Ultra-small metal polyphenol network targeting mitochondria GA-Fe@TPP, and preparation method and application thereof

Abstract

The application discloses a mitochondrion-targeting ultra-small metal polyphenol network GA-Fe@TPP, which takes gentisic acid as a ligand, Fe 3+ is assembled by nodes, and is surface-decorated by 4-carboxybutyl triphenylphosphonium bromide. The application further provides a preparation method and application of the mitochondrion-targeting ultra-small metal polyphenol network GA-Fe@TPP. The mitochondrion-targeting ultra-small metal polyphenol network GA-Fe@TPP can not only inhibit crystal growth, but also block ROS explosion and reshape mitochondrial redox homeostasis by virtue of the synergistic effect of the strong free radical scavenging activity of GA and the Fe-coordination network, and can be used for effectively treating and / or preventing kidney stones, and has extremely high clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mitochondrial-targeting ultrasmall metal polyphenol network GA-Fe@TPP, its preparation method, and its application. Background Technology

[0002] Nephrolithiasis is a prevalent urinary tract disease worldwide, with a high lifetime prevalence and a high five-year recurrence rate after surgery. Recurrent stone attacks, if not effectively controlled, not only severely impact patients' quality of life but also often involve irreversible renal parenchymal damage, making it a significant contributing factor to chronic kidney disease (CKD) and even end-stage renal disease (ESRD). Pathological evidence indicates that stones primarily composed of calcium oxalate are not simply physical precipitates from urine; their formation is a continuous pathological process involving crystal deposition, cell damage, and recrystallization, defined as typical crystal nephropathy. Under conditions of high oxalate load or local microenvironmental imbalance, crystals deposited on the renal tubular lumen and epithelial surface can induce a cascade of oxidative stress and inflammation, accelerating the deterioration of renal function. However, current clinical interventions are mainly limited to surgical physical removal of existing stones, lacking specific drugs capable of blocking crystal-cell interactions at the source and reversing the pathological mineralization microenvironment.

[0003] Numerous basic and translational studies suggest that oxidative stress and mitochondrial dysfunction play pivotal roles in calcium oxalate-related kidney injury. On one hand, high oxalate and calcium oxalate crystals significantly deplete glutathione in renal tubular cell mitochondria, inducing mitochondrial membrane potential collapse and cytochrome c release, accompanied by altered antioxidant enzyme activity and persistent accumulation of reactive oxygen species (ROS), indicating that mitochondrial redox imbalance is a key initiating event in the development of crystalline nephropathy. On the other hand, in both acute kidney injury and other renal pathological states, mitochondrial structural damage, impaired oxidative phosphorylation, and metabolic disorders are closely related to a rapid decline in renal function; therefore, mitochondria are considered a core therapeutic target for blocking the progression of renal pathology. Currently, specific interventions for kidney stones targeting mitochondrial oxidative stress and energy metabolism disorders are extremely limited. Cis-targeting mitochondrial antioxidant strategies have received widespread attention in the field of nephrology in recent years.

[0004] Genisic acid (GA, 2,5-dihydroxybenzoic acid) is a naturally occurring bioactive small molecule widely found in plants of the Gentianaceae family, and is also an important metabolite of salicylic acid in mammals. Thanks to its unique 2,5-dihydroxybenzoic acid structure, GA exhibits excellent free radical scavenging ability and metal ion chelating properties under physiological conditions. In vitro studies have confirmed that GA can effectively inhibit the generation of various lipid free radicals and peroxide free radicals. In in vivo models such as diabetic nephropathy and drug-induced kidney injury, GA has also been shown to effectively reduce oxidative stress and tissue inflammation by activating the Nrf2 antioxidant pathway and inhibiting NF-κB signaling, demonstrating clear renal protective potential. However, despite its significant pharmacological activity, the clinical application prospects of free GA are severely limited by its inherent pharmacokinetic defects. First, as a hydrophilic small molecule, free GA is readily and rapidly eliminated by renal filtration after entering the body, resulting in a very short biological half-life, making it difficult to maintain effective therapeutic concentrations. Secondly, and more critically, free GA lacks specific tissue or subcellular targeting capabilities and cannot penetrate the mitochondrial double membrane structure to achieve effective accumulation in the mitochondrial matrix, the core region of ROS outbreaks. This "systemic non-specific distribution-rapid excretion" characteristic makes it difficult for GA to exert its due therapeutic effect in crystalline nephropathy characterized by mitochondrial dysfunction, and its potential value in hyperoxalate-related kidney injury has not yet been fully explored.

[0005] Metal-phenolic networks (MPNs), as a new class of supramolecular materials constructed by the coordination self-assembly of polyphenol ligands and metal ions, have shown great promise in the biomedical field due to their mild synthetic strategies, highly tunable topologies, and ease of surface functionalization. Particularly in the treatment of kidney diseases, MPNs exhibit unique advantages that traditional carriers cannot match. First, thanks to the dynamic coordination bonds between components, MPNs can be easily regulated to form ultrasmall nanoparticles with a particle size of less than 10 nm; this size advantage allows them to efficiently cross the glomerular filtration barrier (GFB), achieving precise targeting and enrichment of renal tubular regions, overcoming the bottleneck of large-sized nanomedicines' inability to enter the kidneys. Second, MPNs are not inert carriers, but rather a "bioactive platform" that combines the catalytic activity of metal ions with the antioxidant capacity of polyphenols. Its surface has a high density of exposed phenolic hydroxyl and carboxyl groups, which not only endow it with excellent ROS scavenging and microenvironment remodeling capabilities, but also mimic the negatively charged surface of natural macromolecular inhibitors in urine. Through multi-site chelation, it interferes with crystal nucleation and growth, thus showing outstanding crystal regulation potential. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a targeted drug that can effectively treat kidney stones.

[0007] To achieve the above-mentioned objectives, this invention provides an ultrasmall metal polyphenol network GA-Fe@TPP targeting mitochondria, which uses gentianic acid as a ligand and Fe... 3+ It is assembled from nodes and its surface is modified with 4-carboxybutyltriphenylphosphine bromide.

[0008] Preferably, the ultrasmall metal polyphenol network GA-Fe@TPP has an amorphous structure.

[0009] Preferably, the particle size of the ultrasmall metal polyphenol network GA-Fe@TPP is less than 10 nm.

[0010] Preferably, the particle size of the ultrasmall metal polyphenol network GA-Fe@TPP is 7.06±1.78 nm.

[0011] On the other hand, the present invention also provides a method for preparing the ultrasmall metal polyphenol network GA-Fe@TPP, which includes the following steps: Synthesis of S1, GA-Fe MPNs Using gentic acid as an organic ligand, the metal ion Fe³⁺ + Using polyvinylpyrrolidone as an inorganic crosslinking center and as a protective polymer, the phenolic hydroxyl groups in gentian acid molecules react with the Fe³⁺ in the complex. + Fully coordinated crosslinking to construct metal-polyphenol nanonetworks GA-Fe MPNs; S2, TPP-PEG 2000 Synthesis of -NH2 4-Carboxybutyltriphenylphosphine bromide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole were dissolved in ultra-dry chloroform solvent and stirred until homogeneous, then activated; NH2-PEG was then added. 2000 -NH-Boc was dissolved in ultra-dry chloroform and slowly added dropwise to the above reaction solution. The reaction was carried out at room temperature, and the reaction solvent was removed by vacuum distillation. The crude product was dried, dissolved in deionized water, dialyzed, and lyophilized to obtain the product TPP-PEG. 2000 -NH-BOC; subsequent removal of the BOC protecting group yields the product TPP-PEG. 2000 -NH2; Synthesis of S3 and GA-Fe@TPP The GA-Fe MPNs were dispersed in deionized water and ultrasonically dispersed to form a uniform suspension. Under continuous stirring, EDC and NHS were added sequentially to the suspension, and the reaction was carried out at room temperature in a dark environment. Subsequently, TPP-PEG was added to the activated system. 2000 -NH2, continue stirring the reaction under light-protected conditions; after the reaction is complete, purify by dialyzing in deionized water, freeze-dry to obtain the ultrasmall metal polyphenol network GA-Fe@TPP.

