Ferroptosis diagnosis and treatment integrated material, preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and discloses an iron death diagnosis and treatment integrated material, a preparation method and application thereof. The material comprises a methylacrylated hyaluronic acid hydrogel matrix and hybrid molecules dispersed in the hydrogel matrix; the hybrid molecules comprise an octamercaptopropyl polyhedral oligomeric silsesquioxane skeleton and functional molecules grafted on the skeleton through a covalent bond; the functional molecules comprise polyethylene glycol acrylate, gallic acid acrylate and deferoxamine acrylate. The material can not only realize visual monitoring of an iron death process, but also can play an efficient tumor inhibition role by inducing iron death, realizes diagnosis and treatment integration, and provides a novel and efficient diagnosis and treatment integrated solution for iron death related disease diagnosis and treatment.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to an integrated material for the diagnosis and treatment of ferrodeath, its preparation method, and its application. Background Technology

[0002] Ferroprelation is widely involved in various major pathological processes, including osteoarthritis, neurodegenerative diseases, ischemia-reperfusion injury, and tumor drug resistance. Its persistent occurrence exacerbates tissue degeneration, functional loss, and even organ failure. The core mechanism of this process is the abnormal accumulation of intracellular iron ions, which drives an uncontrolled cascade of lipid peroxidation through the Fenton reaction, ultimately leading to damage to key structures such as the cell membrane and mitochondria. Currently, intervention strategies mainly rely on molecular drugs such as iron chelators and antioxidants, as well as nanomaterials designed to improve their delivery. However, these methods have significant limitations in terms of synergistic efficacy, targeting, and visualization. Therefore, developing novel ferroptosis diagnostic and therapeutic materials that can achieve both diagnosis and synergistically efficient iron ion removal and antioxidant effects has become an important and urgent challenge in the biomedical field.

[0003] Current designs for ferroptosis intervention materials primarily follow three empirical pathways: first, directly scavenging iron ions using clinical iron chelators (such as DFO) or their derivatives; second, introducing antioxidant molecules (such as Ferrostatin-1) or mimicking the activity of antioxidant enzymes (such as GPX4) to interrupt the lipid peroxidation chain; and third, constructing nanocarriers to achieve targeted drug delivery and controlled release. However, these strategies have fundamental limitations: small molecule drugs have short-lived effects and lack tissue selectivity; simple antioxidant action cannot fundamentally address iron overload; and most nanosystems are merely physical loads, with their intrinsic electronic structures not rationally designed for iron ion coordination and magnetic regulation. This makes it difficult for materials to synergistically achieve efficient iron removal, long-lasting antioxidant effects, and in vivo visual monitoring.

[0004] Therefore, there is an urgent need in this field for an integrated nanodiagnostic platform that can simultaneously achieve precise iron ion chelation, efficient removal of reactive oxygen species, and visualization via magnetic resonance imaging (MRI), providing a novel solution for the precise diagnosis and treatment of ferroptosis-related diseases. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated material for the diagnosis and treatment of ferroptosis, its preparation method and application, in order to solve the technical problem that existing ferroptosis intervention materials lack the ability to synergistically achieve efficient iron removal and in vivo dynamic monitoring.

[0006] Therefore, this application is based on density functional theory (DFT), that is, DFT calculations can accurately resolve the chelate ligand and Fe from a quantum mechanical perspective. 3+ The essence of interaction, the core advantage of DFT lies in its ability to quantitatively calculate the binding energy (ΔE). bindThis study aims to predict chelation stability and resolve spin states and integrated spin density (ISD) to directly quantify changes in iron ion paramagnetism. This is crucial for designing iron death materials that combine therapeutic (efficient iron removal) and diagnostic (visualization via MRI T2 signal changes) functions. However, the field has long lacked a method to integrate the electronic structure parameters (ΔE) calculated by DFT. bind The quantitative structure-property relationship, which unifies the magnetic properties (T2 relaxation rate) and biological efficacy (ability to inhibit ferroptosis) of materials, means that material development remains in the "trial and error" stage, making it impossible to carry out forward-looking rational design.

[0007] This application first integrates DFT calculations with in vitro magnetic resonance imaging (MRI) to systematically analyze the correlation between the electronic structure characteristics of different iron ion chelators and their corresponding in vitro MRI signals. It is the first to establish a mechanism to weaken Fe... 3+ The quantitative relationship between key electronic structure parameters of paramagnetism and in vitro imaging signal enhancement, i.e., when the binding energy ΔE bind < 5.8 eV and integral spin density ISD < 3.2 At a ratio of / 2, the signal contrast of T2-weighted imaging can be maximized. Furthermore, this application designed and prepared hyaluronic acid hydrogel microspheres (HAMA@PPDG) containing the iron chelating agent DFO and the antioxidant gallic acid (GA). Spectroscopic and in vitro cell experiments verified that HAMA@PPDG can efficiently chelate iron ions and inhibit lipid peroxidation. In an in vivo model of ferroptosis osteoarthritis, it was shown that HAMA@PPDG not only effectively reversed the ferroptosis phenotype of chondrocytes and alleviated disease progression through a synergistic effect of "iron chelation-antioxidation," but also enabled dynamic visualization monitoring of the iron ion clearance process and treatment response via in vivo MRI. This application fully constructs a closed-loop diagnostic and therapeutic system integrating "theoretical prediction—material design—disease intervention—imaging verification," breaking through the limitations of traditional ferroptosis intervention materials that rely on empirical design, and providing an important basis for developing controllable ferroptosis diagnostic and therapeutic materials.

[0008] Specifically, this application adopts the following technical solution:

[0009] Firstly, this application provides an integrated material for the diagnosis and treatment of ferritinosis, including... Methacrylamide hyaluronic acid hydrogel matrix; and Hybrid molecules dispersed within the hydrogel matrix; The hybrid molecule includes an octamercaptopropyl polyhedral oligosilsesquioxane backbone and functional molecules grafted onto the backbone via covalent bonds. The functional molecules include polyethylene glycol acrylate, gallic acid acrylate, and deferoxamine acrylate.

[0010] Secondly, this application provides a method for preparing the integrated diagnostic and therapeutic material for ferritin, comprising the following steps: Step 1: Prepare polyethylene glycol acrylate, gallic acid acrylate and deferoxamine acrylate; Step 2: The polyethylene glycol acrylate, gallic acid acrylate and deferoxamine acrylate are covalently grafted onto the octamercaptopropyl polyhedral oligomeric silsesquioxane backbone to obtain a hybrid molecule, named PPDG. Step 3: Prepare methacrylamide hyaluronic acid lyophilized hydrogel, named HAMA; Step 4: The PPDG obtained in Step 2 and the HAMA obtained in Step 3 are mixed as the aqueous phase, and the oily medium and emulsifier are used as the oil phase. The integrated material for the diagnosis and treatment of ferroptosis is prepared by microfluidic interface solidification method and named HAMA@PPDG.

[0011] Furthermore, in step 1, the preparation method of polyethylene glycol acrylate is as follows: Polyethylene glycol was dissolved in dichloromethane, an acid-binding agent was added, and acryloyl chloride was slowly added dropwise under ice bath conditions. The reaction system was stirred at 0°C. After the reaction was completed, the reaction solution was washed, dried, filtered, and evaporated to dryness to obtain the polyethylene glycol acrylate.

[0012] Further, in step 1, the preparation method of gallic acid acrylate is as follows: Gallic acid was dissolved in anhydrous ethanol, an acid-binding agent was added, and acryloyl chloride was slowly added dropwise under ice bath conditions. The reaction system was stirred at 0°C. After the reaction was completed, the reaction solution was washed, dried, filtered, and evaporated to dryness to obtain the gallic acid acrylate.

[0013] Further, in step 1, the preparation method of deferoxamine acrylate is as follows: Deferroamine was dissolved in anhydrous N,N-dimethylformamide, an acid-binding agent was added, and acryloyl chloride was slowly added dropwise under ice bath conditions. The reaction system was stirred at 0°C, and then the temperature was raised to room temperature to continue the reaction. After the reaction was completed, the reaction solution was slowly poured into a large amount of ice-cold anhydrous diethyl ether, the precipitate was collected by centrifugation, washed, and freeze-dried to obtain the deferroamine acrylate.

[0014] Furthermore, in step 2, the molar ratio of octamercaptopropyl polyhedral oligomeric silsesquioxane, polyethylene glycol acrylate, gallic acid acrylate, and deferoxamine acrylate is 1:4:0.25:3.

[0015] Furthermore, step 3 consists of the following steps: Hyaluronic acid was dissolved in preheated phosphate-buffered saline solution and stirred continuously until a clear and homogeneous solution was formed. Then, methacrylic acid was slowly added, and the reaction system temperature was controlled at 50°C. After the reaction was completed, excess PBS was added to the system to terminate the reaction, and the dilution-displacement operation was repeated several times. The reaction solution was then transferred to a dialysis bag for dialyzing and freeze-dried to obtain the methacrylamide hyaluronic acid lyophilized hydrogel, named HAMA.

[0016] Furthermore, step 4 consists of the following steps: The hybrid molecule PPDG was dissolved in phosphate buffered saline, and then pre-dissolved lyophilized hydrogel HAMA was added. After thorough stirring, a homogeneous and stable aqueous phase was formed. Paraffin oil is mixed with an emulsifier to form an oil phase; During the microfluidic preparation process, the aqueous phase and oil phase are injected through the internal and external phase channels of the microfluidic chip, respectively, where the internal phase is the aqueous phase and the external phase is the oil phase. The flow rates are set to 0.8 mL·h for the aqueous phase. -1 oil phase 16 mL·h -1 The generated droplets further cross-link and solidify at 80°C to form microspheres. The obtained microspheres are collected by centrifugation and washed repeatedly with petroleum ether and deionized water to finally obtain the HAMA@PPDG integrated material for the diagnosis and treatment of ferrodeogenesis.

