the physiological barrier of the blood-brain barrier (BBB), with its tight junction structure and efflux pump system, significantly limits the delivery efficiency of nanozymes in the brain and their accumulation at the tumor site; second, traditional nanozymes have limited catalytic activity and low atom utilization; and third, the deep immunosuppression of the tumor microenvironment (TME), such as hypoxia and high lactate levels, can inhibit macrophage polarization and cytotoxic T cells (CD8+). + The activity of T cells, in contrast to that of immunosuppressive regulatory T cells (T cells) regs The cells exhibit high lactate tolerance, which further activates and maintains their immunosuppressive function. This imbalanced immune microenvironment severely weakens the anti-tumor immune response, thereby affecting the therapeutic effect of GBM.
[0005] The brain's blood vessel barrier (BBB) is composed of endothelial cells, the basement membrane, pericytes, and astrocytes, forming a highly precise bidirectional selective barrier that can selectively regulate the transport of substances to the central nervous system (CNS). Currently, existing strategies for crossing the BBB are mainly based on three directions: barrier opening modification, carrier-mediated transmembrane transport, and optimized drug delivery routes. Barrier opening strategies, such as using hypertonic mannitol and focused ultrasound-microbubbles to instantaneously open the tight junctions of cerebral vascular endothelium, can increase drug delivery to the brain, but generally suffer from drawbacks such as non-specific opening, easy damage to brain tissue and blood vessels, and increased systemic toxicity. Carrier-mediated transmembrane transport utilizes drug modification and various nanocarriers to achieve BBB transport, but it still struggles to avoid inherent problems such as liver and kidney accumulation after intravenous entry, first-pass metabolic loss, and peripheral off-target toxicity. Optimized drug delivery routes can bypass the BBB to achieve central delivery, with brain parenchyma, ventricle, and intrathecal puncture injections directly reaching the lesion; however, invasive techniques carry clinical risks such as intracranial infection and brain tissue damage. In contrast, the nasobrain pathway (NBP) shows great potential as a novel non-invasive route for brain drug delivery. This pathway not only avoids gastrointestinal degradation and the first-pass effect of the liver, but also reduces systemic drug exposure levels, thereby reducing off-target toxicity. Based on the advantages of NBP drug delivery, developing functional nanozymes adapted for nasal administration and possessing both catalytic therapy and brain-targeting capabilities has become an important research direction for overcoming the bottleneck of GBM drug delivery.
[0006] Meanwhile, research shows that exogenous stimulation can enhance the catalytic efficiency of nanozymes. For example, magnetic nanozymes can generate magnetoelectric effects (ME) under magnetic field stimulation, including magnetocaloric, mechanical, and electrical effects, which have great potential in the treatment of deep tumors. Low-frequency magnetic fields (LFMF), as a non-ionizing radiation physical therapy method, have opened up new avenues for GBM treatment through their mediated ME and enzyme activity regulation mechanisms. ME refers to the energy conversion phenomenon between magnetism and electricity. Magnetoelectric nanomaterials have attracted much attention due to their unique ability to convert externally applied magnetic fields into nanoscale polarization, achieving highly efficient energy conversion without bulk thermal effects or traditional electromagnetic induction. When acting at the tumor site, they do not require physical electrode intervention and have no systemic toxicity risks. Since catalytic reactions are often accompanied by electron transfer, reshaping the internal electrostatic field of enzymes using external electric fields and regulating the electronic structure of nanocatalysts has become a potentially effective way to enhance their enzyme activity. Compared with conventional radiotherapy, chemotherapy, and MHT, this LFMF-driven synergistic strategy, through a non-thermal physical mechanism, can achieve precise targeting and highly efficient catalytic therapy of deep brain tissue while avoiding significant tissue heating and the potential damage caused by it, demonstrating higher biocompatibility. This strategy is expected to overcome the shortcomings of MHT, SDT and other therapies, such as insufficient precision and low response rate of immunotherapy.
[0007] Therefore, integrating the ME and nanozyme catalytic functions of LFMF to construct a therapeutic platform that combines BBB crossing capability, magnetic field response to enhance enzyme activity, and TME remodeling synergistic effect is of great significance for breaking through the current bottleneck in GBM treatment. Summary of the Invention
[0008] In view of this, the present invention provides a low-frequency magnetic field triggered magnetic nanozyme, its preparation method and application. The low-frequency magnetic field triggered magnetic nanozyme provided by the present invention has both targeted delivery and synergistic catalytic functions, and can be used to prepare GBM drugs or construct GBM targeting systems.
[0009] This invention provides a low-frequency magnetic field-triggered magnetic nanozyme, comprising a core and a mouse glioma cell membrane coated on the surface of the core; the core comprises a magnetic titanium-doped Fe-CN nanozyme (magnetic Ti / Fe-CN nanozyme).
[0010] This invention also provides a method for preparing low-frequency magnetic field-triggered magnetic nanozymes according to the above scheme, comprising the following steps: (1) Prussian blue was doped with titanium and calcined to obtain magnetic Ti / Fe-CN nanozymes; (2) Mouse glioma cell membranes were coated on the surface of magnetic Ti / Fe-CN nanozymes to obtain low-frequency magnetic field triggered magnetic nanozymes.
[0011] Preferably, the Prussian blue doping with titanium includes the following steps: premixing polyvinylpyrrolidone and a portion of hydrochloric acid to obtain a polyvinylpyrrolidone premix, premixing potassium ferricyanide and the remaining hydrochloric acid to obtain a potassium ferricyanide premix, then adding the potassium ferricyanide premix dropwise to the polyvinylpyrrolidone premix and mixing to obtain a premixed solution, adding a titanium source to the premixed solution, and coprecipitating reaction.
[0012] Preferably, the mass ratio of polyvinylpyrrolidone to potassium ferricyanide is 1:0.9~1.1.
[0013] Preferably, the molar ratio of titanium in the titanium source to iron in potassium ferricyanide is 9~11:100.
[0014] Preferably, the coprecipitation reaction is carried out under oil bath conditions; the temperature of the oil bath is 78~82 degrees Celsius; and the coprecipitation reaction time is 18~25 hours.
[0015] Preferably, the calcination temperature is 660~730 degrees Celsius and the time is 1.8~2.2 hours; the calcination is carried out in a protective atmosphere.
[0016] Preferably, the process of coating mouse glioma cell membranes includes the following steps: mixing magnetic Ti / Fe-CN nanozymes and solvents to obtain a magnetic Ti / Fe-CN nanozyme dispersion; mixing the cell membrane dispersion and the magnetic Ti / Fe-CN nanozyme dispersion and then centrifuging, discarding the supernatant, and freeze-drying.
[0017] Preferably, the method for preparing the cell membrane dispersion includes the following steps: digesting the substrate with trypsin containing ethylenediaminetetraacetic acid, collecting GL261 cells, resuspending them, freezing and thawing them, centrifuging them to obtain the supernatant, centrifuging the supernatant to discard the supernatant, collecting the precipitate, and dispersing the precipitate in water.
[0018] This invention also provides the application of the above-described scheme of low-frequency magnetic field triggering magnetic nanozymes in the preparation of GBM drugs or the construction of GBM targeting systems.
