IMSFs hydrogel, ferroptosis induction system, preparation method and application

Abstract

The invention relates to the field of biological medicine, in particular to IMSFs hydrogel, a ferroptosis induction system, a preparation method and application. The IMSFs hydrogel comprises a hydrogel body, and ferroferric oxide nanoparticles and sorafenib are loaded in the hydrogel body in a wrapping manner. The IMSFs hydrogel can be used as an injectable cascade ferroptosis anti-cancer drug. The preparation method of the IMSFs hydrogel comprises the following steps: firstly, preparing a silk fibroin-hyaluronic acid hydrogel body: dissolving silk hydrogel and hyaluronic acid in a lithium bromide solution; bDDE is added into the dissolved solution, and incubation is carried out; then dialyzing and washing with deionized water to obtain a silk fibroin-hyaluronic acid hydrogel body; and then loading the sorafenib nanoparticles and the ferroferric oxide nanoparticles into the silk fibroin-hyaluronic acid hydrogel body. The IMSFs hydrogel disclosed by the invention integrates magnetic thermal response and drug controlled release, and can be injected to a tumor site, so that the treatment effect on TNBC is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an IMSFs hydrogel, a ferroptosis inducing system, a preparation method and applications. Background Art

[0002] Triple-negative breast cancer (TNBC) is an extremely aggressive subtype of breast cancer. Its treatment faces great challenges due to the lack of specific therapeutic targets.

[0003] The main treatments for TNBC currently have the following drawbacks: The primary treatments for TNBC are surgery and chemotherapy, but these are often associated with a high risk of recurrence and metastasis. Chemotherapy (primarily apoptosis-inducing) is prone to drug resistance, and traditional drug delivery systems are unable to precisely target drug delivery at specific locations, making it difficult to accurately meet individual needs.

[0004] Ferroptosis, a cell death mechanism involving iron-dependent lipid peroxidation, offers a novel approach for the anticancer treatment of highly malignant tumors. However, current treatments using ferroptosis are primarily achieved through oral administration of ferroptosis inducers (such as sorafenib). However, ferroptosis inducers, due to their first-pass metabolism and rapid clearance, result in insufficient effective concentrations, and systemic administration is associated with severe side effects. Consequently, the therapeutic strategy of oral ferroptosis inducers is limited by their low bioavailability and significant systemic toxicity.

[0005] 3. Although magnetic hyperthermia can induce ferroptosis through local thermal effects, traditional magnetic hyperthermia has a high temperature and may damage normal tissues.

[0006] Therefore, the treatment of TNBC in the existing technology is greatly limited. Summary of the Invention

[0007] The present invention aims to provide an IMSFs hydrogel, a ferroptosis-inducing system, a preparation method, and an application to overcome the shortcomings of the existing technology, improve the treatment effect of refractory tumors (such as TNBC), and provide a new treatment strategy for refractory tumors.

[0008] To achieve the above objectives, on the one hand, the present invention adopts the following technical solution: an IMSFs hydrogel, comprising a hydrogel body, wherein the interior of the hydrogel body contains ferrosoferric oxide nanoparticles and sorafenib.

[0009] The IMSFs hydrogel in this protocol contains ferroferric oxide nanoparticles. By loading the ferroferric oxide nanoparticles, the IMSFs hydrogel achieves the dual functions of localized magnetothermal therapy and iron ion release. Once the IMSFs hydrogel enters the tumor site, a magnetic field is applied, generating a magnetothermal effect under an alternating magnetic field (AMF). This magnetic field triggers the release of iron ions from the ferroferric oxide, leading to the accumulation of reactive oxygen species (ROS) via Fenton-induced ferroptosis. Furthermore, the magnetothermal effect enables precise temperature control (42–45°C) at the tumor site for mild hyperthermia, preventing thermal damage to surrounding healthy tissue.

[0010] At the same time, IMSFs hydrogel releases sorafenib at the tumor site, and the released sorafenib enters the cells. Sorafenib promotes the formation of the Beclin1-SLC7A11 complex through the AMPK-Beclin1-SLC7A11 pathway to inhibit the XC⁻ system, induce GSH and GPX4 downregulation, and destroy oxidative defense. The combined effect of sorafenib and iron ions expands the effectiveness of ferroptosis. Iron ions and sorafenib synergize to achieve cascade ferroptosis by actively increasing oxidative stress and destroying oxidative defense, resulting in better ferroptosis effect and better tumor treatment effect.

[0011] Preferably, as an improvement, the weight ratio of sorafenib to ferrosoferric oxide nanoparticles is 1:5-7.