[0012] Preferably, in step S1, the metal ions Fe³⁺ + It comes from FeCl3·6H2O, with a mass ratio of gentic acid to FeCl3·6H2O of 1:1.

[0013] More preferably, step S1 is as follows: at room temperature, 66 mg of PVP is completely dissolved in 5 mL of methanol. While stirring continuously, 1 mL of methanol solution containing 20 mg of FeCl3·6H2O is slowly added dropwise, and the reaction is stirred for 30 min to form a PVP-Fe precursor complex. Subsequently, 1 mL of gentianic acid methanol solution with a concentration of 20 mg / mL is added dropwise to the above mixture, and the reaction is maintained at 600 rpm for 3 h to allow the phenolic hydroxyl groups in the gentianic acid molecules to fully coordinate and crosslink with the metal ions in the complex. The dispersion is then dialyzed to obtain the metal-polyphenol nanonetwork GA-Fe.

[0014] Preferably, in step S3, 4-carboxybutyltriphenylphosphine bromide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and NH2-PEG are present. 2000 The molar ratio of -NH-Boc is 5:5:5:1.

[0015] Preferably, in step S3, GA-Fe MPNs are reacted with EDC, NHS, and TPP-PEG. 2000 The mass ratio of -NH2 is 1:1:1:1.

[0016] On the other hand, the present invention also provides the use of the ultrasmall metal polyphenol network GA-Fe@TPP in the preparation of medicaments for the treatment and / or prevention of kidney stones.

[0017] Preferably, the effective concentration of the ultrasmall metal polyphenol network GA-Fe@TPP is 25-400 μg / mL.

[0018] Preferably, the ultrasmall metal polyphenol network GA-Fe@TPP promotes the decomposition of hydrogen peroxide.

[0019] Preferably, the ultrasmall metal polyphenol network GA-Fe@TPP scavenge free radicals and has antioxidant properties.

[0020] Preferably, the ultrasmall metal polyphenol network GA-Fe@TPP inhibits the nucleation, growth, and aggregation of calcium oxalate crystals.

[0021] Preferably, the ultrasmall metal polyphenol network GA-Fe@TPP inhibits high oxalate-induced excessive mitochondrial cleavage and / or cell damage.

[0022] The ultra-small metal polyphenol network GA-Fe@TPP of this invention uses natural GA as a ligand and Fe as a ligand. 3+ This invention presents a mitochondrial-targeting metal-polyphenol network (MPN) assembled from nodes. By surface-modifying with TPP, this MPN is endowed with membrane potential-driven targeting capabilities, aiming to precisely locate the mitochondrial matrix of damaged proximal tubular epithelial cells. It not only inhibits crystal growth but also, through the potent free radical scavenging activity of GA and the synergistic effect of the Fe-coordination network, blocks ROS bursts at the source and reshapes mitochondrial redox homeostasis. This invention provides a theoretical basis and technical support for the prevention and treatment of crystalline nephropathy through this nanotherapeutic strategy integrating "precise targeting—microenvironment remodeling—key enzyme regulation." Attached Figure Description

[0023] Figure 1 Characterization of GA-Fe@TPP is shown. A) Synthesis flowchart of GA-Fe@TPP; B) Transmission electron microscopy (TEM) analysis of GA-Fe@TPP; C) Atomic force microscopy (AFM) analysis of GA-Fe@TPP; D) FT-IR analysis; E) XPS spectra of Fe 2p in GA-Fe@TPP; F) Particle size distribution; G) XPS full spectrum and P 2p / Fe 2p spectrum of GA-Fe@TPP.

[0024] Figure 2 The in vitro antioxidant capacity of GA-Fe@TPP was demonstrated. AC) UV-Vis detection of the scavenging capacity of different concentrations of GA-Fe@TPP against ABTS+·DPPH· and ·OH free radicals; DF) Quantitative analysis of the scavenging rates of different concentrations of GA-Fe@TPP against ABTS+·DPPH· and ·OH free radicals.

[0025] Figure 3The in vitro antioxidant scavenging capacity of GA-Fe@TPP is evaluated. A) Absorption spectral changes of different concentrations of GA-Fe@TPP (0–400 μg / mL) in the CAT system, used to characterize its scavenging capacity for hydrogen peroxide (H2O2); B) Absorption spectral changes of different concentrations of GA-Fe@TPP (0–400 μg / mL) in the DTNB system, used to characterize its thiol-related reducing capacity; C) Quantitative analysis of the H2O2 scavenging capacity of the samples determined by the CAT method; D) Quantitative analysis of the thiol-related reducing capacity of the samples determined by the DTNB method.

[0026] Figure 4 This section evaluates the DPPH· scavenging ability of GA-Fe@TPP. A) Comparison of DPPH· scavenging abilities between GA-Fe and GA-Fe@TPP at the same concentration, measured by UV-Vis. B) Quantitative analysis of DPPH· scavenging rates between GA-Fe and GA-Fe@TPP at the same concentration. Figure 5 This study demonstrates the regulatory effect of GA-Fe@TPP on calcium oxalate crystals. A) Observation of the regulatory effect of different concentrations of GA and GA-Fe@TPP on calcium oxalate crystals using ordinary optical microscopy; B) Analysis of the area, quantity, and size of CaOx crystals after GA-Fe@TPP regulation; C) SEM observation of the regulatory effect of GA-Fe@TPP on calcium oxalate crystals; GA-Fe@TPP: 0-200 μg / mL. Crystallization time: 24 h; D) Density functional theory (DFT) simulation calculations of the interaction between GA and crystal planes, where red, gray, green, and white spheres represent O, C, Ca, and H atoms, respectively.

[0027] Figure 6 This demonstrates the precise mitochondrial targeting and functional homeostasis remodeling of GA-Fe@TPP. A, B) Flow cytometry analysis of GA-Fe and GA-Fe@TPP binding to mitochondria; C) Confocal microscopy observation of cellular uptake of GA-Fe@TPP (green) in NRK-52E cells and its co-localization with mitochondria (Mito-Tracker, red); D) Line scan image of a linear region spanning NRK-52E cells; E) Fluorescence microscopy observation of mitochondrial membrane potential levels and quantitative analysis of fluorescence intensity; F) Intracellular Mito ROS levels and quantitative analysis. Compared with the model group, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns P > 0.05.

[0028] Figure 7The results show that GA-Fe@TPP restores mitochondrial calcium homeostasis and ultrastructural integrity. A) Fluorescence spectrum of mitochondrial calcium concentration; B) Quantitative analysis of mitochondrial calcium concentration; C) Flow cytometry analysis of mitochondrial calcium; D) Quantitative analysis of mitochondrial calcium by flow cytometry; E) Transmission electron microscopy observation of cell and organelle damage; F) Super-resolution confocal microscopy of mitochondria. Compared with the model group, ** P < 0.01, **** P < 0.0001, ns P > 0.05. Figure 8 The results show the cytotoxicity levels of GA-Fe@TPP against different kidney-derived cell lines.

[0029] Figure 9 This study demonstrates the effects of GA-Fe and GA-Fe@TPP protection on NRK-52E cell viability. Figure 10 This section compares the inhibition of ROS induced by high oxalate by GA, GA-Fe, and GA-Fe@TPP. A) ROS levels observed under a fluorescence microscope; B) Flow cytometry analysis of ROS; C) Statistical analysis and quantification of ROS. Compared with the normal group, ns indicates P>0.05, *** indicates P<0.001, and **** indicates P<0.0001.