[0017] Thirdly, this application provides the application of the integrated ferroptosis diagnostic and therapeutic material in the preparation of diagnostic and therapeutic products for ferroptosis-related diseases.

[0018] Furthermore, the iron death-related diseases include osteoarthritis, neurodegenerative diseases, ischemia-reperfusion injury, and tumors.

[0019] In summary, this application has the following beneficial effects: This application proposes a method based on binding energy (ΔE) at the electronic structure level. bind A novel strategy for designing ferroptosis diagnostic and therapeutic materials based on integral spin density (ISD). Through density functional theory calculations, a quantitative criterion was established for the first time: when ΔE bind < 5.8 eV and ISD < 3.2 At / 2, the material can efficiently chelate Fe 3+Simultaneously, it weakens the paramagnetism of the material, achieving effective modulation of T2-MRI signals and meeting the synergistic requirements of "strong chelation—weak paramagnetism—imaging capability." Guided by this theory, a multifunctional microsphere system, HAMA@PPDG, was constructed, integrating iron chelation, anti-oxidation, and MRI imaging. Experiments show that this system can effectively inhibit iron overload-induced lipid peroxidation, alleviate ferroptosis-related cartilage degeneration in cell and animal models, and achieve dynamic visualization monitoring of the treatment process via T2-MRI. This application establishes the intrinsic correlation between electronic structural parameters, material function, biological effects, and imaging response, providing a predictable and controllable theoretical framework for the rational design and application of ferroptosis diagnostic and therapeutic materials. Attached Figure Description

[0020] Figure 1 Different ligands and Fe under their typical coordination ratios 3+ The resulting complex structure was optimized, along with the corresponding frontier molecular orbitals (HOMO and LUMO) and band gap difference. Specifically, (A)DFP and its Fe... 3+ (A) Optimized configuration of the complex and corresponding HOMO and LUMO molecular orbital distributions; (B) DFS and its Fe 3+ Optimized configuration of the complex and corresponding HOMO and LUMO molecular orbital distributions; (C) DFO and its Fe 3+ The optimized configuration of the complex and the corresponding HOMO and LUMO molecular orbital distributions.

[0021] Figure 2 Different chelating agents affect Fe 3+ The binding stability and paramagnetic regulation of Fe were verified by T2-MRI. Among them, (AE)EDA, GA, DFP, DFS, and DFO played a role in chelating Fe. 3+ Distribution of molecular electrostatic potential (ESP) before and after; Fe calculated by (F)DFT. 3+ – Ligand binding energy (ΔE_bind); (G) Integral spin density (ISD) calculated by DFT; (H) Relationship between different chelating agents and Fe 3+ In vitro T2-MRI images after the formation of the complex (FeCl3 is the control).

[0022] Figure 3 Synthesis, characterization and Fe of multifunctional PPDG 3+ Chelating properties. (A) Schematic diagram of the one-pot thiol-alkenyl click chemistry synthesis of the multifunctional POSS molecule PPDG; (B) Synthetic intermediates and PPDG... 1 (C) Fourier transform infrared (FTIR) spectral characterization of PPDG; (D) POSS, DFO, Fe3+ Comparison of UV-Vis absorption spectra of PPDG; (E) PPDG with the gradual addition of different equivalent amounts of Fe 3+ (1-5 eq) UV-Vis absorption spectrum changes; (F) POSS-PEG and PPDG nanoparticle size analysis; (G) PPDG chelated Fe 3+ Changes in Zeta potential before and after; (H)PPDG chelates Fe 3+ The frontier molecular orbital (HOMO / LUMO) distribution; (I) PPDG chelates Fe 3+ The frontier molecular orbital (HOMO / LUMO) distribution after (J) PPDG chelation of Fe 3+ Distribution of molecular surface electrostatic potential (ESP) before and after.

[0023] Figure 4 Morphology, elemental composition, chemical state, and sustained-release / degradation performance of HAMA@PPDG microspheres. The images include: (A) SEM images of microspheres at different magnifications (surface morphology and porous structure); (B, D) EDS mapping and elemental superposition diagrams (C, O, N, S, Si) of individual microspheres; (C) Statistical distribution of microsphere particle size (d = 310.6 ± 6.2 μm); (E) XPS full spectrum; (F–J) High-resolution XPS and fitting of C1s, O1s, N1s, S2p, and Si2p; (K) Drug release curves; (L) Microsphere degradation curves; (M) Mercury intrusion porosimetry pore size distribution and porosity (Porosity = 65.7%, Pore size = 485 nm).

[0024] Figure 5 The inhibitory effect of HAMA@PPDG on ferroptosis in chondrocytes and its biosafety assessment. (A) Live / Dead fluorescence staining images of chondrocytes after different material treatments to assess cell biosafety (Control, PPD, HAMA, PPDG, HAMA@PPDG); (B) Quantitative analysis of cell density in different treatment groups after 1–3 days of culture; (C) CCK-8 cell viability assay results in different treatment groups after 1–3 days of culture; (D) Detection of reactive oxygen species (ROS) levels in chondrocytes of different treatment groups using the DCFH-DA fluorescent probe (Control, Erastin, HAMA, PPD, PPDG, HAMA@PPDG); (E) Detection of lipid peroxidation levels in chondrocytes of different treatment groups using the C11-BODIPY fluorescent probe; (F) Fe in chondrocytes of different treatment groups. 2+Quantitative analysis of intracellular ROS fluorescence intensity; (G) quantitative analysis of oxidized C11-BODIPY fluorescence intensity; (H) quantitative analysis of OX-C11 / NO-OX-C11 fluorescence intensity ratio; (I) quantitative analysis of intracellular Fe... 2+ Quantitative analysis of fluorescence intensity.

[0025] Figure 6 : Real-time MRI monitoring of the alleviating effect of HAMA@PPDG on degenerated cartilage. Among them, (A) short-term (within 8 days) ultra-high field MRI imaging results of knee cartilage under HAMA@PPDG, HAMA, PPDG, Erastin and PBS intervention; (B) long-term progression of knee cartilage degeneration after intervention with HAMA@PPDG and PPDG control group; (C) quantitative analysis based on the data in Figure A: dynamic change of contrast-to-noise ratio (CNR) of cartilage relative to subchondral bone in each treatment group over time (sample size n=3 per group); (D–F) trend of MRI quantitative indicators based on Figure B: where D is the cartilage thickness, E is the cartilage area and F is the cartilage volume change curve over time (sample size n=3 per group).

[0026] Figure 7 The study investigated the ameliorative effect of HAMA@PPDG on cartilage degeneration. (A) Micro-CT three-dimensional reconstruction (top) and sagittal view (bottom) of the knee joints in the Sham, Erastin, HAMA, PPDG, and HAMA@PPDG groups (scalebar=5mm); (B) Quantitative analysis of the total osteophyte volume in each group's knee joints; (C) OARSI scores of cartilage in each group (n=5, data expressed as mean±SD). (P<0.001); (D) Histological staining and immunofluorescence results of cartilage in each group include: Safranin O-Fixed Green (SF), Alcian Blue (AB), DABPB staining, Masson trichrome staining, and immunofluorescence staining of antioxidant-related proteins GPX4, ACSL4 and cartilage matrix protein COLII (green for target protein, blue for cell nucleus), magnification = 200x. Detailed Implementation

[0027] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0028] Example Experimental materials: Octadec-mercaptopropyl polyhedral oligomeric silsesquioxane (POSS-SH, purity ≥95%) was synthesized in our laboratory and used after characterization. The preparation method was as follows: 20 mL of γ-mercaptopropyltrimethoxysilane (KH590), 40 mL of methanol, and 20 mL of concentrated hydrochloric acid were added to a 250 mL three-necked flask equipped with a magnetic stir bar and a spherical condenser. The mixture was heated to 90 °C with stirring and reacted for 24 hours. Heating was stopped when the solution changed from colorless to milky white, and the mixture was allowed to cool to room temperature for at least 6 hours. At this point, a white viscous substance precipitated at the bottom of the flask. The supernatant was discarded, and the product was washed three times with 30 mL of methanol. Then, 60 mL of dichloromethane was added to fully dissolve the product. The product was extracted four times with 10 mL × 4 saturated saline solution. The lower milky white solution was collected, and an appropriate amount of anhydrous magnesium sulfate was added. The mixture was dried overnight, filtered to remove magnesium sulfate, and the filtrate was rotary evaporated to remove dichloromethane, finally yielding a colorless, viscous mercaptocage-type semisiloxane.

[0029] Polyethylene glycol 400 (PEG400, molecular weight 400, purity ≥98%) was purchased from Sigma-Aldrich.

[0030] Deferoxamine (DFO, purity ≥99%) was purchased from MedChemExpress (MCE).

[0031] Gallic acid (GA, purity ≥99%) was purchased from Sigma-Aldrich.

[0032] Acryloyl chloride (AC, purity ≥98%) was purchased from Adamas.

[0033] The photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA, purity ≥99%) was purchased from Sigma-Aldrich.

[0034] LAP photoinitiator (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate, purity ≥98%) was purchased from Aladdin.