[0012] Preferably, as an improvement, the hydrogel is a silk fibroin-hyaluronic acid hydrogel (SF-HA hydrogel). This hydrogel exhibits superior mechanical properties and stability compared to hyaluronic acid (HA) hydrogels alone, and superior biocompatibility compared to silk fibroin (SF) hydrogels alone. Both are readily available, with a simple preparation process and ease of clinical translation. Furthermore, the use of a silk fibroin-hyaluronic acid hydrogel provides excellent injectability for IMSFs hydrogels, and its porous sponge structure protects the loaded drug from shear forces, enabling minimally invasive local injection and achieving in situ tumor delivery. Nanoparticles are not used as drug-encapsulating carriers in this approach because they suffer from circulatory clearance and low local accumulation efficiency due to the EPR effect. Using a silk fibroin-hyaluronic acid hydrogel as a drug-encapsulating carrier eliminates these issues.

[0013] Preferably, as an improvement, the ferroferric oxide nanoparticles in the IMSFs hydrogel are replaced with iron-based magnetocaloric materials or iron-based photothermal materials. Iron-based magnetocaloric materials, such as Fe-MNPs and Fe3-xCoxO4, can also produce similar effects to ferroferric oxide nanoparticles. Iron-based photothermal materials induce heat through lasers, promoting the release of iron ions. Combined with sorafenib, the release of iron ions under thermal action can also achieve the cascading ferroptosis effect of IMSFs.

[0014] Preferably, as an improvement, sorafenib is replaced with a GPX4 inhibitor and / or a GSH inhibitor. Examples of GPX4 inhibitors include RSL3, ML162, and DPI; and examples of GSH inhibitors include fenbendazole. Thus, similar effects can be achieved by replacing sorafenib with either.

[0015] On the other hand, the present invention also adopts the following technical scheme: a method for preparing IMSFs hydrogel, preparing a silk fibroin-hyaluronic acid hydrogel body: dissolving silk hydrogel and hyaluronic acid in a lithium bromide solution; adding BDDE to the dissolved solution and incubating; then dialyzing and washing with deionized water to obtain a silk fibroin-hyaluronic acid hydrogel body; Sorafenib and ferroferric oxide nanoparticles were loaded into the silk fibroin-hyaluronic acid hydrogel. Thus, the IMSFs hydrogel was prepared by this method.

[0016] On the other hand, the present invention also adopts the following technical solution: the use of the above-mentioned IMSFs hydrogel as an injectable cascade ferroptosis anti-cancer drug.

[0017] In summary, through the above scheme, the IMSFs hydrogel described in this patent is used as an injectable cascade-induced ferroptosis anticancer drug. This allows for minimally invasive local injection, achieving in situ tumor delivery without the need for oral administration. Compared to oral medications, it avoids the problems of first-pass metabolism and rapid clearance that lead to insufficient effective concentrations, as well as the serious side effects associated with systemic administration. It also exhibits magnetothermal response and controlled drug release. For tumor treatment, a magnetic field is simply applied to the tumor site in need. Compared to traditional drug delivery systems, this allows for precise targeting of tumor sites, enabling personalized treatment. Furthermore, it can generate a magnetothermal effect under an alternating magnetic field. Compared to other effects (such as photothermal effects), it is less affected by tissue depth and allows for precise temperature control (42-45°C) at the tumor site for mild hyperthermia, avoiding thermal damage to surrounding healthy tissue. Furthermore, the synergistic effect of sorafenib and iron ions amplifies the efficacy of ferroptosis. Iron ions and sorafenib synergize to induce cascade-induced ferroptosis by actively increasing oxidative stress and disrupting oxidative defenses, resulting in enhanced ferroptosis efficacy and tumor treatment.

[0018] On the other hand, the present invention also adopts the following technical solution: the ferroptosis induction system includes a first single-use part used first and a second single-use part used later, the first single-use part includes the above-mentioned IMSFs hydrogel, and the second single-use part includes TAT-Beclin1.

[0019] Therefore, the ferroptosis induction system includes two parts, which are used separately. After the first part is injected into the tumor site, the second part is injected. The reason why the second part is additionally designed in this solution is because the inventors took into account the poor immune microenvironment of the tumor and the possibility of drug release exhaustion, which leads to high recurrence and metastasis rates of TNBC, which is still a key factor in the poor prognosis of patients. In addition, most of the current tumor environments are low immunogenic environments. Most studies have changed the local microenvironment by adding PD-1 / PD-L1 and other methods to induce immune activation. However, this solution is expensive and has side effects caused by excessive activation of autoimmune diseases such as severe enteritis and myasthenia gravis, so its application is limited.

