A tumor microenvironment-responsive iron overload hydrogel as well as a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a tumor microenvironment responsive iron overload hydrogel, a preparation method and application thereof. The hydrogel anchors deferoxamine conjugate in the hydrogel network through thiol-maleimide click chemistry, does not affect intracellular Fe 2+ The Fenton reaction process occurs, and the excreted Fe 3+ is specifically captured and locked, iron ion loss and systemic diffusion are effectively prevented, and a long-acting iron overload microenvironment is constructed locally in the tumor; iron oxide nanoparticles release Fe 2+ in response to a weak acid microenvironment, hydroxyl radicals are catalytically generated, and the effect of the iron death inducer on the antioxidant pathway is synergistic, effectively inducing tumor cell ferroptosis and overcoming treatment resistance; the hydrogel has excellent biocompatibility, blood compatibility and targeted tumor inhibition activity, inhibits the growth of solid tumors while reducing systemic toxic side effects, and thus realizes efficient and safe local treatment of tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a tumor microenvironment-responsive iron-overloaded hydrogel, its preparation method, and its application. Background Technology

[0002] Surgery, chemotherapy, and radiotherapy are the main methods of clinical cancer treatment, but each has certain limitations. Surgery is difficult to remove microinvasive lesions and metastases, which are important causes of postoperative recurrence; radiotherapy and chemotherapy lack tumor targeting, and can cause systemic toxic side effects while damaging normal tissues. Moreover, long-term treatment can easily lead to drug resistance in tumor cells, resulting in a significant decline in clinical treatment efficacy.

[0003] The tumor microenvironment exhibits characteristics such as weak acidity and enrichment in hydrogen peroxide (H2O2), significantly differing from normal tissues. This provides a biological basis for constructing microenvironment-responsive catalytic therapy systems. Iron-based nanomaterial-mediated Fenton-like catalytic therapy (CDT) can be specifically activated at tumor sites and is less prone to drug resistance, making it a hot topic in anti-tumor research in recent years. Among these, iron(III) oxide (Fe3O4) nanoparticles demonstrate good biocompatibility, enable magnetic imaging, and have low preparation costs; their lattice contains Fe... 2+ / Fe 3+ Fe3O4 exhibits mixed valence states and good Fenton-like catalytic activity. In the acidic tumor microenvironment, Fe3O4 can dissociate and release active iron ions, catalyzing the generation of a large number of reactive oxygen species (ROS) such as hydroxyl radicals (·OH) from locally high concentrations of H2O2. This damages the lipid, protein, and DNA structures of tumor cells, disrupts the redox balance, and induces apoptosis and ferroptosis. Simultaneously, tumor cells generally exhibit iron-dependent metabolism and are more sensitive to iron-mediated oxidative damage. Fe3O4 can further enhance its anti-tumor effect by increasing the unstable iron pool in cells.

[0004] However, the single Fe3O4 Fenton therapy system still has obvious technical shortcomings: on the one hand, the free iron generated by dissociation is easily lost with the tissue fluid and it is difficult to maintain a continuous iron overload state in the tumor, resulting in a rapid decay of catalytic efficacy over time; on the other hand, the free iron that diffuses into normal tissues may induce oxidative stress damage and has potential biotoxicity.

[0005] Erastin can inhibit System Xc on the surface of tumor cells. - It acts as a transporter protein, blocking cysteine ​​uptake, reducing intracellular glutathione (GSH) levels, inactivating the key lipid peroxidation scavenger GPX4, weakening the antioxidant capacity of tumor cells, and forming a synergistic effect with Fe3O4-mediated ROS generation, effectively promoting tumor ferroptosis. However, Erastin's poor water solubility, short in vivo half-life, limited systemic targeting, and easy metabolism limit its further clinical application.

[0006] Existing research on ferroptosis generally suffers from inherent limitations: deferoxamine (DFO), as a classic iron chelating agent, can chelate free iron and reduce the body's iron levels, thereby inhibiting the Fenton reaction and ferroptosis process. Currently, most published studies use free DFO, which can freely penetrate tissues and cells, producing systemic iron-lowering and antioxidant effects.

[0007] Therefore, how to provide a hydrogel system that can maintain local iron overload in tumors, enhance the therapeutic effect of ferroptosis, and reduce systemic toxicity is a technical problem that urgently needs to be solved. Summary of the Invention

[0008] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide a method for preparing a tumor microenvironment-responsive iron overload hydrogel; the second objective is to provide a tumor microenvironment-responsive iron overload hydrogel; and the third objective is to provide an application of the tumor microenvironment-responsive iron overload hydrogel.

[0009] To achieve the first objective, the technical solution adopted by this invention is as follows: A method for preparing a tumor microenvironment-responsive iron-overloaded hydrogel includes: DFO-PEG-SH was prepared by chemical reaction using DFO and SH-PEG-COOH as raw materials. DFO stands for deferoxamine, and its structural formula is shown below: ; SH-PEG-COOH is thiolized polyethylene glycol carboxylic acid, and DFO-PEG-SH is deferoxamine-polyethylene glycol-thiol coupling compound; Using DFO-PEG-SH as the functional unit, and thiol-functionalized polyethylene glycol and maleimide-functionalized polyethylene glycol as the crosslinking backbone unit, the functional unit and the crosslinking backbone unit are crosslinked to form DMS hydrogel by utilizing the click chemical reaction between the thiol group in DFO-PEG-SH and the maleimide in the crosslinking backbone unit. Iron oxide nanoparticles and ferroptosis inducers were loaded into the DMS hydrogel network to obtain the tumor microenvironment-responsive iron overload hydrogel.

