Preparation method and application of Fe3O4-based active oxygen responsive probe

Abstract

The present invention discloses an application of an iron-based probe responsive to active oxygen, which can disintegrate and form clusters after responding to hydrogen peroxide, and can enhance 1 H T2-weighted magnetic resonance imaging contrast effect, using liposome modification, through the high permeability and retention effect of solid tumors, the probe can passively target the lung cancer area, and the loaded glucose oxidase catalyzes glucose to produce hydrogen peroxide, thereby enhancing the contrast of the lung cancer area. 1 H T2-weighted magnetic resonance imaging effect. In addition, the probe can also induce ferroptosis in lung cancer cells. Combined with the loaded immune-activating peptide Tuftsin, the system can activate the local immune response of the tumor, and finally combine with the immune checkpoint blocker α-PD-L1 to enhance the immunotherapy effect of lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of magnetic resonance imaging and anti-tumor technology, and specifically relates to a method based on Fe 3 O 4 Active oxygen responsive probe and its preparation method and application. Background Art

[0002] Lung cancer seriously endangers human life and health, and early and accurate detection can effectively improve its treatment effect. At present, the main means of clinical lung cancer detection are CT, PET and magnetic resonance imaging. Studies have shown that the false positive rate of low-dose spiral CT for lung cancer detection is as high as 96.4%, and there are disadvantages of ionizing radiation. Compared with CT, MRI has the advantages of high spatial resolution, no ionizing radiation, and strong soft tissue contrast, which is suitable for early detection of lung cancer. However, the US Food and Drug Administration has issued a safety warning for the use of gadolinium in patients with renal insufficiency, because the free gadolinium ions in gadolinium can cause nephrogenic systemic fibrosis. The US FDA emphasizes that for patients with acute kidney injury or chronic severe kidney disease, gadopentetate dimeglumine, gadodiamide, and gadofosamine contrast agents should not be used. Iron is an endogenous substance in the human body with good safety. Excessive iron can also be taken up and utilized by the human body and metabolized and cleared. Therefore, the construction of iron-based magnetic resonance contrast agents has safety guarantees in human applications. In addition, the construction of tumor microenvironment activated probes can help improve the contrast effect of the tumor area and is more conducive to distinguishing normal tissue from tumor tissue.

[0003] The tumor microenvironment is characterized by slight acidity, hypoxia, GSH overexpression, multiple protease overexpression, H 2 O 2 However, although GSH, proteases or H 2 O 2 However, for molecular probes that respond to the tumor microenvironment, there is a high probability that the response source is insufficient. 2 O 2 The content is only 0.002μM, while the content in tumor tissue reaches 100μM, which is a difference of 4 to 5 orders of magnitude. This feature can be used to develop H 2 O 2 However, H overexpressed in the tumor area 2 O 2 In most cases, it is still insufficient to respond to the molecular probe, resulting in insufficient activation of the imaging signal in the tumor area, which in turn fails to enhance the imaging contrast of the tumor area to a greater extent, and is not conducive to the accurate identification of the tumor.

[0004] In addition to accurate imaging of tumors, the ultimate goal is to intervene in them and achieve effective treatment of tumors. At present, clinical treatments for tumors include chemotherapy, radiotherapy, surgery, immunotherapy, etc. For tumors diagnosed at an early stage, surgical treatment is the most effective way to cure the tumor, which requires accurate tumor imaging. For other treatments, there are problems such as tumor resistance or ineffective treatment. Among them, immunotherapy has made major breakthroughs, and many drugs based on immune checkpoint blockade have been developed, which are expected to eradicate advanced malignant tumors. However, due to many factors, such as individual differences, genotype differences, and tumor immunosuppressive microenvironment, the effect rate of immunotherapy based on immune checkpoint blockade is low.

[0005] In current research, the tumor immunosuppressive microenvironment is considered to be the key reason for the low efficiency of immunotherapy. How to reverse the immunosuppressive microenvironment and increase the response rate of immunotherapy drugs is the key to improving the effectiveness of immunotherapy. Summary of the invention

[0006] Based on the above prior art, the present invention provides a Fe-based 3 O 4 The present invention assembles a large-size Fe 3 O 4 Nanoparticles, the probe does not disassemble in normal tissues, but in tumor tissues overexpressing H 2 O 2 Under these conditions, the probes disintegrate and form clusters, which can enhance 1 HT 2 -weighted MRI contrast effect. In addition, under the slightly acidic conditions of the tumor, the Fe 2+ Can and H 2 O 2 The Fenton reaction produces hydroxyl radicals, which induce ferroptosis in lung cancer cells. Combined with the loaded immune-activating peptide Tuftsin, it can reverse the immunosuppressive microenvironment. Combined with the immune checkpoint blocker α-PD-L1, it can enhance the immunotherapy effect of lung cancer.

