TAT-beclin1, ferroptosis induction system and application

Abstract

The invention relates to the field of biological medicine, in particular to TAT-beclin1, a ferroptosis induction system and application. Through research, the inventor finds that after ferroptosis is started, the effect of enhancing ferroptosis is achieved by injecting TAT-beclin1, and therefore the TAT-beclin1 is applied to the medicine for enhancing ferroptosis and used for treating cancers. According to the scheme, the ferroptosis effect is amplified, and the ferroptosis enhancement effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically relates to TAT-beclin1, a ferroptosis induction system and applications thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer. Due to the lack of specific therapeutic targets, its treatment faces great challenges. Currently, the main treatment methods for triple-negative breast cancer are surgery and chemotherapy. However, the treatment methods of surgery and chemotherapy are often accompanied by a high risk of recurrence and metastasis.

[0003] Ferroptosis, as a mechanism of iron-dependent lipid peroxidation cell death, provides new ideas for the anti-cancer treatment of highly malignant tumors. However, the current ferroptosis effect is limited. For example, for low-immunogenic tumors such as TNBC, the ability to trigger immunogenic cell death (ICD) by limited ferroptosis is limited. Combined with the loss effect of drug release by ferroptosis, it leads to a high recurrence rate and metastasis rate of TNBC, which is still a key factor for poor prognosis of patients. If the ferroptosis effect can be further amplified, it can bring new hope for tumor treatment.

[0004] TAT-Beclin1 is a fusion protein composed of the TAT (Trans-Activator of Transcription) peptide and the Beclin1 protein. It combines the cell-penetrating ability of the TAT peptide with the important function of Beclin1 in the process of autophagy. TAT-beclin1 in the prior art is used as an autophagy activator, and there is no relevant report on the role of TAT-beclin1 in amplifying ferroptosis. Summary of the Invention

[0005] The present invention aims to provide TAT-beclin1, a ferroptosis induction system and applications thereof to amplify the ferroptosis effect and play a role in enhancing ferroptosis.

[0006] To achieve the above object, on the one hand, the present invention adopts the following technical solution: the application of TAT-beclin1 in enhancing ferroptosis drugs.

[0007] On the other hand, the present invention also adopts the following technical solution: the application of TAT-beclin1 in anti-cancer drugs, and TAT-beclin1 has the effect of enhancing ferroptosis.

[0008] The inventors found through experiments that after inducing ferroptosis in tumors, by injecting TAT-beclin1, 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 cooperating with autophagy to expand the ferroptosis effect, the ferroptosis effect is further enhanced (the first enhancement of ferroptosis). Therefore, TAT-beclin1 has the effect of enhancing ferroptosis. At the same time, strong ferroptosis and autophagy promote the formation of immunogenic cell death (ICD), thereby activating tumor immunity, inducing the maturation of immune cells and releasing INF-r, thereby inhibiting the XC - transporter, and the ferroptosis effect is further enhanced (the second enhancement of ferroptosis), forming a cyclic positive feedback loop, paralyzing the oxidative defense system and exerting excellent anti-cancer efficacy.

[0009] In the prior art, TAT-beclin1 is used as an autophagy activator, and the sequential use of TAT-beclin1 and a ferroptosis inducer and the effect of enhancing ferroptosis have not been reported. Therefore, the inventors found that TAT-beclin1 has the effect of enhancing ferroptosis, and thus applied for a patent for it to protect the innovation.

[0010] In addition, as a small molecule activating peptide, TAT-beclin1 has good membrane penetration characteristics, can overcome tumor heterogeneity and reach the reaction center directly.

[0011] Through the above scheme, the ferroptosis effect is enhanced, overcoming the problems of poor tumor immune microenvironment and the high recurrence rate and metastasis rate of TNBC caused by the possible release and depletion of ferroptosis inducers, and greatly improving the treatment effect of tumors. In addition, TAT-beclin1 promotes immune activation. Compared with the traditional methods of using PD-1 / PD-L1, etc. to change the local microenvironment to promote immune activation, it will not cause side effects induced by overactivation of autoimmunity such as severe enteritis and myasthenia gravis.

[0012] On the other hand, the present invention also adopts the following technical solution: a ferroptosis induction system, including a first separately used part used first and a second separately used part used later. The first separately used part has the effect of inducing ferroptosis, and the second separately used part includes TAT-Beclin1 with the effect of enhancing ferroptosis.