[0010] Polyethylene glycol (PEG)-based hydrogels possess excellent biocompatibility, low immunogenicity, in-situ molding capability, and controllable structure, making them ideal local drug co-delivery carriers. Three-dimensional cross-linked PEG hydrogels constructed based on thiol-maleimide click chemistry exhibit mild reaction conditions and stable cross-linked structures, enabling efficient loading of nanoparticles and small molecule drugs. This achieves in-situ retention and slow drug release at the tumor site, effectively addressing the issues of rapid diffusion and poor targeting of small molecule drugs.

[0011] DFO was covalently grafted into a PEG hydrogel three-dimensional network, anchoring it within the tumor extracellular gel region. This anchored DFO cannot penetrate the cell membrane into tumor cells, nor does it diffuse into normal tissue. Therefore, it does not interfere with the Fe²⁺ within tumor cells. 2+ Instead of undergoing the Fenton reaction process, it specifically captures Fe leaked from the dissociation of iron oxides or the rupture of tumor cells. 3+ Iron ions. This mechanism effectively prevents iron ions from being lost through body fluids or diffusing throughout the body, maintaining a long-term iron overload microenvironment by anchoring iron ions to the tumor site. This invention successfully achieves enhanced iron accumulation at the tumor site through covalent anchoring and remodeling of DFO function.

[0012] Preferably, the thiolized polyethylene glycol is a multi-arm thiolized polyethylene glycol, and the maleimide-functionalized polyethylene glycol is a multi-arm maleimide-functionalized polyethylene glycol.

[0013] Preferably, the multi-arm thiolized polyethylene glycol is a four-arm PEG-thiol, and the multi-arm maleimide-functionalized polyethylene glycol is a four-arm PEG-maleimide.

[0014] Preferably, the molecular weight of the four-armed polyethylene glycol-thiol group is 2000-10000 Da, and the molecular weight of the four-armed polyethylene glycol-maleimide is 2000-10000 Da.

[0015] Preferably, the molar ratio of DFO to SH-PEG-COOH is 1:1 to 1:1.5.

[0016] Preferably, the iron oxide nanoparticles are iron(III) oxide (Fe3O4) nanoparticles, and the ferroptosis inducer is Erastin.

[0017] Fe3O4 induces the Fenton reaction and generates reactive oxygen species (ROS) in response to the tumor microenvironment. Fe3O4 nanoparticles loaded onto a gel network dissociate in the weakly acidic tumor microenvironment, releasing a large amount of reactive Fe. 2+ These Fe 2+It catalyzes the Fenton reaction of H2O2 highly expressed in tumors, generating a large number of hydroxyl radicals (·OH), which attack the lipids, proteins and DNA of tumor cells, induce oxidative stress damage, and thus initiate the ferroptosis program of tumor cells.

[0018] Erastin blocks the tumor's antioxidant defense system, sensitizing ferroptosis. Gel-released Erastin specifically inhibits System Xc on the surface of tumor cells. - Transport proteins block cysteine ​​uptake, leading to a significant depletion of intracellular glutathione (GSH). GSH deficiency inactivates the lipid peroxidation scavenger GPX4, causing tumor cells to lose their ROS scavenging ability and resulting in a large accumulation of lipid peroxides. This process synergistically interacts with Fe3O4-mediated Fenton oxidative damage, effectively overcoming tumor resistance to antioxidant therapy.

[0019] Preferably, the loading of the iron oxide nanoparticles is 0.1–1 mg / mL, and the loading of the ferroptosis inducer is 10–50 μmol / L.

[0020] To achieve the second objective, the technical solution adopted by this invention is as follows: A tumor microenvironment-responsive iron overload hydrogel is prepared using any of the above-described methods for preparing tumor microenvironment-responsive iron overload hydrogels. The matrix of the tumor microenvironment-responsive iron overload hydrogel is formed by crosslinking thiol-modified polyethylene glycol with maleimide-functionalized polyethylene glycol, and the network is covalently grafted with DFO-PEG-SH functional conjugates. The tumor microenvironment-responsive iron overload hydrogel is loaded with iron oxide nanoparticles and ferroptosis inducers.

[0021] To achieve the third objective, the technical solution adopted by this invention is as follows: An application of a tumor microenvironment-responsive iron overload hydrogel, wherein the application is to use the tumor microenvironment-responsive iron overload hydrogel to prepare a drug or carrier, wherein the drug is a drug for treating tumors.

[0022] Furthermore, the tumor-treating drug is used in any one or more of the following: Maintaining an iron-overloaded microenvironment in the tumor region; Induces oxidative damage in tumor cells; Induces ferroptosis in tumor cells; It depletes GSH in tumor cells and inactivates GPX4; GSH is reduced glutathione, and GPX4 is glutathione peroxidase 4.