[0030] Figure 11 This study demonstrates that GA-Fe@TPP alleviates oxalate-induced cell necrosis and apoptosis. A) Staining results of live and dead cells; green represents live cells, and red represents dead cells. B) Flow cytometry analysis of cell apoptosis and necrosis. C) Semi-quantitative results of live and dead cells. D) Quantitative analysis of cell apoptosis rate. Compared with the oxalate-treated group, ** indicates P<0.01, **** indicates P<0.0001.

[0031] Figure 12 This diagram illustrates how GA-Fe@TPP reduces crystal load in a Drosophila crystallization model. A) Schematic diagram of Drosophila Malpighian tubules; B) Schematic diagram of experimental design; C) Images showing the specific state of dissected Drosophila Malpighian tubules under bright-field and polarized light microscopy; D) Statistical results of the number of crystals, crystal perimeter, and crystal area in the polarized light microscopy images. Compared with the model group, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns P > 0.05. Compared with the model group, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns P > 0.05.

[0032] Figure 13This diagram shows the distribution and metabolism of GA-Fe and GA-Fe@TPP in mice. A) Distribution of GA-Fe and GA-Fe@TPP in mice at different time points; B) Distribution of GA-Fe and GA-Fe@TPP in the heart, liver, spleen, lung, and kidney of mice at different time points; C) Fluorescence observation of the distribution of GA-Fe@TPP-C6 in mouse kidney tissue at different time points; D) Statistical results of fluorescence intensity of GA-Fe and GA-Fe@TPP in mice at different time points; E) Semi-quantitative results of fluorescence intensity of GA-Fe and GA-Fe@TPP in mouse kidney.

[0033] Figure 14 This diagram illustrates how GA-Fe@TPP reduces CaOx deposition and kidney damage in a mouse model. A) Schematic diagram of the animal experimental design; B, C) HE staining and statistical analysis of kidney damage; D, E) PAS staining observation of tissue condition and semi-quantitative analysis of damage; F) Observation of calcium oxalate crystal deposition in the kidney using polarized light microscopy; G) Observation of calcium oxalate crystals deposited in the kidney using SEM; H) Statistical results of the number, perimeter, and area of ​​crystals in the polarized light microscopy images; Compared with the model group, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, ns P>0.05.

[0034] Figure 15 This shows the changes in the levels of CR (A), UA (B), and BUN (C) in mouse serum.

[0035] Figure 16 Images of HE staining of mouse heart, liver, spleen, and lung tissue sections are shown. A) HE staining image of the complete tissue; B) Magnified HE staining image of the tissue. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0038] 1. Experiment 1.1 Materials Rat renal tubular epithelial cells (NRK-52E), human proximal renal tubular epithelial cells (HK-2), and Madin-Darbycanine kidney cells (MDCK) were all purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. High-glucose DMEM medium, fetal bovine serum, and phosphate-buffered saline (PBS) were purchased from Gibco (USA). Penicillin-streptomycin solution was purchased from Solarbio Technology Co., Ltd. (Beijing, China). 4-Carboxybutyltriphenylphosphonium bromide (TPP), NH2-PEG 2000-NH2, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), methylene blue (MB), 3,3',5,5'-tetramethylbenzidine (TMB), and 2,2-bis[4-(1,1,3,3-tetramethylbutyl)phenyl]-1-(2,4,6-trinitrophenyl)hydrazyl radical (DPPH) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Gentian acid (GA), ferric chloride (FeCl3), sodium oxalate, coumarin 6 (C6), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Reactive oxygen species (ROS) assay kit, acridine orange (AO) fluorescent staining kit, and Annexin V-FITC / PI apoptosis / necrosis assay kit were purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. (Nanjing, China). Lactate dehydrogenase assay kit, lipid oxidation (MDA) assay kit, HE staining kit, Actin-Tracker Deep Red-633 fluorescent probe, JC-1 mitochondrial membrane potential assay kit, enhanced ATP assay kit, Calcein AM / PI live / dead cell assay kit, and mitochondrial calcium ion fluorescent probe Rhod-2 AM were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). MitoOrange Fix mitochondrial fluorescent probe staining working solution was purchased from Genvivo Biotech (Nanjing, China). Monoamine oxidase (MAO) activity assay kit was purchased from Solarbio Biotechnology Co., Ltd. (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Guangzhou EcoTop Biotechnology Co., Ltd. (Guangzhou). Methanol, xylene, calcium chloride, paraformaldehyde, and anhydrous ethanol were all analytical grade (Sinopharm Chemical Reagent Co., Ltd., China). The water used in the experiment was prepared using the Milli-Q ultrapure water system.

[0039] 1.2 Experimental Methods 1.2.1 Synthesis of GA-Fe A one-step assembly strategy involving coordination of phenolic hydroxyl groups and metal ions was employed to construct an ultra-small particle size metal-polyphenol nanonetwork with controllable dimensions.

[0040] Gentianic acid (GA) was selected as the organic ligand, and the metal ion Fe³⁺ was used. + Using polyvinylpyrrolidone (PVP, molecular weight 10000) as an inorganic crosslinking center and as a protective polymer, its amide groups can coordinate with metal ions, effectively regulating the nucleation and growth process of the nanonetwork.

[0041] The specific experimental steps are as follows: At room temperature, 66 mg of PVP was completely dissolved in 5 mL of methanol. While continuously stirring, 1 mL of a methanol solution containing 20 mg of FeCl3·6H2O was slowly added dropwise, and the reaction was stirred for 30 min to form a PVP-Fe precursor complex. Subsequently, 1 mL of a 20 mg / mL GA methanol solution was added dropwise to the above mixture, and the reaction was maintained at 600 rpm for 3 h to allow the phenolic hydroxyl groups in the GA molecules to fully coordinate and crosslink with the metal ions in the complex, constructing a metal-polyphenol nanonetwork. The resulting mixture was placed in a dialysis bag with a molecular weight cutoff (MWCO) of 30 kDa and dialyzed in deionized water for 72 h (with the dialyzing water changed every 6–8 h) to remove small molecules and residual organic solvents. After dialysis, the dispersion was collected and stored at 4°C in the dark to obtain GA-Fe MPNs for later use.

[0042] 1.2.2 TPP-PEG 2000 Synthesis of -NH2 Synthesis of TPP-PEG 2000 The -NH2 procedure is as follows: 4-Carboxybutyltriphenylphosphine bromide (TPP, 221.7 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (95.85 mg, 0.5 mmol), and 1-hydroxybenzotriazole (76.5 mg, 0.5 mmol) were dissolved in 5 mL of ultra-dry chloroform solvent and stirred until homogeneous, allowing the carboxyl groups to activate for 6 h. Then, NH2-PEG was... 2000 -NH-Boc (200 mg, 0.1 mmol) was dissolved in 1 mL of ultra-dry chloroform and slowly added dropwise to the above reaction solution. The reaction was carried out at room temperature for 48 h, after which the solvent was removed by vacuum distillation. The crude product was dried, dissolved in 5 mL of deionized water, and dialyzed against deionized water for 3 days. Finally, it was lyophilized to obtain the product TPP-PEG. 2000-NH-BOC. The product was then deprotected by dissolving it in 5 mL of dichloromethane (DCM) solvent, adding 1 mL of excess trifluoroacetic acid, stirring at room temperature for 6 h, removing the solvent by vacuum distillation, and extracting twice with DCM to obtain the final product TPP-PEG. 2000 -NH2.