[0019] Compared with the prior art, the present invention has achieved the following beneficial effects: The low-frequency magnetic field-triggered magnetic nanozyme provided by this invention is a multifunctional magnetic nanozyme derived from Prussian blue (PB). This invention uses PB as a precursor, which is calcined to form an Fe-CN (FC) framework. The electronic structure is improved by incorporating titanium (Ti), enhancing the magnetoelectric responsiveness (especially LFMF response) and catalytic activity of the low-frequency magnetic field-triggered magnetic nanozyme. Furthermore, it is coated with mouse glioma cell (GL261) membranes (CM) to improve the biocompatibility and tumor-specific recognition ability of the low-frequency magnetic field-triggered magnetic nanozyme, thereby constructing a low-frequency magnetic field-triggered magnetic nanozyme with both targeted delivery and synergistic catalytic functions.
[0020] The low-frequency magnetic field-triggered magnetic nanozyme provided by this invention possesses multiple synergistic functions: First, it accumulates in the brain region under the mediation of a static magnetic field (SMF), and simultaneously, under the homologous targeting effect of CM, it enters the brain tumor region via NBP, increasing the accumulation of the low-frequency magnetic field-triggered magnetic nanozyme at the brain tumor site; second, under LFMF stimulation, the low-frequency magnetic field-triggered magnetic nanozyme responsively generates ROS through magnetoelectric coupling effect, achieving targeted killing of tumor cells; in addition, the low-frequency magnetic field-triggered magnetic nanozyme also possesses nicotinamide adenine dinucleotide (NADH) oxidase (NO... The activities of NADH-like, catalase-like, and lactate oxidase-like enzymes were significantly enhanced by LFMF stimulation, which significantly improved the catalytic performance of these three enzymes: lactate oxidase-like activity efficiently cleared high concentrations of lactate in the tumor microenvironment and blocked lactate metabolism; catalase-like activity catalyzed the decomposition of hydrogen peroxide (H2O2) to produce oxygen (O2), improving the hypoxic state of the tumor; and NADH-like oxidase-like activity synergistically amplified oxidative stress, jointly triggering immunogenic cell death (ICD) in tumor cells to activate the systemic anti-tumor immune response.
[0021] The low-frequency magnetic field-triggered magnetic nanozyme provided by this invention possesses magnetoelectric coupling properties. Under LFMF (10Hz, 600rpm) stimulation, the low-frequency magnetic field triggers the magnetic nanozyme to generate a local electrical effect in the target area. Through electrocatalysis, it induces specific kinetic reactions within tumor cells, efficiently generating ROS, ultimately achieving non-thermal, highly selective killing of tumor cells. The low-frequency magnetic field-triggered magnetic nanozyme-mediated magnetoelectrodynamic therapy (MEDT) and immunomodulation synergistic strategy provided by this invention represent a comprehensive breakthrough and optimization, and are expected to provide a new multi-modal synergistic approach to overcome the dual barriers of low delivery efficiency and immunosuppressive microenvironment in GBM treatment.
[0022] In summary, this invention pioneers a new MEDT (Multi-Active Therapy) modality by constructing a low-frequency magnetic field-triggered magnetic nanozyme. Leveraging the LFMF-mediated magnetoelectric coupling effect, it eliminates the need for high temperatures, using a non-thermal electrocatalytic mechanism to target and generate ROS to kill tumor cells, effectively addressing the issues of poor precision and easy damage to normal tissues inherent in traditional exogenous stimulation therapies. This invention employs a multi-pronged non-invasive targeted delivery strategy. First, it avoids gastrointestinal degradation and the first-pass effect in the liver through NBP (Natural Biofilm Blocker). Then, through the homologous targeting recognition characteristics of CM (Cellular Rooting Cell) and the brain enrichment effect of SMF (Small Intensive Fiber Foam), the nanozyme can achieve non-invasive, safe, efficient, and specific penetration of the BBB (Body-Body Blocker), precisely accumulating at GBM lesions, significantly improving drug delivery efficiency and reducing systemic off-target toxicity. Compared to traditional nanozymes with low atom utilization and limited catalytic activity, this invention innovatively introduces Ti doping to optimize the electronic structure, enhancing the magnetoelectric response capability of the low-frequency magnetic field-triggered magnetic nanozyme. In addition to catalytic ability, this invention utilizes external physical stimulation of LFMF to further regulate the electronic arrangement of magnetic nanozymes triggered by low-frequency magnetic fields, synergistically enhancing multiple enzyme activity characteristics. Furthermore, by leveraging the triple enzyme activity of lactate oxidase-like, catalase-like, and NADH oxidase-like magnetic nanozymes triggered by low-frequency magnetic fields, this invention achieves immunosuppressive microenvironment remodeling and immune system activation. Through lactate oxidase-like activity, it degrades high lactate levels within the tumor, while catalase-like activity decomposes endogenous H2O2 to generate O2, improving the hypoxic tumor microenvironment, amplifying oxidative stress, leading to mitochondrial damage, inducing ICD in tumor cells, and activating systemic anti-tumor immunity. This relieves the immunosuppressive state of GBM, solving the key problems of weak immune response and limited therapeutic effects in traditional treatments. It forms a precise, safe, and efficient multimodal synergistic therapeutic approach, overcoming multiple core barriers in the clinical treatment of glioblastoma. Attached Figure Description
[0023] To more clearly illustrate the technical solutions of this invention, the accompanying drawings used in the embodiments of this invention or in the prior art are briefly described below. For those skilled in the art, other drawings can be derived from the following drawings without creative effort, and all such drawings are within the protection scope of this invention.