[0023] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides a tumor microenvironment-responsive iron overload hydrogel and its preparation method. Through molecular design and material construction, it achieves long-term accumulation of iron ions in the tumor local area, effective blocking of antioxidant defense, and efficient synergistic drug delivery, thereby significantly improving the efficacy and safety of ferroptosis therapy. Specifically, this is reflected in the following aspects: First, this invention overcomes the traditional misconception that deferoxamine (DFO), as an iron chelating agent, scavenges iron ions and thus inhibits ferroptosis. By covalently grafting DFO into a three-dimensional network of polyethylene glycol (PEG) hydrogel, it is anchored in the tumor extracellular matrix region. This spatially confined DFO cannot penetrate the cell membrane to enter the tumor cell interior, thus not interfering with intracellular iron production. 2+ The Fenton reaction process occurs; simultaneously, it does not diffuse to normal tissues to cause systemic iron deprivation. Instead, anchored DFO specifically captures Fe leaked from the dissociation of iron oxide nanoparticles or the rupture of tumor cells. 3+ The ions effectively prevent iron ions from being lost with body fluids or spreading throughout the body, locking iron ions firmly in the tumor site and successfully constructing a long-lasting iron overload microenvironment, providing a material basis for the continuous induction of ferroptosis.

[0024] Secondly, this hydrogel achieves synergistic therapy between iron oxides and ferroptosis inducers. Iron oxide nanoparticles loaded in the gel network can dissociate in response to the weakly acidic tumor microenvironment, releasing active Fe. 2+ The catalytic Fenton reaction of endogenous H2O2 generates a large number of hydroxyl radicals (·OH), which directly attack the lipids, proteins, and DNA of tumor cells, inducing oxidative stress damage. Simultaneously, the gel-released ferroptosis inducer specifically inhibits System Xc on the surface of tumor cells. - Transport proteins block cysteine ​​uptake, leading to depletion of intracellular glutathione (GSH) and inactivation of GPX4 enzyme, causing tumor cells to lose their ability to scavenge ROS and accumulate lipid peroxides. These two mechanisms promote each other, forming a dual effect of oxidative damage generation and antioxidant defense collapse, thereby effectively preventing tumor resistance to single therapy and efficiently initiating ferroptosis.

[0025] Furthermore, the PEG hydrogel constructed based on thiol-maleimide click chemistry possesses a stable CS covalent cross-linked structure that remains intact in normal physiological environments, ensuring local retention of the drug and iron ions, and slow degradation in the tumor microenvironment for controlled sustained release. This design reduces systemic toxicity, thereby effectively improving treatment safety. Experiments have demonstrated that this hydrogel has no significant toxicity to normal cells, while exhibiting highly efficient targeted killing activity against 4T1 tumor cells. In vivo experiments show that it significantly inhibits the growth of solid tumors, induces large-area tumor cell apoptosis and necrosis, and demonstrates excellent organ safety and blood compatibility. In summary, this invention provides a highly efficient and safe new strategy for precision tumor treatment by reshaping DFO function, optimizing iron metabolism regulation, and achieving multi-mechanism synergy.

[0026] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0027] Figure 1 The 1H NMR spectra of DFO and DFO-PEG-SH provided in Example 1 of this invention are as follows ( 1 H NMR).

[0028] Figure 2 This is the Fourier transform infrared spectrum of the MS and DMS hydrogels provided in Embodiment 2 of the present invention.

[0029] Figure 3 This is a frequency scan rheological diagram of hydrogels with different components provided in Embodiment 2 of the present invention.

[0030] Figure 4 This is a time-scan rheological diagram of different component hydrogels provided in Embodiment 2 of the present invention.

[0031] Figure 5 These are scanning electron microscope images (scale bar: 50 μm) of different component hydrogels provided in Example 2 of this invention.

[0032] Figure 6 This is the in vitro static chelation of iron ions provided in Example 1 of the present invention.

[0033] Figure 7 This is the in vitro iron ion slow-release situation provided in Example 1 of the present invention.

[0034] Figure 8 This is the cytotoxicity result of the DMSE@Fe3O4 hydrogel provided in Example 2 of this invention.

[0035] Figure 9 This is a graph showing the apoptosis-killing effect of different hydrogels on 4T1 tumor cells provided in Example 2 of this invention.

[0036] Figure 10 The images shown are fluorescent staining images of iron accumulation in 4T1 cells from different groups, provided in Example 2 of this invention.

[0037] Figure 11 This is a bar chart showing the GPX4 activity in different groups of tumor cells provided in Example 2 of this invention.

[0038] Figure 12 This is a fluorescence image of ROS generation in different groups of tumor cells provided in Example 2 of the present invention.

[0039] Figure 13 This is the accumulation of lipid peroxides in different groups of tumor cells provided in Example 2 of this invention.

[0040] Figure 14 This is a diagram showing the solid tumors in each group of mice after 21 days of treatment, provided in Example 3 of this invention.

[0041] Figure 15 This is a graph showing the changes in tumor growth in mice in each group after 21 days of treatment, as provided in Example 3 of this invention.

[0042] Figure 16 This is a graph showing the change in body weight of mice in each group after 21 days of treatment, as provided in Example 3 of this invention.

[0043] Figure 17 These are images of Prussian blue iron ion stained sections of tumor tissue from each group after 21 days of treatment, provided in Example 3 of this invention.

[0044] Figure 18 These are HE pathological staining slides of tumor tissues from each group 21 days after treatment, provided in Example 3 of this invention.

[0045] Figure 19 This is a TUNEL in situ apoptosis fluorescence staining image of tumor tissues in each group after 21 days of treatment, provided in Example 3 of this invention.

[0046] Figure 20 These are HE-stained sections of the heart, liver, spleen, lungs, and kidneys of mice in each group after 21 days of treatment, as provided in Example 3 of this invention.