[0043] 1.2.3 Synthesis of GA-Fe@TPP 30 mg of GA-Fe MPNs were weighed and dispersed in 30 mL of deionized water. The dispersion was then sonicated (80 kHz, 5 min) to ensure complete dispersion and form a homogeneous suspension. Under continuous stirring (600 rpm), 30 mg of EDC and 30 mg of NHS were added sequentially to the suspension. The reaction was carried out at room temperature for 3 hours in the dark to activate the carboxyl groups (–COOH) in the GA units on the GA-Fe surface, generating a highly reactive ester intermediate. Subsequently, 30 mg of TPP-PEG was added to the activated system. 2000 -NH2, continue stirring the reaction under light-protected conditions for 3 hours to make TPP-PEG 2000 -NH2 reacts with the carboxyl group of GA-Fe via the amino group. After the reaction, the mixed solution is transferred to a dialysis bag with a molecular weight cutoff of 30 kDa and purified by dialyzing in deionized water for 48 hours to completely remove unreacted small molecules and byproducts. Finally, the purified solution is freeze-dried to obtain the final product GA-Fe@TPP, which is then sealed and stored for subsequent experiments.

[0044] 1.2.4 Characterization of GA-Fe@TPP 1.2.4.1 Transmission Electron Microscopy (TEM) Observation The GA-Fe@TPP material was uniformly dispersed in ultrapure water and sonicated for 10 min. A suitable amount of sample was then transferred and loaded onto a copper grid (with an ultrathin carbon support film on the surface). After the solvent was allowed to evaporate completely at room temperature, the sample was transferred to a TEM for microscopic morphology observation.

[0045] 1.2.4.2 Atomic Force Microscopy (AFM) Observation Freshly peeled mica sheets (10 mm × 10 mm) were fixed onto the sample stage, and the surface was cleaned with deionized water and dried under nitrogen. 10 μL of GA-Fe@TPP MPNs suspension (concentration: 0.01 mg / mL, solvent: ultrapure water) was dropped onto the mica substrate surface and allowed to stand at room temperature for 10 min for adsorption. Unadsorbed particles were gently rinsed three times with ultrapure water, dried under nitrogen, and then ready for analysis.

[0046] 1.2.4.3 Fourier Transform Infrared Spectroscopy (FT-IR) Detection Weigh 2.0 mg of dried GA, GA-Fe, and GA-Fe@TPP samples and mix them thoroughly with 400 mg of KBr. The mixture was then ground and compressed into tablets. The tablets were then scanned using FT-IR with a wavenumber range of 4000 to 400 cm⁻¹. - ¹, resolution of 2 cm - ¹.

[0047] 1.2.4.4 X-ray photoelectron spectroscopy (XPS) detection Approximately 20 mg of vacuum-dried GA-Fe and GA-Fe@TPP samples were weighed for XPS analysis. The X-ray source was set to Al Ka, with an energy of 1486.6 eV and a step value of 6 mA × 12 kV. Binding energy correction was performed using C1s surface contamination (BE = 284.8 eV). The full-spectrum energy scan range was 0 eV–1300 eV, with a step value of 1.0 eV. The high-resolution Fe spectrum energy scan range was 700 eV–735 eV, with a step value of 0.1 eV.

[0048] 1.2.4.5 Particle size detection GA-Fe and GA-Fe@TPP were dispersed in deionized water to prepare a dispersion of 0.20 mg / mL, and their particle size distribution was detected using a nanoparticle size analyzer.

[0049] 1.2.5 In vitro antioxidant activity assay 1.2.5.1 ABTS Free Radical Scavenging Ability Test A 7 mM ABTS stock solution was mixed with a 2.45 mM potassium persulfate solution at a 1:1 (v / v) ratio and incubated at room temperature in the dark for 12 h. 3 mL of the above solution was then reacted with 1 mL of GA-Fe@TPP solutions of various concentrations (final concentrations of 0, 25, 50, 100, 200, and 400 μg / mL) and a positive control group (VC) at room temperature for 20 min. The ABTS+· scavenging rate was calculated by scanning the entire wavelength range (400-900 nm) using a microplate reader and measuring the absorbance at 734 nm.

[0050] ABTS+·Clearance Rate%=[(A ABTS+· -A 样品 ) / A ABTS+· ]×100. In the formula, A ABTS+· This represents the OD value of unprocessed ABTS+·; A 样品 This indicates the OD value of ABTS+ after adding GA-Fe@TPP.

[0051] 1.2. 5.2 Hydroxyl radical (•OH) scavenging ability test The in vitro scavenging ability of GA-Fe@TPP to remove •OH was detected using the methylene blue (MB) system method. Gradient concentrations of GA-Fe@TPP (final concentrations of 0, 25, 50, 100, 200, and 400 μg / mL) and a positive control group (VC) were mixed with an equal volume of MB solution (100 μM), and then 1 mL of Fenton's solution (Fe...) was added. 2+ Mix the 1 mM H2O2 and 10 mM H2O2, vortex, and incubate at 37 °C for 15 min. Perform a full-wavelength scan (400-900 nm) using a microplate reader, and calculate the •OH scavenging rate by observing the absorbance change at the 665 nm characteristic peak.

[0052] •OH removal rate % = [(A 样品 A MB ) / A0]×100.

[0053] In the formula, A0 represents the OD value of the MB without additional processing; A MB This indicates the OD value of MB without the addition of a sample. A 样品 This indicates the OD value of MB after adding GA-Fe@TPP.

[0054] 1.2.5.3 DPPH free radical scavenging ability test Prepare a 0.4 mmol / L DPPH solution (solvent: anhydrous ethanol). Mix 3 mL of GA-Fe@TPP solution (final concentrations of 0, 25, 50, 100, 200, and 400 μg / mL) and the positive control group (VC) thoroughly with 1 mL of the 0.4 mmol / L DPPH solution in a test tube and react for 30 min. Perform a full-wavelength scan (300-700 nm) using a microplate reader, and calculate the DPPH scavenging rate based on the absorbance at 517 nm.

[0055] DPPH· clearance rate % = [(A DPPH A 样品 ) / A DPPH ]×100.

[0056] In the formula, A DPPH Indicates the OD value of untreated DPPH·; A 样品 This indicates the OD value of DPPH· after adding GA-Fe@TPP.

[0057] 1.2.5.4 Detection of catalase (CAT)-like activity 150 μL of 10 mM H2O2 and 150 μL of GA-Fe@TPP solution (final concentrations of 0, 25, 50, 100, 200, and 400 μg / mL) were mixed with 2.75 mL of PBS buffer (pH 7.4) in the dark and reacted at 25 °C for 1 h. Then, 50 μL of 1 mM MFeSO4 solution was added, and the reaction was continued in the dark for another 1 h. Finally, 50 μL of 30 mM terephthalic acid (dissolved in 0.1 M NaOH) was added, and the mixture was allowed to stand in the dark for 1 h. The fluorescence intensity was measured using a microplate reader (excitation wavelength 330 nm, emission wavelength 425 nm).

[0058] CAT-like activity % = [(F 对照 F 样品 ) / F Control ] × 100, where F 对照 The fluorescence value of the untreated system; F 样品 The fluorescence value of the system after adding GA-Fe@TPP is shown.

[0059] 1.2.5.5 Detection of Glutathione peroxidase (GPx-like) activity The assay was performed using a DTNB-containing glutathione peroxidase (GSH) assay kit. First, 5 μL of 10 mM GSH solution was added to 183 μL of buffer, followed by GA-Fe@TPP solution (final concentrations of 0, 25, 50, 100, 200, and 400 μg / mL) and 12 μL of 15 mM t-Bu-OOH. The mixture was incubated at 37°C for 20 min. Immediately afterwards, 6.6 μL of 7.6 mM DTNB solution was added, and the reaction was carried out in the dark for 10 min. The absorbance at 412 nm was recorded after a full-wavelength scan (300–500 nm) using a microplate reader.

[0060] GPx-like activity % = [(A 对照 A 样品 ) / A 对照 ]×100, where A 对照 Absorbance of the untreated system; A 样品 The absorbance of the system after adding GA-Fe@TPP is given.