[0035] Ferroptosis inducer (Erastin, purity ≥99.6%) was purchased from MCE China Reagents.

[0036] Fetal bovine serum (FBS) was purchased from Sigma-Aldrich.

[0037] DMEM / F-12 medium (containing penicillin and streptomycin antibiotics) and phosphate-buffered saline (PBS, pH 7.4) were purchased from Gibco.

[0038] The pancreatic enzyme digestion solution (0.25%, containing EDTA and phenol red) was purchased from Adamas Life.

[0039] Unless otherwise specified, all chemical and biological reagents were used directly without further purification after purchase. The water used in the experiments was deionized water, and all organic solvents were of analytical grade or higher.

[0040] It is worth noting that all experiments in this application were repeated at least three times, and each assay was performed with three parallel samples. Data are expressed as mean ± standard deviation (mean ± SD). Differences between groups were assessed using one-way ANOVA combined with Tukey's post-hoc test. Statistical significance is determined by... P<0.05 P<0.01 P<0.001 and P < 0.0001 indicates a negative result. All analyses were performed using SPSS 26.0 (IBM, USA) software.

[0041] Example 1: Determination of DFO for chelate iron module (1) To explore the design principles of magnetically responsive materials, this application first uses density functional theory (DFT) to systematically calculate various ligands and their complexes with iron ions. Specifically, the Dmol3 module in Materials Studio (MS) platform was first used to calculate the complexes of EDA, GA, DFP, DFS, DFO and their complexes with Fe. 3+ The chelated structure underwent geometric optimization, and its electronic structure characteristics, including HOMO and LUMO energy levels and the HOMO-LUMO band gap, were analyzed. The binding energy and electrostatic potential distribution of the ligands and iron ions were calculated to evaluate the ligands' electron-donating ability and stability. Furthermore, the magnetic properties of the system, including magnetic moment, spin integral density, and initial spin polarization, were calculated using the CASTEP module, revealing the influence of iron ion coordination on the material's paramagnetism and magnetic response. These calculations aim to clarify the effects of different ligands and their structural characteristics on iron ion binding and magnetic response performance, providing theoretical guidance for the design of magnetically responsive materials.

[0042] Specifically, this application selects DFP, DFS, and DFO as representative model ligands, corresponding to typical coordination ratios of 1:3, 1:2, and 1:1, respectively. Figure 1 The electronic structure response of Fe was analyzed by combining the A–C values ​​of the frontier molecular orbitals (HOMO / LUMO) and band gap changes. From the perspective of structure and coordination, Fe… 3+As a typical hard acid, it tends to form a six-coordinate octahedral configuration. DFO, composed of three hydroxamic acid groups, can provide six O donor atoms, achieving Fe under 1:1 conditions. 3+ Complete occupancy of coordination sites leads to the formation of highly pre-organized encapsulated intra-coordination complexes. Figure 1 In contrast, DFS can only participate in coordination in a tridentate manner, requiring two ligands in synergy (1:2) to satisfy the hexacoordination requirement. Figure 1 B), while bidentate DFP requires three ligands (1:3) to assemble together to construct a complete coordination environment ( Figure 1 (A) EDA, like GA, relies on multi-molecular assembly, and its complex structure is relatively open. This evolution of the coordination ratio from 1:3 to 1:2 to 1:1 reflects the relationship between the number of ligand teeth and Fe. 3+ The degree of matching between coordination requirements and the difference in coordination saturation lay the foundation for the electronic structure response. Further analysis of the coordinating atom types reveals that DFO, DFS, DFP, and GA mainly participate in coordination through O donors. Among them, the O atom of the hydroxamic acid in DFO is similar to that of Fe³⁺ in both spatial configuration and electronic properties. + The hard acid properties of the two molecules are highly compatible, enabling multi-point synergistic chelation within a single molecule; while EDA mainly relies on N donors and reacts with Fe. 3+ The matching degree is relatively low. Frontier molecular orbital analysis shows that DFP and DFS have relatively low matching degree in chelating Fe. 3+ Subsequently, their HOMO–LUMO band gaps decreased from 3.416 eV to 1.824 eV and from 1.823 eV to 1.379 eV, respectively. GA and EDA also showed a similar band gap decreasing trend. Figure 1 The A and B values ​​in the figure indicate that the metal d orbitals introduce low-energy metal-ligand mixed orbitals, making the electronic structure of the system more open. It is noteworthy that DFO is the only one found in chelated Fe... 3+ The system subsequently exhibited a reverse increase in the band gap, with ΔE increasing from 0.129 eV to 0.328 eV. Figure 1 The presence of C in the figure reflects the complete coordination saturation achieved by the 1:1 hexadentate chelation, which causes the metal-ligand orbitals to split sufficiently and move away from the Fermi level, thereby significantly improving the stability of the electronic structure. Considering the overall structural configuration, coordination ratio, donor atom type, and frontier orbital response, it can be seen that DFO achieves Fe at the single-molecule level through a hexadentate O donor. 3+ The complete chelation of the complex structure and electronic stability of the complex are significantly superior to those of the other ligands.

[0043] (2) To verify the theoretically calculated and predicted magnetic response, this application conducted multimodal magnetic resonance imaging (MRI) experiments in vitro. Specifically, T1- and T2-weighted imaging was performed on samples before and after chelating iron ions with ligands such as EDA, GA, DFP, DFS, and DFO to systematically evaluate the influence of ligand structure and chelation state on MRI signals. In the experiment, the iron ion concentration and sample ratio were controlled to determine the -Fe content of each ligand. 3+ The relaxation rates (r1, r2) and signal enhancement efficiency of the system were analyzed and compared with electronic and magnetic parameters such as the HOMO-LUMO band gap, binding energy, magnetic moment, and spin integral density obtained by DFT calculation. The results show that the theoretically predicted paramagnetic enhancement or weakening trend is highly consistent with the changes in in vitro MRI signal, providing direct experimental verification for understanding the magnetic response behavior of ligand structure to iron ions, and providing both theoretical and experimental support for the multimodal imaging design of magnetically responsive materials.

[0044] To further elucidate the effects of different chelating agents on Fe from the perspective of real space charge distribution 3+ The coordination differences were analyzed, and the electrostatic potential (ESP) distributions of each ligand before and after complexation were calculated and compared. Figure 2 (A–E in the text). For systems requiring coordination through 1:3 multi-molecule assembly, the maximum negative electrostatic potential of EDA and DFP decreases further after complexation (EDA: 0.0728→ 0.0817 Ha; DFP: 0.0848→ 0.102Ha), indicating that the system surface still retains or even enhances local strong negative potential "hot spots", Fe 3+ The surrounding high negative potential sites were not fully consumed, and the overall potential distribution exhibited a clear openness characteristic. As a bidentate ligand with O donor as the main component, GA's complexation behavior showed a different moderate charge compensation characteristic compared to EDA / DFP: GA complexed Fe... 3+ Then its most negative electrostatic potential is 0.0581 rebounded to 0.0494 Ha., while ΔESP decreased significantly, indicating that GA and Fe 3+ Coordination can weaken the local negative potential and achieve partial charge neutralization to some extent. However, due to the reliance on multi-molecule cooperative assembly and lack of spatial pre-organization ability, the distribution of its negative electrostatic potential remains relatively dispersed, making it difficult to form a continuous, closed potential enclosure environment. In contrast, the maximum negative electrostatic potential of DFS (1:2 coordination) after complexation is reduced from... 0.0869 rebounded to The value of 0.0644 indicates a more effective charge compensation capability than GA, but a certain degree of potential openness still exists. It is worth noting that DFO-Fe...3+ The complex exhibits the weakest negative electrostatic potential "hot spot" among all systems, with its ESPmin being only [missing value]. 0.0447, significantly higher than the other complexes, indicating that Fe 3+ The surrounding strongly negative potential sites are most fully consumed and neutralized, and the surface potential of the system tends to be highly uniform. This characteristic reflects that DFO achieves the most complete charge compensation and spatial closure after complexation, exhibiting typical coordination saturation and encapsulation-type internal coordination complexation behavior. This electrostatic potential evolution law is consistent with... Figure 1 The frontline trajectory analysis results are highly consistent: DFO achieves Fe chelation through a 1:1 hexadecanthogenic hydroxamic acid O donor. 3+ Six-coordinate saturation facilitates the formation of stable internally encapsulated complexes; while systems relying on multi-molecule assembly, such as GA, DFP, and EDA, are more prone to residual negative potential hotspots, manifesting as open potentials and electronic structures. Overall, ESP analysis further validates the effect of DFO on Fe from the perspective of real-space charge distribution. 3+ It has the strongest and most stable chelating ability.

[0045] To elucidate how electronic structure modulates magnetism, we selected the aforementioned typical chelate iron molecules as model ligands, including the nitrogen-containing bidentate ligand EDA, the polyphenol oxygen-containing ligand GA, and the clinically representative chelate iron preparations DFP, DFS, and DFO (with the number of electron-donating atoms and the number of coordinating teeth increasing progressively). We systematically calculated their interactions with Fe. 3+ Binding energy and related magnetic parameters were calculated and compared with in vitro T2-MRI signals for verification. Binding energy calculation results ( Figure 2 As shown in F), ΔE_bind exhibits a monotonically negative trend from EDA to DFO (approximately). 4.7 5.9 8.0 9.2 to (12.4 eV), indicating that chelation stability continuously increases with increasing coordination ability. Among them, DFO–Fe 3+ Having the largest absolute value of binding energy indicates the highest thermodynamic stability, providing the optimal molecular basis for efficient iron removal. Furthermore, the integrated spin density (ISD) is introduced as a paramagnetic descriptor (…). Figure 2 The results showed that ISD decreased synchronously with increasing chelating ability (EDA ~ 3.95). / 2→DFO~1.86 / 2), indicating that a strong coordination field and high-tooth coordination can effectively confine Fe. 3+ The 3d unpaired electrons have significantly reduced spin degrees of freedom, thus achieving paramagnetic passivation.