[0024] Figure 1 Basic characterization of PB prepared for Comparative Example 1; where a is the XRD pattern of PB, b is the SEM pattern of PB, c is the DLS size distribution map of PB, and d is the FTIR pattern of PB. Figure 2 The magnetic responsiveness characterization results are for the PB prepared in Comparative Example 1. Figure 3 Basic characterization of Fe-CN nanozymes prepared in Comparative Examples 1-6 and PB prepared in Comparative Example 1; where a is the XRD pattern of Fe-CN nanozymes prepared in Comparative Examples 1-6 and PB prepared in Comparative Example 1, b is the SEM pattern of Fe-CN nanozymes prepared in Comparative Examples 1-6, c is the DLS size distribution map of Fe-CN nanozymes prepared in Comparative Examples 1-6, and d is the FTIR pattern of Fe-CN nanozymes prepared in Comparative Examples 1-6. Figure 4 Basic characterization of the magnetic Ti / Fe-CN (TFC) nanozymes prepared in Examples 1 and Comparative Examples 7-10; wherein, a is the XRD pattern of the magnetic Ti / Fe-CN nanozymes prepared in Examples 1 and Comparative Examples 7-10, b is the SEM pattern of the magnetic Ti / Fe-CN nanozymes prepared in Examples 1 and Comparative Examples 7-10, c is the DLS size distribution map of the magnetic Ti / Fe-CN nanozymes prepared in Examples 1 and Comparative Examples 7-10, and d is the FTIR pattern of the magnetic Ti / Fe-CN nanozymes prepared in Examples 1 and Comparative Examples 7-10; Figure 5 The magnetic Ti / Fe-CN nanozymes of H2O, PB, FC, Comparative Examples 7-10 and Example 1 1 O2 production capacity; where a is the H2O production capacity. 1 O2 generation capacity, b is PB 1 O2 generation capacity, c is FC 1 O2 generation capacity, d represents the comparative example 7 magnetic Ti / Fe-CN nanozyme (Ti-2%). 1 O2 generation capacity, e represents the comparative example 8 magnetic Ti / Fe-CN nanozyme (Ti-5%). 1 O2 generation capacity, f is the magnetic Ti / Fe-CN nanozyme (Ti-10%) of Example 1. 1 O2 generation capacity, g is the comparative example 9 magnetic Ti / Fe-CN nanozyme (Ti-15%) 1 O2 generation capacity, h is the comparative example of 10 magnetic Ti / Fe-CN nanozymes (Ti-20%). 1 O2 generation capability; Figure 6Characterization of the magnetic Ti / Fe-CN nanozyme and TFC@CM nanozyme in Example 2; where a is a transmission electron microscope (TEM) image of TFC@CM nanozyme, b is an energy dispersive spectroscopy (EDS) elemental mapping spectrum of TFC@CM nanozyme, c is a gel electrophoresis image of CM, TFC and TFC@CM nanozyme, d is a FTIR spectrum of FC, TFC and TFC@CM nanozyme, and e is a Zeta potential map (n=3) of FC, TFC and TFC@CM nanozyme. Figure 7 The basic characterization of TFC@CM nanozymes is shown below; where a is the XRD pattern of FC, TFC, and TFC@CM nanozymes, b is the DLS size distribution of FC, TFC, and TFC@CM nanozymes, c is the XPS full spectrum of TFC@CM nanozymes, d is the XPS pattern of Fe 2P orbitals of TFC@CM nanozymes, e is the XPS pattern of Ti 2P orbitals of TFC@CM nanozymes, and f is the stability of TFC@CM nanozymes in H2O, PBS, and DMEM, respectively. The inset shows the stability characterization of TFC@CM nanozymes in H2O, PBS, and DMEM on day 7 (n=3). Figure 8 The figures show the hysteresis loops at 300K as a function of the magnetic field; where a is the hysteresis loop of FC and TFC@CM nanozymes, the inset is the magnetic adsorption of TFC@CM nanozymes in water, b is the magnified hysteresis loop of FC, and c is the magnified hysteresis loop of TFC@CM nanozymes. Figure 9 The magnetoelectric dynamic properties of FC and TFC@CM nanozymes from Example 2 are characterized; where a is the UV-Vis DRS spectrum of FC, b is the UV-Vis DRS spectrum of TFC@CM nanozymes, c is the VB-XPS spectrum of TFC@CM nanozymes, d is the electrochemical impedance spectroscopy (EIS) of FC and TFC@CM nanozymes, e is the magneto-induced current spectrum of FC and TFC@CM nanozymes under LFMF (10Hz, 600rpm) stimulation, and f is the UV-Vis absorption curves of FC and TFC@CM nanozymes with DPBF as the trapping agent. Figure 10The following data were used to detect the NADH-like oxidase catalytic activity of TFC@CM nanozymes: a) NADH-like oxidase activity of TFC@CM nanozymes at pH 7.4; b) NADH-like oxidase activity of TFC@CM nanozymes at pH 6.5; c) NADH-like oxidase activity of TFC@CM nanozymes at pH 5.5; d) UV-Vis absorption spectra of FC, TFC@CM nanozymes, and NADH stimulated by LFMF (10 Hz, 600 rpm); e) Michaelis-Menten kinetic analysis of TFC@CM nanozymes and LFMF stimulation; and f) Lineweaver-Burk double reciprocal kinetic curves (n=3) of TFC@CM nanozymes with NADH as the substrate and LFMF stimulation. Figure 11 The catalase-like catalytic activity of TFC@CM nanozymes was detected. Among them, a is the oxygen content of mixed solutions containing H2O2 and TFC@CM nanozymes under different pH conditions, b is the oxygen content of mixed solutions containing 1mM H2O2 at different TFC@CM nanozyme concentrations, c is the oxygen content of mixed solutions containing 200μg / mL TFC@CM nanozymes at different H2O2 concentrations, and d is the oxygen content of FC, TFC@CM nanozymes and TFC@CM nanozymes after exposure to LFMF (10Hz, 600rpm) for 10 minutes. Figure 12 The lactate oxidase-like activities of FC, TFC, and TFC@CM nanozymes were tested. In this study, a represents the consumption of L-lactate substrate by different concentrations of TFC@CM nanozyme solutions, and b represents the lactate consumption of FC, TFC, and TFC@CM nanozymes after 10 minutes of exposure to LFMF (10 Hz, 600 rpm) (n=3). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Figure 13 This is an evaluation of the cell safety performance of the TFC@CM nanozyme in Example 2; where a is the toxicity test of L929 cells under different concentrations of TFC@CM nanozyme, and b is the toxicity test of Raw264.7 cells under different concentrations of TFC@CM nanozyme (n=3). Figure 14 This is an evaluation of the cell-killing performance of the TFC@CM nanozyme in Example 2; where a represents the survival rate of GL261 cells under different concentrations of TFC@CM nanozyme (n=3), b represents the survival rate of cells in different treatment groups by trypan blue staining, G1 represents PBS, G2 represents LFMF, G3 represents TFC@CM nanozyme, and G4 represents TFC@CM nanozyme + LFMF (10Hz, 600rpm) (n=3). Figure 15 To assess the cellular uptake capacity of TFC@CM nanozyme by GL261 cells; where a is the fluorescence image captured by GLSM at different time points, b is the quantitative analysis at different time points captured by GLSM (n=3), ns indicates no significant difference, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001; Figure 16 Fluorescence images and quantitative statistics of ROS generated by DCFH-DA probe for TFC@CM nanozyme after different treatment groups (n=3), scale bar is 400μm; where G1 represents PBS, G2 represents LFMF, G3 represents TFC@CM nanozyme, G4 represents TFC@CM nanozyme + LFMF (10Hz, 600rpm), * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001; Figure 17 To assess mitochondrial membrane potential damage in GL261 cells under different treatment groups; where a) is JC-1 staining CLSM imaging of mitochondrial membrane potential in GL261 cells under different treatment groups; b) is the corresponding quantitative analysis of fluorescence intensity (n=3). Figure 18 To evaluate the effect of intranasal administration in tumor-bearing mice; where a is the brain enrichment bioimaging at different time points after intranasal administration, and b is the quantitative fluorescence statistical graph of the brain at different time points after intranasal administration (n=3). Figure 19 To evaluate the therapeutic effect in tumor-bearing mice; where a represents the bioluminescent signal in the brain of tumor-bearing mice after intranasal injection of TFC@CM nanozyme; b represents the corresponding quantitative fluorescence statistics (n=6). Detailed Implementation
[0025] This invention provides a low-frequency magnetic field-triggered magnetic nanozyme, comprising a core and a mouse glioma cell membrane coated on the surface of the core; the core comprises a magnetic titanium-doped Fe-CN nanozyme (magnetic Ti / Fe-CN nanozyme).
[0026] This invention also provides a method for preparing low-frequency magnetic field-triggered magnetic nanozymes according to the above scheme, comprising the following steps: (1) Prussian blue was doped with titanium and calcined to obtain magnetic Ti / Fe-CN nanozymes; (2) The surface of the magnetic Ti / Fe-CN nanozyme was coated with the cell membrane of mouse glioma cells to obtain a low-frequency magnetic field triggered magnetic nanozyme.