[0007] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0008] A Fe-based 3 O 4 An active oxygen responsive probe, wherein the probe is Fe-linked to 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid 3 O 4 The assembly is the core, the liposome is the modified outer layer, and the Fe3 O 4 The assembly was loaded with glucose oxidase and immune-activating peptide Tuftsin.

[0009] Furthermore, the probe has a spherical structure and a particle size of 53.6±12.2 nm.

[0010] A Fe-based 3 O 4 The method for preparing an active oxygen responsive probe comprises the following steps:

[0011] S1, mixing the ferrous source solution and the 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid solution evenly to obtain a mixed solution;

[0012] S2, adding a stabilizer polyvinyl pyrrolidone, a pH adjuster triethylamine and anhydrous ethanol to the mixed solution, mixing evenly to obtain a precursor, at which point the pH value of the precursor is 10-12;

[0013] S3, the precursor is subjected to a solvothermal reaction, and after the reaction is completed, the obtained reaction product is centrifuged, washed and dried to obtain Fe 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid-linked 3 O 4 Assemblies;

[0014] S4, Fe connected to 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid 3 O 4 The assembly, glucose oxidase and immune-activating peptide Tuftsin were dispersed in PBS, stirred and reacted under light protection, and then centrifuged and washed to obtain the nanomaterial Fe 3 O 4 @GOx@Tuftsin;

[0015] S5, dissolving distearoylphosphatidylcholine, PEGylated distearoylphosphatidylethanolamine and cholesterol in an organic solvent, adding nanomaterial Fe 3 O 4 @GOx@Tuftsin, mix well, spin dry to remove organic solvent, add PBS to the residue, sonicate, and then pass through a gel microcolumn to obtain Fe-based 3 O 4 Reactive oxygen species responsive probe Fe 3 O 4 @GOx@Tuftsin@Lipsomes.

[0016] Furthermore, the ferrous source is ferrous chloride or its hydrate.

[0017] Furthermore, the solvents of the ferrous source solution and the 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid solution are both N,N-dimethylformamide.

[0018] Furthermore, the molar ratio of the ferrous source to 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid is 1-5:1.

[0019] Furthermore, the temperature of the solvent thermal reaction is 150-200° C., and the reaction time is 12-24 hours.

[0020] Furthermore, in step S4, the stirring reaction temperature is 4-8°C and the time is 12-24h.

[0021] Furthermore, the organic solvent is dichloromethane.

[0022] Furthermore, the mass ratio of the distearoylphosphatidylcholine, PEGylated distearoylphosphatidylethanolamine and cholesterol is 65:10:25.

[0023] A Fe-based 3 O 4 Application of reactive oxygen species-responsive probes in the preparation of magnetic resonance imaging agents.

[0024] A Fe-based 3 O 4 Application of reactive oxygen species-responsive probes in the preparation of anti-tumor drugs that induce ferroptosis of tumor cells and reverse the tumor immunosuppressive microenvironment.

[0025] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0026] 1. The probe of the present invention can realize ROS response disassembly into small-size nanoparticles and form larger clusters, thereby achieving 1 HT 2 -weighted MRI contrast enhancement.

[0027] 2. The probe of the present invention can catalyze glucose to produce hydrogen peroxide, self-supply the response source, ensure that the probe is fully disintegrated, promote drug release, and enhance ROS production through Fenton reaction, which is used to induce ferroptosis of tumor cells.

[0028] 3. The probe of the present invention can induce ferroptosis of tumor cells, and the loaded immune-activating peptide Tuftsin can reverse the tumor immunosuppressive microenvironment, promote the infiltration of effector T cells in tumor tissues, and combine with the immune checkpoint blocker α-PD-L1 to enhance the immunotherapy effect of lung cancer.