[0061] 1.2.5.6 Comparative Detection of DPPH· Radical Scavenging Ability of GA-Fe and GA-Fe@TPP Prepare a 0.4 mmol / L DPPH solution (solvent: anhydrous ethanol). Mix 3 mL of GA-Fe and GA-Fe@TPP solutions (final concentration 100 μg / mL) thoroughly with 1 mL of the 0.4 mmol / L DPPH solution in a test tube and react for 30 min. Perform a full-wavelength scan (300-700 nm) using a microplate reader, and calculate the DPPH scavenging rate based on the absorbance at 517 nm.

[0062] DPPH· clearance rate % = [(A DPPH A 样品 ) / A DPPH ]×100.

[0063] In the formula, A DPPH Indicates the OD value of untreated DPPH·; A 样品 This indicates the OD value of DPPH· after adding GA-Fe / GA-Fe@TPP.

[0064] 1.2.6 Crystallization regulation of CaOx crystals by GA-Fe and GA-Fe@TPP Prepare 2 mmol / L CaCl2 solution, 10 mmol / L sodium oxalate (Na2Ox) solution, and GA-Fe@TPP solutions of various concentrations using Tris buffer as the solvent. Place 12 mm circular glass slides at the bottom of the wells of a 12-well plate. Take a centrifuge tube and add 800 μL of Tris buffer, 50 μL of CaCl2 solution, and 50 μL of GA-Fe@TPP solution sequentially, vortexing to mix. Add 100 μL of Na2Ox solution to the centrifuge tube, vortex quickly to mix, and immediately add the mixture to the 12-well plate with the glass slides placed on it. Incubate the 12-well plate at 37°C for 12 h. After incubation, wash three times each with double-distilled water and anhydrous ethanol. Dry the washed samples in a 37°C oven. The dried samples can be directly observed under an optical microscope. After gold sputtering, the morphological characteristics of CaOx crystals are recorded using a scanning electron microscope (SEM). In addition, the same procedure was used to compare the regulatory effects of GA-Fe and GA-Fe@TPP on CaOx crystals and the results were recorded under a microscope.

[0065] 1.2.7 Density Functional Theory Calculations All theoretical analyses are based on density functional theory. Exchange-correlated interactions are described using the generalized gradient approximation combined with Perdew–Burke–Ernzerhof functionals. The electronic structure is described using a combination of DFT half-core pseudopotentials and dual numerical polarized basis sets. The real-space cutoff radius is set to 4.6 Å. The total energy convergence tolerance of the Kohn-Sham self-consistent field calculation is 1 × 10⁻⁶.-6 The adsorption energy (Eab) of Ha. GA on the COM (100) or (010) surface is calculated using the following formula: E ab = E AB – E A – E B (1) Among them, E AB E represents the total energy of the GA–COM (100) or (010) surface system. A For the energy of the COM(100) or (010) surface, E B The energy for an isolated GA molecule.

[0066] 1.2.8 Cell Culture and Grouping NRK-52E cells, HK-2 cells, and MDCK cells were cultured in DMEM or DMEM / F12 medium supplemented with 10% v / v fetal bovine serum at 37°C and 5% CO2. A concentration of 5×10⁶ cells was used. 4 NRK-52E cell suspension at a concentration of cells / mL was seeded into well plates, and 0.1-2 mL of cell suspension per well was added depending on the size of the well plate.

[0067] The subsequent cell experiments were grouped as follows: a) Control group: serum-free culture medium was added only; b) Damage group: Incubate with serum-free medium containing 1.0 mmol / L sodium oxalate for 24 h; c) GA protection group: Incubate with serum-free medium containing 1.0 mmol / L sodium oxalate and 50 μg / mL GA for 24 h; d) GA-Fe protection group: Incubate with serum-free medium containing 1.0 mmol / L sodium oxalate and 50 μg / mL GA-Fe for 24 h; e) GA-Fe@TPP protection group: Incubate with serum-free medium containing 1.0 mmol / L sodium oxalate and 50 μg / mL GA-Fe@TPP for 24 h.

[0068] 1.2.9 Cell viability assay 1.2.9.1 Cell safety assay of GA-Fe@TPP 100 μL of a concentration of 5 × 10 4 NRK-52E, HK-2 and MDCK cell suspensions were seeded in 96-well plates. When the cells adhered and reached 70% confluence, the culture medium was removed and the cells were washed three times with PBS buffer.

[0069] The cells were divided into two groups: 1) Blank control group: only serum-free culture medium was added; 2) GA-Fe@TPP group: serum-free culture medium containing GA-Fe@TPP solution at concentrations of 1, 2.5, 5, 10, 20, 50, 75, 100 and 200 μg / mL, respectively.

[0070] After 24 hours of incubation, 10 μL of CCK-8 reagent was added to each well, and the absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0071] 1.2.9.2 Detection of the effects of GA-Fe and GA-Fe@TPP on the viability of oxalate-damaged cells The cells were divided into 3 groups: 1) Control group: only serum-free culture medium was added; 2) Injury group: Serum-free culture medium containing 1.0 mmol / L sodium oxalate was added; 3) GA-Fe protection group: serum-free culture medium containing 1.0 mmol / L sodium oxalate and 1, 2.5, 3.125, 5, 6.25, 10, 20, 50, 75 and 100 μg / mL GA-Fe@TPP was added; 4) GA-Fe@TPP Protected Group: Serum-free culture medium containing 1.0 mmol / L sodium oxalate and 1, 2.5, 3.125, 5, 6.25, 10, 20, 50, 75 and 100 μg / mL GA-Fe@TPP was added.

[0072] After incubation for 24 hours, 10 μL of CCK-8 was added to each well, and the cells were incubated for 2 hours. The absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0073] 1.2.10 Measurement of intracellular ROS levels After the treatment time was reached, the cells were washed twice with PBS, and 1 mL of DCFH-DA staining solution diluted 1:1000 with culture medium was added to each well. The cells were incubated at 37 °C in the dark for 30 min, washed three times with PBS, and then observed under a fluorescence microscope. Fluorescence intensity was quantified using ImageJ software. Simultaneously, to further quantitatively assess intracellular ROS levels, flow cytometry was used for detection. After DCFH-DA staining incubation, the staining solution was aspirated, the cells were washed twice with PBS, and then gently digested with EDTA-free trypsin to collect the cells. The cells were centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in PBS. The cell suspension was transferred to flow cytometry tubes and analyzed using a flow cytometer.

[0074] 1.2.11 TEM observation of organelle damage To observe the ultrastructure of mitochondria, cells were collected and fixed overnight at 4°C with 2.5% glutaraldehyde. After washing with PBS, the samples were post-fixed with 1% osmium tetroxide for 1 hour, dehydrated by a series of ethanol gradients, and embedded in epoxy resin. Ultrathin sections of approximately 70 nm thickness were prepared using an ultramicrotome, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope with an accelerating voltage of 80 kV.

[0075] 1.2.12 Live / Dead Cell Staining After the cells reached the predetermined time according to each group treatment, the culture medium was aspirated, and 600 μL of Calcein AM / PI detection working solution was added to each well. After incubation in the dark for 30 min, the cells were observed under a fluorescence microscope. The fluorescence intensity was quantified using ImageJ software. 1.2.13 Apoptosis / Necroptosis Rate Detection After the cells reached the predetermined time according to each group treatment, the culture medium was aspirated, and the cells were collected by trypsin digestion without EDTA. The cells were washed twice with PBS, and 500 μL of binding buffer was added to each well to resuspend the cells. Then, 5 μL of Annexin V-FITC and propidium iodide solution were added sequentially and mixed well. After incubation at room temperature in the dark for 15 min, the cells were analyzed by flow cytometry.

[0076] 1.2.14 Mitochondrial targeting ability assay Mitochondria were isolated strictly according to the instructions of the mitochondrial isolation kit. After cell collection and homogenization, differential centrifugation was performed: first, centrifugation at 600 g for 10 min (4℃) removed the nuclei and unlyzed cells; the supernatant was then centrifuged at 11,000 g for 10 min to precipitate the mitochondria. The isolated mitochondria were incubated with coumarin 6 (C6)-labeled GA-Fe / GA-Fe@TPP at 37°C for 1 hour. Subsequently, the mitochondria were washed three times with PBS to remove unbound assemblages. The fluorescence intensity of the mitochondrial suspension was measured by flow cytometry.