[0027] This invention involves calcining Prussian blue and doping it with titanium to obtain magnetic Ti / Fe-CN nanozymes. The titanium doping of Prussian blue in this invention may include the following steps: premixing polyvinylpyrrolidone (PVP) with a portion of hydrochloric acid (HCl) to obtain a PVP premix; premixing potassium ferricyanide (K3[Fe(CN)6]) with the remaining hydrochloric acid to obtain a potassium ferricyanide premix; then adding the potassium ferricyanide premix dropwise (referred to as the first dropwise addition) to the PVP premix and mixing (referred to as the first mixing) to obtain a premixed solution; and adding a titanium source to the premixed solution for a co-precipitation reaction.
[0028] In this invention, the weight-average molecular weight of polyvinylpyrrolidone can be 9900~10100, specifically 10000.
[0029] In this invention, the mass ratio of polyvinylpyrrolidone to the volume of a portion of hydrochloric acid can be (8~10) g:140 mL, specifically 9 g:140 mL; the concentration of hydrochloric acid can be 1 mol / L.
[0030] In this invention, the mass ratio of polyvinylpyrrolidone to potassium ferricyanide can be 1:0.9~1.1, specifically 1:1.
[0031] In this invention, the mass ratio of potassium ferricyanide to the volume of the remaining hydrochloric acid can be (0.8~1) g:20 mL, specifically 0.88 g:20 mL or 0.95 g:20 mL; the concentration of hydrochloric acid can be 1 mol / L.
[0032] In this invention, the rate of the first drop addition can be 9 to 10 drops / minute, specifically 9 drops / minute or 10 drops / minute; the first drop addition can be carried out under stirring conditions; the stirring device can be a magnetic stirrer.
[0033] In this invention, the first mixing can be stirring; the first mixing time can be more than 1 hour.
[0034] In this invention, the titanium source can be titanium sulfate.
[0035] In this invention, the molar ratio of titanium in the titanium source to iron in potassium ferricyanide can be 9~11:100, specifically 10:100.
[0036] In this invention, the coprecipitation reaction can be carried out under oil bath conditions; the temperature of the oil bath can be 78~82 degrees Celsius, specifically 80 degrees Celsius; the coprecipitation reaction time can be 18~25 hours, specifically 20 hours.
[0037] In this invention, the coprecipitation reaction may further include centrifuging the resulting product, collecting the precipitate, washing the precipitate, and freeze-drying it sequentially.
[0038] In this invention, the centrifugation speed can be 10,000~12,000 rpm, specifically 11,000 rpm, and the time can be 20~25 minutes, specifically 20 minutes.
[0039] In this invention, the washing can be alcohol washing; the alcohol used for alcohol washing can be anhydrous ethanol; the number of washing cycles can be more than one, specifically three or four.
[0040] In this invention, the freeze-drying temperature can be -56 to -52 degrees Celsius, specifically -54 degrees Celsius, and the time can be 10 to 14 hours, specifically 12 hours.
[0041] In this invention, the calcination temperature can be 660~730 degrees Celsius, specifically 680 degrees Celsius, 700 degrees Celsius or 720 degrees Celsius, and the time can be 1.8~2.2 hours, specifically 2 hours; the calcination can be carried out in a protective atmosphere; the protective atmosphere can be an inert gas; the inert gas can be argon.
[0042] After obtaining the magnetic Ti / Fe-CN nanozyme, this invention coats the surface of the magnetic Ti / Fe-CN nanozyme with mouse glioma cell membranes to obtain a low-frequency magnetic field-triggered magnetic nanozyme. In this invention, coating the mouse glioma cell membrane may include the following steps: mixing the magnetic Ti / Fe-CN nanozyme with a solvent (denoted as the second mixture) to obtain a magnetic Ti / Fe-CN nanozyme dispersion; mixing the cell membrane dispersion and the magnetic Ti / Fe-CN nanozyme dispersion (denoted as the third mixture); sequentially centrifuging (denoted as the first centrifugation); discarding the supernatant; and freeze-drying.
[0043] In this invention, the solvent can be water.
[0044] In this invention, the second mixing can be ultrasonic dispersion; the ultrasonic dispersion device can include a cell disruptor; the ultrasonic dispersion power can be 110~130W, specifically 120W, and the time can be 4~6 minutes, specifically 5 minutes.
[0045] In this invention, the concentration of the magnetic Ti / Fe-CN nanozyme dispersion can be 240~260 μg / mL, specifically 250 μg / mL.
[0046] In this invention, the preparation method of cell membrane dispersion may include the following steps: digesting the substrate with trypsin containing ethylenediaminetetraacetic acid (EDTA), collecting GL261 cells, resuspending, freezing and thawing, centrifuging (referred to as the second centrifugation) to obtain the supernatant, centrifuging the supernatant (referred to as the third centrifugation) to discard the supernatant, collecting the precipitate, and dispersing the precipitate in water.
[0047] In this invention, the mass ratio of ethylenediaminetetraacetic acid (EDTA) to trypsin can be 2.5 to 12.5:1, specifically 12.5:1.
[0048] In this invention, the substrate may include one or more of fibronectin, laminin, E-cadherin and collagen.
[0049] In this invention, the volume ratio of trypsin to substrate can be 0.5 to 1:5, specifically 1:5.
[0050] In this invention, the digestion time can be 35 to 45 seconds, specifically 40 seconds.
[0051] In this invention, the volume of GL261 cells collected can be 2 dishes (10 cm in diameter).
[0052] In this invention, resuspension may include the following steps: resuspending GL261 cells in deionized water; the volume of deionized water may be 1 mL.
[0053] In this invention, the freezing temperature for freeze-thaw cycles can be -78 to -82 degrees Celsius, specifically -80 degrees Celsius; the freezing time can be 5 to 7 minutes, specifically 6 minutes; the thawing temperature can be 25 to 30 degrees Celsius, specifically 27 degrees Celsius; and the thawing time can be 10 to 15 minutes. The number of freeze-thaw cycles can be more than 3.
[0054] In this invention, the temperature of the second centrifugation can be 3-5 degrees Celsius, specifically 4 degrees Celsius; the centrifugal force can be 280-320g, specifically 300g; and the time can be 8-12 minutes, specifically 10 minutes.
[0055] In this invention, the temperature of the third centrifugation can be 3-5 degrees Celsius, specifically 4 degrees Celsius; the centrifugal force can be 13000-15000g, specifically 14000g; and the time can be 25-35 minutes, specifically 30 minutes.
[0056] In this invention, the concentration of the cell membrane dispersion can be 70~90μg / mL, specifically 80μg / mL.
[0057] In this invention, the volume ratio of the cell membrane dispersion to the magnetic Ti / Fe-CN nanozyme dispersion can be 0.9~1.1:1, specifically 1:1.
[0058] In this invention, the third mixing can be ultrasonic mixing; the power of ultrasonic mixing can be 350~450W, specifically 400W; the temperature of the third mixing can be 3~5 degrees Celsius, specifically 4 degrees Celsius; and the time can be 15~30 minutes, specifically 20 minutes.
[0059] In this invention, the rotation speed of the first centrifugation can be 10,000 to 12,000 rpm, specifically 11,000 rpm, and the time can be 10 to 20 minutes, specifically 15 minutes.
[0060] In this invention, the freeze-drying temperature can be -56 to -52 degrees Celsius, specifically -54 degrees Celsius, and the time can be 11 to 13 hours, specifically 12 hours.
[0061] This invention also provides the application of the above-described scheme of low-frequency magnetic field triggering magnetic nanozymes in the preparation of GBM drugs or the construction of GBM targeting systems.