[0020] In this scheme, the inventors separately introduced the intratumoral injection of TAT-beclin1 (Beclin1 activator / autophagy activator) for enhancement. After the second partial injection, it has the following effects: 1. TAT-beclin1, as a small molecule activating peptide, has good membrane-penetrating properties, can overcome tumor heterogeneity and directly reach the reaction center; 2. TAT-beclin1 can activate beclin1, thereby activating autophagy. TAT-beclin1 can also further promote the formation of the Beclin1-SLC7A11 complex. In this way, by promoting the formation of the Beclin1-SLC7A11 complex and coordinating autophagy to expand the ferroptosis effect, the ferroptosis effect is further enhanced. Therefore, TAT-beclin1 has the effect of enhancing ferroptosis; 3. Powerful ferroptosis and autophagy promote the formation of immunogenic cell death (ICD), thereby activating tumor immunity, inducing immune cell maturation and releasing INF-r, thereby inhibiting XC again. - The ferroptosis effect is further enhanced, forming a circular positive feedback loop, which paralyzes the oxidative defense system and exerts excellent anti-cancer effects.

[0021] The order of use of the first and second independently used parts in this application is crucial. The first independently used part is used before the second independently used part. In this way, after the IMSFs hydrogel is injected, the IMSFs hydrogel takes effect first, which will first initiate ferroptosis, and then by injecting TAT-beclin1, TAT-beclin1 initiates autophagy, avoiding TAT-beclin1 being injected first and premature autophagy affecting the process of ferroptosis. Through the use order of this application, ferroptosis is achieved first, and then the autophagy flux is increased, thereby promoting the degradation of ferroptosis products, promoting the process of ferroptosis, and enhancing ferroptosis.

[0022] In the prior art, TAT-beclin1 is used as an autophagy activator, but there are no reports on the effect of using TAT-beclin1 and IMSFs hydrogel successively to enhance ferroptosis. Therefore, the inventors believe that injecting TAT-beclin1 after using IMSFs hydrogel is also an important innovation of this application.

[0023] On the other hand, the present invention also adopts the following technical solution: the above-mentioned ferroptosis induction system is used as an injectable cascade ferroptosis anti-cancer drug.

[0024] Preferably, as a refinement, the cancer is triple-negative breast cancer.

[0025] The patent focuses on triple-negative breast cancer because it is highly malignant and prone to metastasis. As a vital organ in women, minimally invasive injection therapy is highly recommended. Of course, the invention's application in tumor treatment is not limited to triple-negative breast cancer; it can also be used for any tumor that is sensitive to ferroptosis, such as liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Electron micrographs of SF-HA hydrogel, Fe3O4, sorafenib, and IMSFs hydrogel.

[0027] Figure 2 A diagram demonstrating the injectability of IMSFs hydrogel.

[0028] Figure 3 The drug encapsulation status of IMSFs hydrogel is shown schematically, where a is the electron microscope energy spectrum of IMSFs hydrogel; b is the Fourier spectrum of IMSFs hydrogel; c is the X-ray diffraction analysis diagram of IMSFs hydrogel; and d is the X-ray photoelectron energy spectrum of IMSFs hydrogel.

[0029] Figure 4 Schematic diagram of the hysteresis curve.

[0030] Figure 5 Schematic diagram of thermogravimetric analysis curve.

[0031] Figure 6 Schematic diagram of the release curve of sorafenib at different pH values under the action of a magnetic field.

[0032] Figure 7 Schematic diagram of the release curve of iron ions at different pH values under the action of a magnetic field.

[0033] Figure 8 Fluorescence images of IMSFs hydrogel after co-culture with 4T1 cells.

[0034] Figure 9 Schematic diagram of temperature-time variation curve.

[0035] Figure 10 Fe detected by each group of iron ion detection kit 2+ Concentration diagram.

[0036] Figure 11 a is a schematic diagram of the fluorescence microscopy evaluation of intracellular ROS levels in each group; Figure 11 b is the positive analysis diagram of DCF reagent test results.

[0037] Figure 12 a is a schematic diagram of the formation of the Beclin1-SLC7A11 complex; Figure 12 b and Figure 12 c shows the expression of SLC7A11 and GPX4 proteins in each group; Figure 12 d shows the GSH detection results of each group.

[0038] Figure 13 a is a transmission electron microscopy image of cells; Figure 13 b is the fluorescence image of each group after staining with BODIY-C11; Figure 13 c is the live-dead double-stained fluorescence image of each group.