[0077] 1.2.15 Mitochondrial colocalization analysis NRK-52E cells were seeded in confocal microplates (Glass-bottom dishes, 3×10⁶ cells / year). 4 The culture medium (cells / mL) was incubated overnight for adhesion. The medium was then replaced with fresh serum-free medium containing C6-labeled GA-Fe or GA-Fe@TPP nanocomposite (50 μg / mL), and incubated at 37°C for 6 h. After washing with PBS, mitochondria were specifically stained with MitoTracker™ Red strictly according to the manufacturer's instructions. Finally, imaging was performed using a laser confocal microscope (CLSM), and the fluorescence colocalization coefficient (Pearson's coefficient) was calculated to evaluate targeting efficiency.

[0078] 1.2.16 Detection of mitochondrial membrane potential levels After the cells reached the predetermined time according to each group treatment, the culture medium was aspirated, and 500 μL of JC-1 staining working solution prepared according to the instructions was added to each well. The mixture was thoroughly mixed, and the cells were incubated at 37°C in the dark for 20 min. The cells were washed twice with buffer, and 500 μL of fresh serum-free culture medium was added to each well. The cells were then observed under a fluorescence microscope. The membrane potential level was quantified by calculating the intensity ratio of red fluorescence (polymer, representing high potential) to green fluorescence (monomer, representing low potential / depolarization).

[0079] 1.2.17 Mitochondrial superoxide (Mito ROS) detection MitoSOX™ Red fluorescent probes were used to assess mitochondrial superoxide levels. After cell treatment, cells were washed twice with PBS. After removing the washings, 3 μM MitoSOX™ Red working solution (300 μL / well) prepared with serum-free medium was added, and cells were incubated at 37°C in the dark for 30 min. After incubation, cells were washed twice with PBS to remove unbound probes. Live-cell imaging was then performed under a fluorescence microscope to observe and acquire red fluorescence signals.

[0080] 1.2.18 Mitochondrial Ca² + Horizontal detection Mitochondrial calcium ion levels were detected using the Rhod-2 AM fluorescent probe. After treatment, cells were washed with PBS and incubated with Rhod-2 AM working solution prepared in serum-free medium at 37°C for 30 min in the dark. The staining solution was discarded, and fresh medium was added for another 30 min to promote complete deesterification of the probe within the cells. Cells were then washed three times with PBS. Confocal imaging: Cells were stained with DAPI (5 min) and washed before observation under a laser confocal microscope. Flow cytometry: The medium was aspirated, and cells were digested with trypsin without EDTA to avoid interference with the calcium ion signal. Cells were collected and centrifuged (200 × g, 5 min), the supernatant was discarded, and the cells were resuspended in PBS. Rhod-2 fluorescence intensity was detected using flow cytometry, and the mean fluorescence intensity was analyzed using FlowJo software to quantify mitochondrial Ca²⁺. + level.

[0081] 1.2.19 Stimulated Emission Depletion Microscopy (STED) for Observing Mitochondrial Morphology To observe the fine structure of mitochondria, NRK-52E cells were seeded in high-precision glass-bottomed culture dishes. After cell adhesion and reaching the predetermined time according to each treatment group, the cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. After washing three times with PBS, the mitochondrial fluorescent probe PK Mito Orange Fix working solution was added, and the cells were incubated at room temperature in the dark for 30 min. The staining solution was discarded, and the cells were washed three times with PBS. Subsequently, the cell nuclei were counterstained with DAPI working solution for 5 min, washed with PBS, and then mounted with an appropriate amount of anti-fluorescence quenching mounting medium. Images were acquired using a super-resolution STED microscope.

[0082] 1.2.20 Establishment of the Drosophila crystallization model and crystal deposition analysis Wild-type fruit flies (3-5 days after emergence) were selected. Drosophila melanogaster Female adult fruit flies were reared on standard cornmeal medium (25 ℃, RH 50%–60%, 12 h light / dark cycle). Modeling and drug administration: A hyperoxaluria model was established by adding 0.1 wt% Na2Ox to the diet. Four groups were established (n=15 / group): normal control group, model group, low-dose GA-Fe@TPP group, and high-dose GA-Fe@TPP group. The intervention lasted for one week, with the diet containing the drug / inducer changed every two days. Sampling and observation: After the experiment, fruit flies were anesthetized with CO2. Under a stereomicroscope, the abdomen was cut open ventrally in a dissecting dish containing PBS, and Malpighian tubules were carefully pulled and completely separated. The Malpighian tubules were transferred to a glass slide, a small amount of PBS was added, and a coverslip was placed over them and gently flattened. Observation was performed under a polarized light microscope, and the morphology and distribution of crystals within the lumen were recorded using the birefringence property of the crystals.

[0083] 1.2.21 Establishment of a mouse model of hyperoxalate-induced kidney stones and evaluation of drug efficacy 1.2.21.1 In vivo biodistribution and renal targeting analysis of GA-Fe@TPP To evaluate the pharmacokinetic behavior of GA-Fe@TPP in vivo and its ability to target and enrich pathological kidney tissue, this invention uses GA-Fe@TPP-C6 loaded with fluorescent probes for in vivo imaging analysis in small animals.

[0084] Male C57BL / 6 mice aged 6–8 weeks (weighing 18 ± 2 g) were randomly divided into two groups after acclimatization: (a) Normal control group: intraperitoneal injection of normal saline daily for 7 consecutive days; (b) Glyoxylic acid stone model group: Glyoxylic acid (70 mg / kg) was injected intraperitoneally daily for 7 consecutive days to induce kidney stone formation. After the model was established, both groups of mice were injected once via tail vein with GA-Fe@TPP-C6 (10 mg / kg).

[0085] Following drug administration, whole-body fluorescence signals were acquired using a small animal in vivo imaging system (IVIS) under isoflurane inhalation anesthesia at different time points (0, 1, 2, 4, and 8 h) to dynamically monitor the in vivo distribution and renal enrichment trend of GA-Fe@TPP. Mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were rapidly collected for in vitro fluorescence imaging and semi-quantitative analysis. Subsequently, kidney tissue was embedded in OCT and frozen sections were prepared. Renal tubular epithelial cells were specifically labeled with anti-CK-18 antibody, and the cell nuclei were counterstained with DAPI. The co-localization of the nanodrug fluorescence and renal tubular markers was observed using fluorescence microscopy to verify the targeted uptake ability of GA-Fe@TPP on renal tubular epithelial cells.

[0086] 1.2.21.2 Evaluation of the anti-calculi and renal protective efficacy of GA-Fe@TPP All animal experimental protocols of this invention have been approved by the Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University and strictly comply with relevant animal welfare and management regulations.

[0087] Male C57BL / 6 mice aged 6–8 weeks (weighing 18 ± 2 g) were randomly divided into 5 groups (n = 7) after acclimatization: Normal group: Standard feeding, free access to water; Model group: Daily intraperitoneal injection of glyoxylic acid (70 mg / kg) for 7 consecutive days to induce hyperoxaluria and renal crystallization model; GA treatment group: GA (10 mg / kg) was injected into the tail vein daily during modeling. GA-Fe treatment group: GA-Fe (10 mg / kg) was injected into the tail vein daily during modeling. GA-Fe@TPP treatment group: GA-Fe@TPP (10 mg / kg) was injected daily via the tail vein during modeling. All treatments lasted for 7 days. After the experiment, mice were euthanized by cervical dislocation, and blood and major organs were collected for subsequent analysis.