[0062] To further illustrate the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments.
[0063] Comparative Example 1: A Fe-CN nanozyme was prepared in this comparative example, and its characterization and testing were performed as follows: 1) Preparation of PB: Preparation of PB by coprecipitation method: Accurately weigh 9g of polyvinylpyrrolidone (PVP, M w A 10000 g potassium ferricyanide solution was dissolved in 140 mL of 1 mol / L hydrochloric acid to obtain a PVP solution. Separately, 0.88 g of potassium ferricyanide was dissolved in 20 mL of 1 mol / L hydrochloric acid to obtain a potassium ferricyanide solution. The PVP solution was stirred on a magnetic stirrer, while the potassium ferricyanide solution was added dropwise to the PVP solution at a rate of 10 drops / min. After the addition was complete, stirring was continued for 1 hour to ensure thorough mixing, resulting in a mixture. The mixture was then transferred to a round-bottom flask and subjected to a co-precipitation reaction at 80°C in an oil bath for 20 hours. After the reaction, the product was centrifuged at 11000 rpm for 20 minutes, the precipitate was collected, washed three times with anhydrous ethanol, and freeze-dried at -54°C for 12 hours to ensure complete drying. The powder was collected to obtain PB.
[0064] 2) Characterization of PB: The PB prepared in this comparative example was characterized by XRD, SEM, DLS, and FTIR. The results are as follows: Figure 1 As shown. The magnetism of PB was then tested, and the results are as follows. Figure 2 As shown.
[0065] according to Figure 1 As can be seen, the XRD pattern of PB shows that the diffraction peaks of PB are consistent with the characteristic peaks of the standard card, and it is a cubic particle with a particle size of 110 nm; the FTIR results of PB show that at 2070 cm⁻¹... -1 The presence of the characteristic C≡N absorption peak at this location further confirms the successful synthesis of PB. Based on... Figure 2 The magnetic response test results of PB show that PB is not magnetic.
[0066] 3) Preparation of Fe-CN nanozymes: PB was calcined at 700°C for 2 hours under an argon atmosphere to obtain Fe-CN nanozyme.
[0067] Comparative Example 2: The preparation method of this comparative example is the same as that of comparative example 1, except that in step 3), the calcination temperature is 300 degrees Celsius.
[0068] Comparative Example 3: The preparation method of this comparative example is the same as that of comparative example 1, except that in step 3), the calcination temperature is 400 degrees Celsius.
[0069] Comparative Example 4: The preparation method of this comparative example is the same as that of comparative example 1, except that in step 3), the calcination temperature is 500 degrees Celsius.
[0070] Comparative Example 5: The preparation method of this comparative example is the same as that of comparative example 1, except that in step 3), the calcination temperature is 600 degrees Celsius.
[0071] Comparative Example 6: The preparation method of this comparative example is the same as that of comparative example 1, except that in step 3), the calcination temperature is 800 degrees Celsius.
[0072] Test Example 1: The Fe-CN nanozymes and PB prepared in comparative examples 1-6 were characterized by XRD, SEM, DLS, and FTIR, and their magnetic properties were determined. The results are as follows: Figure 3 As shown.
[0073] according to Figure 3As can be seen from the XRD pattern of Fe-CN nanozymes obtained by calcination of PB, the intensity of the characteristic diffraction peaks of PB gradually decreases with increasing calcination temperature, indicating that the PB framework begins to undergo thermal decomposition. When the temperature rises above 600 degrees Celsius, the characteristic peaks of PB completely disappear, and new diffraction peaks appear. Furthermore, it was found that PB exhibits strong magnetism at calcination temperatures above 600 degrees Celsius, proving that PB completely decomposes at high temperatures, the oxidation of CN groups is inhibited, and it is more likely to coordinate with iron ions to form Fe-CN. SEM images and DLS results show that after the calcination temperature gradually increases, the morphology of Fe-CN nanozymes becomes spherical and agglomerates, with an increased particle size. FTIR spectroscopy indicates that the intensity of the C≡N peak gradually weakens with increasing temperature, while the intensity at 1600 cm⁻¹... -1 The presence of a stretching vibration peak of the C=C bond at this point proves that the cyano framework of PB has completely decomposed, generating a composite product of Fe-CN and amorphous carbon.
[0074] Comparative Example 7: This comparative example prepared a magnetic Ti / Fe-CN nanozyme. The preparation method of this comparative example is the same as that of Comparative Example 1, except that in step 1), potassium ferricyanide solution is added to the PVP solution at a rate of 10 drops / minute. After the addition is completed, stirring is continued for 1 hour to ensure thorough mixing. Titanium sulfate is then added, and the molar ratio of titanium in titanium sulfate to iron in potassium ferricyanide solution is 2:100 to obtain a mixed solution.
[0075] Comparative Example 8: The preparation method of this comparative example is the same as that of comparative example 7, except that in step 1), the molar ratio of titanium to iron in the potassium ferricyanide solution is 5:100.
[0076] Comparative Example 9: The preparation method of this comparative example is the same as that of comparative example 7, except that in step 1), the molar ratio of titanium to iron in the potassium ferricyanide solution is 15:100.
[0077] Comparative Example 10: The preparation method of this comparative example is the same as that of comparative example 7, except that in step 1), the molar ratio of titanium to iron in the potassium ferricyanide solution is 20:100.
[0078] Example 1: The preparation method in this embodiment is the same as that in Comparative Example 7, except that in step 1), the molar ratio of titanium to iron in the potassium ferricyanide solution is 10:100. The resulting magnetic Ti / Fe-CN nanozyme (denoted as FC) is the final product of this embodiment.
[0079] Test Example 2: 1) The magnetic Ti / Fe-CN nanozymes prepared in Example 1 and Comparative Examples 7-10 were characterized by XRD, SEM, DLS, and FTIR. The results are as follows: Figure 4 As shown.
[0080] according to Figure 4 It can be seen that this invention introduces titanium in the PB synthesis stage. XRD characterization shows that the crystallinity of the product gradually decreases with increasing titanium doping ratio. High doping levels form new diffraction peaks. Some titanium reacts with O2 during calcination, possibly resulting in TiO2. SEM and DLS results show that titanium doping has no significant effect on the particle morphology and size of the magnetic Ti / Fe-CN nanozyme. FTIR spectroscopy shows that at 2089 cm⁻¹... -1 The reappearance of the C≡N characteristic peak may lead to the formation of a Ti-C≡N bond with higher bond energy. This bond is more difficult to cleave at high temperatures, thus protecting some of the C≡N triple bonds from decomposition.
[0081] 2) Based on structural characterization, the magnetic Ti / Fe-CN nanozymes prepared in Example 1 and Comparative Examples 7-10 were stimulated with 1,3-diphenylisobenzofuran (DPBF) to produce... 1 The performance of O2 was characterized, with H2O, PB, and FC used as controls. The results are as follows: Figure 5 As shown.
[0082] according to Figure 5 It can be seen that magnetic Ti / Fe-CN nanozymes are produced after different proportions of titanium doping. 1 O2, but the magnetic Ti / Fe-CN nanozyme of Example 1 1 The O2 generation capacity is strongest because as the proportion of titanium increases, a high proportion of Ti... 4+ The introduction of Fe induces severe lattice distortion, leading to disorder in the crystal structure of Fe-based particles. This structural imbalance disrupts the continuous pathways for electron transfer, reducing the electron transport efficiency required for O2 activation and ultimately inhibiting its activation. 1 The generation of O2.
