Embolism emulsion capable of regulating and controlling tumor pH and inducing potent ferroptosis and preparation method of embolism emulsion

Abstract

The invention discloses an embolism emulsion capable of regulating and controlling tumor pH and inducing potent ferroptosis and a preparation method of the embolism emulsion, and belongs to the technical field of biological medicines.The emulsion is prepared by combining surface-modified iron nanoparticles (FeNPs), iodized oil and a sodium-hydrogen exchanger inhibitor (cariposide) through a Pickering emulsion technology; the problems that a traditional iodinated oil emulsion is poor in stability and insufficient in drug slow release are solved. FeNPs is used as a Fenton reaction catalyst, hydroxyl radicals are efficiently generated in a tumor acidic microenvironment, and ferroptosis is induced; the cariposide inhibits a sodium-hydrogen exchange body, reduces the intracellular pH value, relieves extracellular acidification, and synergistically enhances the Fenton reaction activity. The emulsion is administrated through arterial embolism, and can remarkably inhibit tumor growth, activate anti-tumor immunity and reverse an immunosuppression microenvironment. In-vitro and animal experiments show that the compound has excellent stability, slow release characteristic (the drug release speed is reduced by 50%) and treatment efficiency, and is suitable for treating liver cancer through transcatheter arterial chemoembolization (TACE).

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically discloses an embolic emulsion capable of regulating tumor pH and inducing potent ferroptosis, and a preparation method thereof. Background Art

[0002] Currently, iodized oil is the most common embolic material used in clinical transcatheter arterial chemoembolization for the treatment of liver cancer patients. The main principle is to form an embolic emulsion by simply mixing with chemotherapy drugs, blocking the tumor's blood supply artery to induce ischemic necrosis while further enhancing the anti-tumor effect through chemotherapy. However, its limited stability can lead to rapid diffusion of the drug from the embolization site, thus limiting its efficacy. Although a variety of drug-loaded microspheres have been developed to achieve sustained drug release, they are still limited by low drug loading and limited drug types. In addition, regardless of the embolic material used, vascular embolization will significantly aggravate tumor hypoxia and acidification, increase the immunosuppressive nature of the tumor microenvironment, further reduce the therapeutic effect of simple chemotherapy embolization, fail to induce sufficient and effective immunogenic death, and ultimately lead to treatment resistance. Therefore, developing a new generation of embolic materials with significantly superior pharmacological properties and tumor microenvironment regulation is a reasonable strategy to further improve the therapeutic effect of TACE for HCC patients.

[0003] Ferroptosis is a recently discovered non-apoptotic form of programmed cell death that kills tumor cells through an iron-dependent lipid peroxidation process. Several ferroptosis inducers, including erastin and RSL3, have been reported and are actively being explored in preclinical studies. However, these molecular agents have also exhibited significant limitations in preclinical analysis, such as intrinsic metabolic instability, lack of tumor selectivity, and poor in vivo circulation. Recent studies have demonstrated that targeted tumor delivery of agents containing iron / ferrous ions is an alternative approach to induce ferroptosis, which can be achieved through the Fenton reaction, which promotes the generation of highly reactive hydroxyl radicals from endogenous hydrogen peroxide. Currently, several iron-based agents, including ferrocene, iron oxide nanoparticles, and iron nanoparticles, have been used as Fenton reaction catalysts. However, the limited catalytic activity of current iron-based agents in biological environments makes achieving efficient tumor killing challenging. Therefore, achieving efficient Fenton reaction catalysis is of great significance for improving the efficacy of cancer treatment. Summary of the Invention

[0004] To solve the above problems, the present invention discloses an embolic emulsion that can regulate tumor pH and induce potent ferroptosis, and a preparation method thereof. The novel embolic emulsion achieves sustained drug release through Pickering emulsion technology, solving the problem of poor stability of traditional iodized oil emulsions; at the same time, the novel embolic emulsion kills tumors by regulating the pH in tumor cells and enhances the catalytic activity of the Fenton reaction to induce potent ferroptosis, thereby achieving efficient tumor killing, thereby solving the problems of insufficient tumor killing effect and treatment resistance of simple chemotherapy embolization; in addition, the novel embolic emulsion can effectively alleviate the acidic microenvironment of the tumor, eliminate the negative effects of acidification, relieve immunosuppression, and induce immunogenic death in combination with ferroptosis, thereby achieving anti-tumor immune activation, solving the problem of tumor immunosuppression limiting efficacy.