[0029] 4. The probe of the present invention can be synthesized by a solvothermal method, which is simple and convenient, can be synthesized quickly, has low preparation cost, and has good biosafety, further expanding its application prospects in magnetic resonance imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The Fe-based 3 O 4 Schematic diagram of the structure of the reactive oxygen species responsive probe FGTL.

[0031] Figure 2 The Fe-based 3 O 4 TEM image of the active oxygen responsive probe FGTL.

[0032] Figure 3 The Fe-based 3 O 4 The reactive oxygen species-responsive probe FGTL was 2 O 2 TEM image after response.

[0033] Figure 4 The Fe-based 3 O 4 Reactive oxygen species responsive probes FGTL and Fe 3 O 4 Zeta potential diagram.

[0034] Figure 5 The Fe-based 3 O 4 The reactive oxygen species-responsive probe FGTL was detected at different concentrations 1 HT 2 -weighted MRI and transverse relaxation rate plots.

[0035] Figure 6 The Fe-based 3 O 4 The active oxygen responsive probe FGTL before and after active oxygen response 1 HT 2 -weighted MRI comparison images.

[0036] Figure 7 The Fe-based 3 O 4 Western blot experimental results of GPX4 and β-actin in tumor cells induced by the reactive oxygen species responsive probe FGTL and the corresponding gray value quantitative analysis results: Figure 7(a) is the Western blot image of GPX4 protein and internal standard β-actin protein. Figure 7 (b) is the result of quantitative grayscale analysis of GPX4 protein bands.

[0037] Figure 8 The Fe-based 3 O 4 A diagram showing the therapeutic effect of the reactive oxygen species-responsive probe FGTL on ferroptosis in LLC cells.

[0038] Fig. 9 The Fe-based 3 O 4 Reactive oxygen species responsive probe FGTL 1 HT 2 -weighted MRI images.

[0039] Fig.10 The Fe-based 3 O 4 Reactive oxygen species responsive probe FGTL 1 HT 2 -weighted MR signal maps.

[0040] Fig.11 The Fe-based 3 O 4 Figure 2 shows the effect of the reactive oxygen species-responsive probe FGTL on the reversal of the immunosuppressive microenvironment of a living lung cancer model: Fig.11 (a) is a comparison of the proportion of helper T cells in tumor tissues of LLC subcutaneous lung cancer model mice treated with different agents; Fig.11 (b) Comparison of the proportion of cytotoxic T cells in tumor tissue of LLC subcutaneous lung cancer model mice under treatment with different agents.

[0041] Fig.12 The Fe-based 3 O 4 Figure 12(a) is a comparison of the tumor sizes of LLC subcutaneous lung cancer model mice in different groups after treatment with different drugs; Figure 12(b) is a curve showing the changes in tumor volume over time in transplanted tumor model mice under different drug treatments. DETAILED DESCRIPTION

[0042] The present invention is described in detail below with reference to specific embodiments.

[0043] Example 1

[0044] 1. Add 107 μL FeCl 2 ·4H 2 O DMF solution (397.3mM) and 437μL 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid DMF solution (59.4mM) were added to the reactor and mixed evenly. Then, 300mg polyvinyl pyrrolidone and 50μL triethylamine were added to the reactor. Then, 6.5mL DMF and 6.5mL anhydrous EtOH were added to the reactor respectively. Ultrasonication was performed for 5min. Then, the reactor was transferred to a 150℃ oven for reaction for 24h. After the reaction was completed, it was cooled to room temperature. The obtained mixed product was centrifuged at 10000rpm for 10min. The obtained precipitate was washed with water three times and finally freeze-dried to obtain 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid-linked Fe 3 O 4 Assembly.

[0045] 2. 10 mg of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid was linked to Fe 3 O 4 The assembly, 5 mg of glucose oxidase GOx, and 1 mg of immune-activating peptide Tuftsin were dispersed in 10 mL of PBS, stirred at 4 °C in the dark for 24 h, and then centrifuged at 10,000 rpm for 10 min. The resulting precipitate was washed three times with ultrapure water to obtain the nanomaterial Fe 3 O 4 @GOx@Tuftsin, denoted as FGT, was dispersed in PBS for later use.

