Application of Fe-TCPP nanoparticles and drug delivery platforms in the preparation of formulations that sensitize the antitumor effects of TTF.

Abstract

This invention belongs to the field of biomedicine and tumor treatment technology, and relates to the application of Fe-TCPP nanoparticles and a drug-loading platform in the preparation of formulations that sensitize the antitumor effect of TTF. The tumor is selected from any one of glioblastoma, lung cancer, and drug-resistant glioma. This invention is the first to discover that Fe-TCPP can undergo structural cleavage and release the loaded drug under a TTF electric field, and that this cleavage process has a synergistic effect with TTF-induced cell death. This invention achieves active targeting based on the active site of Fe-TCPP and chemotherapeutic drugs. Experimental results show that the targeted combination group showed a 2-fold decrease in tumor fluorescence signal compared to the non-targeted combination group, and a nearly 5-fold decrease compared to the control group, with a significantly prolonged median survival. It can effectively kill drug-resistant / resistant tumor cells and effectively reverse drug resistance. Simultaneously, the combination strategy of this invention also has a synergistic killing effect on other solid tumors such as lung cancer, and has the potential for universal application across cancer types.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and tumor treatment technology, and relates to the application of Fe-TCPP nanoparticles and drug delivery platform in the preparation of formulations that enhance the antitumor effect of TTF. Background Technology

[0002] Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant tumor of the central nervous system, characterized by high proliferation, extensive infiltration, and a high recurrence rate. The current standard clinical treatment is surgical resection combined with temozolomide (TMZ) chemotherapy and radiotherapy, but the median survival of patients is low.

[0003] Tumor Treating Fields (TTF) is an innovative physical therapy targeting tumors. It was the first FDA-approved treatment in recent years for glioblastoma (GBM), a refractory tumor, extending median survival from 14.4 months to 19 months and more than doubling the 5-year survival rate. Its mechanism involves using low-intensity, mid-frequency alternating electric fields to precisely block tumor cell mitosis with minimal detectable biological damage to normal tissues. This mechanism allows TTF to seamlessly integrate with multiple modalities of therapy, including radiotherapy, chemotherapy, targeted therapy, and immunotherapy, demonstrating significant synergistic potential. Although it has been included in first-line treatment, GBM remains difficult to cure due to its refractory and aggressive nature, and its location within the blood-brain barrier, which hinders drug penetration.

[0004] Existing technology CN112569363A discloses a lead compound for a tumor therapeutic electric field and its preparation method, as well as a tumor therapeutic electric field sensitization method. The lead compound for the tumor therapeutic electric field is composed of a tumor antibody (cetuximab), graphene oxide, and iron oxide linked by non-covalent bonds. Although this lead compound can inhibit glioma cell proliferation under a mid-frequency, low-intensity electric field, its composition is complex (involving antibodies, graphene, and magnetic nanoparticles), its preparation process is cumbersome, and it mainly relies on antibody-mediated targeting, lacking a design for drug release responsive to the tumor microenvironment.

[0005] Fe-TCPP (iron-tetra(4-carboxyphenyl)porphyrin coordination nanoparticles) is a known metal-organic framework material that can be prepared by conventional methods such as solvothermal methods. Prior art CN120535767A discloses a porphyrin-based iron metal-organic framework (Fe...) for improving photosensitivity. The preparation of Fe-TCPP nanocatalysts involved a simple solvothermal method using an iron source and centrifugal tetra(4-carboxyphenyl)porphyrin (TCPP) as precursors, with polyvinylpyrrolidone (PVP) as the assisted coordination agent. After ultrasonic dispersion, the reaction was carried out in a closed system to obtain a purple suspension. This suspension was then centrifuged, washed, and dried. Previous studies have reported that Fe-TCPP possesses peroxidase-like activity and photocatalytic properties, and its applications in biomedical fields such as photodynamic therapy and nanocatalysis have been explored. However, no studies have yet documented the combined use of Fe-TCPP and TTF. Summary of the Invention

[0006] The purpose of this invention is to provide an application of Fe-TCPP nanoparticles and a drug delivery platform in the preparation of formulations that enhance the antitumor effect of TTF.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Application of Fe-TCPP nanoparticles in the preparation of formulations that enhance the antitumor effect of TTF, wherein the tumor is selected from any one of glioblastoma, lung cancer, and drug-resistant glioma.

[0008] In the prior art, Fe-TCPP has only been used as a photosensitizer or drug carrier, while TTF has been used as an independent physical therapy, and no synergistic effect between the two has ever been reported. This invention is the first to discover that Fe-TCPP can undergo structural cleavage and release the loaded drug under a TTF electric field, and that this cleavage process has a synergistic mechanism with TTF-induced cell death.

[0009] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows: In one preferred embodiment, Fe-TCPP nanoparticles were prepared by solvothermal synthesis.

[0010] In one preferred embodiment, the method for preparing Fe-TCPP nanoparticles includes the following steps: The iron source, tetra(4-carboxyphenyl)porphyrin, and competing ligand were dissolved in a solvent, and then stirred at 80-1000℃ for 6-12 hours. After purification, the Fe-TCPP nanoparticles were obtained.

[0011] In one preferred embodiment, the iron source is any one or more of ferric chloride, ferrous sulfate, ferric nitrate, or their hydrates.

[0012] In one preferred embodiment, the competing ligand is benzoic acid or a lower fatty acid.

[0013] In one preferred embodiment, the lower fatty acid is one or more of formic acid, acetic acid, butyric acid, and caprylic acid.