[0005] To achieve the above object, the present invention includes the following technical solutions:

[0006] A method for preparing an embolic emulsion capable of regulating tumor pH and inducing potent ferroptosis comprises the following steps:

[0007] 1) Synthesis of iron nanoparticles FeNPs as Fenton reaction catalyst and emulsion stabilizer:

[0008] 11) dissolving ferric chloride and a surfactant in ultrapure water to form solution A;

[0009] 12) dissolving the reducing agent in ultrapure water to form solution B;

[0010] 13) Under inert gas protection, slowly inject solution B into solution A and react at 40-60°C for 20-40 minutes to generate FeNPs;

[0011] 14) Add ethanol to eliminate bubbles, separate and wash FeNPs by magnetic separation;

[0012] 2) FeNPs surface modification: The surface modifier is mixed with FeNPs, stirred under inert gas for 20-40 minutes, magnetically separated and washed;

[0013] 3) Preparation of Pickering emulsion: The modified FeNPs were mixed with iodized oil, and then mechanically mixed with an aqueous solution containing a sodium hydrogen exchanger inhibitor at a volume ratio of 1:1 to 3:1 to form a stable emulsion.

[0014] Furthermore, in the above preparation method, the surfactant in step 11) is Pluronic F-127, and its amount is 30%-50% of the mass of ferric chloride.

[0015] Furthermore, in the above preparation method, the reducing agent in step 12) is sodium borohydride, and the molar ratio of sodium borohydride to ferric chloride is 1:1 to 1:2.

[0016] Furthermore, in the above preparation method, the surface modifier in step 2) is sodium octanoate, and its amount is 3-5 times the mass of FeNPs, and the thickness of the sodium octanoate modification layer is 5-15 nm.

[0017] Furthermore, in the above preparation method, the mass ratio of FeNPs to iodized oil in step 3) is 1:50 to 1:200.

[0018] Furthermore, in the above preparation method, the sodium hydrogen exchanger inhibitor in step 3) is cariporide, and its concentration in the aqueous phase is 100-300 μM.

[0019] The present invention also discloses an embolic emulsion capable of regulating tumor pH and inducing potent ferroptosis, which is prepared by any of the methods described above.

[0020] The present invention also discloses the use of the embolic emulsion in preparing a drug for treating liver cancer. The embolic emulsion is administered through arterial embolization to regulate the pH in tumor cells, induce ferroptosis, and activate anti-tumor immunity.

[0021] Furthermore, in the above-mentioned use, the dosage of the embolic emulsion is:

[0022] FeNPs: 1-5 mg / kg;

[0023] Iodized oil: 1-5 mg / kg;

[0024] Cariporide: 5-15mg / kg.

[0025] The present invention also discloses a pharmaceutical composition comprising the embolic emulsion, further comprising pharmaceutically acceptable excipients, and is used for transcatheter arterial chemoembolization (TACE).

[0026] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0027] The novel embolic emulsion disclosed in this invention, after entering liver cancer tumors via arterial embolization, releases cariporide, which inhibits sodium-hydrogen exchangers on the tumor cell membrane, reducing hydrogen ion efflux, thereby lowering intracellular pH and alleviating the acidic microenvironment outside the tumor cells. FeNPs taken up by tumor cells act as Fenton reaction catalysts, inducing ferroptosis in tumor cells. The intracellular acidic environment further enhances the catalytic activity of the Fenton reaction, thereby inducing even more potent ferroptosis. Furthermore, this novel embolic emulsion reverses immunosuppression by alleviating the acidic tumor microenvironment and effectively activates anti-tumor immunity by inducing immunogenic death, further enhancing the therapeutic efficacy of embolic therapy.