[0046] 3. Dissolve 52 mg of distearoylphosphatidylcholine, 8 mg of PEGylated distearoylphosphatidylethanolamine and 20 mg of cholesterol in dichloromethane, and sonicate at 4°C. After sonication, add 20 mg of nanomaterial FGT, vortex evenly, and then transfer to a rotary evaporator and spin dry at 40°C. Add PBS to the residue, sonicate, and then pass through a gel microcolumn to remove free lipids to obtain Fe-based 3 O 4 Reactive oxygen species responsive probe Fe 3 O 4 @GOx@Tuftsin@Lipsomes, denoted as FGTL, were dispersed in PBS for later use.

[0047] The Fe-based 3 O 4 The schematic diagram of the active oxygen responsive probe FGTL is shown in Figure 1 As shown by Figure 1It can be seen that the core of the prepared probe FGTL is Fe 3 O 4 The assembly is loaded with glucose oxidase GOx and immune-activating peptide Tuftsin, and the outer layer is a modified liposome.

[0048] The Fe-based 3 O 4 The active oxygen responsive probe FGTL was scanned by a transmission electron microscope, and the obtained TEM image is shown in Figure 2 As shown by Figure 2 It can be seen that the prepared probe FGTL has a spherical structure and an average particle size of about 61 nm.

[0049] The Fe-based 3 O 4 The reactive oxygen species-responsive probe FGTL was 2 O 2 After the response, the transmission electron microscope was used to scan and the EM image was as follows: Figure 3 As shown by Figure 3 It can be seen that the prepared probe FGTL is broken down into particles with smaller particle size and forms a cluster structure, which is expected to be used to enhance 1 HT 2 -weighted MRI contrast effect.

[0050] The Fe-based 3 O 4 The Zeta potential measurement results of the active oxygen responsive probe FGTL are shown in Figure 4 As shown by Figure 4 It can be seen that the Zeta potential of the unmodified liposomes was 5.1 mV, while the Zeta potential of the FGTL after modification was -38.3 mV. The main purpose of modifying liposomes with nanomaterials is to convert the surface charge into negative charge to prevent the nanoparticles from being quickly cleared by the reticuloendothelial system.

[0051] Experiment 1: Enhanced active oxygen response of the probe FGTL of the present invention 1 HT 2 -weighted MRI test

[0052] Test method:

[0053] 1. The probe FGTL prepared in Example 1 was dispersed in sterile PBS to prepare probe solutions with Fe concentrations of 0.0125, 0.025, 0.05, 0.1 and 0.2 mM, respectively, and the probe solutions were subjected to 9.4T micro-imaging system. 1 HT2 -weighted MRI.

[0054] 2. Mix the probe solution with a Fe concentration of 0.025 mM in 1 mM H 2 O 2 The cells were incubated for 4 h under the same conditions and then imaged in a 9.4T micro-imaging system. 1 HT 2 -weighted MRI.

[0055] all 1 H MRI used the RARE imaging sequence with the following parameters: TR was 2500 ms, TE was 77 ms, FOV size was 4 cm × 4 cm, slice thickness was 1.0 mm, matrix size was set to 256 × 256, acceleration factor was set to 4, and sampling was 16 times.

[0056] Test results:

[0057] Different concentrations of probe FGTL solution 1 HT 2 -weightedMRI and transverse relaxivity as Figure 5 As shown by Figure 5 It can be seen that as the Fe concentration in the probe FGTL increases, 1 HT 2 -weighted MRI contrast effect gradually increased, and in the corresponding transverse relaxation rate test, the r of the probe FGTL was measured. 2 29.88mM -1 s -1 , suitable for use as 1 HT 2 -weighted MRI contrast agent.

[0058] FGTL probe solution with Fe concentration of 0.025 mM before and after enhanced reactive oxygen species response 1 HT 2 -weighted MRI images Figure 6 As shown by Figure 6 It can be seen that after the FGTL probe responds to active oxygen, 1 HT 2 -weighted MRI contrast effect was significantly enhanced, indicating that the constructed probe FGTL can achieve enhanced reactive oxygen response 1 HT 2 -weighted MRI contrast.

