Preparation method of tumor magnetic targeting guided magnetic heat self-enhanced nanodiagnosis and treatment agent

Abstract

The application discloses a preparation method of a tumor magnetic targeting guided magnetic heat self-enhancing nano diagnosis and treatment agent, and belongs to the technical field of cancer diagnosis and treatment. The preparation method comprises the following steps: S1, preparing NaGdF4:Nd / Yb Tm nanoparticles; and S2, preparing NaGdF4:Nd / Yb Tm@WFe2O4-NH2-PEG magnetic nanoparticles. The tumor magnetic targeting guided magnetic heat self-enhancing nano diagnosis and treatment agent provided by the application has good dispersity, strong magnetic attraction, can be efficiently enriched at a tumor site under magnetic guidance, can generate a large amount of heat in an alternating magnetic field to realize MHT treatment of the tumor, has near-infrared II region fluorescence imaging and MRI dual-mode imaging effects, thereby improving the diagnosis and treatment accuracy of the tumor and realizing diagnosis and treatment integration, has high biological safety and small toxic and side effects, and the preparation method is simple and easy to operate.

Description

Technical Field

[0001] This invention relates to the field of cancer diagnosis and treatment technology, and in particular to a method for preparing a tumor magnetically targeted, magnetothermal self-enhanced nanotherapeutic agent. Background Technology

[0002] With the continuous improvement of people's living standards, cancer has gradually become one of the leading causes of morbidity and mortality worldwide. According to IARC statistics, in 2021 alone, the number of new cancer cases worldwide exceeded 21 million, and the number of deaths from cancer exceeded 12 million. Traditional diagnostic methods lack specificity, sensitivity, and resolution, meaning most cancer patients are diagnosed at middle or late stages, missing the optimal treatment period. Furthermore, current clinical treatments (including chemotherapy, radiotherapy, and surgery) are disconnected from diagnosis and have invasive and side effects, further limiting the efficiency and accuracy of cancer treatment.

[0003] Currently, novel cancer treatments have been developed, including immunotherapy, gene therapy, photodynamic therapy, photothermal therapy, sonodynamic therapy, and chemodynamic therapy. Among these, magnetothermal therapy and chemodynamic therapy are considered alternatives to traditional cancer treatments due to their non-invasiveness, low toxicity, and negligible drug resistance, and have received increasing attention.

[0004] However, due to the influence of the solid tumor microenvironment (such as overexpression of glutathione, high levels of hydrogen peroxide, low pH, and insufficient oxygen content), a single treatment cannot completely cure cancer. Furthermore, current clinical assessment of cancer efficacy primarily relies on cytology, histopathology, and imaging, separating treatment from efficacy evaluation. Therefore, integrating cancer treatment and efficacy assessment in clinical practice remains a significant challenge.

[0005] As a new generation of artificial enzymes, nanozymes show great promise in tumor catalytic therapy. In particular, peroxidase activity has been used to catalyze the production of highly toxic hydroxyl radicals (·OH) from hydrogen peroxide to kill tumor cells. However, due to the low affinity between nanozymes and H₂O₂, peroxidase nanozymes typically produce insufficient ·OH to kill tumor cells. Currently, most effective nanozyme-based tumor therapy strategies are combined with other therapeutic interventions, such as photodynamic therapy, sonodynamic therapy, and immunotherapy.

[0006] In recent years, magnetothermal therapy (MHT) has attracted increasing attention due to its good therapeutic effects, high tissue penetration, and non-invasiveness. The therapeutic effect of MHT on tumors stems from the heat generated by magnetothermal materials under an alternating magnetic field (AMF), which rapidly raises the temperature of tumor tissue to a certain level, killing or inducing tumor cell apoptosis. However, compared to photothermal materials used in photothermal therapy, the thermal efficiency of magnetothermal materials has always been relatively low. To achieve the required therapeutic temperature, the dosage of magnetic nanoparticles needs to be significantly increased during treatment; however, increasing the dosage of magnetic nanoparticles inevitably leads to cytotoxicity issues. Furthermore, although in situ injection can deliver materials directly to superficial tumors, deep tumors remain unreachable. While MHT based on intravenously injected magnetic materials has clinical application potential for deep-seated tumors, the low magnetothermal efficiency and insufficient material accumulation at the lesion site severely limit its application in clinical tumor treatment. Therefore, developing an MHT material with excellent magnetothermal and high targeting properties is of great significance.

[0007] Fluorescence imaging, as an emerging diagnostic technique, has attracted considerable research interest due to its high specificity and sensitivity. With the continuous development of optical imaging technology, the bioimaging window has gradually expanded from the visible light region (400–700 nm) to the first near-infrared region (NIR-I, 700–900 nm) and the second near-infrared region (NIR-II, 1000–1700 nm). Currently, visible light imaging typically has some drawbacks, such as strong absorption by endogenous substances, high tissue scattering, and autofluorescence interference, limiting its penetration depth to 1 mm. Due to the lower tissue absorption / scattering and autofluorescence effect in the NIR region, NIR light can achieve deeper tissue penetration. Compared to NIR-I FLI, NIR-II FLI has longer excitation and emission wavelengths, thus offering advantages in spatiotemporal resolution, signal-to-noise ratio, and imaging. Therefore, NIR-II fluorescence imaging has broad application prospects in the accurate diagnosis and treatment guidance of early-stage tumors. Therefore, the development of nanomedicines that combine NIR-II fluorescence imaging with multiple treatment methods is of great significance for improving the efficiency and accuracy of tumor treatment and increasing the cure rate and survival rate of patients.