[0047] Figure 21 The results of the in vitro hemolysis experiment of DMSE@Fe3O4 hydrogel provided in Example 3 of this invention are shown in Figure A, which is a quantitative bar chart of hemolysis rate, and Figure B is a physical picture of the hemolysis situation. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0049] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0050] The raw materials used in the following examples mainly include the following products: Four-arm-polyethylene glycol-thiol (4arm-PEG-SH, molecular weight 5000Da): purchased from Bid Pharmaceutical, specification 1g / bottle; Four-arm polyethylene glycol-maleimide (4arm-PEG-MAL, molecular weight 5000Da): purchased from Bid Pharmaceutical, specification 1g / bottle; Thiol-modified polyethylene glycol carboxylic acid (SH-PEG-COOH, molecular weight 5000 Da): purchased from Yusi Pharmaceutical, specification 1g / bottle; Deferoxamine (DFO): Purchased from Macklin, 100mg / vial, structural formula shown below: ; Erastin: Purchased from Beyotime Biotechnology, 5mg / vial; Doxorubicin (DOX): Purchased from Macklin, 100mg / bottle; Iron oxide nanoparticles (Fe3O4NPs): purchased from XFNANO, 5mL / bottle (aqueous dispersion with a concentration of 10mg / mL and an average particle size of approximately 10nm).

[0051] Example 1 The preparation process of the DFO-PEG-SH functional conjugate is as follows: 100 mg of SH-PEG-COOH powder was added to 100 mL of 2-morpholinoethanesulfonic acid (MES) buffer at pH 6.0 and magnetically stirred at room temperature until the powder was completely dissolved. Then, 4.6 mg of N-hydroxysuccinimide (NHS) and 7.7 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) were added sequentially and stirred at room temperature for 15 min to activate the PEG carboxyl group. Then, 7.2 mg of deferoxamine (DFO) was added to the activated PEG reaction solution and stirred continuously at room temperature in the dark for 4 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 72 h. The dialysate was changed every 8 h to remove unreacted small molecule impurities and residual reagents. After dialysis, the dialysate was freeze-dried to obtain a white powdery DFO-PEG-SH covalent conjugate.

[0052] To verify the successful preparation of the conjugate, pure DFO raw material and DFO-PEG-SH product were dissolved in a deuterated solvent and then subjected to 1H NMR spectroscopy. 1 H NMR (H NMR) tests were performed, and the covalent coupling effect was determined by the characteristic peak shift and integration ratio. The spectrum is shown below. Figure 1 As shown in the figure, this confirms that the DFO-PEG-SH conjugate was successfully synthesized.

[0053] Example 2 A series of gradient hydrogels were prepared under mild physiological conditions based on the thiol-maleimide click chemistry reaction, as follows: I. Preparation of blank maleimide-thiol (MS) hydrogel.

[0054] 50 mg each of 4arm-PEG-SH and 4arm-PEG-MAL with a molecular weight of 5000 Da were dissolved in 1 mL of PBS buffer with pH 7.4. After complete dissolution, they were mixed in a 1:1 volume ratio. After cross-linking and formation, a blank hydrogel was obtained, denoted as MS.

[0055] 0.4 mg of Fe3O4 nanoparticles were added to 1 mL of MS hydrogel, and the mixture was ultrasonically dispersed in an ice-water bath. After in-situ crosslinking at room temperature, the resulting hydrogel was denoted as MS@Fe3O4.

[0056] II. Preparation of DMS hydrogel.

[0057] 15 mg of DFO-PEG-SH was dissolved in 1 mL of PBS buffer, and then mixed with blank hydrogel MS at a 1:1 volume ratio. After cross-linking and molding, a hydrogel grafted with DFO functional groups was prepared, denoted as DMS.

[0058] Fourier transform infrared (FTIR) spectroscopy was performed on the MS blank hydrogel and the DMS hydrogel, and the results are as follows: Figure 2 As shown in the figure, the DFO grafting and hydrogel cross-linking structure were successfully formed, confirming the successful preparation of the DMS hydrogel.

[0059] III. Preparation of tumor microenvironment-responsive iron overload hydrogel (DMSE@Fe3O4).

[0060] 0.4 mg of Fe3O4 nanoparticles (i.e., the loading of Fe3O4 nanoparticles is 0.4 mg / mL) and 20 nmol of Erastin (i.e., the loading concentration of Erastin is 20 μmol / L) were added to 1 mL of DMS hydrogel, and the mixture was uniformly dispersed by ultrasonication in an ice-water bath. After in-situ crosslinking at room temperature, a tumor microenvironment-responsive iron-overloaded hydrogel was obtained, denoted as DMSE@Fe3O4.

[0061] Add 0.4 mg of Fe3O4 nanoparticles to 1 mL of DMS hydrogel, disperse evenly by ultrasonication in an ice-water bath, and crosslink in situ at room temperature. The resulting hydrogel is denoted as DMS@Fe3O4.

[0062] Add 20 nmol Erastin to 1 mL of DMS hydrogel, disperse evenly by ultrasonication in an ice-water bath, and crosslink in situ at room temperature. The resulting hydrogel is denoted as DMSE.

[0063] IV. Testing of the rheological properties of hydrogels.

[0064] The rheological properties of the hydrogel were tested using a rheometer to verify its molding performance and structural stability. The test temperature was 25℃, and the shear strain was fixed at 1%.

[0065] Frequency scanning test: The frequency scanning range is 1-10Hz. The changes in the energy storage modulus G' and loss modulus G'' at different frequencies are tested and recorded. The test results are as follows: Figure 3 As shown in the figure, within the scanning frequency range, the storage modulus (G') of the DMSE@Fe3O4 hydrogel is greater than the loss modulus (G''), indicating that the hydrogel structure has excellent stability.