[0014] In one preferred embodiment, the mass ratio of the iron source, tetra(4-carboxyphenyl)porphyrin, and competing ligand is 1:(0.2-1.0):(5-25), preferably 1:(0.3-0.6):(8-15), and more preferably 1:0.4:10.

[0015] In one preferred embodiment, the TTF frequency is 50-250kHz and the electric field strength is 0.1-2.0V / cm.

[0016] This invention demonstrates through frequency and field strength gradient experiments that the synergistic effect of TTF and Fe-TCPP-TMZ is parameter-dependent, with the optimal synergistic window being a frequency of 150-200kHz and a field strength of 0.5-1.0V / cm.

[0017] This invention validated the synergistic effect in multiple cell models, including TTF-sensitive (GL261, U87MG, A549) and drug-resistant (U251_Resis) tumor cells. Particularly in TMZ-resistant glioma cells, Fe-TCPP-TMZ combined with TTF resulted in increased intracellular plasma volume (ICV) of 100%. 50 It decreased by nearly four times, and the synergy index was greater than 20.

[0018] In one preferred embodiment, when the Fe-TCPP nanoparticles are used in combination with TTF, the concentration of the Fe-TCPP nanoparticles is 5-100 μg / mL.

[0019] Based on the same inventive concept, this invention also claims the use of a drug delivery platform in the preparation of formulations that enhance the antitumor effect of TTF, wherein the drug delivery platform comprises Fe-TCPP nanoparticles and a chemotherapeutic drug, and the tumor is selected from any one of glioblastoma, lung cancer, and drug-resistant glioma.

[0020] In one preferred embodiment, the chemotherapy drug is temozolomide.

[0021] In one preferred embodiment, the ratio of Fe-TCPP nanoparticles to chemotherapeutic drugs in the drug delivery platform is 4-10:1; preferably 5-8:1; and more preferably 5:1.

[0022] In one preferred embodiment, the drug delivery platform further includes transferrin.

[0023] Transferrin receptors are highly expressed on glioma cells and the blood-brain barrier. Transferrin modification endows Fe-TCPP nanoparticles with active targeting capabilities. In vivo experiments showed that the tumor accumulation of the non-targeted Fe-TCPP-TMZ was reduced by 2-fold compared to the targeted version, and the median survival was significantly shortened.

[0024] In one preferred embodiment, the transferrin is selected from one or more of serum transferrin, milk transferrin, oocyte transferrin, melanin transferrin, human recombinant transferrin, or deferrotransferrin; preferably deferrotransferrin or human recombinant transferrin.

[0025] Compared with the prior art, the beneficial effects of the present invention are: (1) Through extensive experiments, this invention demonstrates that Fe-TCPP (tetra(p-carboxyphenyl)iron porphyrin) can be activated and cleaved by TTF, resulting in a significant reduction in particle size, with some particles decreasing to below 10 nm. On the one hand, this provides a potent killing effect on tumor cells, achieving a strong synergistic sensitization effect. On the other hand, it can also achieve responsive release of encapsulated drugs, achieving significant synergistic killing even at low drug concentrations. The underlying mechanism is that under the action of an electromagnetic field, the structure of Fe-TCPP is partially destroyed, leading to the release of Fe ions from the porphyrin ligand, promoting ferroptosis in GBM cells. Furthermore, the released iron ions can act as catalysts to achieve radio-Fenton reactions and charge transfer, catalyzing the decomposition of oxygen and hydrogen peroxide (H2O2) to generate hydroxyl radicals (·OH) and superoxide anions (·O2). - Reactive oxygen species (ROS) such as temozolomide (TTF) can further kill tumors. Based on the characteristic that Fe-TCPP can be activated by TTF, spatiotemporally controllable drug release can be achieved, and reactive oxygen species can be catalyzed at the tumor site to achieve a multi-faceted synergistic anti-GBM effect. This invention reveals and confirms for the first time that Fe-TCPP can be activated by an alternating electric field, thereby cleaving and releasing drugs. The mechanism may be through electric field-induced interfacial polarization and charge migration to regulate the Fe(III) / Fe(II) redox cycle and catalytic activity of the Fe center, thereby amplifying the ROS / oxidative stress effect. In the prior art, porphyrin materials as photothermal photosensitizers first convert light energy into local heating and are dominated by thermal damage, which is a completely different process and mechanism from electromagnetic response. Fe-TCPP does not produce local tissue heating or thermal damage under an electric field, making it safer and more controllable. Based on the principle of this invention, in addition to temozolomide, other common chemotherapy drugs, such as carmustine, paclitaxel, nivolumab, and ozone, can be used to treat tumors. 6 Any of the benzylguanine derivatives can also achieve similar synergistic effects based on the principles of this invention.

[0026] (2) This invention utilizes the active site of Fe-TCPP to efficiently load transmembrane peptides (transferrin, TRF) and the chemotherapeutic drug temozolomide. Leveraging the properties of nanoparticles and their ability to penetrate the blood-brain barrier, the nano-immunodelivery system is retained within the GBM due to the EPR effect of the nanoparticles, thus achieving active targeting. In vivo fluorescence imaging showed that the targeted combination group exhibited a 2-fold decrease in tumor fluorescence signal compared to the non-targeted combination group, and a nearly 5-fold decrease compared to the control group, with a significantly prolonged median survival.