[0008] Ferroprelation is a newly discovered non-apoptotic form of cell death, distinct from necrosis and autophagy. Its primary inducing factor is the excessive accumulation of iron ions in cells, where glutathione and other reducing agents cannot reduce the reactive oxygen species (ROS) generated by the Fenton reaction, leading to a significant increase in lipid peroxides (LPO) and ultimately cell death. Although the exact mechanism of iron in ferroptosis remains unclear, various studies have shown that highly tumorigenic, invasive, and metastatic tumor cells are particularly sensitive to ferroptosis. In recent years, strategies for inducing ferroptosis using iron-based nanomaterials to treat tumors have been extensively studied. However, due to the high permeability and retention effect (EPR) of solid tumors, most iron-based nanomedicines are difficult to effectively treat tumors. Therefore, developing effective targeted nanomaterial carriers is particularly important for the clinical treatment of solid tumors. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing a tumor magnetically targeted and guided magnetothermal self-enhanced nanotherapeutic agent, in order to solve the technical problems of low thermal efficiency, poor targeting performance, large toxic side effects on patients due to increased dosage, and the disconnect between cancer treatment assessment methods and diagnosis.

[0010] To achieve the above objectives, this invention provides a method for preparing a tumor magnetically targeted, magnetothermal self-enhanced nanotherapeutic agent, comprising the following steps:

[0011] S1. Preparation of NaGdF4:Nd / Yb Tm nanoparticles

[0012] The prepared rare earth raw materials were added to a mixed solvent of oleic acid and octadecene. Under the protection of an inert gas, the temperature was raised to 110°C and held for 10-20 minutes to remove water from the solution. Then the temperature was raised to 155°C until the raw materials were completely dissolved. The system temperature was then lowered to 75°C, sodium fluoride was slowly added dropwise, and the temperature was maintained for 1 hour. The temperature was then rapidly raised to 310°C and held for 1 hour. After cooling to room temperature, the product was washed three times each with cyclohexane and anhydrous ethanol. The obtained NaGdF4:Nd / Yb Tm nanoparticles were dispersed in cyclohexane and kept at room temperature.

[0013] S2. Preparation of NaGdF4:Nd / YbTm@WFe2O4--NH2-PEG magnetic nanoparticles

[0014] The nanoparticle dispersion prepared in step S1 was slowly added dropwise to sodium citrate aqueous solution under ultrasonication. After magnetic stirring for 24 hours, cyclohexane was evaporated and removed. After centrifugation, the precipitate was taken and freeze-dried.

[0015] Take the freeze-dried solid sample and add it to deionized water, then add ferrous sulfate, stir overnight at 65°C, then add sodium tungstate dihydrate, and after it is completely dissolved, add polyacrylamide to the system and stir at room temperature for 30 min.

[0016] After adjusting the pH of the system to 9, the mixture is placed in a high-pressure reactor with a polytetrafluoroethylene liner and kept at 200°C for 12 hours. The product is then dialyzed and freeze-dried.

[0017] Preferably, in step S1, the molar ratio of rare earth raw materials Gd:Yb:Nd:Tm is 0.6:0.9:0.48:0.02, the volume ratio of oleic acid to octadecene is 2:5, and the molar-volume ratio of rare earth raw materials to oleic acid is 1 mmol:6 mL.

[0018] Preferably, in step S1, the slow addition of sodium fluoride refers to adding it at a rate of 10 drops / min; the rapid heating to 310℃ refers to heating at a rate of 10℃ / min; when the NaGdF4:Nd / YbTm nanoparticles are dispersed in cyclohexane, the molar-volume ratio of NaGdF4:Nd / YbTm nanoparticles to cyclohexane is 1 mmol:10 mL.

[0019] Preferably, in step S2, the ultrasonic power during the process of adding the dispersion to the sodium citrate aqueous solution is 300W, and the ultrasonication continues until the dispersion is completely added; slow addition refers to adding at a rate of 2 drops / min, and the concentration of the sodium citrate aqueous solution is 2M.

[0020] Preferably, in step S2, the magnetic stirring rate is 600 r / min, the evaporation temperature is 50°C, and the centrifugation conditions are centrifugation at 11000 rpm / min for 20 min.

[0021] Preferably, in step S2, the mass-to-volume ratio of the freeze-dried solid sample to deionized water is 0.325 g: 10 mL, and the mass ratio of the freeze-dried solid sample to ferrous sulfate, sodium tungstate dihydrate, and polyacrylamide is 325:432:329:566.

[0022] Preferably, in step S2, the pH of the mixture is adjusted using 1M NaOH solution, and the high-pressure reactor is lined with polytetrafluoroethylene; in both steps S1 and S2, the mixture needs to be pre-frozen at -20°C for 3 hours before freeze-drying.

[0023] Preferably, in step S2, dialysis is performed using a dialysis bag with MD=8000, and the dialysis solution is pure water containing 0.01M NH2-PEG (Mn=2000). The dialysis solution is changed every 2 hours, and the dialysis time is 12 hours.

[0024] A tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent prepared by the method described above.