[0013] Thus, the first individually used part acts on the tumor, inducing ferroptosis. Then, by injecting TAT-beclin1, autophagy is initiated, preventing premature autophagy caused by prior injection of TAT-beclin1 from affecting the process of ferroptosis. Through the usage sequence of the ferroptosis induction system of the present application, ferroptosis occurs first, followed by an increase in autophagic flux, thereby promoting the degradation of ferroptosis products, facilitating the process of ferroptosis, and enhancing ferroptosis. Therefore, the use of TAT-beclin1 after the first individually used part in the present application has not been reported in the prior art and is also an innovation of the present application.

[0014] Preferably, as an improvement, the first individually used part includes a carrier, and the carrier internally encapsulates magnetic iron oxide nanoparticles. The magnetic iron oxide nanoparticles can induce ferroptosis.

[0015] Preferably, as an improvement, the carrier also internally encapsulates sorafenib.

[0016] In this solution, the release of sorafenib into cells promotes the formation of the Beclin1-SLC7A11 complex through the AMPK-Beclin1-SLC7A11 pathway to inhibit the XC⁻ system, inducing the downregulation of GSH and GPX4 and disrupting the oxidative defense. The combined action of sorafenib and iron ions expands the ferroptosis effect. The iron ions and sorafenib synergistically achieve cascading ferroptosis by actively increasing oxidative stress and disrupting oxidative defense in two aspects. Therefore, the first individually used part in the present application also has a good ferroptosis effect. Coupled with the continuous enhancement of ferroptosis by the second individually used part, the tumor treatment effect will be better. The co-use of sorafenib and magnetic iron oxide nanoparticles to achieve cascading ferroptosis has not been reported in the prior art. Therefore, the combination of sorafenib and magnetic iron oxide nanoparticles is also an innovation of this patent. Preferably, as an improvement, the carrier is a hydrogel.

[0017] Of course, for the first individually used part, it can also be a drug that inhibits the XC system such as Erastin or SAS (sulfasalazine). Alternatively, these drugs can replace the sorafenib encapsulated inside the carrier. These drugs also have the effect of initiating ferroptosis, so they can be used as the first individually used part.

[0018] On the other hand, the present invention also adopts the following technical solution: the application of the ferroptosis induction system in ferroptosis anti-cancer drugs.

[0019] Preferably, as an improvement, the cancer is triple-negative breast cancer. The reason why triple-negative breast cancer is the research object in this patent is that it is highly malignant and prone to metastasis. Moreover, as an important organ of women, it is of great significance to choose injectable minimally invasive treatment. Of course, the application of this invention to tumor treatment is not limited to triple-negative breast cancer, and any tumor that is sensitive to ferroptosis can be used, such as liver cancer, etc.

[0020] In addition, this patent discloses another invention. For the first separately used part for initiating ferroptosis mentioned above, it is an IMSFs hydrogel, which includes a silk fibroin-hyaluronic acid hydrogel body, and the interior of the silk fibroin-hyaluronic acid hydrogel body is loaded with iron oxide nanoparticles and sorafenib.

[0021] In this solution, the silk fibroin-hyaluronic acid hydrogel body is adopted, which has higher mechanical properties and stability compared with the single hyaluronic acid (HA) hydrogel body, and has more excellent biocompatibility compared with the single silk fibroin (SF) hydrogel body; and both of them are simple in source and easy to obtain, and the preparation process is simple and easy for clinical transformation. At the same time, the adoption of the silk fibroin-hyaluronic acid hydrogel body makes the IMSFs hydrogel have excellent injectability, and the porous sponge structure can protect the drugs loaded inside from shear force, with the ability of minimally invasive local injection, realizing in-situ tumor injection.

[0022] The IMSFs hydrogel in this patent, as the first separately used part, has the following advantages: it can be used for minimally invasive local injection to realize in-situ tumor injection without oral administration. Compared with oral drugs, there will be no problems such as first-pass metabolism and rapid clearance resulting in insufficient effective concentration, and at the same time, systemic drug administration is accompanied by serious side effects. At the same time, it also has the functions of magnetic heat response and drug controlled release. When treating tumors, only a magnetic field needs to be applied to the tumor site to be treated. Compared with traditional drug release systems, it can act precisely on the tumor site and can precisely meet personalized needs. At the same time, it can also generate a magnetic heat effect under an alternating magnetic field. Compared with other effects (such as photothermal effect), it is less affected by tissue depth and can precisely control the temperature (42-45°C) at the tumor site for mild hyperthermia, avoiding thermal damage to surrounding healthy tissues. At the same time, the combined action of sorafenib and iron ions expands the ferroptosis effect. The iron ions and sorafenib cooperate to achieve cascading ferroptosis by actively increasing oxidative stress and destroying oxidative defense. It has a good ferroptosis effect at the initiation stage of ferroptosis, and the tumor treatment effect is improved.