[0027] (3) Experimental data from this invention demonstrate that in the TMZ-resistant cell line U251_Resis, TTF combined with Fe-TCPP-TMZ resulted in IC50. 50 The drug resistance decreased by nearly four times, and the synergy index was greater than 20, indicating that the combined strategy of this invention can effectively kill drug-resistant / resistant tumor cells and effectively reverse drug resistance. Meanwhile, in A549 human lung cancer cells, the synergy index of TTF combined with Fe-TCPP-TMZ was greater than 10, indicating that the combined strategy of this invention also has a synergistic killing effect on lung cancer and other solid tumors, and has the potential for universal application across cancer types.

[0028] (4) The in vivo decomposition pathway of Fe-TCPP in this invention: Fe-TCPP → TCPP + Fe 2+ / Fe 3+ →Small molecule metabolites. In the tumor microenvironment (acidic + high GSH), the metal-organic framework structure of Fe-TCPP is disrupted, releasing: TCPP ligands (porphyrin ring structure) and Fe... 2+ / Fe 3+ The Fe ions are endogenous porphyrin analogs that can be metabolized by hepatocytes into water-soluble small molecules and excreted in urine (67%) and feces (22%). Fe ions, on the other hand, are regulated by iron metabolism pathways (such as the transferrin-ferritin system) and do not remain in the body for extended periods. Therefore, the Fe-TCPP (iron-tetracarboxyphenyl porphyrin) of this invention, when activated by an electric field, can be broken down into smaller nanoparticles, which are mainly excreted in the body via the kidneys in the form of urine. More than 89% is cleared within 72 hours, with no significant bioaccumulation and low toxicity. Attached Figure Description

[0029] Figure 1 This is a SEM image showing the pyrolysis results of Fe-TCPP particles treated with TTF; among them, Figure 1 In the image, 'a' represents the SEM image of Fe-TCPP particles in the control group within a 1µm scale field of view. Figure 1 In the image, b represents the SEM image of the Fe-TCPP particles in the control group at a 100 nm scale. Figure 1 In the image, c represents the SEM image of the Fe-TCPP particles in the control group at a 200 nm scale bar. Figure 1 The image shows SEM images of Fe-TCPP particles in the TTF-treated group within a field of view with d as a 1µm scale. Figure 1 In the image, 'e' represents the SEM image of the Fe-TCPP particles in the TTF-treated group within a 100 nm scale field of view. Figure 1 f in the image is a SEM image of the Fe-TCPP particles in the TTF-treated group in a 200 nm field of view.

[0030] Figure 2This is a line graph showing the inhibition rate of GL261 by different concentrations of Fe-TCPP.

[0031] Figure 3 This is a line graph showing the suppression rate of GL261 by Fe-TCPP under different electric field intensities.

[0032] Figure 4 This is a combined index diagram of various concentration / intensity combinations in the Fe-TCPP synergistic sensitization TTF experiment.

[0033] Figure 5 This is a line graph showing the inhibition rate of TMZ on GL261 under different concentrations and electric field strengths; where... Figure 5 In the figure, 'a' represents a line graph showing the inhibition rate of GL261 by different concentrations of TMZ. Figure 5 In the figure, b is a line graph showing the suppression rate of TMZ on GL261 under different electric field intensities.

[0034] Figure 6 This is a heatmap of the combined index of various concentration / intensity combinations in the TMZ synergistic sensitization TTF experiment.

[0035] Figure 7 This is a line graph showing the inhibition rate of GL261 by different concentrations of Fe-TCPP-TMZ.

[0036] Figure 8 This is a line graph showing the suppression rate of GL261 by Fe-TCPP-TMZ under different electric field intensities.

[0037] Figure 9 This is a heatmap of the combined index of various concentration / intensity combinations in the Fe-TCPP-TMZ synergistic sensitization TTF experiment.

[0038] Figure 10 This is a median effect diagram of various concentration / intensity combinations in the Fe-TCPP-TMZ synergistic sensitization TTF experiment.

[0039] Figure 11 This is a combined index diagram of various concentration / intensity combinations in the Fe-TCPP-TMZ synergistic sensitization TTF experiment.

[0040] Figure 12 This is a bar graph showing the apoptosis rate of cells 24 hours after intervention with TTF 0.5V / cm combined with Fe-TCPP-TMZ 10.42μg / mL.

[0041] Figure 13 This is a line graph showing the inhibition rate of TMZ on the drug-resistant cell line (U251_Resis) and the parent cell line U251_Parental.

[0042] Figure 14This is a line graph showing the inhibition rate of Fe-TCPP-TMZ on U251_Resis with and without TTF.

[0043] Figure 15 This is a heatmap of the combined index of various concentration / intensity combinations in the Fe-TCPP-TMZ synergistic sensitization TTF experiment in U251_Parental.

[0044] Figure 16 This is a heatmap of the combined index of various concentration / intensity combinations in the Fe-TCPP-TMZ synergistic sensitization TTF experiment in U251_Resis.

[0045] Figure 17 This is a heatmap of the combined index of various concentration / intensity combinations of Fe-TCPP-TMZ synergistic enhancement TTF in the human lung cancer cell line A549.

[0046] Figure 18 This is a TUNEL staining image of mouse glioma cell line GL261 after intervention with TTF 0.5V / cm (200Hz) combined with Fe-TCPP-TMZ 100μg / mL.

[0047] Figure 19 This is a staining image of phalloidin in mouse glioma cell line GL261 after intervention with TTF 0.5V / cm (200Hz) combined with Fe-TCPP-TMZ 100μg / mL.

[0048] Figure 20 This is a graph showing the results of differentially expressed genes between the combined treatment group and the control group.

[0049] Figure 21 This is a graph showing the comparison results of the enrichment scores of the four groups of ferroptosis pathways in Example 9.

[0050] Figure 22 These are in vivo fluorescence imaging images of the mice in each group of Example 10.