[0028] The method of the present invention is convenient and quick. Pickering embolic emulsion is prepared by simply mixing iron nanoparticles with iodized oil and cariporide solution (for example, in clinical preparation, the modified FeNPs can be mixed with iodized oil, and then the mixture is mixed with ultrapure water pre-dissolved with cariporide at a volume ratio of 2:1 and quickly pushed through a three-way valve 40 times ( Figure 1 ), thereby preparing Pickering embolic emulsion for clinical use), and the embolic emulsion of the present invention has significantly better stability than traditional iodized oil emulsion (such as Figure 2 The invention also has the functions of enhancing ferroptosis induction, tumor microenvironment regulation, and immune activation, thereby effectively improving the efficacy of TACE. The invention has a high reference value for TACE treatment and clinical application of liver cancer and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Schematic diagram of the preparation of Pickering emulsion using a three-way valve (the left syringe is iodized oil containing FeNPs, and the right syringe is ultrapure water containing cariporide);

[0030] Figure 2 :The changes of traditional iodized oil emulsion (top) and new iodized oil Pickering emulsion (bottom) over time;

[0031] Figure 3 : Schematic diagram of the preparation process of Pickering emulsion;

[0032] Figure 4 : Schematic diagram of the final Pickering emulsion obtained;

[0033] Figure 5 : Particle size range of FeNPs (164-265 nm);

[0034] Figure 6 : XRD detection of FeNPs;

[0035] Figure 7 :FeNPs generate hydroxyl radicals in vitro under different pH conditions;

[0036] Figure 8 : Stability of Pickering emulsions after adding different amounts of FeNPs (2.5 mg (A), 5 mg (B), and 10 mg (C));

[0037] Figure 9 : In vitro drug release curve;

[0038] Figure 10: Changes in intracellular and extracellular pH of N1S1 cells under different treatments (BCECCF-AM and BCECF were used as pH probes, and relative fluorescence intensity was measured by microplate reader to evaluate the effects of different treatments on intracellular (left) and extracellular (right) pH);

[0039] Figure 11 : Confocal images of ROS generation in N1S1 cells under different treatments;

[0040] Figure 12 : Confocal images of lipid peroxidation in N1S1 cells under different treatments (using BODIPY-C11 as a lipid peroxidation probe);

[0041] Figure 13 : Confocal images of the evaluation of immunogenic death of N1S1 cells under different treatments (CRT, HMGB1);

[0042] Figure 14 : Changes in pH in H22 subcutaneous tumor cells of mice after different treatments;

[0043] Figure 15 : Changes in extracellular pH of H22 subcutaneous tumor cells in mice after different treatments;

[0044] Figure 16 : Confocal images of lipid peroxidation in H22 subcutaneous tumors of mice receiving different treatments;

[0045] Figure 17 : Confocal images (left) and semi-quantitative analysis of fluorescence intensity of GPX4, a ferroptosis marker, in the H22 subcutaneous tumor model of mice after different treatments;

[0046] Figure 18 :Evaluation of the efficacy of different treatments in the mouse H22 subcutaneous tumor model (tumor volume changes (left) and survival (right));

[0047] Figure 19 : Flow cytometry was used to evaluate the effects of different treatments on the infiltration of different immune cells and cytokine secretion levels in the mouse H22 subcutaneous tumor model;

[0048] Figure 20 : Evaluation of tumor volume changes in response to different treatments in the rat N1S1 orthotopic hepatocellular carcinoma model. DETAILED DESCRIPTION

[0049] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0050] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0051] Table 1 Raw materials

[0052] Ferric chloride Sigma Aldrich Pluronic F-127 Sigma Aldrich Sodium borohydride Sigma Aldrich Sodium octanoate Sigma Aldrich cariporide Biyuntian Biological Iodized oil (poppy seed ethiodized oil injection) Hengrui Medicine

[0053] Example 1

[0054] Preparation Example

[0055] This embodiment is the preparation of embolic emulsion (iodized oil Pickering emulsion), and its preparation process diagram is as follows: Figure 3 The specific steps are as follows:

[0056] (1) Reaction solution: Solution A was composed of 40 mL of ultrapure water, 1.35 g of ferric chloride, and 0.5 g of F127; Solution B was prepared by dissolving 0.95 g of sodium borohydride in 10 mL of ultrapure water.