[0066] Time-scan test: With fixed test parameters, a continuous 300-second scan was performed to monitor the hydrogel modulus stability over a short period. The test results are as follows: Figure 4 As shown in the figure, it can be seen that the various hydrogels remain stable within 5 minutes, and the storage modulus (G') is greater than the loss modulus (G''). This figure confirms that the DMSE@Fe3O4 hydrogel is stable in short-term molding and does not collapse.

[0067] Scanning electron microscopy (SEM) images of hydrogels with different components, such as Figure 5As shown in the figure, it can be seen that the MS hydrogel, DMS hydrogel and DMSE@Fe3O4 hydrogel all exhibit a three-dimensional porous network structure.

[0068] Test Example 1 The iron ion trapping and iron ion releasing capabilities of the hydrogel were evaluated through in vitro static iron chelation experiments and in vitro sustained-release iron ion experiments, as follows: I. Investigate the static chelation of iron ions in vitro.

[0069] Prepare a 0.1 mM FeCl3 standard aqueous solution as an iron ion supply solution for later use.

[0070] 1 g each of MS hydrogel and DMS hydrogel were rinsed three times with deionized water to remove free water from the surface and avoid interference from residual impurities. Both hydrogels were then immersed in 5 mL of FeCl3 standard aqueous solution and incubated at 37°C with continuous shaking for 24 h until both hydrogels reached saturation for iron ion adsorption. 100 μl of the supernatant from each group was taken, and the residual iron ion concentration was determined by UV spectrophotometry. Combined with the initial iron ion concentration of the FeCl3 stock solution, the saturated iron ion chelation capacity per unit mass of MS hydrogel and DMS hydrogel was calculated. The results are as follows: Figure 6 As shown in the figure, the DMS hydrogel modified with DFO has a stronger iron chelating ability than the unmodified MS hydrogel.

[0071] II. Investigate the sustained release of iron ions in vitro.

[0072] The MS and DMS iron-loaded hydrogels that had undergone iron saturation chelation were removed, and the free FeCl3 solution adhering to the gel surface was gently wiped away. Then, both groups of gels were immersed in 5 mL of deionized water release medium, sealed, and allowed to stand for iron ion release experiments. At ten time points (1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, and 24 h), 100 μL of supernatant was quantitatively sampled. Immediately after each sampling, 100 μL of fresh deionized water was added to maintain a constant total volume of the release system, avoiding detection errors caused by changes in solution volume. The iron ion concentration in the supernatant at each time point was detected by ultraviolet spectrophotometry. Using the iron ion standard curve, the cumulative iron ion release rate of the two groups of hydrogels at different time points was calculated, and a cumulative iron ion release kinetic curve over 24 h was plotted. The results are shown below. Figure 7 The figure confirms that the DFO-modified DMS hydrogel has enhanced iron anchoring ability compared to the unmodified MS hydrogel. The DMS hydrogel can retain dissolved iron in the gel network and reduce the diffusion of iron to the external medium.

[0073] Test Example 2 To verify the biocompatibility, tumor-targeting killing ability, and ferroptosis activation of the hydrogel provided by this invention, the following experiments were conducted.

[0074] I. CCK-8 assay for the determination of cell compatibility and cytotoxicity of hydrogels.

[0075] Normal mouse fibroblasts (3T3) and mouse breast cancer tumor cells (4T1) were selected as experimental cells, with 5 × 10⁶ cells per well. 3 100 μL of each culture solution was seeded into 96-well plates and cultured for 24 h. The original culture medium was then discarded, and 100 μL of hydrogel extract from each group was added to each well. Incubation continued for another 24 h. After the initial culture, the mixed culture medium (containing 10 μL of CCK-8 reagent and 90 μL of 1640 basal medium per well) was replaced, and the plates were incubated in the dark for 2 h. The absorbance of each well was measured at 450 nm using a full-wavelength microplate reader, and cell viability was calculated using the following formula: ; Cell survival rate (%): Reflects the proportion of cells that survive. The higher the value, the better the cell survival status. If the survival rate of tumor cells is significantly reduced, it indicates that the hydrogel has an anti-tumor effect. If the survival rate of normal cells is close to 100%, it means that the hydrogel has good biocompatibility and no obvious cytotoxicity.

[0076] OD sampl The absorbance value of the sample group is the absorbance value measured at 450nm in the cell pores after treatment with hydrogel leachate. The more live cells there are, the higher the OD value. OD blank : Absorbance values ​​of the blank group, including 1640 culture medium and CCK-8 reagent, and absorbance of cell-free blank wells, used to subtract background absorbance interference from the culture medium and reagents themselves; OD PBS The absorbance value of the PBS control group is a normal cell well with only PBS buffer added and no hydrogel leachate treatment, serving as a baseline control for 100% cell viability.

[0077] The biosafety and antitumor activity of the hydrogel were examined by analyzing the difference in survival rates between normal cells and tumor cells. The results are as follows: Figure 8 As shown, the results confirm that DMSE@Fe3O4 hydrogel has good safety and anti-tumor effects.

[0078] 2. Flow cytometry was used to examine cell apoptosis.

[0079] Group setup: blank control PBS group, DMS group, DMS@Fe3O4 group, DMSE group, DMSE@Fe3O4 experimental group.