[0039] Figure 14 The diagram shows the overall anti-tumor efficacy in mice, where a is a schematic diagram of the infrared temperature detection curve; b is a schematic diagram of the tumor size of each group of mice on the 25th day; c is the time change curve of the tumor volume of each group; d is the tumor inhibition status of each group; and e is the time change curve of the weight of each group of mice.

[0040] Figure 15 The results of WB detection of tumor tissue proteins are shown.

[0041] Figure 16 a Quantitative analysis of LC3II / GAPDH in each group; Figure 16 b Quantitative analysis of P62 / GAPDH in each group; Figure 16 c Quantitative analysis of LC3-II / P62 in each group.

[0042] Figure 17 These are the immunofluorescence images of GPX4 and SLC7A11 in tumor tissues of each group.

[0043] Figure 18 The immune activation and tumor metastasis inhibition of each group are shown, where a shows the concentrations of IL-6, IL-12, TNF-α, and IFN-γ detected by Elisa in each group; b is the CD8 immunohistochemistry image of the tumor tissue in each group; and c is the solid image of the lung tissue in each group.

[0044] Figure 19 Schematic diagram of the release curve of iron ions at different pH values in the absence of a magnetic field.

[0045] Figure 20 Schematic diagram of the release curve of sorafenib at different pH values without the action of a magnetic field. DETAILED DESCRIPTION

[0046] The following is further described in detail through specific implementation methods: Example 1 This embodiment specifically discloses an IMSFs hydrogel, including a hydrogel body, which is a silk fibroin-hyaluronic acid hydrogel body (SF-HA hydrogel body). The interior of the hydrogel body contains ferroferric oxide nanoparticles and sorafenib, and the weight ratio of sorafenib to ferroferric oxide nanoparticles is 1:5-7, preferably 1:6.

[0047] This embodiment discloses a specific preparation method of IMSFs hydrogel: S1. Boil 30g of silk in 6L of a 0.5% w / v aqueous Na2CO3 solution for 30 minutes. Rinse thoroughly with deionized (DI) water three times and dry overnight in a fume hood to obtain degummed silk fibers. Add 2g of the degummed fibers to 10mL of a 9.3M lithium bromide solution and dissolve in a 60°C water bath for 2 hours to obtain a 20% silk solution. Finally, add 400µL of butanediol diglycidyl ether (BDDE) to the silk solution, and incubate the mixture at 60°C for 3 hours to obtain a silk fibroin hydrogel. The silk fibroin hydrogel is then dialyzed in deionized water for 3 days, changing the water 15 times to remove the crosslinker.

[0048] S2. Freeze-dry the silk fibroin hydrogel and grind it into powder. Dissolve the freeze-dried silk hydrogel (1g) and hyaluronic acid (1g) in 10ml of 9.3M lithium bromide solution at 60°C for 3 hours. Supplement the solution with 400µL of BDDE and incubate at 60°C for 3 hours. Then, dialyze and wash with deionized water for 3 days, changing the water 15 times, to obtain the silk fibroin-hyaluronic acid hydrogel (SF-HA).

[0049] S3. Finally, sorafenib (1 wt%) and ferroferric oxide nanoparticles (6 wt%) were added to the SF-HA hydrogel and thoroughly stirred until a uniform brown color was obtained, thereby forming the IMSFs hydrogel. In this example, the amount of sorafenib added per 1 g of hydrogel was 0.0107 g, and the amount of ferroferric oxide added was 0.0645 g. The density of the hydrogel in this example was 1 mg / mL.

[0050] The IMSFs hydrogel in this embodiment can be used as an injectable cascade ferroptosis anti-cancer drug.

[0051] The IMSFs hydrogel in this example is used as an injectable cascade-induced ferroptosis anticancer drug. This allows for minimally invasive local injection, achieving in situ tumor administration without the need for oral administration. Compared to oral medications, this drug avoids the problems of first-pass metabolism and rapid clearance that lead to insufficient effective concentrations, while also avoiding the serious side effects associated with systemic administration. It also exhibits magnetothermal response and controlled drug release. For tumor treatment, a magnetic field is simply applied to the tumor site in need of treatment. Compared to traditional drug delivery systems, this system can precisely target the tumor site and meet individual needs. It can also generate a magnetothermal effect under an alternating magnetic field. Compared to other effects (such as photothermal effects), this is less affected by tissue depth and allows for precise temperature control (42-45°C) at the tumor site for mild hyperthermia, avoiding thermal damage to surrounding healthy tissue. Furthermore, the combined action of sorafenib and iron ions amplifies the efficacy of ferroptosis. Iron ions and sorafenib synergize to achieve cascade-induced ferroptosis by actively increasing oxidative stress and disrupting oxidative defenses, resulting in enhanced ferroptosis efficacy and tumor treatment.