[0057] (2) Solution A was placed in a 100 mL three-necked flask and stirred at 50°C under nitrogen for 1 hour. Solution B was then slowly injected into solution A. A large amount of bubbles were generated during the reaction, and the solution eventually turned black.

[0058] (3) After the reaction was carried out for 30 minutes, 10 mL of anhydrous ethanol was added to eliminate bubbles. The obtained Fe NPs were separated by magnet and washed three times with ultrapure water.

[0059] (4) FeNPs surface modification: 10 mL of ultrapure water containing 800 mg of sodium octanoate was added to 20 mL of ultrapure water containing 200 mg of FeNPs. The mixture was stirred under nitrogen for 30 minutes, separated by a magnet, and washed three times with ultrapure water to obtain surface-modified FeNPs.

[0060] (5) Preparation of Pickering emulsion: First, 10 mg of FeNPs was mixed with 1 ml of iodized oil, and then the mixture was thoroughly mixed with ultrapure water pre-dissolved with cariporide at a volume ratio of 2:1 under magnetic stirring to prepare the iodized oil Pickering emulsion (the final product is shown in FIG. Figure 4 shown).

[0061] Example 2

[0062] In this example, the physical and chemical properties of the products prepared in Example 1 were tested, mainly including in vitro characterization of FeNPs and Pickering emulsion.

[0063] (1) Experimental items and methods: The hydrated particle size of FeNPs was determined by dynamic light scattering (DLS).

[0064] Result analysis: Figure 5 As shown, the particle size of FeNPs ranges from 164 to 265 nm.

[0065] (2) Experimental items and methods: X-ray diffraction (XRD) analysis of FeNPs crystal form.

[0066] Result analysis: Figure 6 As shown, XRD showed that the FeNPs sample was amorphous.

[0067] (3) Experimental project and methods: Using methylene blue (MB) as a hydroxyl radical (·OH) detector, the potential of FeNPs as Fenton's reagent to induce ·OH generation was investigated. FeNPs were added to a MB solution (30 μg / ml) containing H2O2 (1 mM) and incubated for 5 min at different pH values ​​(pH = 7.4, 6.5, 5.4). The absorbance of the above solution at 665 nm was measured to monitor the degradation of MB. Before measurement, high-speed centrifugation was performed to sediment the FeNPs to the bottom of the tube to prevent them from interfering with the absorbance reading.

[0068] Result analysis: The results are as follows Figure 7 As shown in the figure, the color of MB gradually changes from blue to colorless, indicating the generation of ·OH. At the same time, the lower the pH, the more MB is degraded, indicating the generation of more ·OH, indicating that low pH is conducive to the generation of ·OH by FeNPs.

[0069] (4) Experimental items and methods: Use a digital camera to monitor the separation of water and oil phases at defined intervals to evaluate the stability of LPE;

[0070] Result analysis: Figure 8 As shown in Figure 3, the water-oil separation was slowest when 10 mg FeNPs were added to 1 ml iodized oil, indicating higher stability.

[0071] (5) Experimental items and methods: To study the release of cariporide, 1.2 ml of traditional lipiodol emulsion (LE) and lipiodol Pickering emulsion containing FeNPs (CFe-LPE) were immersed in 5 mL of PBS (pH 6.5) and incubated at 37°C. At predetermined time intervals, the supernatant was collected and its characteristic absorbance at 254 nm was measured using a UV-visible spectrophotometer (Lambda 750, PerkinElmer) to quantify the amount of cariporide released.

[0072] Result analysis: Figure 9 As shown, the CFe-LPE group exhibited a significantly reduced drug release rate.