[0023] Therefore, applying the IMSFs hydrogel to the ferroptosis induction system can integrate magnetic heat response, drug controlled release and immune enhancement, can significantly improve the treatment effect on refractory tumors such as TNBC, and provides a new treatment plan for refractory tumors. Description of the Drawings

[0024] Figure 1 Electron micrographs of SF-HA hydrogel body, Fe3O4, sorafenib, and IMSFs hydrogel.

[0025] Figure 2 Demonstration diagram of the injectability of IMSFs hydrogel.

[0026] Figure 3 Schematic diagram showing the drug encapsulation of IMSFs hydrogel, where a is the energy spectrum diagram of the electron microscope of IMSFs hydrogel; b is the Fourier spectrum diagram of IMSFs hydrogel; c is the X-ray diffraction analysis diagram of IMSFs hydrogel; d is the X-ray photoelectron energy spectrum diagram of IMSFs hydrogel.

[0027] Figure 4 Schematic diagram of the hysteresis curve.

[0028] Figure 5 Schematic diagram of the thermogravimetric analysis curve.

[0029] Figure 6 Schematic diagram of the release curve of sorafenib at different pH values under the action of magnetic field.

[0030] Figure 7 Schematic diagram of the release curve of iron ions at different pH values under the action of magnetic field.

[0031] Figure 8 Fluorescence image of IMSFs hydrogel co-cultured with 4T1 cells.

[0032] Figure 9 Schematic diagram of the temperature-time change curve.

[0033] Figure 10 Fe detected by iron ion detection kits for each group 2+ Concentration schematic diagram.

[0034] Figure 11 Schematic diagram of the intracellular ROS level evaluated by fluorescence microscopy for each group (a); Positive analysis diagram of the detection result of DCF reagent (b).

[0035] Figure 12 Schematic diagram of the formation of Beclin1-SLC7A11 complex (a); Expression of SLC7A11 and GPX4 proteins in each group (b and c); Detection results of GSH in each group (d).

[0036] Figure 13 Cell transmission electron micrograph (a); Fluorescence image of each group after staining with BODIY-C11 (b); Fluorescence image of live-dead double staining in each group (d).

[0037] Figure 14 Shows the overall anti-tumor efficacy in mice. Among them, a is the schematic diagram of the infrared temperature detection curve; b is the schematic diagram of the tumor size of each group on the 25th day of the mice; c is the curve of the time change of the tumor volume of each group; d is the tumor inhibition of each group; e is the curve of the time change of the weight of each group of mice.

[0038] Figure 15 Shows the WB detection of tumor tissue proteins.

[0039] Figure 16 Is the quantitative analysis of LC3II / GAPDH for each group (a); the quantitative analysis of P62 / GAPDH for each group (b); the quantitative analysis of LC3-II / P62 for each group (c).

[0040] Figure 17 Are the immunofluorescence images of GPX4 and SLC7A11 in tumor tissues of each group.

[0041] Figure 18 Shows the immune activation and tumor suppression and metastasis of each group. Among them, a shows the concentration of each group detected by Elisa of IL-6, IL-12, TNF-α, IFN-γ; b is the immunohistochemical image of CD8 in tumor tissues of each group; c is the solid image of lung tissues of each group.

[0042] Figure 19 Is the schematic diagram of the release curve of iron ions at different pH values without the action of magnetic field.

[0043] Figure 20 Is the schematic diagram of the release curve of sorafenib at different pH values without the action of magnetic field. Specific implementation mode

[0044] The following is a further detailed description through specific implementation modes: Example 1 Because the IMSFs hydrogel, which acts as the first independent part to induce ferroptosis, is also an invention of the inventor, this example specifically describes the IMSFs hydrogel, mainly describing the structure and preparation method of the IMSFs hydrogel and proving its effects through experiments.