[0059] Experiment 2: Experiment on the ferroptosis of lung cancer cells induced by the probe FGTL of the present invention

[0060] Test method:

[0061] 1. Weigh 1 mg of the probe FGTL prepared in Example 1 and disperse it in 10 ml of sterile PBS to prepare a probe FGTL solution with a FGTL concentration of 100 μg / mL; weigh pure Fe 3 O 4 1 mg was dispersed in 10 mL of sterile PBS to prepare Fe 3 O 4 A solution with a concentration of 100 μg / mL; 1 mg of the ferroptosis inhibitor Ferrostasin-1 (Fer-1) was dispersed in 3.812 mL of sterile PBS to obtain a 1 mM concentration of Fer-1 solution, which was then diluted 100 times with sterile PBS to prepare a 10 μM concentration of the ferroptosis inhibitor Fer-1 solution;

[0062] 2. Add mouse LLC cells to DMEM culture medium (containing 10% fetal bovine serum and 1% double antibody (penicillin 100U / mL, streptomycin 100U / mL). When the cells grow to occupy about 90% of the culture dish area, add 2mL of PBS solution (control), Fe 3 O 4 Solution (100 μg / mL), probe FGTL solution (100 μg / mL) and probe FGTL solution (100 μg / mL) + Fer-1 solution (10 μM) were co-incubated for 12 hours, and Western blot experiments were performed to determine the effect of probe FGTL on the expression of GPX4, a key protein in ferroptosis. In addition, flow cytometry analysis was performed on the cells after incubation to evaluate the effect of probe FGTL in inducing ferroptosis in LLC cells.

[0063] Test results:

[0064] PBS, Fe 3 O 4 Four different inducers induced ferroptosis in mouse LLC tumor cells, including probe FGTL and probe FGTL+ferroptosis inhibitor Fer-1. The Western blot of GPX4 protein and β-actin protein in mouse LLC tumor cells is shown in Figure 2. Figure 7 As shown in (a), the grayscale quantitative analysis of GPX4 protein bands is as follows Figure 7 As shown in (b), Figure 7 (a) and Figure 7 (b) It can be seen that compared with PBS and Fe 3 O 4 After incubation induction, the GPX4 protein expression level was significantly reduced after incubation of LLC cells with probe FGTL, and the protein expression level of GPX4 was restored after the addition of ferroptosis inhibitor Fer-1, indicating that the probe FGTL can induce ferroptosis in tumor cells.

[0065] PBS, Fe 3 O 4 The mortality rate of LLC cells induced by four different inducers: probe FGTL, probe FGTL and probe FGTL + ferroptosis inhibitor Fer-1. Figure 8 As shown by Figure 8 It can be seen that in PBS and Fe 3 O 4 Under incubation induction, the mortality rates of LLC cells were 6.7% and 40.0% respectively. Under incubation induction with probe FGTL, the iron death rate of LLC cells was 81.5%. After adding the iron death inhibitor Fer-1, the mortality rate of LLC cells was 21.0%. This shows that the probe FGTL can effectively induce iron death in tumor cells and cause effective damage.

[0066] Experiment 3: In vivo expression of the probe FGTL of the present invention 1 HT 2 -weighted MRI test

[0067] Test method:

[0068] 1. Disperse the probe FGTL prepared in Example 1 in sterile PBS to prepare an FGTL contrast agent with a FGTL concentration of 100 μg / mL;

[0069] 2. Add mouse LLC cells to DMEM culture medium (containing 10% fetal bovine serum and 1% double antibody (penicillin 100U / mL, streptomycin 100U / mL). When the cells grow to about 90% of the culture dish area, digest them with trypsin and inject the digested LLC cells into the right hind leg of C57BL / 6 mice by subcutaneous injection to obtain a subcutaneous lung cancer model.

[0070] 3. When LLC tumor volume grows to 100mm 3 The FGTL contrast agent was injected into C57BL / 6 mice via tail vein injection at a dose of 20 mg / kg. The mice were imaged in vivo on a 9.4T micro-imaging system before and 4, 8, 12, and 24 hours after injection. 1 HT 2 -weighted MRI, the experiment selected the RARE imaging sequence with the following imaging parameters: TR was 2500ms, TE was 36ms, FOV size was 3.5cm×3.5cm, slice thickness was 1.0mm, matrix size was set to 256×256, acceleration factor was set to 8, and sampling times was 10.

[0071] Test results:

[0072] LLC subcutaneous lung cancer model before and after injection of probe FGTL contrast agent1 HT 2 -weighted MRI Fig. 9 As shown, the MR signal intensity of LLC subcutaneous lung cancer model before and after injection of probe FGTL contrast agent is shown in Fig.10 As shown by Fig. 9 and Fig.10 It can be seen that after the LLC subcutaneous lung cancer model mice were injected with the probe FGTL contrast agent through the tail vein, the tumor area 1 HT 2 -weighted MRI contrast effect gradually increases, reaching its strongest effect at 12 hours, and 24 hours 1 HT 2 -weighted MRI contrast effect returned to the initial value, indicating that the probe FGTL can effectively achieve lung cancer area 1 HT 2 -weighted MRI imaging is expected to be used for tumor detection.