[0080] The apoptosis status of cells was investigated using a flow cytometry assay with Annexin V-FITC / PI double staining. The procedure is as follows: 4T1 tumor cells were used at a rate of 2×10 5 Cells were seeded at a density of [number] cells / well in six-well plates and cultured for 24 hours until complete cell adhesion. The original culture medium was discarded, and 2 mL of different hydrogel extraction solutions were added to each well. The cells were then cultured at 37°C for another 24 hours. After culture, the cell supernatant and adherent cells were collected from each well. Cells were gently digested with trypsin, centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended twice with PBS buffer to remove residual culture medium and impurities. Following the Annexin V-FITC / PI apoptosis detection kit instructions, 0.5 mL of PBS buffer was added to each cell pellet for resuspending, followed by Annexin V-FITC and PI staining solutions. The cells were incubated at room temperature in the dark for 15 minutes. Flow cytometry was used to detect the apoptosis rate in each group. The ability of different experimental groups to induce tumor cell apoptosis was quantitatively compared by measuring the proportions of early apoptosis, late apoptosis, and total apoptosis. The results are shown below. Figure 9 As shown, the results confirm that DMSE@Fe3O4 hydrogel has excellent cell-killing effects.

[0081] III. Detection of intracellular iron overload using ferrous ion fluorescent (FerroOrange) probe.

[0082] Group setup: blank control PBS group, DMS group, DMS@Fe3O4 group, DMSE group, DMSE@Fe3O4 experimental group.

[0083] 4T1 tumor cells were injected at a dose of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / dish in confocal culture dishes and cultured for 24 h. After the cells were stably adherent, 1 mL of different group hydrogel extraction solutions was added to each dish for 24 h. The culture medium was then discarded, and the cells were thoroughly washed three times with PBS buffer. Subsequently, 1 mL of 1640 medium containing FerroOrange iron ion fluorescent probe was added to each dish, and the cells were incubated at 37°C in the dark for 30 min. The cumulative fluorescence intensity of intracellular iron ions in each group was observed by laser confocal microscopy to verify the effect of Fe3O4 releasing iron ions to induce iron overload in tumor cells. Cell imaging results are shown below. Figure 10 As shown, the results confirm that DMSE@Fe3O4 hydrogel has a good intracellular iron overloading effect.

[0084] IV. Detection of the activity of GPX4, a key biomarker of ferroptosis.

[0085] 4T1 tumor cells were injected at a rate of 5 × 10 5Cells were seeded at a density of [number] cells / dish in 6cm cell culture dishes and cultured adherently for 24 hours. Then, 4 mL of different group hydrogel extraction buffer was added to each dish for 24 hours. Cells from each group were collected, centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and cell lysis buffer was added. Cells were lysed on ice for 30 minutes, centrifuged again, and the supernatant was collected. Following the instructions of the GPX4 detection kit, the changes in intracellular GPX4 activity in each group were quantitatively detected to examine the inhibitory effect of Erastin-mediated antioxidant system. The results are shown below. Figure 11 As shown, the results indicate that the DMSE@Fe3O4 hydrogel treatment resulted in the lowest content of the cell ferroptosis marker GPX4, confirming that the DMSE@Fe3O4 hydrogel can effectively activate ferroptosis.

[0086] V. Intracellular ROS levels were investigated using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe.

[0087] Group setup: blank control PBS group, DMS group, DMS@Fe3O4 group, DMSE group, DMSE@Fe3O4 experimental group.

[0088] 4T1 tumor cells were injected at a dose of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / dish in confocal culture dishes. After 24 hours of culture and adherence, 1 mL of different group hydrogel extraction solutions was added to each dish for 24 hours. The culture medium was then discarded, and the cells were washed three times with PBS buffer. Subsequently, 1 mL of culture medium containing the DCFH-DA reactive oxygen species fluorescent probe was added, and the cells were incubated at 37°C for 30 minutes. After incubation, residual probes were washed away with PBS buffer, and the intracellular ROS fluorescence intensity of each group was observed using a laser confocal microscope to verify the ability of Fe3O4 to produce ROS via the Fenton reaction and its synergistic sensitization effect. The imaging results are shown below. Figure 12 As shown, this result indicates that the intracellular ROS level was highest in cells treated with DMSE@Fe3O4 hydrogel, confirming that the Fenton reaction of the DMSE@Fe3O4 hydrogel group produced ROS significantly.

[0089] VI. Intracellular lipid peroxide accumulation was investigated using a lipid peroxide fluorescence (C11-BODIPY) probe.

[0090] Group setup: blank control PBS group, DMS group, DMS@Fe3O4 group, DMSE group, DMSE@Fe3O4 experimental group.

[0091] 4T1 tumor cells were injected at a dose of 1.5 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / dish in confocal culture dishes. After 24 hours of culture and adherence, 1 mL of different group hydrogel extraction solutions was added for 24 hours of treatment. After aspirating the culture medium, the cells were thoroughly washed with PBS buffer, and then culture medium containing the probe was added. The cells were incubated in the dark for 30 minutes. After incubation, the free probes were removed by washing. The accumulation of lipid peroxides in each group of cells was observed using a laser confocal microscope. The synergistic anti-tumor effect was verified through the terminal ferroptosis pathway. Imaging results are shown below. Figure 13 As shown in the figure, the cells treated with DMSE@Fe3O4 hydrogel accumulated the most lipid peroxides, confirming that the ferroptosis pathway was highly activated in the DMSE@Fe3O4 hydrogel group.

[0092] Test Example 3 To investigate the in vivo antitumor effects and biosafety of hydrogels.