[0025] Application of a tumor magnetically targeted, magnetothermal self-enhanced nanotherapeutic agent prepared by the method described above in tumor diagnosis and treatment.

[0026] This invention discloses a magnetocalorically responsive nanozyme composed of rare earth elements and variable-valence metal elements. This nanozyme exhibits both peroxidase-like (POD-like) and glutathione oxidase (GSH-Ox) activities. Under the influence of an applied alternating magnetic field (AMF), it accelerates the release and accumulation of iron ions in tumor cells, thereby inducing ferroptosis in tumor cells. This invention also provides a method for preparing a magnetically targeted, magnetocalorically enhanced nanozyme therapeutic agent. Under the influence of an applied static magnetic field, the nanoparticles are effectively enriched at the tumor site, thereby enhancing the diagnostic and therapeutic effects of tumors. Specifically, NaGdF4:Nd / Yb Tm rare earth nanoparticles (DUCNPs) with both upconversion and downconversion properties are synthesized via high-temperature decomposition. Furthermore, NaGdF4:Nd / Yb Tm@WFe2O4 nanoparticles with a core-shell structure are prepared using in-situ growth technology. The biocompatibility of the nanoparticles is further improved through surface modification with amino-modified polyethylene glycol (NH2-PEG). Under the influence of an alternating magnetic field, heat can be generated in a relatively short time to achieve MHT treatment. Simultaneously, the acidic tumor microenvironment with high hydrogen peroxide (H2O2) expression and the combined effect of magnetothermal action accelerate the Fe... 2+ / W 5+ The release of [a substance] generates a large amount of ·OH through the Fenton reaction. The generated high-valence metal ions (Fe2+)... 3+ / W 6+ The rare earth nanoparticles are reduced by endogenous glutathione (GSH), further inactivating glutathione-dependent peroxidase (GPX4), thereby increasing the efficiency of ferroptosis by disrupting the redox balance. Simultaneously, under 808nm laser irradiation, the rare earth nanoparticles achieve dual-mode fluorescence emission at 475nm upconversion and 1350nm downconversion. The 475nm light energy is absorbed by the shell through fluorescence energy resonance transfer, generating a PDT therapeutic effect. The introduction of Yb / Tm elements effectively enhances the T2-weighted MRI imaging signal of DCF-NPs. Reactive oxygen species obtained from the combined therapy can effectively trigger immunogenic cell death through synergistic therapy based on induced anti-tumor immunity, thereby inhibiting primary and distant tumors and providing highly efficient tumor treatment effects. Using MRI and near-infrared two-zone fluorescence multimodal imaging, more precise cancer diagnosis and treatment monitoring can be achieved. Magnetothermia further amplifies the combined therapeutic effects of chemodynamic therapy, photodynamic therapy, and ferroptosis, thus realizing the integration of multimodal imaging and multimodal combined therapy.

[0027] Therefore, the preparation method of the tumor magnetically targeted magnetothermal self-enhanced nanotherapeutic agent provided by the present invention has the following specific technical effects:

[0028] (1) The DCF-NPs prepared by this invention have good dispersibility and good magnetic attraction ability. Under the action of an external magnetic field, they can achieve efficient enrichment at the tumor site through magnetic guidance. At the same time, the up-conversion fluorescence of NaGdF4:Nd / Yb Tm can be used to realize the integrated diagnosis and treatment of tumors, thereby improving the accuracy, efficiency and effect of diagnosis and treatment.

[0029] (2) The DCF-NPs prepared in this invention can generate a large amount of heat in an alternating magnetic field to kill tumor cells and achieve the MHT treatment effect. The heat generation increases significantly with the increase of DCF-NPs solution concentration and the extension of alternating magnetic field application time.

[0030] (3) The DCF-NPs prepared in this invention can accelerate Fe under the dual effects of magnetothermal activity and acidic tumor microenvironment with high hydrogen peroxide expression. 2+ / W 5+ The release of [the substance] generates a large amount of ·OH(CDT) through the Fenton reaction, while the generated high-valence metal ions (Fe) [are also present]. 3+ / W 6+ It is reduced by endogenous glutathione (GSH) and further inactivates glutathione-dependent peroxidase (GPX4), thereby increasing the efficiency of tumor cell ferroptosis by disrupting the redox balance.

[0031] (4) The NaGdF4:Nd / Yb Tm downconversion fluorescence in this invention can realize near-infrared II region fluorescence imaging for tumor treatment, and the rare earth elements therein can further enhance the MRI imaging capability of DCF-NPs, realizing the dual-mode imaging effect of near-infrared II region fluorescence imaging and MRI, thereby achieving accurate diagnosis and treatment of tumors.

[0032] (5) The present invention improves the biosafety of DCF-NPs by surface-aminated polyethylene glycol (NH2-PEG) modification. When the concentration is 200 μg / mL, the cell survival rate is still above 90%.

[0033] (6) The DCF-NPs provided by this invention have shown good anti-tumor effects in both cellular and mouse in vivo experiments. When CDT, PDT and MHT are used in combination for treatment, they show complete inhibition of tumor cells and are expected to cure breast cancer.

[0034] (7) This invention synthesizes NaGdF4:Nd / YbTm rare earth nanoparticles (DUCNPs) with both up- and down-conversion properties through high-temperature decomposition, and further successfully prepares NaGdF4:Nd / YbTm@WFe2O4 nanoparticles with core-shell structure through in-situ growth technology. The preparation method is simple, easy to operate, safe and efficient.