[0073] Example 3

[0074] In this example, a cell experiment was performed on the product prepared in Example 1.

[0075] (1) Experimental project and method: N1S1 cells were pre-seeded in 12-well plates (10 cells per well). 5 N1S1 cells were incubated with PBS, FeNPs (Fe), cariporide (Ca), or a combination thereof (CFe) for 6 hours (FeNPs = 50 μg mL-1, cariporide = 200 μM). After washing with PBS, cells were incubated in fresh culture medium with BCECF-AM and BCECF as pH probes for 30 minutes. The fluorescence intensity inside and outside the N1S1 cells treated with the different treatments was analyzed using a microplate reader (Bio-tek Synergy H1, USA) to assess their ability to condition intracellular and extracellular pH.

[0076] Result analysis: Figure 10 As shown, intracellular pH measurements using the BCECF-AM probe revealed weaker fluorescence intensities in the cariporide-alone and combined-treatment groups, indicating lower intracellular pH. Extracellular pH measurements using the BCECF probe revealed higher fluorescence intensities in the cariporide-alone and combined-treatment groups, indicating higher extracellular pH. These findings suggest that cariporide creates an acidic intracellular environment that favors the Fenton reaction and provides a promising opportunity to alleviate the acidic tumor microenvironment.

[0077] (2) Experimental items and methods: N1S1 cells were pre-seeded in 12-well plates (10 cells per well). 5 Cells were incubated with PBS, FeNPs (Fe), cariporide (Ca), or their combination (CFe) (FeNPs = 50 μg mL-1, cariporide = 200 μM) for 6 h. After washing with PBS, cells were incubated in fresh medium containing DCFH-DA dye for 30 min and imaged using a confocal laser scanning microscope (CLSM, Zeiss, LSM 800) and a flow cytometer (BD, Accuri) TM C6Plus) was used to analyze DCFH-DA staining in N1S1 cells treated with different methods to evaluate their ability to induce the production of intracellular reactive oxygen species.

[0078] Result analysis: Figure 11 As shown, co-incubation of N1S1 cells with cariporide and FeNPs promoted efficient intracellular ROS generation, which was significantly higher than that of the FeNPs alone group.

[0079] (3) Experimental items and methods: N1S1 cells were pre-seeded in 12-well plates (10 cells per well). 5 Cells were incubated with PBS, FeNPs (Fe), cariporide (Ca), or their combination (CFe) (FeNPs = 50 μg mL-1, cariporide = 200 μM) for 6 h. After washing with PBS, cells were incubated in fresh medium containing BODIPY-C11 dye for 30 min and imaged using a confocal laser scanning microscope (CLSM, Zeiss, LSM 800) and a flow cytometer (BD, Accuri) TM C6Plus) was used to analyze BODIPY-C11 staining of N1S1 cells treated with different methods to evaluate their ability to induce cellular lipid peroxidation.

[0080] Result analysis: Figure 12 As shown, N1S1 cells co-incubated with cariporide and FeNPs exhibited more obvious intracellular lipid peroxidation, which was significantly higher than that of the FeNPs alone group.

[0081] (4) Experimental project and method: N1S1 cells were first seeded in a 12-well plate (10 cells per well). 5 Cells were then incubated with the various materials for 24 hours. Subsequently, treated N1S1 cells were washed twice with PBS, fixed with 4% formaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, blocked with 5% FBS for 30 minutes, and stained with an anti-HMGB1 primary antibody at a dilution of 1:1000 for 1 hour, followed by staining with an Alexa 488-conjugated secondary antibody at a dilution of 1:500 for 30 minutes, following the manufacturer's instructions. Cells were then counterstained with DAPI for 10 minutes and assessed by CLSM. CRT expression was analyzed using a similar protocol.

[0082] Result analysis: Figure 13 As shown, the HMGB1 fluorescence intensity of the combined treatment group was the lowest and the CRT fluorescence intensity was the highest. The surface combined treatment significantly increased the release of HMGB1 and the exposure of calreticulin (CRT), confirming its efficient ability to release damage-associated molecular patterns.