[0088] Histopathological and Crystal Deposition Assessment: Kidney tissue was fixed in 4% paraformaldehyde, dehydrated with graded ethanol, and embedded in paraffin to prepare 5 μm serial sections. After staining with hematoxylin-eosin (H, E) and periodic acid-Schiff (PAS), the sections were subjected to high-resolution imaging using the PathScope™ 4S whole-slide scanning system (DigiPath, USA) to assess pathological changes such as renal tubular dilatation, epithelial cell shedding, and necrosis.

[0089] Crystal deposition observation: Unstained paraffin sections were observed using a polarized light microscope. The birefringence of calcium oxalate crystals was used to visually assess the area and distribution of crystal deposition in the kidney tissue.

[0090] Scanning electron microscopy (SEM) ultrastructural analysis: To further observe crystal morphology and its interaction with epithelial cells at the subcellular level, SEM was performed on partially dewaxed kidney sections. Paraffin sections were thoroughly dewaxed with xylene, rehydrated with graded ethanol (100%–70%), and dried. They were then cut to appropriate sizes and fixed onto conductive adhesive. After being sputtered with a gold film (5–10 nm thick), they were observed under a scanning electron microscope, focusing on recording the aggregation state of microcrystals within the renal tubules and the microstructural damage characteristics on the surface of epithelial cells.

[0091] Serum biochemical index detection: Mouse serum was separated and serum creatinine (Cr), blood urea nitrogen (BUN) and uric acid (UA) levels were detected using a fully automated biochemical analyzer to assess the renal function status of mice in each group.

[0092] Immunohistochemical (IHC) staining: Paraffin sections of the kidneys were dewaxed and antigen-retrieved, then incubated overnight at 4°C with the following primary antibodies: kidney injury markers (KIM-1), adhesion proteins (OPN, CD44), key proteins of the antioxidant pathway (Nrf2, Keap1), and inflammatory factors (IL-6, MCP-1). After incubation, staining was performed according to standard procedures. Sections were scanned using the PathScope™ 4S system, and semi-quantitative analysis of positively stained areas was performed using ImageJ software.

[0093] 1.2.22 Statistical Analysis All quantitative data are expressed as mean ± standard deviation (mean ± SD) and were derived from at least three independent biological replicates. Statistical analysis was performed using GraphPad Prism 9.0 software. Differences between groups were assessed using Student's t-test and one-way ANOVA. p A value <0.05 is considered statistically significant. * indicates p <0.05, ** indicates p<0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0094] 2. Results and Discussion 2.1 Design, synthesis and characterization of GA-Fe@TPP Targeting the key link of mitochondrial oxidative stress in the pathology of kidney stones, this invention designs and constructs an ultrasmall metal-polyphenol network (MPN) (GA-Fe@TPP) with mitochondrial targeting function. Gentian acid (GA), which possesses both carboxyl and diphenol structural characteristics, is selected as the ligand, utilizing its excellent reactive oxygen species scavenging ability and coordination properties with metal ions. Figure 1 As shown in Figure A, the synthesis of GA-Fe@TPP employs a one-pot self-assembly combined with a surface functionalization strategy: First, under the condition of polyvinylpyrrolidone (PVP) as a stabilizer, GA molecules are deprotonated with Fe³⁺ ions via phenolic hydroxyl / carboxyl group deprotonation. + Multi-point chelation coordination occurred, and the solution color rapidly changed from light yellow to dark brown-black, confirming the formation of Fe-O coordination bonds and the construction of ultrasmall GA-Fe nanocores. Subsequently, to endow the nanoparticles with precise mitochondrial targeting capabilities, the carboxyl groups on the GA-Fe surface were activated using EDC / NHS, and the targeting ligand TPP-PEG was added via an amidation reaction. 2000 -NH2 is covalently coupled to the core surface. The introduction of the PEG spacer not only effectively reduces steric hindrance and enhances the binding efficiency of TPP to mitochondria, but also further improves the colloidal stability of the nanoparticles. The GA-Fe@TPP solution after dialysis purification exhibits a significant Tyndall effect. Figure 1 The results (A) indicate the formation of a homogeneous colloidal system. This synthetic route utilizes mild conditions, readily available raw materials, and is easily scaled up, fully demonstrating the design advantages of the "modular assembly" of the MPNs system.

[0095] Morphological and structural characterization results confirmed the successful construction of the nanoplatform. Transmission electron microscopy (TEM) images showed that GA-Fe@TPP exhibited a well-dispersed morphology of ultrasmall nanodots. Figure 1 B). Atomic force microscopy (AFM) further confirmed its highly uniform morphology and regular surface profile. Figure 1 No obvious diffraction peaks were found in the X-ray powder diffraction (XRD) pattern. Figure 1 The presence of E indicates that GA-Fe@TPP has an amorphous, non-crystalline structure. Dynamic light scattering (DLS) was used to determine its hydrated particle size, and the average particle size of GA-Fe@TPP increased slightly to 7.06 ± 1.78 nm. Figure 1It is worth noting that the modified particle size was still strictly controlled within the renal filtration threshold (<10 nm), which laid a key structural foundation for its ability to overcome the glomerular filtration barrier and efficiently accumulate in damaged renal tubular epithelial cells.

[0096] Spectroscopic and surface chemical analyses further clarified the chemical composition of GA-Fe@TPP. FT-IR characterization was performed on GA, GA-Fe, TPP, and GA-Fe@TPP. Figure 1 (D). GA at 1258 cm - A distinct C–O stretching vibration peak appears at position ¹. Compared to GA, this peak is significantly weakened in GA-Fe, indicating that the phenolic hydroxyl group participates in the Fe³⁺ reaction. + Coordination. TPP at 1086 cm⁻¹ - A distinct characteristic peak of P=O stretching vibration appears at position ¹, and at 1025 cm⁻¹... - ¹ A P–O-related absorption peak exists in this region. In contrast, GA-Fe@TPP shows an absorption peak at 1086 cm⁻¹. - A new characteristic absorption peak appears at ¹, and at 1025 cm⁻¹ - ¹The significantly enhanced regional absorption indicates that TPP has been successfully modified. Further X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition and chemical state of GA-Fe@TPP and GA-Fe. Figure 1 XPS analysis showed that the Fe 2p fine spectrum of GA-Fe@TPP exhibited characteristic double peaks at ~710 eV (Fe 2p3 / 2) and ~724 eV (Fe 2p1 / 2), and the binding energy positions were consistent with those of standard Fe(III). Figure 1 (G), indicating that the assembly process did not change the oxidation state of iron ions, Fe³ + It retains its chemical stability as a structural node. The high-resolution P 2p spectrum of GA-Fe@TPP shows peaks at approximately 132 eV and 138 eV, corresponding to P 2p3 / 2 and P 2p1 / 2, respectively, further indicating the successful modification of TPP.

[0097] Furthermore, no obvious turbidity or precipitation was observed in GA-Fe@TPP after standing for several days in water, PBS, and DMEM media, indicating that it has good physiological stability.

[0098] 2.2 Broad-spectrum free radical scavenging and multi-enzyme mimicry activities of GA-Fe@TPP Oxidative stress is one of the key molecular drivers in the pathogenesis of kidney stones. Extensive evidence suggests that exposure to high oxalate and CaOx crystals induces renal tubular epithelial cells to produce excessive reactive oxygen species (ROS)—including hydroxyl radicals (·OH) and superoxide anions (O2··OH). -The oxidation process involves the production of lipid peroxidation and hydrogen peroxide (H2O2). This oxidative burst triggers lipid peroxidation, DNA damage, and amplified local inflammation, thus weaving a vicious cycle of "crystal deposition - epithelial damage - recrystallization." Therefore, restoring renal redox homeostasis is a prerequisite for halting the progression of kidney stones.