[0046] To verify the correlation between "electronic structure-magnetism-imaging", we further investigated different chelating agents –Fe 3+ The system underwent in vitro T2-MRI imaging ( Figure 2 The results showed that with increasing chelation ability, the brightness and contrast of the T2 signal gradually increased: weak coordination systems (such as EDA) still retained a strong paramagnetic attenuation effect, while the T2 signal significantly increased when ΔEbind further decreased and entered the DFP / DFS / DFO range. This trend is highly consistent with the changes in binding energy and ISD, indicating that stronger chelation (more negative ΔEbind) and weaker paramagnetism (lower ISD) are synergistic necessary conditions for improving T2-MRI contrast. Based on the cross-validation of DFT calculation and MRI imaging, we further proposed an imaging criterion: when ΔEbind < 5.8 eV and ISD < 3.2 At / 2, chelating agents can significantly weaken Fe 3+ Paramagnetic properties effectively enhance T2-MRI imaging contrast, thus simultaneously meeting the design requirements of "strong chelation, weak paramagnetism, and imaging capability" for ferroptosis therapeutic materials. Within this framework, DFO possesses both the strongest binding energy and the lowest ISD, and exhibits optimal T2 contrast performance in in vitro MRI, thus being identified as the best chelate iron module for subsequent therapeutic material construction.

[0047] Example 2: Preparation method of HAMA@PPDG microspheres Step 1: Synthesis of polyethylene glycol 400 acrylate (PEG400-Acrylate): Polyethylene glycol 400 (PEG400, 5.00 g, 12.5 mmol) was dissolved in dry dichloromethane (DCM, 25 mL) and stirred under nitrogen protection until a homogeneous solution was formed. Triethylamine (TEA, 0.25 g, 5 wt%) was then added as an acid-binding agent. Acryloyl chloride (Acryloyl chloride, 1.50 g, 13.8 mmol) was slowly added dropwise under ice bath conditions (0°C) to reduce the reaction rate and inhibit side reactions. The reaction system was stirred at 0°C for 1–2 h to allow the terminal hydroxyl groups of PEG400 molecules to undergo complete esterification. After the reaction was complete, the reaction solution was washed several times with deionized water to remove unreacted acryloyl chloride and the generated triethylamine salt. After separating the organic phase, anhydrous sodium sulfate was added for drying, and the solvent was removed by filtration and rotary evaporation to obtain a colorless to pale yellow transparent liquid PEG400-Acrylate in approximately 95% yield.

[0048] Step 2, Synthesis of Gallic Acid Acrylate (GA-Acrylate): Gallic acid (GA, 0.50 g, 2.94 mmol) was dissolved in anhydrous ethanol (EtOH, 25 mL) and stirred at room temperature until completely dissolved. Triethylamine (TEA, 0.025 g, 0.247 mmol) was then added as an acid-binding agent. Acryloyl chloride (0.32 g, 3.53 mmol, approximately 1.2 equivalents) was slowly added dropwise under ice bath conditions (0°C) to avoid excessive esterification initiated by the polyhydroxy structure. The reaction system was continuously stirred at 0°C for 1–2 h to allow the phenolic hydroxyl groups in the GA molecule to undergo esterification with acryloyl chloride. After the reaction was complete, the reaction solution was repeatedly washed with deionized water to remove residual reagents and salt byproducts. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a light yellow solid, GA-Acrylate, with a yield of approximately 92%.

[0049] Step 3, Synthesis of Deferroamine (DFO) Acrylate: Deferoxamine (DFO, 0.50 g, 0.892 mmol) was dissolved in anhydrous N,N-dimethylformamide (DMF, 20 mL) and stirred under nitrogen protection until completely dissolved. Triethylamine (TEA, 0.025 g, 0.267 mmol) was then added as an acid-binding agent and reaction promoter. Acryloyl chloride (0.12 g, 1.34 mmol, 1.5 equivalents) was slowly added dropwise to the reaction system under ice bath conditions (0°C) to control the reaction rate and reduce the possibility of over-acylation at multiple sites. The reaction system was stirred at 0°C for 1 h, then raised to room temperature and reacted for another 2 h to ensure that the hydroxamic acid or amino groups in the DFO molecule fully participated in the acylation reaction. After the reaction was complete, the reaction solution was slowly poured into a large amount of ice-cold anhydrous diethyl ether, precipitating a pale yellow solid. The precipitate was collected by centrifugation and washed successively with diethyl ether and deionized water to remove unreacted reagents and byproducts. Finally, it was freeze-dried to obtain DFO-Acrylate with a yield of about 85%.

[0050] Step 4: Synthesis of the target hybrid molecule PPDG: Octadec-mercaptopropyl polyhedral oligomeric silsesquioxane (POSS-SH, 1.0 mmol, 1.01 g) was dissolved in dry tetrahydrofuran (THF, 20 mL) and stirred at room temperature until completely dissolved. Then, acrylated functional molecules were added sequentially according to the designed proportions: gallic acid acrylate (GA-Acrylate, 0.25 mmol, 0.06 g), polyethylene glycol acrylate (PEG400-Acrylate, 4.0 mmol, 1.67 g), and deferoxamine acrylate (DFO-Acrylate, 3.0 mmol, 1.68 g). Following this, the photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA, 0.10 mmol, 0.023 g) was added. The reaction system was stirred at room temperature for 2 hours, and then a thiol-ene click reaction was initiated under ultraviolet light irradiation to achieve one-pot covalent grafting of various functional molecules with the POSS core, generating the multifunctional POSS hybrid molecule PPDG. After the reaction, the reaction solution was transferred to a dialysis bag (MWCO 1000Da) and dialyzed in deionized water for 48 hours, with the dialysis medium being changed multiple times to remove unreacted small molecules and residual photoinitiator. Finally, the product PPDG was obtained as a light yellow solid after freeze-drying.

[0051] Step 5: Preparation of lyophilized methacryloyl hyaluronic acid (HAMA) hydrogel: 1.0 g of hyaluronic acid (HA) was dissolved in 10 mL of PBS solution preheated to 60°C to a concentration of 10% (w / v), and the solution was stirred continuously until a clear and homogeneous solution was formed. Then, 0.8 mL of methacrylic anhydride (MA) was slowly added dropwise, and the pH of the reaction system was maintained at 8.0–8.5 using NaOH solution. The reaction temperature was controlled at 50°C, and the reaction was continued for 1 h to achieve methacrylylation modification of the hydroxyl groups in the HA molecule. After the reaction, the reaction solution was placed in a dialysis bag and dialyzed against deionized water for 48 h, with the dialysate changed multiple times to remove unreacted small molecules and residual reagents. Finally, the solution was freeze-dried to obtain the methacrylylated hyaluronic acid hydrogel precursor HAMA.

[0052] Step 6: Preparation of HAMA@PPDG microspheres: HAMA@PPDG microspheres were prepared using a microfluidic interface solidification method. First, 200 mg of PPDG was dissolved in 1 mL of PBS, followed by the addition of 100 mg of pre-dissolved HAMA. The mixture was thoroughly stirred to form a homogeneous and stable aqueous precursor solution. The oil phase consisted of paraffin oil and 2 wt% Span 80 emulsifier, and was maintained at a constant temperature of 80°C to ensure good flowability and stability of the continuous phase. During the microfluidic preparation process, the aqueous and oil phases were injected through the internal and external phase channels of the microfluidic chip, respectively, with the flow rates of the internal phase (aqueous phase) and the external phase (oil phase) set to 0.8 mL·h. -1 and 16 mL·h -1 Under shear force, the aqueous phase is stably segmented by the continuous oil phase to form uniformly sized droplets, which are then rapidly embedded in the oil phase. The resulting droplets undergo further cross-linking and solidification at 80°C, ultimately forming structurally stable and morphologically complete HAMA@PPDG microspheres. After preparation, the microspheres are collected by centrifugation and washed repeatedly with petroleum ether and deionized water to thoroughly remove residual oil phase and unreacted components. Finally, the microspheres are redispersed in PBS for subsequent physicochemical characterization and biological experiments.