[0083] Example 4

[0084] In this example, animal experiments were conducted on the product prepared in Example 1.

[0085] (1) Experimental items and methods: In order to evaluate the effect of materials on the intracellular pH value of tumor cells by in vitro fluorescence imaging (IVIS) in vivo, different materials (control group (G1), cariporide-loaded lipiodol emulsion group (G2), FeNPs-stabilized Pickering emulsion group (G3), and cariporide-loaded FeNPs-stabilized Pickering emulsion (G4)) were injected intratumorally 24 hours later. Then, BCECF-AM (20 μM, 50 μL) was injected subcutaneously into H22 tumor-bearing mice. The doses of cariporide, FeNPs, and lipiodol injected into the tumor were 9.36 mg / kg, 2.5 mg / kg, and 2.5 mg / kg, respectively. The injection volume of the emulsion was 75 μL. After 0.5 hours, the mice were sacrificed, and their tumors were collected and imaged using IVIS under the conditions of excitation wavelengths of 440 and 480 nm and emission wavelength of 535 nm. The IVIS results were then semi-quantitatively analyzed.

[0086] Result analysis: Figure 14 The results showed that CFe-LPE significantly reduced the BCECF-AM fluorescence intensity, indicating successful induction of intracellular acidification in tumors in vivo.

[0087] (2) Experimental items and methods: In order to evaluate the effect of materials on the pH value of the tumor microenvironment in vivo by in vitro fluorescence imaging (IVIS), different materials (control group (G1), cariporide-loaded lipiodol emulsion group (G2), FeNPs-stabilized Pickering emulsion group (G3), and cariporide-loaded FeNPs-stabilized Pickering emulsion (G4)) were injected intratumorally 24 hours later. Then, BCECF (20 μM, 50 μL) was injected subcutaneously into H22 tumor-bearing mice. The doses of cariporide, FeNPs, and lipiodol injected into the tumor were 9.36 mg / kg, 2.5 mg / kg, and 2.5 mg / kg, respectively. The injection volume of the emulsion was 75 μL. After 0.5 hours, the mice were sacrificed, and their tumors were collected and imaged using IVIS under the conditions of excitation wavelengths of 440 and 480 nm and emission wavelength of 535 nm. The IVIS results were then semi-quantitatively analyzed.

[0088] Result analysis: Figure 15 , the results showed that CFe-LPE significantly increased the BCECF signal intensity, indicating that the alkalinization of the tumor microenvironment was successfully induced in tumors in vivo.

[0089] (3) Experimental items and methods: To evaluate the level of lipid peroxidation in H22 tumors after different treatments, mice bearing subcutaneous H22 tumors were randomly divided into four groups, each receiving intratumoral injection of different substances (control group (G1), cariporide-loaded lipiodol emulsion group (G2), FeNPs-stabilized Pickering emulsion group (G3), and cariporide-loaded FeNPs-stabilized Pickering emulsion group (G4)). At 24 hours, 48 ​​hours, and 72 hours after injection, the tumors were harvested and cryosectioned, stained with BODIPY-C11, and then observed by confocal laser scanning microscopy (CLSM) to evaluate the level of lipid peroxidation.

[0090] Result analysis: Figure 16 Confocal microscopy revealed that significant BODIPY-C11 fluorescence signals were only observed in the CFe-LPE and Fe-LPE-treated groups. Furthermore, at all time points, the BODIPY-C11 fluorescence intensity in tumor sections from CFe-LPE-treated mice was significantly higher than that in the Fe-LPE-treated group, indicating a higher degree of lipid peroxidation. These results demonstrate that CFe-LPE can effectively induce lipid peroxidation.

[0091] (4) Experimental items and methods: To evaluate the expression level of GPX4 in H22 tumors after different treatments, mice bearing subcutaneous H22 tumors were randomly divided into four groups, each receiving intratumoral injection of different substances (control group (G1), cariporide-loaded lipiodol emulsion group (G2), FeNPs-stabilized Pickering emulsion group (G3), and cariporide-loaded FeNPs-stabilized Pickering emulsion group (G4)). Tumors were collected 24 hours after injection and cryosectioned and subjected to GPX4 immunofluorescence staining, followed by observation and evaluation by confocal laser scanning microscopy (CLSM).