[0051] Figure 23 This is a line graph showing the total fluorescence intensity of each group of mice in Example 10.

[0052] Figure 24 This is a line graph showing the survival rates of mice in each group in Example 10.

[0053] Figure 25 These are HE staining images of mice from each group in Example 10. Detailed Implementation

[0054] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0055] Example 1 Synthesis of Fe-TCPP nanoparticles and Temozolomide loading Weigh 20 mg of FeCl3·6H2O, 8 mg of tetrakis(4-carboxyphenyl)porphyrin (TCPP), and 200 mg of benzoic acid, and dissolve them in 5 mL of N,N-dimethylformamide (DMF) in a round-bottom flask. React at 90 °C for 8 hours with stirring. After the reaction is complete, collect the product by centrifugation, wash three times each with N,N-dimethylformamide (DMF) and ethanol, to obtain Fe-TCPP nanoparticles. Resuspend the nanoparticles in ethanol and store at 4 °C in the dark.

[0056] The chemotherapy drug temozolomide (TMZ) and the above-mentioned Fe-TCPP nanoparticles were co-dispersed in DMF at a mass ratio of 1:5 and stirred at room temperature in the dark for 24 hours. After the reaction was completed, the mixture was washed three times with DMF and centrifuged at 11,000 rpm for 15 minutes. The precipitate was collected, dispersed in phosphate-buffered saline (PBS), and washed twice with PBS. The final product was dispersed in PBS to obtain a Fe-TCPP-TMZ suspension, which was stored at 4°C in the dark for later use and designated as Fe-TCPP-TMZ.

[0057] Fe-TCPP-TMZ was collected, dispersed in water, and then transferrin (C) was added. 75 H 121 N 23 O 28 The transferrin and Fe-TCPP nanoparticles were mixed in a PBS solution (S, KKL MED, 11096-37-0) at a ratio of 1:2, stirred for 2 hours, centrifuged again at 11000 rpm for 15 minutes, and washed twice with PBS. The final product was dispersed in water and stored at 4°C for later use.

[0058] Example 2 TTF electric field-induced Fe-TCPP cleavage and drug release Six mL of Fe-TCPP-TMZ suspension with a concentration of 50 μg / mL was placed in a tumor therapeutic electric field (TTF) generator, with a frequency of 200 Hz and an electric field strength of 0.5 V / cm, and the intervention lasted for 24 hours (the electric field strength of the control group was 0 V / cm). After the intervention, the results were observed using a transmission electron microscope.

[0059] The results are as follows Figure 1 As shown, where, Figure 1 In the image, 'a' represents the SEM image of Fe-TCPP particles in the control group within a 1µm scale field of view. Figure 1 In the image, b represents the SEM image of the Fe-TCPP particles in the control group at a 100 nm scale. Figure 1 In the image, c represents the SEM image of the Fe-TCPP particles in the control group at a 200 nm scale bar. Figure 1 The image shows SEM images of Fe-TCPP particles in the TTF-treated group within a field of view with d as a 1µm scale. Figure 1 In the image, 'e' represents the SEM image of the Fe-TCPP particles in the TTF-treated group within a 100 nm scale field of view. Figure 1 In the figure, f represents the SEM image of Fe-TCPP particles in the TTF-treated group at a 200 nm scale bar. The results show that the Fe-TCPP particles in the TTF-treated group underwent effective fragmentation, with a significant reduction in particle size, and some particles decreasing to below 10 nm; the control group particles remained intact. These results indicate that under electric field activation, Fe-TCPP can be fragmented into smaller nanoparticles and release the encapsulated drug.

[0060] Example 3 Fe-TCPP synergistic sensitization effect of TTF on GL261 cell killing 3.1 Cell Culture and Intervention The mouse glioma cell line GL261 (purchased from Abiowell) was used at 1×10⁻⁶ cells / year. 5 Cells were seeded at a density of 10 cells / slice in 20 mm diameter cell culture slides and transferred to a TTF generator for intervention after 6 hours of adhesion. TTF parameters were set as follows: frequency 200 Hz; electric field intensity gradients of 0, 0.5, 0.8, 1.1, and 1.4 V / cm; and Fe-TCPP concentration gradients of 9.58, 38.33, 153.33, and 613.33 μg / mL. Single-drug or combined intervention was administered for 24 hours.

[0061] 3.2 Cell viability assay Cell viability and inhibition rate were detected and calculated using the CCK-8 assay. The specific steps were as follows: After intervention, 10 μL of CCK-8 solution (Tongren Chemical, CK04) was added to each well, and the culture plate was gently tapped to mix. The culture plate was then returned to the incubator for another 2 hours of incubation. After incubation, the absorbance (OD450) of each well was measured using a microplate reader at a wavelength of 450 nm, with a reference wavelength of 650 nm. Cell viability was calculated using the following formula: Cell viability (%) = [(OD450 of experimental wells - OD450 of blank wells) / (OD450 of negative control wells - OD450 of blank wells)] × 100%.

[0062] The inhibition rate is calculated using the following formula: Inhibition rate (%) = 100% - Cell viability (%).

[0063] All experiments were independently repeated at least three times, and results are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPadPrism or SPSS software. Intergroup comparisons were performed using t-tests or one-way ANOVA, and p < 0.05 was considered statistically significant.

[0064] The inhibition rates of different concentrations of Fe-TCPP on GL261 are as follows: Figure 2 As shown, the inhibition rate gradually increased with increasing Fe-TCPP concentration, exhibiting a typical S-shaped dose-response curve. At lower concentrations (10.42, 41.67 μg / mL), the single-drug inhibition rate was low (<40%).