[0073] Experiment 4: Experiment on the use of the probe FGTL of the present invention in reversing the tumor immunosuppressive microenvironment

[0074] Test method:

[0075] The LLC subcutaneous lung cancer model was constructed according to the method of Experiment 3. When the LLC tumor volume grew to 100 mm 3 About 10 days, the LLC subcutaneous lung cancer model C57BL / 6 mice were divided into three groups (n=3), and PBS solution, FGL solution (without immune activation peptide Tuftsin) and FGTL solution were injected into the three groups of LLC subcutaneous lung cancer model C57BL / 6 mice by tail vein injection, with an injection dose of 20 mg / kg. Seven days later, each group of LLC subcutaneous lung cancer model C57BL / 6 mice was treated as follows: the C57BL / 6 mice were killed, and the tumor tissue was removed, the tumor tissue was digested into a single cell suspension, and the flow cytometer test was performed after flow antibody staining, and the effector T cells of the tumor tissue were statistically analyzed. Experimental results:

[0076] Comparison of the reversal effects of PBS solution, FGL solution and FGTL solution on the immunosuppressive microenvironment of LLC subcutaneous lung cancer model Fig.11 As shown by Fig.11 It can be seen that in the PBS-treated group, helper T cells (CD3 + CD4 +) and cytotoxic T cells accounted for 16.9% and 17.2%, respectively, while in the FGL-treated group and FGTL-treated group, the proportion of helper T cells increased to 29.1% and 48.2%, respectively, and the proportion of cytotoxic T cells increased to 23.9% and 36.7%, respectively. This shows that both FGL and FGTL probes can effectively reverse the tumor immunosuppressive microenvironment, and the FGTL probe loaded with the immune-activating peptide Tuftsin can reverse the tumor immunosuppressive microenvironment to a greater extent, which helps to improve the response rate of immune checkpoint blockade therapy.

[0077] Experiment 5: Experiment on the application of the probe FGTL of the present invention in lung cancer ferroptosis-enhanced immunotherapy

[0078] Test method:

[0079] Five-week-old C57BL / 6 mice were divided into five groups (n=5), namely control group, α-PD-L1 group, Fe 3 O 4 @GOx(FG) group, FGTL group and FGTL+α-PD-L1 group. Three days after the LLC subcutaneous lung cancer model was established in five groups of C57BL / 6 mice, PBS solution was injected into the model mice of the control group, α-PD-L1 solution was injected into the model mice of the α-PD-L1 group (dose 25μg / time, α-PD-L1 solution was prepared by dissolving α-PD-L1 in PBS), and Fe 3 O 4 Intravenous injection of Fe 3 O 4 @GOx solution (dosage 20 mg / kg, Fe dispersed in PBS 3 O 4 @GOx prepared Fe 3 O 4 @GOx solution), FGTL solution was injected into the FGTL group model mice (dosage 20 mg / kg, FGTL was dispersed with PBS to prepare FGTL solution), and FGTL (dosage 20 mg / kg, FGTL was dispersed with PBS to prepare FGTL solution) + α-PD-L1 solution was injected into the FGTL+α-PD-L1 group model mice (dosage 25 μg / time, α-PD-L1 was dissolved with PBS to prepare α-PD-L1 solution), and the drug treatment was performed by tail vein injection. For the α-PD-L1 group and the FGTL+α-PD-L1 group, α-PD-L1 (25 μg / time) was administered by tail vein injection on the 3rd, 5th, and 7th days after the tail vein injection of the solution. The body weight and tumor volume of each group of mice were monitored every two days, and the tumor volume was calculated according to the formula: volume (mm 3 ) = length × width 2 / 2, and the tumors were dissected and photographed after 14 days of monitoring.