[0053] Example 3: Characterization of HAMA@PPDG microspheres 1 H nuclear magnetic resonance (H nuclear magnetic resonance) 1 H NMR: Using a Bruker Avance III 600MHz NMR spectrometer, the sample was dissolved in DMSO-d6, and the chemical shifts of the intermediate and final target product PPDG were determined at room temperature. By comparing the changes in the position, peak shape evolution, and relative integrated intensity of characteristic peaks before and after the reaction, the coexistence of acryloyl characteristic peaks and POSS backbone-related signals was carefully observed to verify that the acrylated small molecule had been successfully covalently grafted onto the POSS molecular backbone, further confirming the successful synthesis of PPDG. UV-Vis Absorption Spectroscopy: UV-Vis absorption spectra were measured on a Shimadzu UV-3600Plus spectrometer. POSS, GA, DFO, Fe 3+ The test concentration of PPDG solution was controlled at approximately 0.1 mg / mL. -1 A quartz cuvette with an optical path length of 1 cm was used, and the scanning wavelength range was 200-600 nm. PPDG was compared with its Fe... 3+ Complex (PPDG-Fe) 3+ The changes in the position and intensity of characteristic absorption peaks were used to evaluate Fe. 3+ The influence of coordination on the electronic structure and light absorption behavior of the system was investigated, further confirming the formation of the complex. PPDG and its Fe... 3+The hydrodynamic particle size distribution and surface charge properties of the composite (PPDGFE) were determined using a Malvern Zetasizer Nano ZS instrument. The sample concentration was 0.2 mg·mL⁻¹. -1 The experiments were conducted at room temperature. Dynamic light scattering (DLS) was used to obtain the average hydrodynamic diameter and particle size distribution of the system in the aqueous phase, while Zeta potential measurements were used to evaluate the surface charge characteristics and colloidal stability of the composite. Theoretical calculations were performed using Materials Studio software. First, DMol... 3 Module construction of PPDG and its Fe 3+ A molecular model of the composite system (PPDGFE) was developed, and its geometry was optimized at the B3LYP / 6-31G(d,p) basis set level. The binding energy (E_b), HOMO-LUMO energy level distribution, and molecular charge distribution of the system were calculated to theoretically analyze the Fe... 3+ The optimal coordination sites and the stability of the complex were determined, providing theoretical support for the experimental characterization results. Furthermore, the spin polarization of the PPDGFE composite system was calculated using the CASTEP module in Materials Studio, focusing on analyzing the changes in the system's magnetic properties, particularly the evolution of the integral spin density, thereby revealing the Fe... 3+ The regulatory mechanism of paramagnetic properties of the system before and after coordination. Structural and performance characterization of HAMA@PPDG microspheres: The morphology of HAMA@PPDG microspheres was observed using field emission scanning electron microscopy (FE-SEM, Zeiss GeminiSEM 500), including the microstructure of the microsphere surface and cross-section. Simultaneously, energy dispersive spectroscopy (EDS) was used to obtain the mapping distribution of elements such as Si, C, O, N, and S to verify the uniform dispersion of PPDG in the HAMA microspheres. The pore structure of the microspheres was determined using mercury intrusion porosimetry (Micromeritics AutoPore IV 9500). The total porosity, pore volume, and pore size distribution of the microspheres were obtained by recording the high-pressure mercury intrusion-extrusion curves. The in vitro degradation behavior of the microspheres was evaluated in PBS (pH=7.4) under shaking conditions at 37°C. Samples were periodically removed during the experiment, freeze-dried, weighed, and their mass loss ratio was calculated. The degradation supernatant was also collected for PPDG release behavior testing. Release kinetics were quantitatively analyzed using the UV-Vis absorption method, and the cumulative release rate was calculated in conjunction with liquid replenishment correction, thereby systematically evaluating the sustained degradation and sustained release performance of HAMA@PPDG microspheres under physiological conditions.

[0054] The results are as follows Figure 3 As shown, where, Figure 3Figure A shows that SH-POSS serves as an octahedral cage-like rigid framework. Deferroamine (DFO), gallic acid (GA), and polyethylene glycol (PEG) are covalently grafted onto the POSS surface in a one-pot process under mild conditions via a DMPA-initiated thiol-olefin click reaction. In this structure, DFO provides multidentate coordination sites to achieve Fe... 3+ The high efficiency of chelation by GA endows it with continuous antioxidant capacity, while the introduction of PEG significantly improves the water solubility and biocompatibility of the material. 1 H NMR spectroscopy ( Figure 3 In (B) of the spectrum, the -CH2CH2O- signal of the PEG backbone appears at δ 3.50-3.80 ppm, the GA aromatic ring protons are located at 6.60-7.20 ppm, and the -CH2-N- and -CH2-O- signals of DFO are distributed at 2.80-4.10 ppm; simultaneously, the olefin proton peak (5.70-6.40 ppm) essentially disappears, indicating that the click reaction proceeded fully. FTIR spectra ( Figure 3 In C), the characteristic peaks of Si-O-Si are located at 1030-1110 cm⁻¹. -1 -OH (3200-3500cm) -1 ) and C=O (1650-1730cm) -1 The absorption was significantly enhanced, while the -SH characteristic peak was significantly weakened, further verifying the successful construction of the multifunctional molecule. (UV-Vis absorption spectrum) Figure 3 As shown in D), PPDG exhibits a significantly broadened and enhanced absorption characteristic in the 200–350 nm region, distinct from single POSS or DFO, indicating that GA undergoes π→π... Transitions and the n→π transition of DFO The transition achieves cooperative coupling on the POSS framework. With Fe... 3+ With increasing equivalent (1–5 eq), the absorption intensity of PPDG in the 280–350 nm region significantly increased, accompanied by a slight redshift. Figure 3 The E in Fe³ exhibits typical metal-ligand charge transfer (MLCT) characteristics, indicating that multiple coordination sites are synergistically involved in Fe³⁺. + Chelation forms stable complexes. Dynamic light scattering results ( Figure 3 The F in the figure shows that the hydrated particle size of POSS-PEG is approximately 142 nm, while the particle size of PPDG increases to approximately 238 nm, reflecting the change in molecular aggregation behavior after functionalization. Zeta potential measurement ( Figure 3 The G in the figure further indicates that PPDG chelates Fe. 3+ The back surface potential is from The voltage reversed from 25mV to approximately +40mV, directly proving that Fe 3+ It is efficiently captured and deeply involved in interfacial charge reconstruction. This provides a quantum chemical perspective for understanding the effects of chelation behavior on Fe.3+ The influence of electronic structure was further investigated through DFT calculations. Frontier molecular orbital analysis ( Figure 3 The presence of H and I indicates that PPDG chelates Fe. 3+ The HOMO–LUMO band gap decreased significantly from 2.451 eV to 0.398 eV, indicating that coordination significantly enhanced charge transfer capability and weakened Fe. 3+ The degree of localization of unpaired electrons. Electrostatic potential analysis ( Figure 3 J) shows that the PPDG surface forms a continuous negative potential enrichment region around oxygen- and nitrogen-containing coordinating atoms, which is conducive to Fe 3+ It provides preferential binding sites. The above theoretical results are highly consistent with spectroscopic, particle size, and potential changes, systematically demonstrating that PPDG can precisely regulate Fe through strong chelation. 3+ The electronic structure and interfacial electrical properties of the sample lay the molecular basis for subsequent paramagnetic attenuation and T2-MRI visualization monitoring.

[0055] To verify whether the HAMA@PPDG microspheres prepared by microfluidic method possess high uniformity in size, hierarchical porous structure, and uniform composition distribution, this application first performed morphological and elemental distribution characterization. For example... Figure 4 As shown in Figure A, SEM reveals that the microspheres are generally spherical with an average size of approximately 300 μm. They have a rough surface and an interconnected honeycomb-like porous network structure with continuous pore walls and interconnected channels, indicating that the microspheres formed a stable three-dimensional porous structure during cross-linking and swelling. This structure is beneficial for improving the loading capacity of functional components and provides low-resistance channels for the diffusion of water molecules, iron ions, and drugs, laying the morphological foundation for subsequent controllable degradation and sustained release. EDS mapping analysis ( Figure 4 Figures B and D show that C, O, N, S, and Si elements are uniformly distributed within individual microspheres. C and O originate from the HAMA backbone and organic side groups, N and S correspond to the functional units of PPDG, and the Si signal comes from the POSS cage-like framework. The continuous elemental distribution and lack of obvious aggregation indicate that PPDG is uniformly immobilized within the HAMA network. Particle size statistics (…) Figure 4 Further analysis (C) in the image shows that the microspheres exhibit a narrow particle size distribution, with an average particle size of 310.6 ± 6.2 μm, demonstrating the advantages of microfluidic systems in terms of size controllability and batch stability. To further confirm the chemical composition and bonding state of the multifunctional components in the microspheres, we performed XPS analysis. Full spectrum (… Figure 4 Characteristic peaks of C, O, N, S, and Si can be observed in the high-resolution spectrum (E). Figure 4The F–J analysis shows that C1s can decompose into C–C / C–H, C–O / C–N, and C=O components; O1s consists of carbonyl oxygen and hydroxyl / ether oxygen; N1s represents nitrogen-containing chelating units; S2p shows the coexistence of sulfur bonding and partial oxidation states; Si2p mainly corresponds to Si–O and Si–C bonds, directly verifying the stable introduction of the POSS structure into the network. These results are highly consistent with EDS analysis, jointly confirming the successful construction of HAMA@PPDG microspheres from both elemental composition and chemical state perspectives.

[0056] To assess its long-term therapeutic potential, this application further investigated the in vitro release and degradation behavior of the microspheres. Figure 4 As shown in K, the microspheres exhibit a mild initial release followed by a stable and sustained release phase: approximately 20–35% cumulative release in the first 1–2 weeks, 65–70% at week 4, and nearly 90–95% at week 8. The corresponding degradation curves ( Figure 4 The L (in the image) shows that the microsphere mass gradually decreased over time, with approximately 15–20% remaining after 8 weeks, indicating that the system has good degradability. Release and degradation behavior are highly correlated, suggesting that drug release is mainly regulated by a degradation-diffusion synergistic mechanism. Mercury porosimetry results ( Figure 4 Further analysis (M) reveals that the overall porosity of the microspheres is approximately 65.7%, with a characteristic pore size of about 485 nm. This high porosity and concentrated pore size distribution provide a structural basis for their efficient mass transfer and long-term sustained release. In summary, the microfluidically constructed HAMA@PPDG microspheres exhibit significant advantages in size uniformity, component stability, and pore structure regulation. Furthermore, they achieve sustained peri-scale release through a degradation-diffusion synergistic mechanism, providing a reliable material basis for their long-term delivery and therapeutic applications within the joint cavity.