[0083] Example 2: In this embodiment, a cell membrane dispersion of GL261 cell membrane was prepared, and TFC@CM nanozyme was also prepared. The specific steps are as follows: 1) Fibronectin was digested for 40 seconds using trypsin containing EDTA (mass ratio of EDTA to trypsin was 12.5:1). The volume ratio of trypsin to substrate fibronectin was 1:5. Two dishes (10 cm in diameter) of GL261 cells were collected and resuspended in 1 mL of deionized water. After repeated freeze-thaw cycles (freezing at -80°C for 6 minutes and thawing at 27°C for 15 minutes) three times, the supernatant was collected by centrifugation at 4°C and 300 g for 10 minutes. The supernatant was further centrifuged at 4°C and 14000 g for 30 minutes. After discarding the supernatant, the precipitate was dispersed in 5 mL of deionized water to obtain a cell membrane dispersion with a concentration of 80 μg / mL.
[0084] 2) Simultaneously, a magnetic Ti / Fe-CN nanozyme dispersion with a concentration of 250 μg / mL was prepared. The dispersion was ultrasonically dispersed for 5 minutes using a cell disruptor at 120W. After mixing with an equal volume of cell membrane dispersion, the dispersion was ultrasonically dispersed for 20 minutes at 4°C and 400W. Then, it was centrifuged at 11,000 rpm for 15 minutes, the supernatant was discarded, and the product was freeze-dried at -54°C for 12 hours. The product was collected to obtain TFC@CM nanozyme.
[0085] Test Example 3: The magnetic Ti / Fe-CN nanozyme and TFC@CM nanozyme in Example 2 were characterized to compare the changes caused by the introduction of titanium and whether the CM coating was successful.
[0086] The results were obtained through TEM image observation and EDS elemental spectrum analysis. Figure 6 a and Figure 6 As shown in b. According to Figure 6 a and Figure 6 As shown in b, the coating layer was observed through TEM images, confirming that the magnetic Ti / Fe-CN nanozyme was successfully coated with the cell membrane on the surface; the EDS elemental spectrum further showed that C, N, O, Fe, and Ti elements were uniformly distributed in the TFC@CM nanozyme.
[0087] To further confirm the CM coating in the TFC@CM nanozyme, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to analyze the protein composition. The results are as follows: Figure 6 As shown in c in the diagram. According to... Figure 6 As can be seen from 'c', the protein bands of the TFC@CM nanozyme are similar to those of the pure GL261 cell membrane, proving that the cell membrane modification was successful.
[0088] Further characterization was performed using FTIR and Zeta potential measurements, with results as follows: Figure 6 d and Figure 6 As shown in e. Figure 6The FTIR results of d in the middle show that 1640 cm -1 1535cm -1 and 1450cm -1 The presence of typical absorption peaks characteristic of the cell membrane, attributed to the amide I and amide II bands in cell membrane proteins and the CH bending vibration in the protein, respectively, confirms successful cell membrane coating. Based on... Figure 6 The zeta potential measurement results of the TFC@CM nanozyme showed that after titanium doping, the potential increased from -17.6 eV of FC to 13.3 eV of TFC, and then decreased to -18.9 eV of TFC@CM nanozyme after cell membrane coating. All of the above results indicate that CM was successfully coated and TFC@CM nanozyme was successfully constructed.
[0089] Test Example 4: The FC, TFC, and TFC@CM nanozymes of Example 2 were characterized by XRD, DLS, and X-ray photoelectron spectroscopy (XPS). The results are as follows: Figure 7 As shown.
[0090] according to Figure 7 XRD analysis shows that titanium doping may introduce lattice distortion, disrupting the ordered atomic arrangement. An amorphous peak appeared at 28°, a feature consistent with TEM images. DLS measurements revealed the hydrodynamic diameter of the FC nanoparticles to be approximately 73 nm, which increased after titanium doping, ultimately reaching 134 nm after cell membrane coating. This is attributed to the cell membrane forming a continuous shell on the surface of the magnetic Ti / Fe-CN nanozyme particles, increasing the physical size while maintaining good dispersibility. XPS full-spectrum analysis showed that the TFC@CM nanozyme contains Fe 2p, O 1s, C 1s, and Ti 2p orbitals, further confirming successful titanium doping into Fe-CN. Fitting analysis of the Fe 2p and Ti 2p orbitals indicates that iron is incorporated into Fe... 2+ and Fe 3+ Mixed valence states exist, with Ti element in Ti... 4+ The existence of Ti confirms its existence. 4+ Due to its higher charge density and coordination affinity, it preferentially replaces some of the Fe. 2+ / Fe 3+ Site selection. Next, the dispersion stability of TFC@CM nanozymes in H2O, PBS, and DMEM was tested. The results showed that the hydrodynamic diameter of TFC@CM nanozymes did not change significantly over time, indicating that TFC@CM nanozymes exhibited good stability in different media.
[0091] Test Example 5: The TFC@CM nanozyme prepared in Example 2 was magnetically characterized using a vibrating sample magnetometer (VSM) at 300 K. The results are as follows: Figure 8 As shown.
[0092] according to Figure 8 As can be seen from the hysteresis loop analysis, the saturation magnetization of FC is 72.6 emu / g and the coercivity is 195.6 Oe, while the saturation magnetization of TFC@CM nanozyme is 33.7 emu / g and the coercivity is 139.4 Oe. This indicates that the coercivity weakens after titanium doping, suggesting a transition from paramagnetism to superparamagnetism. In addition, when a 0.04T rubidium magnet is placed next to the dispersion of TFC@CM nanozyme, the TFC@CM nanozyme rapidly aggregates after 30 seconds, and the solution becomes clear and transparent. This provides evidence for the excellent magnetic drive and magnetic targeting capabilities of TFC@CM nanozyme.
[0093] Test Example 6: Based on the basic structural characterization of TFC@CM nanozymes, the magnetoelectric and magnetoelectric dynamic properties of the TFC@CM nanozymes in Example 2 were characterized.
[0094] Since the electronic structure of nanomaterials is closely related to their charge separation ability and ROS generation efficiency, the band gap of FC and TFC@CM nanozymes was first evaluated using UV-Vis diffuse reflectance (UV-Vis DRS), and the results are as follows: Figure 9 a and Figure 9 As shown in b, according to Figure 9 a and Figure 9 As shown in b, the band gap of FC is 1.37 eV, while the optical band gap of TFC@CM nanozyme is Eg≈1.32 eV. These results indicate that the band gap narrows after titanium doping. Simultaneously, the valence band potential was measured using VB-XPS spectroscopy, and the results are as follows... Figure 9 As shown in c, according to Figure 9 As shown in 'c', the conduction band potential of the TFC@CM nanozyme was calculated to be -0.11 eV using the x-axis coordinates corresponding to the intersection of the tangents of the VB spectral characteristic curve, indicating that the conduction band electrons have strong reducing power. To evaluate the charge transfer efficiency of FC and TFC@CM nanozymes, electrochemical impedance spectroscopy (EIS) was used for testing, and the results are as follows: Figure 9 As shown in d, according to Figure 9 As can be seen from d, compared with FC, TFC@CM nanozymes exhibit a smaller radius of curvature, indicating that titanium doping further optimizes the electronic structure and increases the carrier transport rate. The generation of magnetic induced current in FC and TFC@CM nanozymes was detected by transient magnetocurrent response, and the results are as follows... Figure 9 As shown in e, according to Figure 9As can be seen from the 'e', the TFC@CM nanozyme generates a stronger magnetically induced current than the FC nanozyme, indicating that the TFC@CM nanozyme produces more current-carrying electrons under LFMF stimulation. Next, this test preliminarily evaluates the MEDT characteristics of the TFC@CM nanozyme by measuring the ROS generated under LFMF stimulation. A 1,3-diphenylisobenzofuran (DPBF) fluorescent probe was used to evaluate the effect of titanium doping level on... 1 The effects of O2 production, as shown in the following results Figure 9 As shown in f in the figure. According to Figure 9 f in combination Figure 5 As can be seen from b, under LFMF stimulation, PB does not produce 1 O2, and after PB calcination, FC degrades DPBF, which indicates that... 1 The generation of O2, and its production after titanium doping 1 The ability to generate O2 is further enhanced, indicating that titanium doping can respond to LFMF to produce more 1 O2 provides support for the MEDT properties of TFC@CM nanozymes.