[0080] Test results:

[0081] The results of using probe FGTL to enhance ferroptosis immunotherapy in tumors are as follows Fig.12 As shown, from Fig.12 As can be seen in (a), the tumor photos of the mice dissected from each group showed that compared with the control group, α-PD-L1 group, Fe 3 O 4 The tumor size in the mice of the @GOx(FG) group, FGTL group, and FGTL+α-PD-L1 group showed a significant trend of shrinking. Compared with the control group and the α-PD-L1 group alone, the tumors in the mice of the FGTL group and FGTL+α-PD-L1 group were significantly smaller. This shows that the probe FGTL-induced tumor ferroptosis can inhibit tumor growth to a certain extent, and further combined with the immune checkpoint blocker α-PD-L1, it can achieve better anti-tumor effects.

[0082] Comparison of the α-PD-L1 group and the FGTL+α-PD-L1 group shows that the response rate of immunotherapy alone is low and it is difficult to inhibit tumor growth. However, after combining the ferroptosis of the probe FGTL and the effect of reversing the tumor immunosuppressive microenvironment, the anti-tumor efficacy of immunotherapy can be significantly improved. In addition, consistent results were also shown in the process of monitoring tumor growth, which fully demonstrates that the probe FGTL combined with α-PD-L1 can enhance the effect of immunotherapy and achieve effective treatment of living tumors.

Claims

1. An active oxygen responsive probe based on Fe3O4, characterized in that: The probe has a Fe3O4 assembly connected with 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid as the inner core and a liposome as the modified outer layer. The liposome is prepared from distearoylphosphatidylcholine, pegylated distearoylphosphatidylethanolamine and cholesterol, and the Fe3O4 assembly is loaded with glucose oxidase and immune-activating peptide Tuftsin.

2. The active oxygen responsive probe based on Fe3O4 according to claim 1, characterized in that: The probe has a spherical structure and a particle size of 53.6 ± 12.2 nm.

3. A method for preparing an active oxygen responsive probe based on Fe3O4 according to claim 1, characterized in that The steps include: S1, mixing the ferrous source solution and the 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid solution evenly to obtain a mixed solution; S2, adding a stabilizer polyvinyl pyrrolidone, a pH adjuster triethylamine and anhydrous ethanol to the mixed solution, mixing evenly to obtain a precursor, at which point the pH value of the precursor is 10-12; S3, subjecting the precursor to a solvothermal reaction, and after the reaction is completed, centrifuging, washing and drying the obtained reaction product to obtain a Fe3O4 assembly connected with 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid; S4, dispersing the Fe3O4 assembly linked by 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid, glucose oxidase and immune-activating peptide Tuftsin in PBS, stirring the reaction under light protection, and then centrifuging and washing to obtain the nanomaterial Fe3O4@GOx@Tuftsin; S5. Distearoylphosphatidylcholine, PEGylated distearoylphosphatidylethanolamine and cholesterol are dissolved in an organic solvent, and the nanomaterial Fe3O4@GOx@Tuftsin is added and mixed evenly. The organic solvent is removed by spin drying, and PBS is added to the residue. Ultrasound is performed and then the mixture is passed through a gel microcolumn to obtain the Fe3O4-based active oxygen responsive probe Fe3O4@GOx@Tuftsin@Lipsomes.

4. The method for preparing the Fe3O4-based active oxygen responsive probe according to claim 3, characterized in that: The ferrous source is ferrous chloride or its hydrate.

5. The method for preparing the Fe3O4-based active oxygen responsive probe according to claim 3, characterized in that: The solvents of the ferrous source solution and the 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid solution are both N , N -Dimethylformamide.

6. The method for preparing the Fe3O4-based active oxygen responsive probe according to claim 3, characterized in that: The molar ratio of the ferrous source to 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid is 1-5:

1.

7. The method for preparing the Fe3O4-based active oxygen responsive probe according to claim 3, characterized in that: The temperature of the solvent thermal reaction is 150-200 ° C, and the reaction time is 12-24 h.

8. The method for preparing the Fe3O4-based active oxygen responsive probe according to claim 3, characterized in that: In the step S4, the stirring reaction temperature is 4-8 ° C and the time is 12-24 h.

9. Use of the Fe3O4-based active oxygen responsive probe according to claim 1 in the preparation of a magnetic resonance imaging agent for lung tumors.

10. Use of the Fe3O4-based reactive oxygen species responsive probe according to claim 1 in the preparation of an anti-lung tumor drug that induces ferroptosis of tumor cells and reverses the tumor immunosuppressive microenvironment.

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