[0057] Example 4: Synergistic inhibitory effect of HAMA@PPDG on chondrocyte ferroptosis To verify the biosafety of the microsphere material at the cellular level and its ability to intervene in chondrocyte ferroptosis, the following experimental methods were used: (1) Evaluation of cytotoxicity and biosafety To eliminate the cytotoxicity of the material itself and ensure the reliability of subsequent ferroptosis assay results, the in vitro cell compatibility of the material was first systematically evaluated. The cytotoxicity of the material to BMSCs was assessed using a cell counting kit (CCK-8, MCE, China). BMSCs were counted at 1×10⁻⁶. 4Cells were seeded at a density of [number] cells / mL in 96-well plates. After cell adhesion, PBS (control group), PPD, HAMA, PPDG, and HAMA@PPDG (final concentration of 0.05 mg / mL) were added to each well. Cells were cultured in a humidified incubator at 37°C and 5% CO2, with the culture medium changed every 2 days. At 24, 48, and 72 h after treatment, 90 μL of LMEM and 10 μL of CCK-8 solution were added to each well, and incubation was continued for 2 h. Subsequently, absorbance was measured at 450 nm using a microplate reader (SpectraMax i3x, MolecularDevices, USA) to evaluate the effect of different treatment groups on cell viability.

[0058] The survival status of BMSCs after material treatment was further visualized using a cell apoptosis detection kit (Beyotime, China). BMSCs were collected at a concentration of 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 0.05 mg / mL in 24-well plates, with the same groupings as above (PBS, PPD, HAMA, PPDG, HAMA@PPDG, 0.05 mg / mL), and incubated at 37°C and 5% CO2 for 24, 48, and 72 h, respectively. After culture, the culture medium was discarded, and the cells were thoroughly washed with PBS. Staining working solution was then added to each well, and the cells were incubated at room temperature for 20 min in the dark. Annexin V labeled live cells showed green fluorescence, while PI labeled dead cells showed red fluorescence. After staining, cell morphology and live / dead cell distribution were observed using an inverted fluorescence microscope (IX73, Olympus, Japan). For quantitative analysis, ImageJ (v1.54, NIH, USA) software was used for semi-automatic analysis of the fluorescence images. Live cell signals were extracted and counted using a standardized image processing workflow. The number of live cells was calculated using the following formula: N live =N counted ×F area ; Where, N live N represents the total number of living cells. counted F represents the number of live cells counted in a single image. area This is the area correction factor.

[0059] (2) Erastin induces ferroptosis. To construct a chondrocyte ferroptosis model, Erastin was selected as a specific ferroptosis inducer in this application. The experiment began with human or mouse chondrocytes cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 until 70–80% confluence. After the cells reached the logarithmic growth phase, Erastin (10 μM) was added for induction for 24 hours to ensure that the cells exhibited typical ferroptosis characteristics, including ROS accumulation, increased lipid peroxidation levels, and Fe2+. 2+ Increased intracellular content. Control group cells were untreated or only supplemented with the appropriate solvent (DMSO) to eliminate solvent effects.

[0060] Next, to evaluate the intervention effect of magnetically responsive materials on ferroptosis, this application set up multiple experimental groups: a PBS control group, an Erastin model group, and four material intervention groups, including HAMA, PPD, PPDG, and HAMA@PPDG. The intervention experiments adopted a sequence of injecting the material first and then inducing ferroptosis; that is, the cells in the intervention groups were first treated with different materials (0.05 mg / mL), incubated for 2 hours, and then Erastin inducer was added. By comparing with the model group, the effects of various treatment group materials on inhibiting Erastin-induced ferroptosis, reducing ROS accumulation, inhibiting lipid peroxidation, and regulating Fe... 2+ The effect on internal content provides experimental evidence for further research on the cell-protective effect of magnetically responsive materials.

[0061] (3) Detection of key indicators of ferroptosis To evaluate the intervention effect of magnetically responsive materials on chondrocyte ferroptosis, this application measured intracellular reactive oxygen species (ROS), lipid peroxidation levels, and Fe2+ levels, respectively. 2+ Accumulation. ROS levels were detected using the DCFH-DA fluorescent probe: After the experiment, cells were washed three times with PBS to remove residual culture medium, then 10 μM DCFH-DA probe was added, and the cells were incubated at 37°C for 30 min to allow the probe to enter the cells and be oxidized by ROS to generate fluorescent products. After incubation, excess probe was removed, and the cells were washed again with PBS. The fluorescence signal was then observed using confocal microscopy. Lipid peroxidation levels were detected using the C11-BODIPY (581 / 591, OX-C11) probe. Cells treated with different materials were added with 2 μM MOX-C11 probe and incubated at 37°C for 30 min to allow the probe to embed into the cell membrane and become sensitive to lipid peroxidation. After incubation, the cells were washed with PBS, and changes in red-green fluorescence signals were detected using confocal microscopy. A decrease in the red-green fluorescence ratio reflects an increase in the degree of cellular lipid peroxidation, which can be used to visually assess ferroptosis and the effect of material intervention. 2+FerroOrange fluorescent probes were used to detect intracellular exchangeable iron ions. After cell incubation, cells were washed three times with PBS, and then the probe was added and incubated for 30 min according to the manufacturer's instructions. After incubation, excess probe was removed, cells were washed with PBS, and the fluorescence signal intensity was measured using a fluorescence microscope. Fe² + Changes in fluorescence intensity can reflect the material's regulatory effect on iron ion homeostasis. All cell assay data were repeated at least three times (n≥3). Fluorescence signal intensity was quantified using ImageJ analysis software. Results are expressed as mean ± standard deviation and statistically analyzed using one-way ANOVA or t-tests. The fluorescence intensity was measured by ROS, lipid peroxidation, and Fe... 2+ A comprehensive analysis of the three indicators can systematically evaluate the inhibitory effect and potential mechanism of magnetically responsive materials on chondrocyte ferroptosis.

[0062] Experimental results: like Figure 5 As shown in Figure A, Live / Dead staining results indicated that after culturing for 1–3 days under Control, PPD, HAMA, PPDG, and HAMA@PPDG conditions, all groups of cells showed predominantly green live cell signals, with almost no red dead cells observed, indicating that the material had no significant cytotoxicity at the tested concentrations. Cell density quantification results ( Figure 5 (B) shows that the cell count in each group increased with increasing culture time, with the cell density in the PPDG and HAMA@PPDG groups being slightly higher than that in the control group. CCK-8 results ( Figure 5 Further analysis (C) showed that the HAMA@PPDG group exhibited the highest metabolic activity (OD≈1.45–1.55) on days 2–3, indicating that the system has certain cell growth-promoting potential while ensuring biocompatibility. After confirming good material compatibility, an Erastin-induced chondrocyte ferroptosis model was constructed, and the regulatory effects of different materials on key ferroptosis phenotypes were evaluated. DCFH-DA probe results ( Figure 5 The results (D) showed that Erastin treatment significantly increased intracellular ROS levels, while HAMA@PPDG treatment significantly reduced ROS signaling, bringing it close to that of the control group. Quantitative analysis ( Figure 5 The results (G) indicate that HAMA@PPDG reduced ROS levels to approximately 20–25% in the Erastin group, significantly better than other treatment groups. Further analysis using the C11-BODIPY probe revealed lipid peroxidation levels. Figure 5 As shown in Figure E, Erastin treatment significantly enhanced the oxidized C11-BODIPY (OX-C11) fluorescence, while HAMA@PPDG treatment almost completely suppressed the OX-C11 signal and restored the fluorescence characteristics dominated by the reduced state (NO-OX-C11). The corresponding quantitative results ( Figure 5 The H and I results showed that the OX-C11 intensity and OX / NO-OX ratio of the HAMA@PPDG group were significantly lower than those of the Erastin group and other material groups, indicating that it has the strongest efficacy in blocking the lipid peroxidation chain reaction.

[0063] Given that iron accumulation is an upstream driver of ferroptosis, intracellular Fe was further examined. 2+ Horizontal. For example... Figure 5 As shown in F, Erastin treatment leads to Fe 2+ The probe fluorescence was significantly enhanced, and both PPDG and HAMA@PPDG treatments significantly reduced Fe. 2+ The signal decreased most significantly in the HAMA@PPDG group. Quantitative results ( Figure 5 J) shows that HAMA@PPDG can increase intracellular Fe 2+ The levels decreased to below 20% of those in the Erastin group. In summary, in vitro experiments systematically demonstrated that HAMA@PPDG, while maintaining excellent biocompatibility, can significantly inhibit key phenotypes of chondrocyte ferroptosis, including Fe, through a synergistic effect of highly efficient iron removal and sustained antioxidant activity. 2+ Accumulation, ROS bursts, and lipid peroxidation effectively restore cellular homeostasis. This result is highly consistent with the aforementioned electronic structure regulation and chelate iron-magnetic response analysis, providing a solid cellular basis for its further in vivo application in ferroptosis-related diseases.