[0065] The suppression rate of GL261 by Fe-TCPP under different electric field intensities is as follows: Figure 3 As shown.

[0066] 3.3 Synergistic Effect Analysis The combined effects were further analyzed using the ZIP model in SynergyFinder software, and the combined index of Fe-TCPP combinations at various concentrations / intensities was plotted. Figure 4 ).

[0067] The results showed that Fe-TCPP and TTF had a synergistic effect, with an average combination index of 5.36. The synergistic effect was strongest in the combination of TTF 0.5 V / cm and Fe-TCPP 38.33 μg / mL (combination index > 10).

[0068] Example 4 TMZ synergistic TTF killing effect on GL261 cells The mouse glioma cell line GL261 was injected with 1×10 5 Cells were seeded at a density of 20 mm in diameter on cell culture slides and allowed to adhere for 6 h before being transferred to a TTF instrument for intervention. TTF parameters were set as follows: frequency 200 Hz, intensity gradients 0, 0.5, 0.8, 1.1, and 1.4 V / cm; free TMZ concentration gradients were 1.56, 6.25, 25.01, and 100.04 μg / mL. Single-drug or combined intervention was performed for 24 h. Cell viability was assessed and inhibition rate calculated using the CCK-8 assay, following the method described in Example 3.

[0069] Dose-effect analysis results as follows Figure 5 As shown, where, Figure 5 In the figure, 'a' represents a line graph showing the inhibition rate of GL261 by different concentrations of TMZ. Figure 5In the figure, b is a line graph showing the suppression rate of TMZ on GL261 under different electric field intensities.

[0070] The combined effects were further analyzed using the ZIP model in SynergyFinder software, and combined index heatmaps for each concentration / intensity combination were plotted. Figure 6 The results showed that Fe_TCPP and TTF had a synergistic effect, with an average joint index of 2.21.

[0071] Example 5 Fe-TCPP-TMZ synergistic sensitization effect of TTF on GL261 cell killing The mouse glioma cell line GL261 (purchased from Abiowell) was used at 1×10⁻⁶ cells / year. 5 Cells were seeded at a density of 1 cell per 20 mm diameter cell culture slides and transferred to a TTF generator for intervention after 6 hours of adhesion. TTF parameters were set as follows: frequency 200 Hz; electric field intensity gradients of 0, 0.5, 0.8, 1.1, and 1.4 V / cm; and Fe-TCPP-TMZ concentration gradients (corresponding to the single-drug doses in Examples 3 and 4) of 10.42, 41.67, 166.67, and 666.67 μg / mL. Single-drug or combined intervention was performed for 24 hours. Cell viability was assessed and inhibition rate calculated using the CCK-8 assay as described in Example 3.

[0072] The inhibition rates of different concentrations of Fe-TCPP-TMZ on GL261 are as follows: Figure 7 As shown, the inhibition rate gradually increased with increasing Fe-TCPP concentration, exhibiting a typical S-shaped dose-response curve. At lower concentrations (10.42, 41.67 μg / mL), the single-drug inhibition rate was low (<40%).

[0073] The suppression rate of GL261 by Fe-TCPP-TMZ under different electric field intensities is as follows: Figure 8 As shown.

[0074] The combined effects were further analyzed using the ZIP model in SynergyFinder software, and combined index heatmaps of Fe-TCPP-TMZ combinations at various concentrations / intensities were plotted. Figure 9 The results showed that the synergistic effect was strongest (synergistic index > 10) in the combination of TTF 0.5V / cm and Fe-TCPP-TMZ 41.67μg / mL, followed by the combination of TTF 0.5V / cm and Fe-TCPP-TMZ 10.42μg / mL. Both showed strong synergistic effects, suggesting that significant synergistic killing can be achieved at low drug concentrations, indicating good safety potential.

[0075] In addition, independent verification was performed using CompuSyn software. Median effect plot ( Figure 10 The data shows that all combined drug data points are located in the Y>0 region and are above the single-drug fitted line, indicating that combined treatment can produce a >50% cell inhibitory effect within the tested concentration range. (Combination index plot) Figure 11 Further, all data points were located below the dotted line where the joint index was 1, confirming that Fe-TCPP-TMZ and TTF exhibited synergistic effects at all tested concentration combinations and effect levels, without any additive or antagonistic effects.

[0076] 3.4 Apoptosis Detection GL261 cells were treated with TTF 0.5V / cm combined with Fe-TCPP-TMZ 10.42μg / mL. Cells were collected 24 hours later for Annexin V / PI double staining flow cytometry analysis. The apoptosis rate was statistically analyzed, as shown below. Figure 12 As shown, the apoptosis level in the combined group was significantly higher than that in either single-drug group and the control group, further confirming the synergistic sensitizing pro-apoptotic effect of the two drugs.

[0077] Example 6 The reversal effect of the combined regimen on the drug-resistant cell line U251_Resis A temozolomide (TMZ)-resistant cell line for human glioma was established using a combination of escalating drug concentration and intermittent induction. Specifically, the human glioblastoma cell line U251 (purchased from Abiowell) was used as the parent (U251_Parental), and it was continuously induced in complete medium containing TMZ. In the initial stage of induction, a low concentration of TMZ (approximately IC50 of the parent) was used. 50 Cells were treated with 1 / 5 to 1 / 4 of the TMZ concentration (10-15 μM) for 72 hours, then the drug-containing medium was discarded. After washing with PBS, the medium was replaced with drug-free complete medium. Once the cells recovered to the logarithmic growth phase and were passaged 2-3 times, the next round of induction began. Subsequently, the TMZ concentration was gradually increased every 2-3 cycles (the gradient increase was approximately 1.5-2.0 times that of the previous cycle). Cell morphology, proliferation rate, and apoptosis were closely monitored during this period, and cell populations that had arrested growth due to excessive drug toxicity were promptly removed. The resulting human glioma drug-resistant cell line, U251_Resis, was finally obtained.