[0035] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 The results are the analytical results of the DUCNPs prepared in Example 1, where part A is the SEM image, part B is the DLS image, and part C is the XRD result image.

[0038] Figure 2 The results are the analysis results of DCF-NPs in Example 2, where part A is the SEM image, part B is the DLS image, and part C is the XRD result image.

[0039] Figure 3 The image shows the Fourier transform infrared spectrum of DCF-NPs, where DCF-NPs-Cia is the product of DCF-NPs prepared in Example 1 coated with a layer of sodium citrate, and DUCNPs-OA is the DUCNPs prepared in Example 1.

[0040] Figure 4 The results are the optical conversion performance test results of DUCNPs and DCF-NPs under 808nm laser irradiation.

[0041] Figure 5 These are the ultraviolet-visible absorption spectra of DUCNPs and DCF-NPs;

[0042] Figure 6 This is the enzyme activity assessment result of DCF-NPs, where Part A is the POD-Like activity result; Part B is the GSH-Ox activity result; and Part C is the effect of alternating magnetic field on the GSH-Ox activity of DCF-NPs.

[0043] Figure 7 These are photographs evaluating the magnetic properties of DCF-NPs;

[0044] Figure 8 This describes the relationship between temperature and time for DCF-NPs under the influence of an alternating magnetic field.

[0045] Figure 9 These are T2-weighted images of WFe2O4 and DCF-NPs at different Fe concentrations;

[0046] Figure 10The T2 relaxation rates of WFe2O4 and DCF-NPs under different Fe concentrations are:

[0047] Figure 11 This section presents the results of the toxicity study of DCF-NPs on normal and tumor cells. Part A shows the effect of different concentrations of DCF-NPs on the viability of normal cells; Part B shows the toxicity effect of DCF-NPs on 4T1 cells under different conditions. PBS is the control group, AMF+NIR is the conditional treatment group, DCF-NPs is the group with DCF-NPs only, CDT is the DCF-NPs+H2O2 group, PDT is the DCF-NPs+NIR group, MHT is the DCF-NPs+AMF group, CDT+PDT is the DCF-NPs+H2O2+NIR group, CDT+MHT is the DCF-NPs+H2O2+AMF group, PDT+MHT is the DCF-NPs+NIR+AMF group, and CDT+PDT+MHT is the DCF-NPs+H2O2+NIR+AMF group.

[0048] Figure 12 These are images of cellular uptake by DCF-NPs after the application of an external magnetic field;

[0049] Figure 13 This is an evaluation of ferroptosis. Part A shows the statistical results of changes in intracellular GSH content, Part B shows the immunofluorescence image of intracellular GPX4, and Part C shows the results of changes in lipid peroxide (LPO) content.

[0050] Figure 14 These are tumor photos and statistical results from different treatment groups 16 days later. Part A shows actual images of tumors in different groups after treatment, and Part B shows the changes in tumor volume during different treatment periods (0-16 days).

[0051] Figure 15 T2-weighted MRI images of WFe2O4 and DCF-NPs under different conditions, where PBS is the control group, WFe2O4 is the tail vein injection group, DCF-NPs is the tail vein injection group, WFe2O4 M is the group where a neodymium magnet (0.03T) is placed at the tumor site after tail vein injection of DCF-NPs, and DCF-NPs M is the group where a neodymium magnet (0.03T) is placed at the tumor site after tail vein injection of DCF-NPs.

[0052] Figure 16 Near-infrared II region imaging of DCF-NPs under different conditions. The DCF-NPs group is the result of DCF-NPs injected into the tail vein only, and the DCF-NPs M is the result of placing a neodymium magnet (0.03T) at the tumor site after injecting the material into the tail vein. Detailed Implementation

[0053] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0054] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0055] The instruments, equipment, and reagents used in the examples were all obtained commercially.

[0056] Example 1

[0057] The following steps were taken to prepare a tumor magnetically targeted, magnetocalorically enhanced, self-reinforcing nanotherapeutic agent:

[0058] S1. Preparation of NaGdF4:Nd / Yb Tm nanomaterials (DUCNPs)

[0059] Two mmol of rare earth raw materials (Gd:Yb:Nd:Tm molar ratio of 0.6:0.9:0.48:0.02) were added to a mixed solvent containing 12 mL of oleic acid and 30 mL of octadecene. Under Ar protection, the mixture was heated to 110 °C and held for 10–20 min to remove water from the solution. The temperature was then increased to 155 °C until the raw materials were completely dissolved. The system temperature was then lowered to 75 °C, and 10 mL of sodium fluoride was added dropwise at a rate of 10 drops / min. The mixture was kept at this temperature for 1 h, and then the temperature was increased to 310 °C at a rate of 10 °C / min and held for 1 h. After cooling to room temperature, the product was washed three times each with cyclohexane and anhydrous ethanol. Finally, the obtained product (DUCNPs) was dispersed in 10 mL of cyclohexane and stored at room temperature.

[0060] The morphology and size of the prepared DUCNPs cyclohexane dispersion were analyzed by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The results are shown in the figure. Figure 1 ,Depend on Figure 1 Part A shows that the prepared DUCNPs have a monodisperse spherical structure with a particle size of about 20 nm. The DLS test results in Part B also confirm this result.