[0092] Result analysis: Figure 17 , the GPX4 activity in the CFe-LPE group was significantly lower than that in the control group, suggesting the key role of ferroptosis in the CFe-LPE-induced cell death pathway.

[0093] (5) Experimental items and methods: The therapeutic effect of CFe-LPE was evaluated in a subcutaneous H22 tumor model. A total of 25 mice bearing H22 tumors were randomly divided into five groups and received intratumoral injections of different treatments, as follows: control group (PBS), traditional iodized oil emulsion (DOX-LE), FeNPs-stabilized Pickering emulsion (Fe-LPE), cariporide-loaded iodized oil emulsion (Ca-LE), and cariporide-loaded FeNPs-stabilized iodized oil emulsion (CFe-LPE). The doses of cariporide, DOX, FeNPs, and iodized oil injected into the tumor were 9.36 mg / kg, 2.88 mg / kg, 2.5 mg / kg, and 2.5 mg / kg, respectively. The therapeutic effect was evaluated by recording the changes in tumor size over time. Mice were judged to be dead when the tumor volume reached 1500 cubic millimeters.

[0094] Result analysis: Figure 18 As shown, we found that the CFe-LPE group had the slowest tumor growth, demonstrating superior anti-tumor efficacy compared to the clinically used DOX lipiodol emulsion. Furthermore, CFe-LPE treatment significantly prolonged the survival of H22 tumor-bearing mice compared to the other four groups.

[0095] (6) Experimental Project and Methods: For the study of anti-tumor immune mechanisms, 25 H22 tumor-bearing mice were selected and treated in the same manner (5 mice per group). On the 4th day after the corresponding treatment, these mice were sacrificed to collect tumors and adjacent lymph nodes for subsequent flow cytometry analysis and ELISA detection, respectively.

[0096] Result analysis: Figure 19 As shown, flow cytometry results demonstrated that CFe-LPE treatment significantly promoted the maturation of dendritic cells (DCs) in lymph nodes. Simultaneously, it increased the number of CD3+CD8+ T cells in tumors and reduced the number of immunosuppressive Tregs, resulting in a significant increase in the ratio of CD3+CD8+ T cells to Tregs. Furthermore, CFe-LPE treatment increased the percentage of cytotoxic NK cells and pro-inflammatory M1 macrophages while reducing the infiltration of anti-inflammatory M2 macrophages within tumors. ELISA results showed that CFe-LPE treatment significantly enhanced IL-12p70 secretion in tumors while reducing IL-10 production, further demonstrating the potent efficacy of CFe-LPE in promoting the conversion of M2 macrophages to M1 macrophages. Furthermore, CFe-LPE-treated tumors secreted significantly higher levels of TNF-α and IFN-γ than untreated control mice. Taken together, these results demonstrate that CFe-LPE can effectively elicit anti-tumor immune responses and reverse the immunosuppressive tumor microenvironment.

[0097] (7) Experimental items and methods: To evaluate the therapeutic effect of CFe-LPE delivered by transarterial embolization (TAE) on orthotopic N1S1 liver cancer, N1S1 cells were injected into the left liver lobe of 20 rats to establish an orthotopic N1S1 tumor model in rats. After 7 days, these rats were randomly divided into four groups and treated with TAE using the following materials: PBS, Fe-LPE, DOX-LE, and CFe-LPE. The doses of FeNPs, DOX, cariporide, and iodized oil per rat were 1 mg, 0.41 mg, 1.33 mg, and 100 μl, respectively. Tumor size was recorded using a small animal ultrasound imaging system on day 0 before various treatments and 3, 7, and 14 days after treatment.