[0078] The drug-resistant cell line (U251_Resis) and the parental cell line (U251_Parental) were seeded into 96-well plates, respectively. The following day, a gradient concentration of TMZ (0, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 1000, 4000 μM) was added. After 72 hours of culture, cell viability was assessed using the CCK-8 assay, and the IC50 was calculated.50 value.

[0079] The results are as follows Figure 13 As shown. The results indicate that the drug-resistant cell line (U251_Resis) has an IC50 of 99.5% against TMZ. 50 The concentration (996.8 μM) was about 20 times higher than that of the parent strain U251_Parental (55.85 μM).

[0080] For both U251_Parental and U251_Resis, the TTF intensity and drug concentration gradient of Example 3 were set for single-drug or combined intervention for 24 hours. Cell viability was detected and inhibition rate was calculated using the CCK-8 assay.

[0081] The results are as follows Figure 14 As shown. The results show that, under TTF 0.5V / cm conditions, U251_Resis supports the Fe-TCPP-TMZ IC. 50 The concentration decreased from 122.6 μg / mL to 34.6 μg / mL, a decrease of nearly 4 times, indicating that TTF combined with Fe-TCPP-TMZ can effectively kill drug-resistant / resistant tumor cells and significantly enhance the drug effect.

[0082] The combined effects were further analyzed using the ZIP model in SynergyFinder software, and combined index heatmaps for each concentration / intensity combination were plotted. The results are as follows: Figure 15 (U251_Parental) and Figure 16 As shown in (U251_Resis), U251_Parental exhibited the highest synergistic index (synergistic index > 10) when combined with Fe-TCPP-TMZ at TTF 0.5V / cm and 10.42 μg / mL, followed by the combination at 41.67 μg / mL (synergistic index > 10). U251_Resis showed the strongest synergistic effect when combined with Fe-TCPP-TMZ at TTF 0.5V / cm and 41.67 μg / mL (synergistic index > 20), followed by the combination at 166.67 μg / mL (synergistic index > 20), both demonstrating strong synergistic effects.

[0083] Example 7 Synergistic killing effect of combined regimen on non-glioma cell line A549 Human lung cancer cell line A549 (purchased from Abiowell) was seeded into plates according to the method in Example 3. After 6 hours of cell adhesion, it was transferred to a TTF generator for intervention. The TTF frequency was adjusted to 150 Hz, and other intervention parameters and detection methods were the same as in Example 3. SynergyFinder ZIP analysis showed that ( Figure 17The highest synergistic index (synergistic index > 10) was achieved when TTF 0.5V / cm was combined with Fe-TCPP-TMZ 10.42μg / mL, followed by the combination at 41.67μg / mL (synergistic index > 10), indicating that this combined strategy can achieve strong synergistic killing at low concentrations and has good safety.

[0084] Example 8 Morphological evidence of apoptosis induced by combination therapy The mouse glioma cell line GL261 was seeded into a plate according to the conditions in Example 3. After 24 hours of intervention with TTF 0.5V / cm (200Hz) combined with Fe-TCPP-TMZ 100μg / mL, TUNEL staining and phalloidin staining were performed. The experimental procedure for TUNEL staining is as follows: Cell fixation Discard the culture medium and gently wash the cell slides twice with pre-cooled PBS for 5 minutes each time. Add 4% paraformaldehyde and fix at room temperature for 30 minutes. Discard the fixative and wash three times with PBS for 5 minutes each time.

[0085] Cell permeability Add 0.1% Triton X-100 (prepared with 0.1% sodium citrate buffer) and incubate on ice for 5 minutes. Discard the permeabilization buffer and wash three times with PBS for 5 minutes each time.

[0086] TUNEL reaction solution preparation Prepare the TUNEL reaction mixture according to the Roche in situ apoptosis detection kit instructions: mix TdT enzyme and dUTP labeling solution at a ratio of 1:9 and operate on ice. Negative control samples do not contain TdT enzyme, only dUTP labeling solution.

[0087] TUNEL labeling reaction Add 50 μL of TUNEL reaction mixture to each sample, ensuring complete coverage. Incubate the culture plate in a humidified chamber at 37°C for 60 minutes. Negative control samples are processed simultaneously.

[0088] Termination of reaction and washing Discard the reaction solution and wash three times with PBS, five minutes each time.

[0089] Nuclear counterstaining Add DAPI staining solution (1 μg / mL) and incubate at room temperature in the dark for 10 minutes. Wash three times with PBS for 5 minutes each time.

[0090] Sealing and Observation Remove the cell slides and mount them onto a glass slide using an anti-fluorescence quenching mounting medium. Observe and photograph them under a fluorescence microscope. TUNEL-positive cell nuclei show green fluorescence, while DAPI-stained cell nuclei show blue fluorescence.

[0091] Result Interpretation TUNEL positivity rate = (Number of TUNEL-positive cells / Total number of DAPI-positive cells) × 100%. Count at least 5 high-power fields (×200) for each group and calculate the apoptosis index.

[0092] The results of TUNEL staining are as follows: Figure 18 As shown in the figure. TUNEL staining results showed a significant increase in apoptotic signals in the combined group.