[0045] An IMSFs hydrogel, including a hydrogel body, the hydrogel body is a silk fibroin-hyaluronic acid hydrogel body (SF-HA hydrogel body), and the inside of the hydrogel body is loaded with iron oxide nanoparticles and sorafenib. The weight ratio of sorafenib to iron oxide nanoparticles is 1:5-7, preferably 1:6.

[0046] This example discloses a specific preparation method of an IMSFs hydrogel: S1. Take 30 g of silk fibroin and boil it in 6 L of an aqueous Na2CO3 solution (0.5%, w / v) for 30 minutes. Then thoroughly rinse it three times with deionized (DI) water and dry it overnight in a fume hood to obtain degummed silk fibers. Then add the degummed fibers (2 g) to 10 mL of a 9.3 M lithium bromide solution and dissolve them in a water bath at 60 °C for 2 hours to obtain a 20% silk solution. Finally, add 400 μL 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. Then dialyze the silk fibroin hydrogel in deionized water for 3 days, changing the water 15 times to remove the cross-linking agent.

[0047] S2. Freeze-dry the silk fibroin hydrogel and then grind it into a powder. Dissolve the freeze-dried silk fibroin hydrogel (1 g) and hyaluronic acid (1 g) in 10 mL of a 9.3 M lithium bromide solution at 60 °C for 3 hours. Supplement the dissolved solution with 400 μL of BDDE and incubate it at 60 °C for 3 hours. Then dialyze and wash it with deionized water for 3 days, changing the water 15 times, to obtain a silk fibroin-hyaluronic acid hydrogel bulk (SF-HA).

[0048] S3. Finally, load sorafenib (1 wt%) and iron oxide nanoparticles (6 wt%) into the SF-HA hydrogel bulk and stir well until it becomes a homogeneous brown color, thus forming an IMSFs hydrogel. For every 1 g of the hydrogel bulk in this example, the corresponding addition amount of sorafenib is 0.0107 g, and the corresponding addition amount of iron oxide is 0.0645 g. The density of the hydrogel bulk in this example is 1 mg / mL.

[0049] The IMSFs hydrogel in this example can be used for the application of an injectable cascade ferroptosis anti-cancer drug.

[0050] In this embodiment, the IMSFs hydrogel is used as an injectable cascading ferroptosis anti-cancer drug, which can be used for minimally invasive local injection to achieve in-situ tumor injection without oral administration. Compared with oral drugs, it will not have the problems of insufficient effective concentration caused by first-pass metabolism and rapid clearance, nor will it have the serious side effects associated with systemic drug administration. At the same time, it also has the functions of magnetic heat response and drug controlled release. When treating tumors, only a magnetic field needs to be applied to the tumor site to be treated. Compared with traditional drug release systems, it can accurately act on the tumor site and precisely meet personalized needs. At the same time, it can also generate a magnetic heat effect under an alternating magnetic field, which is less affected by tissue depth compared with other effects (such as photothermal effect), and can precisely control the temperature (42 - 45 °C) at the tumor site for mild hyperthermia, avoiding thermal damage to surrounding healthy tissues. At the same time, the combined action of sorafenib and iron ions expands the ferroptosis effect. Iron ions and sorafenib synergistically achieve cascading ferroptosis by actively increasing oxidative stress and disrupting oxidative defense, resulting in a better ferroptosis effect and a better tumor treatment effect.

[0051] Experiment The following is a series of experiments to prove that the IMSFs hydrogel prepared in this embodiment has corresponding effects and functions, and it can be used as an injectable cascading ferroptosis anti-cancer drug.

[0052] Experiment 1: Electron microscopy experiment After freeze-drying the SF-HA hydrogel body, Fe3O4, sorafenib, and IMSFs hydrogel respectively, the pictures shown below are obtained after scanning electron microscopy detection; as seen from the results, 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 without obvious influence on the structure of the SF-HA hydrogel body. Figure 1 As shown in the figure, 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 without obvious influence on the structure of the SF-HA hydrogel body.

[0053] Experiment 2: Injectability experiment Load the IMSFs hydrogel into a 1ML syringe. As shown in the figure below, inject it into the liquid in the beaker, and it can be seen that it can be smoothly injected into the beaker liquid through an 18G needle, thus confirming that the IMSFs hydrogel has excellent injectability and can be used for minimally invasive treatment of tumors. Figure 2 As shown in the figure, inject it into the liquid in the beaker, and it can be seen that it can be smoothly injected into the beaker liquid through an 18G needle, thus confirming that the IMSFs hydrogel has excellent injectability and can be used for minimally invasive treatment of tumors.