[0061] The crystal structure of DUCNPs was analyzed using X-ray powder diffraction (XRD), and the results are shown in the figure. Figure 1As shown in section C, the prepared DUCNPs are consistent with the standard card (JCPDS:16-0334) for rare earth-based upconversion nanomaterials, indicating that the NaGdF4:Nd / Yb Tm nanomaterials were successfully prepared.

[0062] S2. Preparation of NaGdF4:Nd / YbTm@WFe2O4--NH2-PEG magnetic nanoparticles (DCF-NPs)

[0063] The DUCNPs cyclohexane dispersion obtained in step S1 was added dropwise to a 2M sodium citrate aqueous solution at a rate of 2 drops / min under ultrasonic (300W) influence. After magnetic stirring (600 rpm) for 24 h, the cyclohexane was removed by rotary evaporation (temperature set to 50℃). The remaining liquid was then centrifuged at 11000 rpm for 20 min to obtain a colorless, transparent solid, which was freeze-dried and weighed to be 0.325 g.

[0064] Add 0.325 g of solid sample to 10 mL of deionized water, along with 0.432 g of ferrous sulfate. Stir overnight at 65 °C. Then add 0.329 g of sodium tungstate dihydrate and stir at 500 rpm / min until completely dissolved. Add 0.566 g of polyacrylamide. Stir at room temperature for 30 min. Adjust the pH of the mixture to 9 with 1 M sodium hydroxide, then transfer it to a high-pressure reactor lined with polytetrafluoroethylene (PTFE) and incubate at 200 °C for 12 h.

[0065] The product was collected and dialyzed using a dialysis bag (MD=8000). The dialysate was pure water containing 0.01M NH2-PEG (Mn=2000). The dialysate was changed every 2 hours and dialyzed continuously for 12 hours. The liquid in the dialysis bag was collected and freeze-dried to obtain brown DCF-NPs solid powder.

[0066] Implementation 2

[0067] Analysis of the prepared DCF-NPs

[0068] (1) The prepared DCF-NPs were analyzed by SEM and DLS, and the results are shown in the figure below. Figure 2 ,Depend on Figure 2 As can be seen from Part A, the prepared DCF-NPs are monodisperse, rough-surfaced spherical nanoparticles with a diameter of approximately 40 nm. Figure 2 As can be seen from Part B, the DLS test results are basically consistent with the SEM test results.

[0069] (2) The crystal structure of DCF-NPs was analyzed by XRD, and the results are shown in [Figure number missing]. Figure 2Part C of the figure shows that the crystal structure of DCF-NPs includes the crystal structure of rare earth upconversion nanomaterials (JCPDS: 16-0334) and ferrite (JCPDS: 002-4872), which indicates that DCF-NPs nanomaterials were successfully prepared.

[0070] (3) The surface structure of nanomaterials at different synthesis stages was analyzed by Fourier transform infrared spectroscopy. The results are shown in [Figure number missing]. Figure 3 ,Depend on Figure 3 It can be seen that when oleic acid is used as a surfactant, 3425cm -1 The peak appears as a stretching vibration of -OH, and at 1621 cm⁻¹ -1 The peak at 2937 cm⁻¹ is caused by the -C=O stretching vibration. -1 The peak is mainly due to the stretching vibration of CH in the oleic acid structure.

[0071] Because the molecular structure of sodium citrate is similar to that of oleic acid, it is at 3414 cm⁻¹ -1 2941cm -1 and 1606cm -1 The peaks at 3329 cm⁻¹ correspond to the stretching vibrations of -OH, CH, and -C=O in the citric acid molecule. When NH₂-PEG is used as the surfactant, the peak at 3329 cm⁻¹ corresponds to these vibrations. -1 The peak appearing nearby is due to absorption by the NH stretching vibration, 1356 cm⁻¹ -1 The peak at 1591 cm⁻¹ is the absorption peak of CN stretching vibration. -1 and 806cm -1 The peaks appearing at the specified locations correspond to the in-plane bending vibration and out-of-plane stretching vibration of NH, respectively. These results indicate that the surfactant was successfully introduced before and after synthesis.

[0072] (4) The optical conversion performance of DUCNPs and DCF-NPs under 808nm laser irradiation was tested respectively. The results are shown in […]. Figure 4 ,Depend on Figure 4 It can be seen that under 808nm laser irradiation, DUCNPs can emit upconversion fluorescence at 475nm and near-infrared II fluorescence at 1350nm. Under the same conditions, the fluorescence intensity of the recombined DCF-NPs in the near-infrared II region does not change significantly compared to DUCNPs, while the fluorescence at 475nm disappears. This is mainly due to the strong ultraviolet absorption of DCF-NPs at 475nm (see...). Figure 5 The 475nm fluorescence emitted by DUCNPs is absorbed by WFe2O4 via fluorescence energy resonance transfer to generate singlet oxygen.

[0073] The above results indicate that the prepared DCF-NPs can simultaneously generate upconversion and downconversion fluorescence.

[0074] Example of effect 1

[0075] The peroxidase-like activity of DCF-NPs was assessed using 3,3',5,5'-tetramethylbenzidine (TMB) as follows:

[0076] First, PBS buffer solutions with different pH values ​​were prepared, and then the nanomaterials were dispersed into the buffer systems. Next, TMB solution was added to a final concentration of 0.4 mM. To further simulate the tumor microenvironment, a certain amount of H2O2 was added to the buffer system to achieve a final concentration of 100 × 10⁻⁶. -6 M). The POD-Like activity of DCF-NPs was evaluated by measuring the UV absorption of the mixed solution.