[0052] experiment The following series of experiments demonstrate that the IMSFs hydrogel prepared in this example has corresponding effects and functions, and can be used as an injectable cascade ferroptosis anticancer drug.

[0053] Experiment 1: Electron Microscope Experiment SF-HA hydrogel, Fe3O4, sorafenib, and IMSFs hydrogel were freeze-dried and examined by scanning electron microscopy. Figure 1 The picture shown; the results show that the SF-HA hydrogel body presents a porous sponge-like structure. By comparison, it can be seen that Fe3O4 and sorafenib particles are successfully loaded into the SF-HA hydrogel body, and have no obvious effect on the structure of the SF-HA hydrogel body.

[0054] Experiment 2: Injectability experiment Load the IMSFs hydrogel into a 1 mL syringe. Figure 2 As shown, it was injected into the liquid in the beaker, and it can be seen that it can be smoothly injected into the liquid in the beaker through an 18G needle, thereby confirming that the IMSFs hydrogel has excellent injectability and can be used for minimally invasive treatment of tumors.

[0055] Experiment 3: Drug loading analysis experiment The electron microscope energy spectrum analysis of IMSFs hydrogel was carried out, and the quantitative results of the corresponding elements of various substances in IMSFs hydrogel divided by energy spectrum are as follows Figure 3 As shown in a, this is basically consistent with the amount of various substances added to IMSFs hydrogel. Figure 3 As shown in b, the Fourier transform infrared (FTIR) spectrum of IMSFs hydrogel showed the characteristic absorption peaks of Fe3O4 and Sorafenib (SOR), confirming the presence of Fe3O4 and SOR molecules. Figure 3 As shown in c, the results of X-ray diffraction (XRD) analysis of IMSFs hydrogels indicate that the chemical structures of Fe3O4 and SOR incorporated into IMSFs hydrogels will not change due to the preparation process of IMSF hydrogels. Figure 3 As shown in (d), X-ray photoelectron spectroscopy (XPS) analysis of IMSFs hydrogel confirmed the presence of elements C, N, O, F, Cl and Fe through C1s, N1s, O1s, F1s, Cl2P and Fe2p.

[0056] Experiment 4: Magnetocaloric Performance Experiment The IMSFs hydrogel and Fe3O4 nanoparticles were measured using the American LakeShore 7404 instrument. Figure 4 As shown, a narrow hysteresis curve for the IMSFs hydrogel was obtained. This indicates that the IMSFs hydrogel is a soft ferrite, with low coercive force and remanent magnetization (saturation magnetization of 15.74 emu / g), similar to that of pure Fe3O4 nanoparticles (saturation magnetization of 63.18 emu / g). This demonstrates that the IMSFs hydrogel is a soft magnetic material with hysteresis, loss characteristics, and coercive force. Because the coercive force is smaller than that of the AMF (number of coils: 2; coil length: 1 cm; coil diameter: 3 cm; frequency: 626 kHz; output current: 28.6 A; field strength: 5.72 kA / m), it is easily triggered by the AMF.

[0057] Experiment 5: Thermal stability experiment The TGA thermogravimetric analysis instrument was used to perform thermogravimetric analysis of the IMSFs hydrogel and the F@SF-HA (only ferroferric oxide was added to the SF-HA hydrogel) hydrogels at 0-800 degrees Celsius. The results are as follows: Figure 5As shown, IMSFs hydrogel exhibits good thermal stability under heating conditions, and there is no obvious thermal decomposition of the material within the magnetocaloric working temperature range (<50°C).

[0058] Experiment 6: Drug release performance experiment The IMSFs hydrogels were not treated with a magnetic field and were subjected to drug release experiments at pH 6 and pH 7.4. At the corresponding time points and within the predetermined time intervals, samples of the release medium were collected for analysis and supplemented with an equal volume of fresh medium. The concentration of sorafenib released in the supernatant was analyzed using a UV-vis spectrophotometer. In addition, the total iron ion concentration was determined after Fe³⁺ was reduced to Fe²⁺ with sodium sulfite, and the Fe²⁺ concentration was detected using a ferrous ion detection kit. The final test results are shown in Figure 2. Figure 19 、 Figure 20 shown.