[0093] The groupings and dosing regimens for each group are as follows: Blank control group (PBS group, denoted as G1): The administration system was sterile PBS buffer at pH 7.4, and the administration volume per mouse was 50 μL.

[0094] Blank carrier gel group (DMS group, denoted as G2): The drug delivery system was DMS hydrogel, and the drug delivery volume per mouse was 50 μL.

[0095] DFO-free iron-loaded hydrogel group (MS@Fe3O4 group, denoted as G3): The drug delivery system was MS blank framework loaded with Fe3O4 nanoparticles. The Fe3O4 fixed loading amount of a single mouse was 50 μg, and the drug delivery volume was 50 μL.

[0096] DFO-modified mono-loaded iron hydrogel group (DMS@Fe3O4 group, denoted as G4): The drug delivery system was DMS hydrogel loaded with Fe3O4 nanoparticles. The Fe3O4 loading amount of a single mouse was 50 μg, and the drug delivery volume was 50 μL.

[0097] DFO-modified drug-loaded hydrogel group (DMSE group, denoted as G5): The drug delivery system was DMS hydrogel loaded with Erastin. The Erastin dosage was 10 mg / kg. The total dosage for a single administration was calculated based on the real-time body weight of the mice, and the administration volume was 50 μL.

[0098] Clinical positive control group (DOX doxorubicin group, denoted as G6): The administration system was a sterile PBS buffer solution of doxorubicin hydrochloride, which was administered via intratumoral injection. The single administration volume was 50 μL, and the DOX dosage was 5 mg / kg, calculated based on the real-time body weight of the mice.

[0099] The DFO-modified dual-load synergistic treatment group (DMSE@Fe3O4, denoted as G7) had a fixed total Fe3O4 load of 50 μg per mouse, an Erastin dose of 10 mg / kg, and the dosage was adjusted according to the real-time weight of the mice. The single administration volume was 50 μL.

[0100] I. Constructing a mouse subcutaneous tumor model.

[0101] Female BALB / c mice aged 6–8 weeks (SPF grade) were selected and housed in temperature- and humidity-controlled, independently ventilated cages. After 7 days of acclimatization, the modeling process was carried out.

[0102] Log-phase 4T1 mouse breast cancer cells were taken, washed twice with sterile PBS buffer, and resuspended to adjust the cell density to 1×10⁻⁶. 7 Cells / mL; using a 1mL sterile syringe, 100μL of cell suspension was slowly injected subcutaneously into the right back of the mouse to complete the construction of subcutaneous solid tumors in mice.

[0103] The tumor's major diameter (L) and minor diameter (W) are measured daily using vernier calipers. The tumor volume (V) is calculated using the following formula: ; When the average tumor volume grows to 100mm 3 After excluding mice with excessively large volume differences, the tumor-bearing mice were randomly divided into the aforementioned 7 groups, with the same number of mice in each group. All groups were treated with intratumoral injection of the drug, and the observation period was 21 days.

[0104] Mice were sacrificed after the experimental period ended, intact tumor tissue was dissected, photographed, and weighed. The inhibitory effects of each group of hydrogels on tumor growth were compared and analyzed. The results are as follows: Figure 14 , Figure 15 and Figure 16 As shown; from Figure 14 It can be seen that the tumors in the DMSE@Fe3O4 group (G7) were the smallest, confirming that it had the best tumor inhibition effect in vivo.

[0105] from Figure 15 It can be confirmed that the tumor size in the DMSE@Fe3O4 group (G7) was significantly smaller than that in other treatment groups, which confirms that DMSE@Fe3O4 hydrogel has excellent long-term tumor proliferation inhibition ability.

[0106] from Figure 16 It can be confirmed that the hydrogel in the experimental group had no obvious systemic toxicity and good safety.

[0107] II. Investigation of iron accumulation in the tumor tissue microenvironment.

[0108] After fixing, embedding in paraffin, sectioning, dewaxing, and hydrating the dissected tumor tissue, it was stained with Prussian blue in the dark, counterstained with nuclear solid red, dehydrated, cleared, and mounted. The distribution and accumulation of iron ions in the tumor tissue were observed under a microscope. The staining results are as follows: Figure 17 As shown in the figure, the amount of iron accumulation in the tumor tissue of the DMSE@Fe3O4 group was significantly higher than that of other groups. This result indicates that the DFO anchoring hydrogel can effectively lock iron ions and successfully construct a local iron overload microenvironment in the tumor.

[0109] III. Examination of tumor pathological damage.

[0110] Tumor tissues from mice in each group were subjected to HE pathological staining to observe tumor cell morphology, density of cell arrangement, nuclear pyknosis, cell rupture, and tissue necrosis. The results are as follows: Figure 18 As shown in the figure, the tumor cells in the control group were densely packed, morphologically intact, and showed very little necrosis; while the tumor cells in the DMSE@Fe3O4 group showed extensive necrosis, fragmented nuclei, and loose tissue structure, demonstrating a significant in vivo tumor-killing effect.

[0111] IV. Examination of tumor tissue apoptosis.

[0112] In situ apoptosis staining was performed on tumor sections from each group using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit to detect the proportion of apoptotic cells in the tumor tissue. The results are as follows: Figure 19 As shown in the figure, the tumor tissue in the DMSE@Fe3O4 group showed the strongest fluorescence apoptosis signal, and the number of apoptotic tumor cells increased significantly. This result indicates that DMSE@Fe3O4 hydrogel can efficiently induce tumor cell apoptosis and ferroptosis in vivo.