[0077] Test results are as follows Figure 6 As shown in Part A, the POD-Like activity of DCF-NPs increases with decreasing pH. This is mainly because under acidic conditions, the release rate of iron ions (II) and tungsten ions (V) in DCF-NPs increases, and the number of hydroxyl radicals generated by the two and hydrogen peroxide per unit time increases, which in turn leads to an increase in the amount of oxidized TMB, resulting in higher UV absorption at 652 nm.

[0078] The results showed that DCF-NPs have excellent peroxidase-like activity, and the enzyme activity was further improved under the action of an external alternating magnetic field.

[0079] Example of effect 2

[0080] The GSH-Ox activity of the DCF-NPs prepared in Example 1 was evaluated by measuring the consumption of glutathione by the DCF-NPs using the DTNB colorimetric method. The results are as follows: Figure 6 As shown in Part B, the content of residual glutathione decreases over time, indicating that DCF-NPs can effectively consume GSH content.

[0081] To further confirm the effect of alternating magnetic fields on the activity of GSH-Ox, an alternating magnetic field with a frequency of 598 kHz and a field strength of 1.38 mT was simultaneously applied to the DCF-NPs during the test. The test results are as follows. Figure 6 As shown in section C, the results indicate that under the action of an external alternating magnetic field, the consumption of glutathione increases per unit time, and the activity of GSH-Ox enzyme is further enhanced.

[0082] Example of effect 3

[0083] The magnetic properties of the DCF-NPs prepared in Example 1 were evaluated as follows: 0.1 g of DCF-NPs was added to 5 mL of PBS and ultrasonically dispersed to prepare a uniformly dispersed DCF-NPs solution in a colorless transparent glass bottle. A neodymium magnet with a magnetic field strength of 0.03 T was placed against one side of the bottle. The experimental results are shown in [Figure number missing]. Figure 7 .

[0084] As can be seen from the figure, the uniformly dispersed DCF-NPs solution becomes clear and transparent 30 seconds after the neodymium magnet is placed, indicating that DCF-NPs have good magnetic attraction ability and can be enriched at the tumor site through magnetic targeting.

[0085] DCF-NPs solutions with different dispersion concentrations were prepared using the same method described above. These solutions were placed in 1.5 mL transparent conical centrifuge tubes and then placed in an alternating magnetic field with a frequency of 598 kHz and a field strength of 23.5 mT to test the temperature-time relationship of DCF-NPs under the influence of the alternating magnetic field. The results are as follows: Figure 8 As shown, under the influence of an alternating magnetic field, the temperature of the DCF-NPs solution gradually increases with time. Furthermore, within the same time interval, the higher the concentration of the DCF-NPs solution, the greater the temperature increase, and this trend becomes more pronounced over time. These results indicate that DCF-NPs possess excellent magnetocaloric properties.

[0086] Example of effect 4

[0087] To investigate whether the DCF-NPs prepared in Example 1 have MRI effects, the method was as follows: DCF-NPs were dispersed in 1.5% agarose gel with iron ion concentrations ranging from 0 to 0.64 mM. Experiments were conducted using a GE 3.0T MRI scanner. T2-weighted images of WFe2O4 and DCF-NPs at different Fe concentrations are shown below. Figure 9 The T2 relaxation rates of WFe2O4 and DCF-NPs under different Fe concentrations are shown in the figure. Figure 10 .

[0088] Depend on Figure 9 and 10 It can be seen that at low iron concentrations, both WFe2O4 and DCF-NPs exhibit significant T2-weighted MRI signals. With increasing iron concentration, the T2 contrast signal gradually increases. This suggests that both WFe2O4 and DCF-NPs nanoparticles can shorten the transverse relaxation time of water protons, and therefore can both serve as MRI contrast agents. At the same iron concentration, the T2 signal of DCF-NPs is significantly enhanced compared to WFe2O4. This is mainly due to the introduction of Yb / Tm elements into the core structure of DCF-NPs, which contain most paramagnetic lanthanide ions, such as Dy... 3+ Ho3+ Tm 3+ Yb 3+ This results in a short electronic transverse relaxation time, affecting T2-weighted MRI. Among these, Yb... 3+ Ions possess relatively short electronic relaxation times and high effective magnetic moments, enabling effective T2 relaxation. The value (1 / T2) derived from the slope of a linear fit between the transverse relaxation rate (r2) and the reciprocal of the transverse relaxation time also demonstrates that DCF-NPs are more effective at producing T2-enhanced magnetic resonance imaging.

[0089] Example of effect 5

[0090] The antitumor effect of the DCF-NPs material prepared in step one at the cellular level was investigated using the following methods:

[0091] First, the biosafety of DCF-NPs to normal mouse 3T3 and RAW 264.7 cells was evaluated using the MTT assay. The results are as follows: Figure 11 As shown in Part A, the cell survival rate remained above 90% with increasing DCF-NP concentration (0-200 μg / mL), indicating that DCF-NPs have good biocompatibility. Therefore, 200 μg / mL was used as a safe dose for subsequent experiments.