[0098] Result analysis: Figure 20 As shown, the results showed that Fe-LPE and DOX-LE could only partially inhibit tumor growth, while CFe-LPE had the most significant tumor inhibitory effect, and almost all tumors disappeared by day 14, demonstrating the excellent therapeutic effect of CFe-LPE delivered by TAE on orthotopic N1S1 liver cancer.

[0099] Summary of Examples 1-4: In this example, a stable Pickering emulsion (embolic emulsion) was prepared by synthesizing and surface-modifying iron nanoparticles (FeNPs) and then combining them with iodized oil and a sodium-hydrogen exchanger inhibitor (cariporide). In vitro, this emulsion demonstrated excellent Fenton reaction catalytic activity (maximum hydroxyl radical generation at pH 5.4), sustained-release properties (50% reduction in drug release rate), and intracellular acidification (1.2 pH units reduction). Animal studies confirmed that intraarterial embolization significantly inhibited tumor growth (80% reduction in volume), induced lipid peroxidation (3-fold increase in BODIPY-C11 fluorescence intensity), and activated anti-tumor immunity (2-fold increase in CD8+ T cell proportion). Furthermore, the emulsion combined with cariporide reversed tumor microenvironment acidification (0.5 extracellular pH increase), enhanced ferroptosis (70% decrease in GPX4 activity), and significantly prolonged the survival of tumor-bearing mice (median survival increased from 14 days to 28 days).

[0100] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. A method for preparing an embolic emulsion capable of regulating tumor pH and inducing potent ferroptosis, characterized in that: The following steps are involved: 1) Synthesis of iron nanoparticles FeNPs as Fenton reaction catalyst and emulsion stabilizer: 11) dissolving ferric chloride and a surfactant in ultrapure water to form solution A; 12) dissolving the reducing agent in ultrapure water to form solution B; 13) Under inert gas protection, slowly inject solution B into solution A and react at 40-60°C for 20-40 minutes to generate FeNPs; 14) Add ethanol to eliminate bubbles, separate and wash FeNPs by magnetic separation; 2) FeNPs surface modification: The surface modifier is mixed with FeNPs, stirred under inert gas for 20-40 minutes, magnetically separated and washed; 3) Preparation of Pickering emulsion: The modified FeNPs were mixed with iodized oil, and then mechanically mixed with an aqueous solution containing a sodium hydrogen exchanger inhibitor at a volume ratio of 1:1 to 3:1 to form a stable emulsion.

2. The preparation method according to claim 1, characterized in that The surfactant in step 11) is Pluronic F-127, and its usage is 30%-50% of the mass of ferric chloride.

3. The preparation method according to claim 1, characterized in that The reducing agent in step 12) is sodium borohydride, and the molar ratio of sodium borohydride to ferric chloride is 1:1 to 1:

2.

4. The preparation method according to claim 1, characterized in that In step 2), the surface modifier is sodium octanoate, and its dosage is 3-5 times the mass of FeNPs. The thickness of the sodium octanoate modification layer is 5-15 nm.

5. The preparation method according to claim 1, characterized in that In step 3), the mass ratio of FeNPs to iodized oil is 1:50 to 1:

200.

6. The preparation method according to claim 1, characterized in that The sodium hydrogen exchanger inhibitor in step 3) is cariporide, and its concentration in the aqueous phase is 100-300 μM.

7. An embolic emulsion that can regulate tumor pH and induce potent ferroptosis, characterized in that: The method is prepared by any one of claims 1 to 6.

8. Use of the embolic emulsion according to claim 7 in preparing a medicament for treating liver cancer, characterized in that: The embolic emulsion is administered through arterial embolization and is used to regulate pH in tumor cells, induce ferroptosis, and activate anti-tumor immunity.

9. The use according to claim 8, characterized in that The dosage of the embolic emulsion is: FeNPs: 1-5 mg / kg; Iodized oil: 1-5 mg / kg; Cariporide: 5-15mg / kg.

10. A pharmaceutical composition comprising the embolic emulsion according to claim 7, characterized in that: The invention further comprises pharmaceutically acceptable excipients for use in transcatheter arterial chemoembolization (TACE).

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