[0059] After the IMSFs hydrogel was exposed to a magnetic field (the magnetic field parameters were the same as the AMF parameters in Experiment 4, and this parameter was used in the magnetic field treatment below) for 400 seconds, drug release experiments were performed under pH 6 and pH 7.4 conditions. At the corresponding time points and within the predetermined time intervals, samples of the release culture medium were collected for analysis, and an equal volume of fresh culture medium was added. The concentration of sorafenib released in the supernatant was analyzed using a UV-vis spectrophotometer. In addition, the total iron ion concentration was determined after Fe³⁺ was reduced to Fe²⁺ with sodium sulfite, and the Fe²⁺ concentration was detected using a ferrous ion detection kit. The results are shown in Figure 2. Figure 6 and Figure 7 As shown, compared Figure 19 and Figure 20 At a pH similar to that of the tumor microenvironment (pH 6), IMSFs hydrogels exhibited a sustained drug release effect after exposure to a magnetic field. However, when the IMSFs hydrogels were not exposed to a magnetic field, they released little or no iron ions or sorafenib. Furthermore, iron ions were almost completely absent at neutral pH, indicating that their responsiveness to the acidic microenvironment can mitigate the risk of systemic iron overload.

[0060] Experiment 7: Biocompatibility Test 50 μL of IMSFs hydrogel was placed in a 24-well plate, and 4T1 cells (5 × 10 3 ) were inoculated on IMSFs hydrogel and incubated together. IMSFs hydrogel was co-cultured with 4T1 cells for 1, 2, 3, 5, and 7 days, and the cells were detected by live-dead double staining (green represents live cells and red represents dead cells). Figure 8 As shown in the figure, it can be seen that IMSFs hydrogel has almost no toxicity to cells and has excellent biocompatibility.

[0061] Experiment 8: Stable and mild magnetocaloric capability experiment This experiment was divided into two groups, with saline (1 mL) and IMSFs hydrogel (100 μL) placed in EP tubes, respectively, and placed in coils. Infrared temperature imaging was used to detect temperature changes in real time. Figure 9 As shown: the saline group showed no obvious temperature change when exposed to the magnetic field (magnetic field parameters were the same as before), while the temperature of the IMSFs group gradually increased when exposed to the magnetic field and remained stable in the range of 42-45 degrees for a period of time, indicating that the IMSFs hydrogel has excellent controllable magnetothermal properties and can implement stable and mild magnetic hyperthermia.

[0062] Experiment 9: Induced ROS accumulation ability experiment The experiment was divided into 5 groups, namely a: Control group, b: F@SF-HA hydrogel group (only ferric oxide was added to the SF-HA hydrogel body, prepared by 64.51 mg of Fe3O4 and 1 mL of SF-HA hydrogel body), c: IMSFs hydrogel group (prepared by 64.51 mg of Fe3O4, 10.75 mg of Sorafenib and 1 mL of SF-HA hydrogel body), d: F@SF-HA hydrogel + AMF treatment group, e: IMSFs hydrogel + AMF treatment group. 4T1 cells were seeded into 12-well plates and cultured for 24 hours. After the 4T1 cells were treated with the above groups, they were washed with PBS. The iron ion content in the cells was detected using an iron ion detection kit. Combined with Figure 10 As shown in the figure, the results showed that the iron ion content in the F@SF-HA hydrogel + AMF treatment group and the IMSFs hydrogel + AMF treatment group was significantly higher than that in the other groups, indicating that the intracellular iron ion content was significantly increased after magnetic field exposure treatment, confirming that magnetic field exposure is the initiating factor triggering the release of iron ions.

[0063] Subsequently, the cells were incubated with DCFH-DA for 30 minutes and stained with Hoechst 33342 for 5 minutes. The intracellular ROS levels were then assessed using fluorescence microscopy. Figure 11 As shown: A significant increase in ROS levels was observed only in the magnetic field exposure groups (F@SF-HA hydrogel + AMF treatment group and IMSFs hydrogel + AMF treatment group), and ROS accumulation was positively correlated with iron ion release.

[0064] Experiment 10: Sorafenib release-induced complex formation weakens oxidative defense experiment 4T1 cells were cultured and divided into IMSFs hydrogel + AMF treatment group and non-treatment group. Cell proteins were extracted for Co-IP detection. The results are shown in Figure 2. Figure 12As shown in a, after the cells were treated with IMSFs hydrogel + AMF, the Beclin1-SLC7A11 complex was formed. In addition, according to the 5 groups in Experiment 9, the cells were treated and proteins were extracted for WB detection. The results are shown in Figure 12 b. Figure 12 c shows that the expression of SLC7A11 and GPX4 proteins was significantly reduced after treatment with IMSFs hydrogel + AMF. Figure 12 As shown in (d), GSH was detected, and the results showed that GSH also decreased after the cells were treated with IMSFs hydrogel + AMF.