[0054] Experiment 3: Drug encapsulation analysis experiment Perform energy spectrum analysis on the IMSFs hydrogel. The corresponding element quantification results of various substances in the IMSFs hydrogel divided by energy spectrum are as shown in a below, which is basically consistent with the addition amounts of various substances in the IMSFs hydrogel. Combining Figure 3 As shown in a, this is basically consistent with the addition amounts of various substances in the IMSFs hydrogel. Combining Figure 3As shown in Fig. b, the Fourier transform infrared (FTIR) spectrum of the IMSFs hydrogel shows characteristic absorption peaks of Fe3O4 and Sorafenib (SOR), confirming the presence of Fe3O4 and SOR molecules. Combining Figure 3 As shown in Fig. c, the results of X-ray diffraction (XRD) analysis of the IMSFs hydrogel indicate that the chemical structures of Fe3O4 and SOR incorporated in the IMSFs hydrogel are not altered by the preparation process of the IMSF hydrogel. Combining Figure 3 As shown in Fig. d, X-ray photoelectron spectroscopy (XPS) analysis of the IMSFs hydrogel confirmed the presence of elements C, N, O, F, Cl, and Fe through C1s, N1s, O1s, F1s, Cl2p, and Fe2p.

[0055] Experiment 4: Magnetothermal performance experiment The IMSFs hydrogel and iron oxide nanoparticles were measured separately using a LakeShore 7404 instrument in the United States. Combining Figure 4 As shown in the figure, a narrow hysteresis curve of the IMSFs hydrogel was obtained. From the figure, it shows that the IMSFs hydrogel is a soft magnetic ferrite with low coercive force and residual magnetization value (saturation magnetization intensity of 15.74 emu / g), similar to that of pure iron oxide nanoparticles (saturation magnetization intensity of 63.18 emu / g). Therefore, it shows that the IMSFs hydrogel is a soft magnetic material with hysteresis, loss characteristics, and coercive force. Since the coercive force is less than that of the AMF (coil turns: 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.

[0056] Experiment 5: Thermal stability experiment Thermogravimetric analysis of the IMSFs hydrogel and the F@SF-HA (only iron oxide was added to the SF-HA hydrogel matrix) hydrogels was carried out using a TGA thermogravimetric analyzer from 0 to 800 degrees Celsius. The results are as Figure 5 shown. The IMSFs hydrogel exhibits good thermal stability under heating conditions, and within the magnetothermal working temperature range (<50 °C), there is no obvious thermal decomposition of the material.

[0057] Experiment 6: Drug release performance experiment The IMSFs hydrogel was not treated with a magnetic field, and drug release experiments were carried out under the conditions of pH 6 and pH 7.4. At corresponding time points within a predetermined time interval, samples of the release medium were collected for analysis, and an equal volume of fresh medium was supplemented. The concentration of sorafenib released in the supernatant was analyzed using a UV-vis spectrophotometer. In addition, after reducing Fe³⁺ to Fe²⁺ with sodium sulfite, the total iron ion concentration was measured, and the Fe²⁺ concentration was detected using an iron ion detection kit. The final test results are as Figure 19 , Figure 20 shown.

[0058] 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 the same parameters were used for magnetic field treatment in the following text) for 400 s, drug release experiments were carried out under the conditions of pH 6 and pH 7.4. At corresponding time points within a predetermined time interval, samples of the release medium were collected for analysis, and an equal volume of fresh medium was supplemented. The concentration of sorafenib released in the supernatant was analyzed using a UV-vis spectrophotometer. In addition, after reducing Fe³⁺ to Fe²⁺ with sodium sulfite, the total iron ion concentration was measured, and the Fe²⁺ concentration was detected using an iron ion detection kit. The results are as Figure 6 and Figure 7 shown. Compared with Figure 19 and Figure 20 , the IMSFs hydrogel after magnetic field exposure has a good drug sustained-release effect under a pH similar to that of the tumor microenvironment (pH 6). Without the action of a magnetic field, very little or almost no iron ions and sorafenib are released from the IMSFs hydrogel. At the same time, almost no iron ions are released under neutral pH conditions, indicating that its acidic microenvironment responsiveness can avoid the risk of systemic iron overload.