[0093] The experimental procedure for phalloidin staining is as follows: Cell fixation Discard the culture medium and gently wash the cell slides twice with pre-warmed PBS (37°C), 5 minutes each time. Add 4% paraformaldehyde and fix at room temperature for 15-20 minutes. Discard the fixative and wash three times with PBS, 5 minutes each time.

[0094] Cell permeability Add 0.1% Triton X-100 (prepared with PBS) and allow permeabilize at room temperature for 10-15 minutes. Discard the permeabilization solution and wash three times with PBS for 5 minutes each time.

[0095] Preparation of Phalloid Peptide Staining Solution Dilute the FITC-Phalloidin fluorescent working solution with PBS according to the specified ratio (refer to the kit instructions) and operate in the dark.

[0096] Phalloid peptide staining Add 100-200 μL of diluted phalloidin staining solution to each sample, ensuring complete coverage of the cell slide surface. Incubate at room temperature in the dark for 30-60 minutes. Wash three times with PBS for 5 minutes each time to remove unbound phalloidin.

[0097] Nuclear counterstaining Add DAPI staining solution (1 μg / mL) and incubate at room temperature in the dark for 5-10 minutes. Wash three times with PBS for 5 minutes each time.

[0098] Sealing and Observation Remove the cell slides and mount them onto a glass slide using an anti-fluorescence quenching mounting medium. Observe and photograph under a confocal microscope or fluorescence microscope. Phalloidin staining: microfilaments (F-actin) appear green (FITC labeled); DAPI staining: cell nuclei show blue fluorescence.

[0099] Result Interpretation Observe and compare the morphology, arrangement, and fluorescence intensity of the cell microfilament network in each group.

[0100] The results of phalloidin staining are as follows: Figure 19 As shown in the figure. The results showed that, compared with the single-drug group, the combination therapy group exhibited more significant cellular microfilament network disorder, edge contraction with microfilament aggregation, perinuclear microfilament rearrangement and depolymerization, and significantly weakened fluorescence signal, confirming that the combination regimen had a significant pro-apoptotic effect at the cytoskeleton level.

[0101] Example 9 Transcriptomics reveals that combination therapy exerts a synergistic effect through the ferroptosis pathway. GL261 cells were fed at a rate of 5 × 10⁻⁶ 5 After seeding at a density of 1 / 2 cells / slice and applying a combined intervention (TTF 0.5V / cm + Fe-TCPP-TMZ 100μg / mL) for 24 hours, RNA was extracted using Trizol, and transcriptome sequencing was performed on the Illumina novaseq x plus platform. Differential gene expression analysis between the combined group and the control group is as follows: Figure 20 As shown in the figure. Pathway enrichment analysis results showed that multiple ferroptosis-related pathways were significantly upregulated in the combined group, including ferroptosis, oxidative stress cellular response, and iron metabolism. The comparison results of ferroptosis pathway enrichment scores among the four groups are shown in the figure. Figure 21 As shown, the results indicated that the combined group was significantly higher than either single-drug group, and the single-drug group was also significantly higher than the control group, suggesting that ferroptosis is an important molecular mechanism of synergistic antitumor effect of TTF combined with Fe-TCPP-TMZ.

[0102] Example 10 In vivo experiments validate the antitumor effects and safety of the combination therapy. Male C57BL / 6 mice (SPF grade), aged 6-8 weeks and weighing 18-22g, were selected as experimental subjects. The mice were fixed in a stereotaxic apparatus. After head preparation and disinfection, the scalp was incised approximately 1cm along the midline to expose the anterior fontanelle and sagittal suture. Using the anterior fontanelle as the origin, the injection site was located 2.0mm posterior to the anterior fontanelle and 2.0mm lateral to the midline, with an injection depth of 2.5mm subdural. The skull was carefully drilled through using a cranial drill, taking care to maintain the integrity of the dura mater. 2μL of cell suspension (containing 5×10⁻⁶ cells) was aspirated using a microsyringe. 4A glioma model was established by slowly injecting 1 GL261-luc cells into the target brain region at a constant rate of 1 μL / min (D0). On day 4 (D4) post-modeling, the first in vivo fluorescence imaging was performed to confirm successful tumor establishment and collect baseline fluorescence signals. On day 5 (D5) post-modeling, the glioma model mice were randomly divided into five groups: control group, TTF group, Fe-TCPP-TMZ group, TTF + non-targeted Fe-TCPP-TMZ (without transferrin modification) group, and TTF + Fe-TCPP-TMZ combined group. On day 14 (D14) post-modeling, in vivo fluorescence imaging was performed again to detect the intensity of intracranial tumor fluorescence signals and evaluate the treatment effect of each group. From the start of the intervention, the following indicators of the mice were observed and recorded daily: general condition: weight, activity level, coat condition, food and water intake; neurological symptoms: presence or absence of epilepsy, hemiplegia, kyphosis, gait abnormalities; death or near-death state. Mice were considered dead (or euthanized and recorded as dead) if any of the following conditions were observed: weight loss exceeding 20% ​​of initial body weight; severe neurological symptoms (such as persistent epilepsy or complete paralysis); inability to eat or drink independently; excessive tumor burden causing significant abdominal distension (subcutaneous model); or natural death. The date of death for each mouse was recorded (in days, calculated from model establishment D0).