[0065] Experiment 11: Ferroptosis-inducing ability experiment like Figure 13 As shown in a, after 4T1 cells were treated with IMSFs hydrogel + AMF, mitochondrial condensation was observed under transmission electron microscopy compared with the normal group, indicating ferroptosis. In addition, 4T1 cells were treated with 5 groups in the same experiment 9 and stained with BODIY-C11 (green fluorescence: LOP positive, red fluorescence: no, blue fluorescence: nuclear staining) to evaluate lipid peroxidation (LOP). Figure 13 As shown in Figure b, the green fluorescence of the IMSFs hydrogel + AMF treatment group (group e) was the strongest, the fluorescence of the F@SF-HA hydrogel + AMF treatment group (group d) was weaker than that of group e, and the fluorescence of other groups was even weaker, thus confirming that the iron ions in IMSFs and sorafenib synergistically enhanced the effect of ferroptosis. Figure 13 As shown in Figure d, the live-dead double staining results of the cells (the red color of the IMSFs hydrogel + AMF treatment group should be stronger than the red fluorescence of the F@SF-HA hydrogel + AMF treatment group) also confirmed the above results.

[0066] Example 2 This embodiment also discloses a ferroptosis inducing system, including a first single-use part used first and a second single-use part used later, wherein the first single-use part includes an IMSFs hydrogel in Example 1, and the second single-use part includes TAT-Beclin1.

[0067] The ferroptosis induction system in this embodiment can be used as an injectable cascade ferroptosis anticancer drug.

[0068] When this ferroptosis induction system is used as an injectable cascade ferroptosis anti-cancer drug, the first single-use part is first injected into the tumor site. At this time, the IMSFs hydrogel initiates ferroptosis, and then the second single-use part is injected. After the second single-use part is injected, it has the following effects: 1. TAT-beclin1, as a small molecule activation peptide, has good membrane-penetrating properties, can overcome tumor heterogeneity and reach the reaction center directly; 2. TAT-beclin1 can activate beclin1, thereby activating autophagy. TAT-beclin1 can also further promote the formation of the Beclin1-SLC7A11 complex. In this way, by promoting the formation of the Beclin1-SLC7A11 complex and coordinating autophagy to expand the ferroptosis effect, the ferroptosis effect is further enhanced. Therefore, TAT-beclin1 has the effect of enhancing ferroptosis; 3. Powerful ferroptosis and autophagy promote the formation of immunogenic cell death (ICD), thereby activating tumor immunity, inducing immune cell maturation and releasing INF-r, thereby inhibiting XC again. - The ferroptosis effect is further enhanced, forming a circular positive feedback loop, which paralyzes the oxidative defense system and exerts excellent anti-cancer effects.

[0069] experiment The following experiments will continue to prove that the ferroptosis-inducing system has corresponding effects and functions, and can also prove the effects and functions of IMSFs hydrogel.

[0070] Experiment 12: In vivo experiments in mice Tumor-bearing mice were randomly divided into 5 groups, namely (1) control group (normal saline injection, injection volume 100uL), (2) IMSFs hydrogel group (injection volume 100uL), (3) F@SH-HA hydrogel (injection volume 100uL) + AMF treatment group, (4) IMSFs hydrogel (injection volume 100uL) + AMF treatment group, (5) IMSFs hydrogel (injection volume 100uL) + AMF treatment + TAT-Beclin1 treatment group. In groups 3-5, F@SH-HA hydrogel and IMSFs hydrogel were injected into the mouse tumors and exposed to AMF on the second day. The magnetic field parameters were the same as before. Under infrared monitoring, the power was adjusted to maintain the magnetic field in the range of 42-45 degrees for 6 minutes during each magnetic field exposure. Subsequently, TAT-Beclin1 was injected intratumorally for 2 consecutive days (injection volume 1.5mg / Kg / d), followed by 3 days of rest, 5 days per cycle, and a total of 5 cycles. The tumor volume and body weight of tumor-bearing mice were monitored every 3 days throughout the treatment cycle. Mice were analyzed for survival and observed until day 40. 3 The mice were euthanized at 4 hr. Lung tissues were collected after necropsy, fixed, and stained with Bouins solution to evaluate lung metastasis in each group.