[0064] Example 5: Real-time MRI Visualization of Cartilage Repair Based on HAMA@PPDG To achieve real-time, non-invasive, and quantifiable monitoring of ferroptosis-related cartilage degeneration and to simultaneously assess the therapeutic response to material interventions, we used ultra-high field MRI for longitudinal follow-up imaging of different treatment groups, and used contrast-to-noise ratio (CNR) and three-dimensional cartilage structural parameters (thickness, area, and volume) as quantitative indicators. Figure 6 As shown in Figure A, during short-term follow-up (Pre, 1D, 2D, 4D, 8D), both the HAMA@PPDG and PPDG groups showed clearer cartilage contours and more defined cartilage-subchondral bone signal boundaries, while the PBS, Erastin, and HAMA groups showed lower overall contrast and blurred cartilage layer visualization. CNR quantitative analysis results ( Figure 6C) further validated the above observations. The PPDG group showed a rapid CNR peak at 1 day, suggesting that free PPDG can rapidly participate in iron ion complexation and alter the local magnetic relaxation environment in the early stages. In contrast, the CNR of the HAMA@PPDG group reached a higher level at 1–2 days and remained significantly higher than the control group at 4–8 days, indicating a longer time window for imaging enhancement. This difference suggests that the HAMA carrier prolongs the residence time of the effective imaging components in the joint cavity through anchoring and sustained release, enabling HAMA@PPDG to maintain a more stable and sustained ability to visualize cartilage layers in the mid-to-late stages. To assess whether the imaging enhancement truly reflects the tissue protection effect, we further conducted mid-to-long-term follow-up analysis of cartilage structural parameters. Figure 6 As shown in Figure B, during imaging at 1–4 weeks, the cartilage layer structure in the HAMA@PPDG group remained intact and its contours were continuous, while the PPDG group gradually showed cartilage thinning and blurred boundaries over time. Quantitative results showed ( Figure 6 In the D–F group, the cartilage thickness of the HAMA@PPDG group showed a continuous increasing or stable trend, and the cartilage area and volume remained at a high level during the follow-up period. In contrast, the improvement in the PPDG group was limited, and some parameters decreased in the later stage, suggesting that its protection of the three-dimensional structure of cartilage was insufficient.

[0065] From a mechanistic perspective, PPDG can rapidly chelate excess iron ions in the lesion microenvironment in the early stages, reshaping the local magnetic relaxation environment and achieving T2 contrast enhancement. HAMA@PPDG, by extending the material's residence time within the joint cavity and providing periodic sustained release, significantly amplifies the chelation of iron and its antioxidant effects over time. This allows it to continuously protect the cartilage matrix and structural stability while inhibiting iron-dependent ROS generation and lipid peroxidation. In summary, HAMA@PPDG not only achieves long-term clear imaging of degenerated cartilage but also effectively slows down the cartilage degeneration process at the structural parameter level, indicating that its imaging enhancement effect is highly consistent with actual tissue protection. Through a chelation strategy with adjustable residence time, HAMA@PPDG effectively decouples early imaging contrast generation from long-term tissue protection over time, providing a new material design paradigm for the integrated visualization and diagnosis of ferroptosis-related diseases.

[0066] Example 6: In vivo efficacy evaluation of HAMA@PPDG in blocking the auxotrophic chondrocyte death process in osteoarthritis (OA). Animal experiments: This experiment was approved by the Animal Research Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, and all surgical procedures were performed in accordance with the guidelines of the National Institutes of Health.

[0067] Eight-week-old male Sprague–Dawley (SD) rats, weighing 220–250 g, were purchased from Zhejiang Landon Biotechnology Co., Ltd., China. In this application, to establish an animal model of ferroptosis-related osteoarthritis (OA), the model was constructed by combining classic anterior cruciate ligament (ACLT) transection with intra-articular Erastin injection, referencing methods reported in existing literature. A total of 25 rats were randomly divided into five groups (n=5 per group): PBS control group (ACLT+PBS), Erastin model group (ACLT+Erastin), PPDG intervention group (ACLT+Erastin+PPDG), gel group (ACLT+Erastin+HAMA), and composite material group (ACLT+Erastin+HAMA@PPDG). Surgical procedures were performed under strict aseptic conditions. Rats were first anesthetized with isoflurane, and the surgical area was routinely disinfected. The anterior cruciate ligament (ACL) was exposed through a knee joint incision and carefully transcribed to establish the osteoarthritis model. Postoperatively, appropriate materials were injected into the joint cavity according to the group for intervention: Erastin was injected once at a dose of 1 mg / kg, while PPDG, HAMA, and HAMA@PPDG were injected twice a week at 200 μL each time for 4 weeks. The PBS control group received an equal volume of physiological saline. At week 8 after model establishment, knee joint specimens were collected by euthanasia for subsequent histological, imaging, and molecular biological analysis. This method can stably induce ferroptosis in OA cartilage and evaluate the role of different materials in alleviating iron-dependent oxidative stress and cartilage degeneration, providing a reliable experimental basis for evaluating the effectiveness of subsequent interventions.

[0068] High-field MRI imaging of rat knee joint: The cartilage of the rat knee joint was imaged using a Bruker 9.4T small animal magnetic resonance imaging system (BAP94 / 31, Bruker, Germany). Before imaging, rats were anesthetized with isoflurane inhalation (induction concentration 3–4%, maintenance concentration 1.5–2.0%, oxygen carrier) and placed in a specialized small animal fixation device, maintaining the knee joint in natural extension. During imaging, the animals' vital signs were monitored in real time using a respiratory monitoring system, and their body temperature was maintained at 37°C. A Bruker-specific small animal knee joint coil was used for scanning. Cartilage structure imaging employed a T2-weighted turboRARE (fast spin echo) sequence with the following main parameters: repetition time (TR) approximately 2500–3000 ms, echo time (TE) approximately 30–40 ms, field of view (FOV) approximately 20 × 20 mm, matrix size 256 × 256, slice thickness 0.7 mm, slice interval 1 mm, and number of excitations (NEX) = 4. The scanning plane was set along the sagittal plane to fully cover the articular cartilage of the femoral condyle and tibial plateau. According to the experimental design, longitudinal repeated scans of the same knee joint were performed at different time points before and after drug administration to dynamically assess changes in cartilage structure and differences in contrast.

[0069] After imaging, all MRI data were imported into Bruker ParaVision 6.0 software for reconstruction and analysis. Regions of interest (ROIs) were defined as the femoral condyle and tibial plateau articular surfaces. The signal intensity of cartilage and subchondral bone was measured, and the contrast-to-noise ratio (CNR) of cartilage relative to subchondral bone was calculated. Simultaneously, quantitative analysis of cartilage thickness, area, and volume was performed based on multi-slice serial sections to evaluate the effects of different treatments on cartilage degeneration and repair in rat knee joints.

[0070] CT imaging of rat knee joint: CT imaging of the rat knee joint was performed using a high-resolution micro-CT system (Skyscan 1176, Bruker, Belgium). At the end of the experiment, rats were sacrificed, the knee joint specimen was dissected, surrounding soft tissue was removed, and the specimen was fixed in 4% paraformaldehyde for 24 hours. After fixation, the sample was placed in a scanning tube and held in a stable position for micro-CT scanning. Scanning parameters were set as follows: tube voltage 50–60 kV, tube current 400–500 μA, pixel resolution approximately 9–18 μm, rotation step angle 0.5°, and scanning angle range 180°. A 0.5 mm aluminum filter was used during scanning to reduce artifacts caused by low-energy X-rays. All data were reconstructed in three dimensions using NRecon software (Bruker). Osteophyte assessment was performed based on the reconstructed three-dimensional image data. Regions of interest (ROIs) were defined as the articular margins of the distal femur and proximal tibia. Osteophytes were defined as newly formed bone structures located at the joint margins, protruding outwards from the joint cavity, and continuous with the existing cortical bone. The CTAn software (Bruker) was used to perform semi-automatic segmentation of osteophytes, and the total osteophyte volume was calculated under a uniform threshold condition to quantitatively compare the differences in the degree of osteophyte formation among different treatment groups.

[0071] Tissue section staining and immunohistochemistry / fluorescence staining: Eight weeks after ACL transection, all rats were sacrificed, and knee joints were harvested for histological analysis. Joint specimens were first fixed in 4% paraformaldehyde solution, decalcified using 10% EDTA solution, then dehydrated via graded ethanol treatment, and embedded in paraffin. Sections were prepared at a thickness of 5 μm, and serial sections were used for subsequent staining and analysis.

[0072] (1) Histopathological staining To assess cartilage degeneration and matrix changes, sections were stained with Alcian Blue (AB), Safranin O / Fast Green (SF), and Masson's trichrome. After staining, changes in cartilage structure were observed using confocal microscopy or optical microscopy. ImageJ software was used for quantitative analysis of cartilage wear depth and glycosaminoglycan content. Simultaneously, the OARSI semi-quantitative scoring system was used to assess the degree of cartilage degeneration. The score comprehensively considered lesion depth (0–6 points) and lesion extent (0–4 points), with the total score being the product of the depth and extent scores, used to compare the degree of cartilage damage among the experimental groups.

[0073] (2) Immunohistochemical staining (IHC) Paraffin sections were digested with 0.5% pepsin, then non-specific binding sites were blocked with 3% H2O2 and 1% BSA solution, and finally blocked with PBS containing 5% bovine serum albumin for 30 min. Primary antibody Prussian Blue (PB) was added, and the sections were incubated overnight at 4°C. The following day, the corresponding secondary antibody was added, and DAB staining was performed followed by hematoxylin counterstaining to observe iron deposition and the distribution of iron-related damage in the tissues.