[0095] Test Example 7: The multi-enzyme catalytic activity of the TFC@CM nanozyme prepared in Example 2 was tested, as follows: 1) Assay for NADH-like oxidase activity: To test the multi-enzyme catalytic activity of TFC@CM nanozymes, the catalytic activity of NADH-like oxidases was first investigated. The NADH-like oxidase activity was preliminarily evaluated by measuring the changes in the UV-Vis absorption spectrum of nicotinamide adenine dinucleotide (NADH) at 340 nm. The catalytic activity of TFC@CM nanozymes at different pH values was also investigated, and the results are as follows: Figure 10 As shown. According to Figure 10 As can be seen from a~c in the data, under the same time conditions, the consumption of NADH gradually increases as the pH value decreases, and the activity of NADH-like oxidases is optimal at a pH value of 5.5; at the same time, according to Figure 10 As can be seen from d, LFMF stimulation further enhanced the activity of NADH-like oxidases, indicating the regulatory and enhancing effect of LFMF on TFC@CM nanozymes. Subsequently, the catalytic efficiency of TFC@CM nanozymes under LFMF stimulation was analyzed using Michaelis-Menten curves and Lineweaver-Burk double reciprocal kinetic curves. The results are as follows: Figure 10 As shown in e~f in the diagram. According to... Figure 10 As can be seen from e~f, the maximum reaction rate (Vf) of the TFC@CM nanozyme in acidic buffer (pH 5.5) is... max ) and Michaelis constant ( KThe values for m are 3.41 × 10⁻⁶. -7 Ms -1 And 0.834mM, while under LFMF stimulation, V max Increased to 3.93 × 10 -7 Ms -1 , K The decrease in m to 0.211 mM indicates that LFMF stimulation significantly improved the catalytic efficiency of NADH-like oxidases and the affinity of TFC@CM nanozymes for the substrate NADH.
[0096] 2) Catalase-like activity test: Since tumor microenvironments (TMEs) typically exhibit hypoxic characteristics, TFC@CM nanozymes with catalase-like catalytic activity can alleviate the hypoxic state in the tumor microenvironment by catalyzing the decomposition of H₂O₂ to generate O₂. Therefore, by measuring catalase-like activity using a dissolved oxygen analyzer, it was found that under the same H₂O₂ concentration, the oxygen content in the solution was closely related to the pH value, as shown in the results. Figure 11 As shown. According to Figure 11 It can be seen that the growth rate of ΔO2 concentration is higher in neutral environments than in acidic environments. This phenomenon stems from the high concentration of H+ in acidic environments. + It hinders the decomposition of H2O2 molecules, while the catalase-like activity is more stable under neutral conditions; as the concentration of TFC@CM nanozyme increases, the O2 content in the mixed solution also increases, and the catalase-like catalytic activity also shows a dependence on H2O2 concentration; at the same time, this test example found that the O2 content of FC increased after titanium doping, and the O2 content further increased significantly under LFMF stimulation. The O2 generated in this process can not only generate the MEDT process 1 O2 provides an O2 source and can also alleviate hypoxia in the TME and improve the tumor immunosuppressive microenvironment. In summary, LFMF stimulation can enhance the catalase-like catalytic activity of TFC@CM nanozymes.
[0097] 3) Lactate oxidase activity test: Finally, this test case investigated the catalytic activity of lactate oxidase. Under O2 conditions, lactate oxidase catalyzes the oxidation and decomposition of lactate (LA) to generate pyruvate and H2O2, effectively reducing LA levels, blocking abnormal lactate metabolism, reversing the immunosuppressive microenvironment, and contributing to the metabolic therapy of tumors. This test case used a WST-8-based L-lactate assay kit to determine the catalytic activity of lactate oxidase, and the results are as follows: Figure 12 As shown. According to Figure 12It can be seen that the higher the concentration of TFC@CM nanozyme, the lower the content of LA substrate. The FC group indicates that it has basic catalytic activity. The TFC@CM nanozyme obtained by titanium doping modification can further improve the LA consumption efficiency. After applying LFMF (10Hz, 600rpm), the LA content further decreased, indicating that LFMF can significantly enhance the lactate oxidase-like activity of TFC@CM nanozyme, so that the LA substrate consumption efficiency reaches the highest level.
[0098] Test Example 8: The in vitro antitumor effect of the TFC@CM nanozyme prepared in Example 2 was tested, as follows: 1) Biosafety of TFC@CM nanozymes: At the in vitro cell experiment level, the biosafety of the TFC@CM nanozyme was first verified using the MTT assay, and the results are as follows: Figure 13 As shown. According to Figure 13 It can be seen that treating L929 and RAW264.7 cell lines with different concentrations of TFC@CM nanozyme (0~500μg / mL) resulted in the cell viability of L929 and RAW264.7 cell lines remaining above 80% regardless of whether the concentration was high or low, indicating that TFC@CM nanozyme exhibits good biocompatibility in vitro.
[0099] 2) Evaluation of the cytotoxic performance of TFC@CM nanozymes: The cytotoxic properties of TFC@CM nanozymes were tested, and the results are as follows: Figure 14 As shown. According to Figure 14 As shown in 'a', for GL261 cells, cell viability remained stable above 80% at low to medium concentrations (0–200 μg / mL), decreasing slightly only at high concentrations (500 μg / mL), but still remaining >50%. Subsequently, the cytotoxic effect of TFC@CM nanozyme on GL261 tumor cells was detected using trypan blue staining, and the results are as follows: Figure 14 As shown in b. According to Figure 14 As shown in b, no significant cytotoxicity was observed in the control group, but cell survival rate decreased significantly when treated with TFC@CM nanozyme. The TFC@CM nanozyme + LFMF group had the lowest cell survival rate, indicating that TFC@CM nanozyme has a significant killing effect on GL261 tumor cells.