[0059] Experiment 7: Biocompatibility experiment 50 μL of the IMSFs hydrogel was placed in a 24-well plate, and 4T1 cells (5×10 3 per well) were inoculated on the IMSFs hydrogel and co-incubated. The IMSFs hydrogel and 4T1 cells were co-cultured for 1, 2, 3, 5, and 7 days. After live / dead double staining (green represents live cells and red represents dead cells), combined with Figure 8 shown, it can be seen that the IMSFs hydrogel has almost no toxicity to cells, and the IMSFs hydrogel has excellent biocompatibility.

[0060] Experiment 8: Stable and mild magnetic heating ability experiment This experiment was divided into two groups. 1 mL of saline and 100 μL of the IMSFs hydrogel were placed in EP tubes and placed in a coil, and an infrared thermal imager was used to detect the temperature change in real time. As Figure 9Shown as follows: The temperature of the saline group did not change significantly under magnetic field exposure (the magnetic field parameters were the same as before). The temperature of the IMSFs group gradually increased under magnetic field exposure and could be stabilized within the range of 42-45 degrees for a period of time, indicating that the IMSFs hydrogel has excellent controllable magnetic thermal properties and can implement stable mild magnetic thermotherapy.

[0061] Experiment 9: Experiment on the ability to induce ROS accumulation The experiment was divided into 5 groups, namely a: Control group, b: F@SF-HA hydrogel group (prepared by adding only 64.51 mg of Fe3O4 to the SF-HA hydrogel body and 1 mL of the SF-HA hydrogel body), c: IMSFs hydrogel group (prepared by 64.51 mg of Fe3O4, 10.75 mg of Sorafenib and 1 mL of the 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, the cells were washed with PBS. An iron ion detection kit was used to detect the intracellular iron ion content. Combining Figure 10 Shown as follows, 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 other groups, indicating that the intracellular iron ion content increased significantly after magnetic field exposure treatment. Here, it was confirmed that magnetic field exposure was the initiating factor for triggering iron ion release.

[0062] Subsequently, the cells were incubated with DCFH-DA for 30 minutes and stained with Hoechst33342 for 5 minutes. Then, fluorescence microscopy was used to evaluate the intracellular ROS level. The results were as Figure 11 Shown as follows: A significant increase in ROS level was observed only in the magnetic field exposure groups (F@SF-HA hydrogel + AMF treatment group and IMSFs hydrogel + AMF treatment group), and the ROS accumulation was positively correlated with iron ion release.

[0063] Experiment 10: Experiment on the reduction of oxidative defense by the formation of the complex induced by sorafenib release 4T1 cells were cultured and divided into the IMSFs hydrogel + AMF treatment group and the non-treatment group. Cell proteins were extracted for Co-ip detection. The results were as Figure 12 Shown in a, after the cells were treated with the IMSFs hydrogel + AMF, the formation of the Beclin1-SLC7A11 complex was visible. In addition, according to the 5-group grouping method in Experiment 9, after the cells were treated, proteins were extracted for WB detection. The results were as Figure 12 b, Figure 12c shows that after treatment with IMSFs hydrogel + AMF, the protein expression of SLC7A11 and GPX4 decreased significantly. At the same time, as Figure 12 shown in d, the GSH was detected, and the GSH detection results showed that the GSH also decreased after the cells were treated with IMSFs hydrogel + AMF.

[0064] Experiment 11: Experiment on the ability to induce ferroptosis As Figure 13 shown in a, after the 4T1 cells were treated with IMSFs hydrogel + AMF, compared with the normal group under transmission electron microscopy, mitochondrial condensation was visible, presenting the manifestation of ferroptosis. In addition, after the 4T1 cells were treated with the 5 groups in Experiment 9 above respectively, after being stained with BODIY-C11 (green fluorescence: LOP positive, red fluorescence: none, blue fluorescence: nuclear staining), the lipid peroxidation (LOP) was evaluated. As Figure 13 shown in b, it can be seen that the green fluorescence of the IMSFs hydrogel + AMF treatment group (group e) is the strongest, the fluorescence of the F@SF-HA hydrogel + AMF treatment group (group d) is weaker than that of group e, and the fluorescence of other groups is weaker, thus confirming the synergistic effect of iron ions and sorafenib in IMSFs to enhance ferroptosis. As Figure 13 shown in d, the live / dead double staining results of the cells (the red fluorescence of the IMSFs hydrogel + AMF treatment group should be stronger than that of the F@SF-HA hydrogel + AMF treatment group) also confirmed the above results.