[0113] V. Examination of in vivo biosafety.

[0114] After the experiment, five major internal organs of the mice—heart, liver, spleen, lung, and kidney—were collected for hematoxylin-eosin (HE) staining analysis. The analysis was conducted to observe the normality of the tissue structure and cell morphology of each organ, as well as the presence of inflammatory infiltration, edema, necrosis, and pathological damage. The results are as follows: Figure 20 As shown in the figure, the visceral tissues in each group are intact, without obvious pathological damage or inflammatory infiltration. This result indicates that DMSE@Fe3O4 hydrogel has high safety and no obvious organ toxicity when applied in vivo.

[0115] VI. Examination of blood compatibility.

[0116] Fresh anticoagulated blood was collected from mice, red blood cells were separated by centrifugation, and diluted with physiological saline for later use.

[0117] A PBS negative control group, a sterile ultrapure water positive control group, and a DMSE@Fe3O4 hydrogel extraction solution experimental group were set up. Red blood cell suspensions from each group were co-incubated with their corresponding sample systems. After incubation, the supernatant was collected by centrifugation, and the hemolysis rate of each group was calculated by detecting the absorbance of the supernatant. The results are as follows: Figure 21 As shown; Figure A is a quantitative bar chart of hemolysis rate, and Figure B is a physical image of the hemolysis situation. from Figure 21 It can be seen that DMSE@Fe3O4 hydrogel has an extremely low hemolysis rate, meets the blood compatibility standards for medical materials, will not cause hemolytic reactions, and has good safety for in vivo application.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a tumor microenvironment-responsive iron-overloaded hydrogel, characterized in that, include: DFO-PEG-SH was prepared by chemical reaction using DFO and SH-PEG-COOH as raw materials. DFO stands for deferoxamine, and its structural formula is shown below: ； SH-PEG-COOH is thiolized polyethylene glycol carboxylic acid, and DFO-PEG-SH is deferoxamine-polyethylene glycol-thiol coupling compound; Using DFO-PEG-SH as the functional unit, and thiol-functionalized polyethylene glycol and maleimide-functionalized polyethylene glycol as the crosslinking backbone unit, the functional unit and the crosslinking backbone unit are crosslinked to form DMS hydrogel by utilizing the click chemical reaction between the thiol group in DFO-PEG-SH and the maleimide in the crosslinking backbone unit. Iron oxide nanoparticles and ferroptosis inducers were loaded into the DMS hydrogel network to obtain the tumor microenvironment-responsive iron overload hydrogel.

2. The method for preparing the tumor microenvironment-responsive iron-overloaded hydrogel as described in claim 1, characterized in that, Thiolized polyethylene glycol is multi-arm thiolized polyethylene glycol, and maleimide-functionalized polyethylene glycol is multi-arm maleimide-functionalized polyethylene glycol.

3. The method for preparing the tumor microenvironment-responsive iron-overloaded hydrogel as described in claim 2, characterized in that, The multi-arm thiolized polyethylene glycol is a tetra-arm-polyethylene glycol-thiol, and the multi-arm maleimide-functionalized polyethylene glycol is a tetra-arm-polyethylene glycol-maleimide.

4. The method for preparing the tumor microenvironment-responsive iron-overloaded hydrogel as described in claim 3, characterized in that, The molecular weight of the four-armed polyethylene glycol-thiol group is 2000-10000 Da, and the molecular weight of the four-armed polyethylene glycol-maleimide group is 2000-10000 Da.

5. The method for preparing the tumor microenvironment-responsive iron-overloaded hydrogel as described in claim 1, characterized in that, The molar ratio of DFO to SH-PEG-COOH is 1:1 to 1:1.

5.

6. The method for preparing the tumor microenvironment-responsive iron-overloaded hydrogel as described in claim 1, characterized in that, The iron oxide nanoparticles are iron(II,III) oxide nanoparticles, and the ferroptosis inducer is ellastatin.

7. The method for preparing the tumor microenvironment-responsive iron-overloaded hydrogel as described in claim 1, characterized in that, The loading of the iron oxide nanoparticles is 0.1–1 mg / mL, and the loading of the ferroptosis inducer is 10–50 μmol / L.

8. A tumor microenvironment-responsive iron-overload hydrogel, characterized in that, The tumor microenvironment responsive iron overload hydrogel was prepared by the preparation method of any one of claims 1 to 7. The matrix of the tumor microenvironment responsive iron overload hydrogel is formed by crosslinking thiolized polyethylene glycol and maleimide-functionalized polyethylene glycol, and the network is covalently grafted with DFO-PEG-SH functional conjugate. The tumor microenvironment-responsive iron overload hydrogel is loaded with iron oxide nanoparticles and ferroptosis inducers.

9. An application of a tumor microenvironment-responsive iron-overloaded hydrogel, characterized in that, The application is to prepare drugs or carriers using the tumor microenvironment responsive iron overload hydrogel as described in claim 8, wherein the drugs are drugs for treating tumors.

10. The application of the tumor microenvironment-responsive iron-overloaded hydrogel as described in claim 9, characterized in that, The drug for treating tumors is used for any one or more of the following: Maintaining an iron-overloaded microenvironment in the tumor region; Induces oxidative damage in tumor cells; Induces ferroptosis in tumor cells; It depletes GSH in tumor cells and inactivates GPX4; GSH is reduced glutathione, and GPX4 is glutathione peroxidase 4.

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