[0100] 3) Assessment of the uptake capacity of GL261 cells by TFC@CM nanozymes: To test the uptake capacity of tumor cells by TFC@CM nanozymes, this test case used Rhodamine B (RHB)-labeled TFC@CM nanozymes to co-incubate GL261 cells for different times. Fluorescence images at different time points were captured using GLSM and their quantitative analysis was performed to evaluate the uptake. The results are as follows: Figure 15 As shown. According to Figure 15 It can be seen that there is no red fluorescence in the cytoplasm of L929 cells, while there is red fluorescence in the cytoplasm of GL261 cells, and the fluorescence intensity increases with the extension of incubation time. The GL261+SMF group showed a significantly stronger red fluorescence than GL261, indicating that under the drive of SMF, TFC@CM nanozyme can be rapidly and effectively internalized by GL261 cells.
[0101] 4) Assessment of the ROS production capacity of GL261 cells for TFC@CM nanozymes: To test the effect of TFC@CM nanozyme on ROS production in cells, the intensity of intracellular green fluorescence signal in PBS, LFMF, TFC@CM nanozyme, and TFC@CM nanozyme + LFMF (10 Hz, 600 rpm) was detected using the fluorescent probe 2,7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The results are as follows: Figure 16 As shown. According to Figure 16 It can be seen that the TFC@CM nanozyme + LFMF group showed the strongest intracellular green fluorescence signal intensity, indicating that TFC@CM nanozyme can catalyze the generation of a large amount of ROS under LFMF stimulation, thereby killing tumor cells.
[0102] 5) Evaluation of the mitochondrial damage effect of TFC@CM nanozymes: The above tests have confirmed that TFC@CM nanozymes can induce GL261 cells to produce large amounts of ROS under LFMF stimulation. To further test whether TFC@CM nanozymes induce mitochondrial membrane potential changes through ROS-mediated oxidative stress, thereby leading to mitochondrial damage, this test case used the JC-1 fluorescent probe. When the mitochondrial membrane potential was normal, the JC-1 probe showed red fluorescence, while when the membrane potential decreased, it showed green fluorescence. The results are as follows: Figure 17 As shown. According to Figure 17 It can be seen that both G1 and G2 groups showed red fluorescence, indicating that the mitochondrial membrane potential was in a normal state; G3 group showed a small amount of green fluorescence, while G4 group showed a large amount of green fluorescence and a decreased mitochondrial membrane potential; this confirms that TFC@CM nanozyme can cause mitochondrial functional damage under LFMF stimulation, and mitochondrial damage will further induce ICD.
[0103] Test Example 9: The in vivo antitumor effect of the TFC@CM nanozyme prepared in Example 2 was tested, and the details are as follows: To test the in vivo therapeutic effect of TFC@CM nanozyme, an orthotopic GL261 tumor-bearing mouse model was established for this test case. First, the targeting efficiency of TFC@CM nanozyme at the tumor site was evaluated using fluorescence imaging after intranasal administration. The results are as follows: Figure 18 As shown. According to Figure 18 It can be seen that the signal intensity of the TFC@CM-ICG group is better than that of the control group, indicating that the homologous targeting effect of tumor cells enhances the targeting efficiency of TFC@CM nanozyme at the tumor site. The fluorescence signal of the TFC@CM-ICG@CM+SMF group reaches its strongest at 2 hours, indicating that the effects of CM and SMF can synergistically enhance the tumor targeting efficiency and in vivo retention time of TFC@CM nanozyme.
[0104] Next, after successfully establishing an orthotopic glioblastoma model, this test case involved LFMF treatment administered via the nasobrain route. The treatment efficacy in tumor-bearing mice was monitored during the treatment cycle using intraperitoneal injection of D-fluorescein potassium, observed in vivo using a small animal bioluminescence imaging system. The groups were as follows: G1: PBS; G2: SMF; G3: LFMF (10Hz, 600rpm); G4: TFC@CM; G5: TFC@CM + LFMF (10Hz, 600rpm). The dynamic monitoring results of in vivo bioluminescence imaging after nasobrain administration in tumor-bearing mice are as follows: Figure 19 As shown.
[0105] according to Figure 19 As can be seen, stable bioluminescent signals were detected in the brains of all tumor-bearing mice, confirming the successful construction of the orthotopic glioma model. The fluorescence signal intensity of the control group (G1, G2, G3) gradually increased over time, showing a strong red fluorescence signal on day 14. The fluorescence signal intensity of the G4 group also increased after drug administration because no stimulation was applied. The signal intensity of the G5 group remained at a very low level with no obvious high-intensity signal, indicating that tumor growth was significantly inhibited and showing a good in vivo tumor treatment effect.
[0106] The embodiments of the present invention have been described above; however, these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the above embodiments of the present invention without inventive effort are within the protection scope of the present invention.
Claims
1. A low-frequency magnetic field triggered magnetic nanoszyme, characterized in that, It includes a core and a mouse glioma cell membrane covering the surface of the core; the core includes a magnetic titanium-doped Fe-CN nanozyme.
2. A method for preparing low-frequency magnetic field triggered magnetic nanoszyme, characterized in that, The low-frequency magnetic field triggered magnetic nanozyme of claim 1 includes the following steps: (1) Prussian blue was doped with titanium and calcined to obtain magnetic Ti / Fe-CN nanozymes; (2) Mouse glioma cell membranes were coated on the surface of magnetic Ti / Fe-CN nanozymes to obtain low-frequency magnetic field triggered magnetic nanozymes.
3. The production method according to claim 2, characterized by, The process of doping Prussian blue with titanium involves the following steps: premixing polyvinylpyrrolidone and a portion of hydrochloric acid to obtain a polyvinylpyrrolidone premix; premixing potassium ferricyanide and the remaining hydrochloric acid to obtain a potassium ferricyanide premix; then adding the potassium ferricyanide premix dropwise to the polyvinylpyrrolidone premix and mixing to obtain a premixed solution; and adding a titanium source to the premixed solution for a coprecipitation reaction.
4. The production method according to claim 3, characterized in that, The mass ratio of polyvinylpyrrolidone to potassium ferricyanide is 1:0.9~1.
1.
5. The production method according to claim 3, characterized by, The molar ratio of titanium in the titanium source to iron in potassium ferricyanide is 9~11:
100.
6. The production method according to claim 3, characterized by, The coprecipitation reaction was carried out under oil bath conditions; the temperature of the oil bath was 78~82 degrees Celsius; and the coprecipitation reaction time was 18~25 hours.
7. The preparation method according to claim 2, characterized in that, The calcination temperature is 660~730 degrees Celsius, and the time is 1.8~2.2 hours; the calcination is carried out in a protective atmosphere.
8. The preparation method according to claim 2, characterized in that, Encapsulating mouse glioma cell membranes The process includes the following steps: mixing magnetic Ti / Fe-CN nanozymes with a solvent to obtain a magnetic Ti / Fe-CN nanozyme dispersion; mixing a cell membrane dispersion and a magnetic Ti / Fe-CN nanozyme dispersion and then centrifuging, discarding the supernatant, and freeze-drying.
9. The preparation method according to claim 8, characterized in that, The preparation method of cell membrane dispersion includes the following steps: digesting the substrate with trypsin containing ethylenediaminetetraacetic acid, collecting GL261 cells, resuspending them, freezing and thawing them, centrifuging them to obtain the supernatant, centrifuging the supernatant to discard the supernatant, collecting the precipitate, and dispersing the precipitate in water.
10. The application of a low-frequency magnetic field-triggered magnetic nanozyme in the preparation of glioblastoma drugs or the construction of glioblastoma targeting systems; characterized in that, The low-frequency magnetic field triggered magnetic nanoenzyme is the low-frequency magnetic field triggered magnetic nanoenzyme of claim 1.