[0103] In vivo fluorescence imaging monitoring results as follows Figure 22 As shown. The results of comparing the total fluorescence signal intensity of intracranial tumors are as follows. Figure 23 As shown. Figure 22 and Figure 23 The results showed that the total fluorescence signal intensity of intracranial tumors in the combination group was the lowest, significantly lower than that in the non-targeted combination group, the single-drug group, and the control group; it decreased by 2 times compared to the non-targeted combination group, indicating that it can effectively cross the blood-brain barrier and tumors; and it decreased by nearly 5 times compared to the control group, demonstrating the powerful effect of the product.

[0104] The survival rate was recorded at each time point within 40 days. The formula for calculating the survival rate is: Survival rate (at a certain time point) = Number of mice surviving at that time point / Total number of mice at the start of the experiment.

[0105] Survival rate results as follows Figure 24 As shown, the results indicate that the combined group of mice had the longest median survival time, which was significantly better than that of the other groups.

[0106] Mouse heart, brain, and kidney tissues were subjected to HE staining. The procedure was as follows: Mouse heart, brain, and kidney tissues were fixed with 4% paraformaldehyde at room temperature for 24 hours. The tissues were then sequentially immersed in the following solutions: 70% ethanol for 2 hours; 80% ethanol for 2 hours; 90% ethanol for 2 hours; 95% ethanol for 1 hour; anhydrous ethanol I for 30 minutes; anhydrous ethanol II for 30 minutes; xylene I for 15 minutes; and xylene II for 15 minutes. The tissues were then immersed in molten paraffin (60°C) for 1 to 2 hours, then embedded in paraffin blocks and allowed to solidify at room temperature. The paraffin blocks were sectioned to a thickness of 4 to 5 micrometers. The sections were spread in a 40°C water bath, retrieved onto glass slides, and baked at 60°C for 30 minutes. The sections were then immersed in the following solutions in sequence: xylene I, 10 minutes; xylene II, 10 minutes; anhydrous ethanol I, 5 minutes; anhydrous ethanol II, 5 minutes; 95% ethanol, 5 minutes; 90% ethanol, 5 minutes; 80% ethanol, 5 minutes; 70% ethanol, 5 minutes; and distilled water, 5 minutes. Next, the sections were immersed in hematoxylin staining solution for 5 to 10 minutes, then rinsed with tap water for 5 minutes. Then, the sections were immersed in 1% hydrochloric acid ethanol for differentiation for 2 to 5 seconds, and rinsed with tap water for 10 minutes. Next, they were immersed in 0.5% ammonia solution for blue reversion for 10 to 30 seconds, and then rinsed with tap water for 5 minutes. Finally, the sections were immersed in 0.5% eosin staining solution for 1 to 3 minutes, and then rinsed quickly with tap water. The sections were then immersed sequentially in the following solutions for dehydration: 80% ethanol for 2 seconds, 90% ethanol for 2 seconds, 95% ethanol I for 2 minutes, 95% ethanol II for 2 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. Finally, neutral resin was added, a coverslip was placed on top, and the sections were allowed to air dry at room temperature before being observed under a microscope.

[0107] The results are as follows Figure 25 As shown, the results indicate that no significant pathological changes were observed in the morphology and structure of the major organs in the TTF+Fe-TCPP-TMZ group, suggesting that Fe-TCPP-TMZ has no significant toxicity to the major organs of mice at an effective antitumor dose, demonstrating good in vivo safety.

[0108] In summary, through various analytical methods, multi-dimensional evaluation indicators, and cross-species and cross-cancer type verification, this invention is significantly superior to existing technologies. It systematically demonstrates that Fe-TCPP-TMZ can decompose and detoxify drugs under the action of a TTF electric field, and significantly synergistically enhances the killing effect of TTF on tumor cells.

[0109] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The application of Fe-TCPP nanoparticles in the preparation of formulations that sensitize the antitumor effect of TTF, characterized in that, The tumor is selected from any one of glioblastoma, lung cancer, or drug-resistant glioma.

2. The application according to claim 1, characterized in that, The preparation method of Fe-TCPP nanoparticles includes the following steps: The iron source, tetra(4-carboxyphenyl)porphyrin, and competing ligand were dissolved in a solvent, and then stirred at 80-1000℃ for 6-12 hours. After purification, the Fe-TCPP nanoparticles were obtained.

3. The application according to claim 2, characterized in that, The iron source is any one or more of ferric chloride, ferrous sulfate, ferric nitrate, or their hydrates; the competing ligand is benzoic acid or a lower fatty acid.

4. The application according to claim 3, characterized in that, The lower fatty acids are one or more of formic acid, acetic acid, butyric acid, and caprylic acid.

5. The application according to claim 2, characterized in that, The mass ratio of the iron source, tetra(4-carboxyphenyl)porphyrin, and competing ligand is 1:(0.2-1.0):(5-25).

6. The application according to claim 1, characterized in that, The frequency of TTF is 50-250kHz, and the electric field strength is 0.1-2.0V / cm.

7. The application according to claim 1, characterized in that, When the Fe-TCPP nanoparticles are used in combination with TTF, the concentration of the Fe-TCPP nanoparticles is 5-100 μg / mL.

8. The application of a drug delivery platform in the preparation of formulations that sensitize the antitumor effect of TTF, characterized in that, The drug delivery platform comprises Fe-TCPP nanoparticles and chemotherapeutic drugs, and the tumor is selected from any one of glioblastoma, lung cancer, and drug-resistant glioma.

9. The application according to claim 8, characterized in that, The chemotherapy drug in question is temozolomide.

10. The application according to claim 8, characterized in that, The drug delivery platform also includes transferrin; the transferrin is selected from one or more of serum transferrin, milk transferrin, oocyte transferrin, melanin transferrin, recombinant transferrin, or deferrotransferrin.

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