[0092] The cytotoxic effects of DCF-NPs on 4T1 breast cancer cells under different conditions were investigated using the MTT assay. The results are as follows: Figure 11 As shown in Part B, the combined treatment of CDT, PDT, and MHT significantly outperformed single-therapy in killing cancer cells, with a survival rate of less than 12% for 4T1 cells during combined therapy, demonstrating highly efficient and significant cancer cell killing capabilities. This indicates that magnetothermal therapy combined with CDT and PDT can effectively cure breast cancer.

[0093] To further explore the tumor mechanism of DCF-NPs combined therapy, the magnetic targeting effect of DCF-NPs was investigated through a cellular uptake experiment. The method involved adding a neodymium magnet to the bottom of a cell culture dish and utilizing the fluorescence properties of Rhodamine B to observe the uptake of each nanoparticle by cells in PBS (control group), DCF-NPs (non-targeting group), and DCF-NPs+M (magnetically targeted group) at 3 h and 6 h. The results are as follows: Figure 12 As shown, there was virtually no drug accumulation in the non-targeted group. Under the influence of an external magnetic field, DCF-NPs achieved significant enhancement in accumulation within a short period of time, indicating that DCF-NPs exhibited excellent cell uptake effect under magnetic targeting.

[0094] Example of effect 6

[0095] The changes in GSH levels in 4T1 breast cancer cells after treatment with DCF-NPs were investigated using the following method:

[0096] The results are as follows Figure 13 As shown: Compared with the control group, the intracellular GPX4 content decreased in the CDT and magnetothermal therapy groups. Simultaneously, under the influence of an external magnetic field, the GPX4 content in the combined therapy group significantly decreased. This is mainly because DCF-NPs can generate a large amount of heat in an alternating magnetic field, which promotes the release of metal ions and accelerates the Fenton reaction to generate a large amount of ·OH. The generated high-valence metal ions are reduced by endogenous glutathione (GSH), further inactivating glutathione-dependent peroxidases. This increases the efficiency of ferroptosis by disrupting the redox balance. The ferroptosis marker LPO was detected... Figure 13 Part C of the study also demonstrated that magnetothermal combined with chemodynamic and photodynamic therapy can effectively improve the efficiency of induced ferroptosis in 4T1 cells.

[0097] Example of effect 7

[0098] The antitumor effect of DCF-NPs in vivo was verified experimentally as follows: An orthotopic breast cancer model was established using 4-week-old female BALB / c mice. The method involved injecting 4T1 cells under the mammary pads after an incision in the abdominal skin of the mice. When the tumor volume reached 100 mm², the antitumor effect was assessed. 3 Mice were treated with PBS, 808nm laser, and an external alternating magnetic field, respectively. The mice were randomly divided into six groups, receiving PBS, 808nm laser, and an external alternating magnetic field treatment, respectively designated as the PBS group, DCF-NPs (non-targeted group), DCF-NPs C (CDT group), DCF-NPs C+P group (CDT and PDT combined treatment group), DCF-NPs C+M group (CDT and MHT combined treatment group), and DCF-NPs C+M+P group (CDT, MHT, and PDT combined treatment group). At a dose of 20 mg / kg body weight, a PBS solution containing DCF-NPs was injected into the tumor-bearing mice via the tail vein. Tumor size was measured 7 days later.

[0099] The results are as follows Figure 14 As shown, compared to the control group, the tumors in the DCF-NPs (non-targeted group) mice were not inhibited, while compared to the DCF-NPs C group, the tumors were inhibited to some extent. Under the action of an external magnetic field, the growth of the tumors in the DCF-NPs C+M group was significantly inhibited. Under the combined action of near-infrared light and magnetic field, the tumors in the DCF-NPs C+M+P group were completely inhibited. The results indicate that under the guidance of a magnetic field, DCF-NPs can effectively accumulate at the tumor site and produce a certain tumor-inhibiting effect. Under the combined action of alternating magnetic field and near-infrared light, the combined treatment of DCF-NPs can completely cure breast cancer.

[0100] Example of effect 8

[0101] Using a GE 3.0T MRI scanner and a near-infrared in vivo imaging system, the dual-modal imaging of DCF-NPs in mice was evaluated. The results are as follows: Figure 15 As shown, without an external magnetic field, no significant changes were observed in the tumor sites of WFe2O4 and DCF-NPs compared to the control group after 6 hours. However, under the influence of an external magnetic field, the tumor sites of both WFe2O4 and DCF-NPs became noticeably darker after 6 hours. This is mainly due to the effective accumulation of magnetic nanoparticles at the tumor site under the guidance of the external magnetic field. With the same iron content, the T2 imaging effect of the DCF-NPs group was significantly better than that of WFe2O4 after 6 hours. This is because DCF-NPs contain a higher content of rare earth elements, and the high content of paramagnetic lanthanide ions Yb / Tm further enhances the T2 imaging effect.

[0102] Example of effect 9

[0103] To evaluate the near-infrared II imaging capability of DCF-NPs, the method involved injecting nanomaterials into the tail vein and then guiding the accumulation of nanoparticles at the tumor site by placing a neodymium magnet (0.03T, 10mm in diameter, and 2mm thick) at the tumor site.