[0074] (3) Immunofluorescence staining (IF) After dewaxing, antigen retrieval, and blocking of paraffin sections, primary antibodies GPX4, ACSL4, and Col2 (type II collagen) were added, and the sections were incubated overnight at 4°C. The next day, fluorescently labeled secondary antibodies were added for development, followed by mounting. Images were acquired using a confocal microscope (PCOM, Nikon, Japan), and the fluorescence signals were quantitatively analyzed using ImageJ software to assess the expression levels of ferroptosis-related proteins and cartilage matrix collagen.

[0075] All staining and immunoassay experiments were performed in five groups: PBS control group, Erastin group, HAMA intervention group, PPDG intervention group, and HAMA@PPDG intervention group. At least three rat sections were used in each group for repeated experiments. By combining histopathological, immunohistochemical, and immunofluorescence analyses, the degree of cartilage degeneration, iron deposition, and the effects of the materials on ferroptosis and cartilage protection could be systematically evaluated.

[0076] Experimental results: like Figure 7 As shown in Figure A, the Micro-CT 3D reconstruction results revealed that the knee joint structure in the Sham group was intact, while the Erastin group exhibited significant osteophyte formation, irregular articular surfaces, and disordered bone structure; HAMA or PPDG treatment alone only partially improved these changes. In contrast, the overall joint morphology of the HAMA@PPDG group was closest to that of the Sham group, with a significant reduction in osteophyte protrusions and a marked restoration of joint structural continuity. Quantitative analysis of osteophyte volume further confirmed this trend. Figure 7 In section B): The total osteophyte volume was significantly increased in the Erastin, HAMA, and PPDG groups, but significantly decreased in the HAMA@PPDG group, with no statistically significant difference compared to the Sham group (ns), indicating that it can effectively inhibit pathological bone remodeling at the structural level. Meanwhile, the OARSI score results ( Figure 7 The results in (C) show that the HAMA@PPDG group had a significantly lower cartilage pathology score than Erastin and other treatment groups, and recovered to a level close to Sham's, indicating that its inhibition of OA progression is not limited to bone structure improvement, but rather achieves substantial blockade of cartilage degeneration.

[0077] Histological staining results ( Figure 7(D) further reveals the histological basis for cartilage matrix preservation. Compared with the Erastin group, which showed cartilage surface damage, GAG loss, and collagen structure disorder, the HAMA@PPDG group maintained the integrity of the cartilage columnar structure, with significantly restored SF and AB staining intensity, and Masson staining showing significantly improved collagen network continuity. Immunofluorescence and immunohistochemical analysis showed that GPX4 expression was significantly upregulated and ACSL4 expression was significantly downregulated in the HAMA@PPDG group, while COLII signal was enhanced, directly indicating that ferroptosis-related lipid peroxidation was inhibited and the characteristic collagen matrix of cartilage was effectively maintained. In summary, Micro-CT and histological evidence systematically demonstrate that HAMA@PPDG can simultaneously inhibit osteophyte formation and cartilage matrix degeneration in vivo, reverse ferroptosis-related molecular phenotypes, and thus effectively block the pathological process of Erastin-induced OA. Combined with the aforementioned dynamic MRI visualization results, HAMA@PPDG achieves synergistic unity of imaging assessment and disease intervention through a chelate iron strategy with adjustable residence time, demonstrating its significant advantages in the precise diagnosis and treatment of ferroptosis-related OA.

[0078] In summary, this application proposes a method based on binding energy (ΔE) at the electronic structure level. bind A novel strategy for designing ferroptosis diagnostic and therapeutic materials based on integral spin density (ISD). Through density functional theory calculations, a quantitative criterion was established for the first time: when ΔE bind < 5.8 eV and ISD < 3.2 At / 2, the material can efficiently chelate Fe 3+ Simultaneously, it weakens the paramagnetism of the material, achieving effective modulation of T2-MRI signals and meeting the synergistic requirements of "strong chelation—weak paramagnetism—imaging capability." Guided by this theory, a multifunctional microsphere system, HAMA@PPDG, was constructed, integrating iron chelation, anti-oxidation, and MRI imaging. Experiments show that this system can effectively inhibit iron overload-induced lipid peroxidation, alleviate ferroptosis-related cartilage degeneration in cell and animal models, and achieve dynamic visualization monitoring of the treatment process via T2-MRI. This application establishes the intrinsic correlation between electronic structural parameters, material function, biological effects, and imaging response, providing a predictable and controllable theoretical framework for the rational design and application of ferroptosis diagnostic and therapeutic materials.

[0079] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A material for the integrated diagnosis and treatment of ferritinia, characterized in that, include Methacrylamide hyaluronic acid hydrogel matrix; and Hybrid molecules dispersed within the hydrogel matrix; The hybrid molecule includes an octamercaptopropyl polyhedral oligosilsesquioxane backbone and functional molecules grafted onto the backbone via covalent bonds. The functional molecules include polyethylene glycol acrylate, gallic acid acrylate, and deferoxamine acrylate.

2. The preparation method of the integrated diagnostic and therapeutic material for ferrodeogenesis as described in claim 1, characterized in that, Includes the following steps: Step 1: Prepare polyethylene glycol acrylate, gallic acid acrylate and deferoxamine acrylate; Step 2: The polyethylene glycol acrylate, gallic acid acrylate and deferoxamine acrylate are covalently grafted onto the octamercaptopropyl polyhedral oligomeric silsesquioxane backbone to obtain a hybrid molecule, named PPDG. Step 3: Prepare methacrylamide hyaluronic acid lyophilized hydrogel, named HAMA; Step 4: The PPDG obtained in Step 2 and the HAMA obtained in Step 3 are mixed as the aqueous phase, and the oily medium and emulsifier are used as the oil phase. The integrated material for the diagnosis and treatment of ferroptosis is prepared by microfluidic interface solidification method and named HAMA@PPDG.

3. The preparation method according to claim 2, characterized in that, In step 1, the preparation method of polyethylene glycol acrylate is as follows: Polyethylene glycol was dissolved in dichloromethane, an acid-binding agent was added, and acryloyl chloride was slowly added dropwise under ice bath conditions. The reaction system was stirred at 0°C. After the reaction was completed, the reaction solution was washed, dried, filtered, and evaporated to dryness to obtain the polyethylene glycol acrylate.

4. The preparation method according to claim 2, characterized in that, In step 1, the preparation method of gallic acid acrylate is as follows: Gallic acid was dissolved in anhydrous ethanol, an acid-binding agent was added, and acryloyl chloride was slowly added dropwise under ice bath conditions. The reaction system was stirred at 0°C. After the reaction was completed, the reaction solution was washed, dried, filtered, and evaporated to dryness to obtain the gallic acid acrylate.

5. The preparation method according to claim 2, characterized in that, In step 1, the preparation method of deferoxamine acrylate is as follows: Deferroamine was dissolved in anhydrous N,N-dimethylformamide, an acid-binding agent was added, and acryloyl chloride was slowly added dropwise under ice bath conditions. The reaction system was stirred at 0°C, and then the temperature was raised to room temperature to continue the reaction. After the reaction was completed, the reaction solution was slowly poured into a large amount of ice-cold anhydrous diethyl ether, the precipitate was collected by centrifugation, washed, and freeze-dried to obtain the deferroamine acrylate.

6. The preparation method according to claim 2, characterized in that, In step 2, the molar ratio of octamercaptopropyl polyhedral oligomeric silsesquioxane, polyethylene glycol acrylate, gallic acid acrylate, and deferoxamine acrylate is 1:4:0.25:

3.

7. The preparation method according to claim 2, characterized in that, The steps in step 3 are as follows: Hyaluronic acid was dissolved in preheated phosphate-buffered saline solution and stirred continuously until a clear and homogeneous solution was formed. Then, methacrylic acid was slowly added, and the reaction system temperature was controlled at 50°C. After the reaction was completed, excess PBS was added to the system to terminate the reaction, and the dilution-displacement operation was repeated several times. The reaction solution was then transferred to a dialysis bag for dialyzing and freeze-dried to obtain the methacrylamide hyaluronic acid lyophilized hydrogel, named HAMA.

8. The preparation method according to claim 2, characterized in that, The steps in step 4 are as follows: The hybrid molecule PPDG was dissolved in phosphate buffered saline, and then pre-dissolved lyophilized hydrogel HAMA was added. After thorough stirring, a homogeneous and stable aqueous phase was formed. Paraffin oil is mixed with an emulsifier to form an oil phase; During the microfluidic preparation process, the aqueous phase and oil phase are injected through the internal and external phase channels of the microfluidic chip, respectively, where the internal phase is the aqueous phase and the external phase is the oil phase. The flow rates are set to 0.8 mL·h for the aqueous phase. -1 oil phase 16 mL·h -1 The generated droplets further cross-link and solidify at 80°C to form microspheres. The obtained microspheres are collected by centrifugation and washed repeatedly with petroleum ether and deionized water to finally obtain the HAMA@PPDG integrated material for the diagnosis and treatment of ferrodeogenesis.

9. The application of the integrated ferroptosis diagnostic and therapeutic material according to claim 1 in the preparation of diagnostic and therapeutic products for ferroptosis-related diseases.

10. The application according to claim 9, characterized in that, The ferroptosis-related diseases include osteoarthritis, neurodegenerative diseases, ischemia-reperfusion injury, and tumors.

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