[0065] In summary, through the above experiments, after the IMSFs hydrogel in this example is used, it not only has the effect of initiating ferroptosis, but also the combination of iron ions and sorafenib synergistically enhances the effect of ferroptosis.

[0066] Example 2 This example combines the IMSFs hydrogel in Example 1 and the corresponding experiments to specifically illustrate another invention creation, that is, to illustrate the new use of TAT-beclin1 in enhancing ferroptosis.

[0067] This example discloses a ferroptosis induction system, which includes a first separately used part used first and a second separately used part used later. The first separately used part includes an IMSFs hydrogel in Example 1, and the second separately used part includes TAT-Beclin1.

[0068] The ferroptosis induction system in this example can be used for the application of an injectable cascading ferroptosis anti-cancer drug.

[0069] When this ferroptosis induction system is used as an injectable cascading ferroptosis anti-cancer drug, the first part for single use is injected into the tumor site. At this time, the IMSFs hydrogel initiates ferroptosis. Then, the second part for single use is injected. After the second part for single use is injected, the following effects are obtained: 1. As a small molecule activating peptide, TAT-beclin1 has good transmembrane 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 synergistically with autophagy, the ferroptosis effect is expanded. On the basis of simply using the IMSFs hydrogel in Example 1, 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 the maturation of immune cells and releasing INF-r, thereby inhibiting XC again - transporter again, and the ferroptosis effect is further enhanced, forming a cyclic positive feedback loop, paralyzing the oxidative defense system, and exerting excellent anti-cancer efficacy.

[0070] Experiment Next, through experiments, it is proved that the ferroptosis induction system has corresponding effects and functions, that TAT-beclin1 has the effect of enhancing ferroptosis after injection, and at the same time, it can also continue to prove the effects and functions of the IMSFs hydrogel.

[0071] Experiment 12: In vivo experiment on mice The tumor-bearing mice were randomly divided into 5 groups, namely (1) control group (injected with normal saline, injection volume 100 uL), (2) IMSFs hydrogel group (injection volume 100 uL), (3) F@SH-HA hydrogel (injection volume 100 uL) + AMF treatment group, (4) IMSFs hydrogel (injection volume 100 uL) + AMF treatment group, (5) IMSFs hydrogel (injection volume 100 uL) + AMF treatment + TAT-Beclin1 treatment group. In groups 3-5, the F@SH-HA hydrogel and the IMSFs hydrogel were injected into the tumors of the mice, and the mice were exposed to AMF the next day. The magnetic field parameters were the same as before. Under the infrared monitoring of each magnetic field exposure, the power was adjusted to maintain the magnetic measurement group at 42-45 °C for 6 minutes. Subsequently, TAT-Beclin1 was injected intratumorally for 2 consecutive days (injection volume 1.5 mg / Kg / d), rested for 3 days, and one cycle was 5 days, for a total of 5 cycles. During the entire treatment cycle, the tumor volume and body weight of the tumor-bearing mice were monitored every 3 days. The mice were subjected to survival analysis and observed until day 40. When the tumor volume reached 1500 mm 3When the time was up, the mice were euthanized. After autopsy, lung tissues were collected, fixed and stained with Bouin's solution to evaluate lung metastasis in each group.

[0072] The results were as Figure 14 shown in a. By comparing Group 1 and Group 4, after the injection of IMSFs hydrogel and under the exposure of magnetic field, the results of the infrared temperature detector showed that the IMSFs hydrogel exhibited good temperature control performance, and the tumor temperature was controlled between 42 - 45 degrees. As Figure 14 shown in b and 14c, Figure 14 in b, the tumor volume of Group 5 was the smallest, and that of Group 4 was the second. It can be seen that the tumor growth in Groups 4 and 5 was significantly inhibited, and the inhibitory effect of Group 5 was the best. At the same time, the tumor inhibition rate (TGItv=(1 - (tumor volume on the last day of the experimental group - tumor volume at the start of treatment) / (tumor volume on the last day of the control group - tumor volume at the start of treatment of the control group))x100%) was calculated. As Figure 14 shown in d, the tumor inhibition rate of Group 5 was the largest, and that of Group 4 was the second. All these indicated that TAT-Beclin1 had the effect of enhancing ferroptosis. Combining Figure 14 with what was shown in e, by monitoring the body weight, it was found that there was no significant difference in body weight among the groups, indicating that the ferroptosis induction system and the IMSFs hydrogel in this example had relatively high safety.