[0104] The results are as follows Figure 16 As shown, without an applied magnetic field, nanoparticles did not accumulate at the tumor site but were metabolized in the liver. After 12 hours, the fluorescence signal in the liver weakened, indicating that the nanoparticles were metabolized and excreted. However, under the influence of a magnet, a strong fluorescence signal appeared at the tumor site after 6 hours, indicating a large accumulation of nanoparticles. After 12 hours, the fluorescence signal at the tumor site weakened, while the fluorescence signal at the liver site strengthened. These results demonstrate that DCF-NPs can effectively accumulate at the tumor site under the guidance of an external magnetic field, enabling near-infrared II fluorescence imaging and MRI of the tumor, thus allowing for more accurate tumor diagnosis.

[0105] Therefore, the tumor magnetically targeted guided magnetothermal self-enhanced nanotherapeutic agent provided by this invention has good dispersibility and strong magnetic attraction, and can achieve efficient enrichment at the tumor site under magnetic guidance; it can generate a large amount of heat in the magnetic field to achieve MHT treatment of tumors; it has the dual-mode imaging effect of near-infrared II region fluorescence imaging and MRI, thereby improving the accuracy of tumor diagnosis and treatment and realizing integrated diagnosis and treatment; it has high biosafety and low toxicity and side effects; and the preparation method is simple and easy to operate.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent, characterized in that, The steps are as follows: S1. Preparation of NaGdF4:Nd / Yb Tm nanoparticles The prepared rare earth raw materials were added to a mixed solvent of oleic acid and octadecene. Under the protection of an inert gas, the temperature was raised to 110°C and held for 10-20 minutes to remove water from the solution. Then the temperature was raised to 155°C until the raw materials were completely dissolved. The system temperature was then lowered to 75°C, sodium fluoride was slowly added dropwise, and the temperature was maintained for 1 hour. The temperature was then rapidly raised to 310°C and held for 1 hour. After cooling to room temperature, the product was washed three times each with cyclohexane and anhydrous ethanol. The obtained NaGdF4:Nd / Yb Tm nanoparticles were dispersed in cyclohexane and kept at room temperature. S2. Preparation of NaGdF4:Nd / YbTm@WFe2O4--NH2-PEG magnetic nanoparticles The nanoparticle dispersion prepared in step S1 was slowly added dropwise to sodium citrate aqueous solution under ultrasonication. After magnetic stirring for 24 hours, cyclohexane was evaporated and removed. After centrifugation, the precipitate was taken and freeze-dried. Take the freeze-dried solid sample and add it to deionized water, then add ferrous sulfate, stir overnight at 65°C, then add sodium tungstate dihydrate, and after it is completely dissolved, add polyacrylamide to the system and stir at room temperature for 30 min. After adjusting the pH of the system to 9, the mixture is placed in a high-pressure reactor with a polytetrafluoroethylene liner and kept at 200°C for 12 hours. The product is then dialyzed and freeze-dried. In step S2, dialysis is performed using a dialysis bag with an MD=8000. The dialysis solution is pure water containing 0.01M NH2-PEG. The dialysis solution is replaced every 2 hours, and the dialysis time is 12 hours. The relative molecular weight of NH2-PEG is 2000.

2. The method for preparing a tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent according to claim 1, characterized in that: In step S1, the rare earth raw materials have a molar ratio of Gd:Yb:Nd:Tm of 0.6:0.9:0.48:0.02, the volume ratio of oleic acid to octadecene is 2:5, and the molar-volume ratio of rare earth raw materials to oleic acid is 1 mmol:6 mL.

3. The method for preparing a tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent according to claim 1, characterized in that: In step S1, the slow addition of sodium fluoride refers to adding it at a rate of 10 drops / min. Rapid heating to 310℃ refers to heating at a rate of 10℃ / min; when NaGdF4:Nd / Yb Tm nanoparticles are dispersed in cyclohexane, the molar-volume ratio of NaGdF4:Nd / Yb Tm nanoparticles to cyclohexane is 1 mmol / L: 10 mL.

4. The method for preparing a tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent according to claim 1, characterized in that: In step S2, the ultrasonic power during the process of adding the dispersion to the sodium citrate aqueous solution is 300W, and the ultrasonication continues until the dispersion is completely added; slow addition refers to adding at a rate of 2 drops / min, and the concentration of the sodium citrate aqueous solution is 2M.

5. The method for preparing a tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent according to claim 1, characterized in that: In step S2, the magnetic stirring rate is 600 r / min, the evaporation temperature is 50℃, and the centrifugation conditions are centrifugation at 11000 rpm / min for 20 min.

6. The method for preparing a tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent according to claim 1, characterized in that: In step S2, the mass-to-volume ratio of the freeze-dried solid sample to deionized water is 0.325 g: 10 mL, and the mass ratio of the freeze-dried solid sample to ferrous sulfate, sodium tungstate dihydrate, and polyacrylamide is 325:432:329:

566.

7. The method for preparing a tumor magnetically targeted, magnetothermal self-enhancing nanotherapeutic agent according to claim 1, characterized in that: In step S2, the pH of the mixture is adjusted using 1M NaOH solution, and the high-pressure reactor is lined with polytetrafluoroethylene. In both steps S1 and S2, the mixture needs to be pre-frozen at -20°C for 3 hours before freeze-drying.

8. A tumor magnetically targeted magnetothermal self-enhancing nanotherapeutic agent prepared by the preparation method according to any one of claims 1-7.

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