[0071] The results are as follows Figure 14 As shown in a, by comparing Group 1 and Group 4, after the IMSFs hydrogel was injected, the infrared temperature detector results under magnetic field exposure showed that the IMSFs hydrogel exhibited good temperature control performance, controlling the tumor temperature between 42-45 degrees. Figure 14 b, as shown in 14c, Figure 14 In b, the tumor volume of group 5 is the smallest, followed by that of group 4. It can be seen that the tumor growth of groups 4 and 5 is significantly inhibited, and the inhibition effect of group 5 is the best. At the same time, the tumor inhibition rate (TGItv=(1-(tumor volume of the experimental group on the last day - tumor volume at the beginning of treatment) / (tumor volume of the control group on the last day - tumor volume of the control group at the beginning of treatment)) x 100%) was calculated, as shown in Figure 2. Figure 14 As shown in d, the tumor inhibition rate of group 5 was the highest, followed by group 4. Figure 14 As shown in Figure e, there was no significant difference in body weight among the groups through body weight monitoring, indicating that the ferroptosis induction system and IMSFs hydrogel in this example are relatively safe.

[0072] The tumor tissues of the mice treated as above were isolated and the tissue proteins were extracted for WB detection. The results were as follows: Figure 15 and Figure 16 As shown, the LC3II protein in Group 5 was significantly increased, P62 was significantly decreased, and the LC3II / P62 ratio was significantly increased, indicating that TAT-Beclin1 injection promoted autophagic flux mainly by enhancing the degradation of autophagosomes rather than their formation. At the same time, the LC3-II / P62 ratio in Group 5 was significantly higher than that in the other groups. This increase in the ratio further demonstrates that TAT-Beclin1 injection significantly enhanced autophagic flux in tumor tissues.

[0073] In addition, the tumor tissues of each group were extracted and subjected to corresponding immunofluorescence detection. The results were as follows: Figure 17 As shown, the expression of GPX4 and SLC7A11 in group 5 was significantly reduced, which was also significantly reduced compared with the IMSFs hydrogel + AMF treatment group, suggesting that the addition of TAT-Beclin1 further enhanced the inhibition of tumor oxidative defense and expanded the ferroptosis effect.

[0074] In addition, serum from each group of mice was extracted and tested by Elisa. Figure 18 As shown in a, the results showed that the immune factors IL-6, IL-12, TNF-α, and IFN-γ in group 5 and group 4 were significantly increased, but the highest level was in group 5, which was significantly different from group 4, indicating that the injection of TAT-Beclin1 had a significant effect on activating immunity. Immunohistochemistry of CD8 in tumor tissues was performed, as shown in Figure 18As shown in b, the histochemical results also confirmed the above conclusion. Figure 18 As shown in c, the solid image of lung tissue shows that lung metastasis in Group 5 was significantly inhibited, indicating that Group 5 had the best treatment effect.

[0075] In addition, blood was collected from mice in Group 4 and Group 5 every 7 days and blood biochemistry and routine blood tests were completed. It was found that there was no significant difference in the values, and there was no obvious abnormality in the HE pathology results of organs such as the heart, liver, spleen, lungs, and kidneys, indicating that the IMSFs hydrogel and ferroptosis induction system are both highly safe and can be used for biological injection.

[0076] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. An IMSFs hydrogel, comprising a hydrogel body, characterized in that: The hydrogel body contains ferroferric oxide nanoparticles and sorafenib.

2. The IMSFs hydrogel according to claim 1, characterized in that: The weight ratio of sorafenib to ferrosoferric oxide nanoparticles is 1:5-7.

3. The IMSFs hydrogel according to claim 1, characterized in that: The hydrogel body is a silk fibroin-hyaluronic acid hydrogel body.

4. The IMSFs hydrogel according to claim 1, characterized in that: The ferroferric oxide nanoparticles are replaced by iron-based magnetocaloric materials or iron-based photothermal materials.

5. The IMSFs hydrogel according to claim 1, characterized in that: The sorafenib is replaced by a GPX4 inhibitor and / or a GSH inhibitor.

6. The method for preparing an IMSFs hydrogel according to claim 3, wherein: Preparation of a silk fibroin-hyaluronic acid hydrogel: dissolving silk hydrogel and hyaluronic acid in a lithium bromide solution; adding BDDE to the dissolved solution and incubating; then dialysis and washing with deionized water to obtain a silk fibroin-hyaluronic acid hydrogel; Sorafenib and ferroferric oxide nanoparticles were loaded into the silk fibroin-hyaluronic acid hydrogel.

7. Ferroptosis induction system, characterized by: The method comprises a first independently used part used first and a second independently used part used later, wherein the first independently used part comprises an IMSFs hydrogel according to any one of claims 1 to 5, and the second independently used part comprises TAT-Beclin 1.

8. Use of the ferroptosis induction system according to claim 7 as an injectable cascade ferroptosis anticancer drug.

9. Use of the IMSFs hydrogel according to any one of claims 1 to 5 as an injectable cascade ferroptosis anticancer drug.

10. The use according to claim 8 or 9, characterized in that: The cancer is triple-negative breast cancer.

Images