[0073] After the tumor tissues of the above-treated mice were isolated, tissue proteins were extracted for WB detection. The results were as Figure 15 and Figure 16 shown. It can be seen that the LC3II protein in Group 5 increased significantly, P62 decreased significantly, and the ratio of LC3II / P62 increased significantly, indicating that the injection of TAT-Beclin1 promoted autophagic flux mainly by enhancing the degradation rather than the formation of autophagosomes. At the same time, the ratio of LC3-II / P62 in Group 5 was significantly higher than that in other groups, and the increase in this ratio further illustrated that the injection of TAT-Beclin1 significantly enhanced the autophagic flux in tumor tissues.

[0074] In addition, after extracting the tumor tissues of each group for corresponding immunofluorescence detection, the results were as Figure 17 shown, suggesting that the expressions of GPX4 and SLC7A11 in Group 5 decreased significantly, and also decreased significantly compared with the IMSFs hydrogel + AMF treatment group, indicating that the addition of TAT-Beclin1 further enhanced the inhibition of tumor oxidative defense and expanded the ferroptosis effect.

[0075] In addition, the sera of the mice in each group were extracted for Elisa detection. As Figure 18As shown in a, the results showed that the immune factors IL-6, IL-12, TNF-α, and IFN-γ in Group 5 were significantly increased compared with those in Group 4. However, Group 5 had the highest level, showing a significant difference compared with Group 4, indicating that the injection of TAT-Beclin1 had a significant effect on activating the immune system. Immunohistochemistry of CD8 in tumor tissues, as Figure 18 shown in b, also confirmed the above conclusion. As Figure 18 shown in c, the solid image of lung tissue showed that lung metastasis in Group 5 was significantly inhibited, indicating that the treatment effect of Group 5 was the best, suggesting that TAT-Beclin1 had the effect of enhancing ferroptosis.

[0076] In addition, blood was taken from the mice in Group 4 and Group 5 every 7 days for blood biochemical and blood routine tests. It was found that there were no significant differences in the values, and the HE pathological results of organs such as the heart, liver, spleen, lungs, and kidneys were not significantly abnormal, suggesting that both the IMSFs hydrogel and the ferroptosis induction system had high safety and could be used for biological injection.

[0077] In the above embodiment, the IMSFs hydrogel was used as the first part used alone to initiate ferroptosis. Of course, in other embodiments, the IMSFs hydrogel can also be replaced with other conventional drugs that have the effect of initiating ferroptosis, such as drugs that inhibit the XC system like Erastin and SAS (sulfasalazine). Or, replace the sorafenib encapsulated inside the IMSFs hydrogel with these drugs.

[0078] The above are only embodiments of the present invention. Specific technical solutions and / or common knowledge such as characteristics known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. Application of TAT-beclin1 in enhancing ferroptosis drugs.

2. Application of TAT-beclin1 in anti-cancer drugs, characterized in that: TAT-beclin1 has the effect of enhancing ferroptosis.

3. Ferroptosis induction system, characterized in that: It includes a first separately used part used first and a second separately used part used later. The first separately used part has the effect of inducing ferroptosis, and the second separately used part includes TAT-Beclin1 with the effect of enhancing ferroptosis.

4. The ferroptosis induction system according to claim 3, characterized in that: The first separately used part includes a carrier, and the carrier internally encapsulates magnetite nanoparticles.

5. The ferroptosis induction system according to claim 4, characterized in that: The carrier also internally encapsulates sorafenib.

6. The ferroptosis induction system according to claim 4, wherein: The carrier is a hydrogel.

7. The ferroptosis induction system according to claim 3, wherein: The first separately used part is a drug that inhibits the XC system.

8. The ferroptosis induction system according to claim 4, wherein: The carrier also internally encapsulates a drug that inhibits the XC system.

9. Application of the ferroptosis induction system according to any one of claims 3-8 in ferroptosis anti-cancer drugs.

10. The application according to claim 2 or 9, characterized in that: The cancer is triple-negative breast cancer.

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