[0099] To verify the designed antioxidant efficacy, the free radical scavenging ability and nanozyme activity of GA-Fe@TPP were systematically evaluated. In chemical scavenging experiments, GA-Fe@TPP exhibited strong, dose-dependent antioxidant activity across a concentration range of 25–400 μg / mL. Figure 2 As shown in A and D, ABTS· + The characteristic absorption of GA-Fe@TPP decreased continuously with increasing GA-Fe@TPP concentration. A similar trend was observed in the DPPH experiment. Figure 2 The absorbance of GA-Fe@TPP gradually increased, indicating a gradual increase in free radical scavenging ability. Furthermore, the ability of GA-Fe@TPP to neutralize highly destructive hydroxyl radicals (·OH) was evaluated. Using a Fenton reaction-driven methylene blue (MB) degradation model, a rapid decrease in MB absorbance was observed under high oxidation conditions. However, the MB signal was significantly preserved after the introduction of GA-Fe@TPP, and this preservation was dose-dependent. Figure 2 (C, F), confirming its effectiveness in capturing ·OH and preventing oxidative degradation.

[0100] In addition to passive free radical scavenging, GA-Fe@TPP also exhibits multi-enzyme mimicry. Its catalase (CAT)-like activity, which promotes the decomposition of H2O2, was investigated. This is evidenced by the rapid decrease in the H2O2-related colorimetric signal. Figure 3 (A, C) GA-Fe@TPP effectively catalyzed the decomposition of H2O2, mimicking the protective function of endogenous CAT. Furthermore, in a glutathione peroxidase (GPx) simulation experiment using the DTNB probe, GA-Fe@TPP significantly reduced glutathione (GSH) consumption in the presence of H2O2. DTNB signal retention ( Figure 3 (B, D) indicates that GA-Fe@TPP can both replace GSH for peroxide reduction and protect the endogenous thiol library from depletion. Furthermore, to investigate whether surface functionalization affects the antioxidant activity of the core, a representative DPPH· scavenging experiment was conducted to compare GA-Fe@TPP with unmodified GA-Fe. The results showed that, at the same concentration, the difference in DPPH· scavenging efficiency between the two was negligible. Figure 4 (A, B). This result strongly confirms that the surface coupling of TPP-PEG does not shield the key phenolic hydroxyl active sites, thus fully preserving the inherent electron / hydrogen donor potential of the metal-polyphenol network.

[0101] These results indicate that GA-Fe@TPP is a multifunctional nanozyme combining direct radical quenching with CAT-like and GPx-like catalytic activities. This dual mechanism provides a solid functional basis for mitigating the pathological cascade of mitochondrial oxidative stress in vivo.

[0102] 2.3 GA-Fe@TPP exhibits excellent CaOx crystallization control ability. The formation of kidney stones is closely related to the nucleation, growth, and aggregation of CaOx crystals. In the CaOx crystallization system, the regulatory effects of GA and GA-Fe@TPP on the CaOx crystal formation process were systematically compared. Figure 5 Under a bright-field microscope, the blank control group showed a large number of typical rhombic hexahedral structures, consistent with the morphological characteristics of the COM crystal form, with large crystal size and dense quantity. After the addition of GA, the number and aggregation of crystals decreased, but in the concentration range of 25-200 μg / mL, the crystals still mainly exhibited the regular COM morphology, suggesting that GA had a limited effect on crystal form transformation. In contrast, GA-Fe@TPP showed a more significant intervention effect on the CaOx crystallization process. With increasing concentration (50–200 μg / mL), the number of crystals in the field of view decreased significantly, the crystal size decreased significantly, and the distribution became more dispersed; at concentrations of 50 μg / mL and above, some crystals were observed to have a tetragonal bipyramidal morphology, which is the characteristic morphology of the COD crystal form with low stone risk ( ). Figure 5 A). Statistical analysis results of crystals ( Figure 5 (B) Further confirmation shows that GA-Fe@TPP exhibits a more significant dose-dependent inhibitory effect, with a greater reduction in crystal number, average particle size, and total crystal area than the GA group.

[0103] Scanning electron microscopy (SEM) results further revealed its morphology regulation characteristics at the ultrastructural level. Figure 5(C). The control group crystals exhibited typical hexagonal rhomboid or prismatic structures with clear crystal faces and sharp edges, characteristic of rapid crystal growth along high-energy crystal faces. However, under GA-Fe@TPP intervention, crystal growth was severely disrupted, gradually evolving into irregular particles with rounded edges, rough surfaces, and even a dissolving appearance. As the concentration of GA-Fe@TPP increased, the crystal morphology gradually transformed into a centrally concave to dumbbell-shaped shape. This significant morphological change indicates that the ultra-small GA-Fe@TPP MPN can preferentially adsorb and "block" the active growth sites of the crystal, effectively suppressing the epitaxial growth and secondary aggregation of the crystal through steric hindrance. Due to the small molecular weight and lack of steric hindrance of free GA, it is difficult to form a stable and durable coating on specific high-energy crystal faces of CaOx, resulting in limited inhibitory effects. In contrast, the unique coordination network structure of GA-Fe@TPP gives it a decisive advantage: the assembly mode with Fe as nodes achieves high-density enrichment and multivalent display of GA ligands at the nanoscale. This structural characteristic exposes a large number of phenolic hydroxyl and carboxyl groups on the MPN surface, creating a microenvironment with a locally high negative potential. This highly negatively charged surface significantly enhances the MPN's attraction to calcium ions (Ca) on the crystal surface. 2+ The electrostatic affinity of GA allows it to firmly adsorb and lock the active growth sites of the crystal. GA-Fe@TPP, with its high specific surface area effect (physical steric hindrance) brought about by its ultra-small nanosize, combined with the strong coordination and charge repulsion ability generated by the enrichment of GA on its surface, achieves a "physical-chemical" dual blockade of the CaOx crystallization process.

[0104] The mechanism of action of the main active component GA in the GA-Fe@TPP system on different crystal planes of COM was further studied using density functional theory (DFT), and its adsorption behavior on the main crystal planes (100) and (010) of COM was compared. Figure 5 , D). Calculation results show that the adsorption energies of GA on the COM (100) and (010) crystal planes are respectively 4.98 eV and The 4.31 eV indicates that GA adsorption on the (100) crystal plane is more stable, exhibiting significant crystal plane selectivity. This suggests that the adsorption of Ca² on each crystal plane may be due to the different crystal planes. +The density difference resulted in varying adsorption capacities of GA on different crystal planes. This result indicates a stronger interaction between GA and the (100) crystal plane. The preferential adsorption of GA on the COM (100) crystal plane and the significant structural distortion it induces may lead to a marked reduction in the growth rate of this crystal plane. This effect manifests macroscopically as increased crystal surface roughness, and under higher dosage conditions, it causes restricted or even "passivated" growth of the (100) crystal plane, thereby promoting relatively rapid growth of other crystal planes, ultimately forming a characteristic dumbbell morphology.

[0105] 2.4 Precise Mitochondrial Targeting and Functional Homeostasis Remodeling by GA-Fe@TPP To verify whether GA-Fe@TPP can penetrate the cell membrane and precisely target the lesion core, a functional mitochondrial in-situ detection platform based on centrifugation was first established. Flow cytometry quantitative analysis showed ( Figure 6 After co-incubation with GA-Fe@TPP labeled with Cumarin-6 (C6), the binding rate of GA-Fe to isolated mitochondria reached 85.75%, while the binding rate of the non-targeted GA-Fe-C6 group under the same conditions was only 9.42%, showing a highly significant statistical difference (P<0.0001). This result directly confirms that the introduction of TPP ligands endows GA-Fe with excellent mitochondrial affinity.

[0106] Furthermore, through confocal imaging and linear intensity analysis of intracellular mitochondrial colocalization ( Figure 6 As shown in C and D, compared with GA-Fe, the GA-Fe@TPP group showed a higher degree of overlap with mitochondria, and the fluorescence peaks of the two groups highly overlapped, indicating that GA-Fe@TPP has a significantly enhanced mitochondrial targeting enrichment ability.

[0107] This precise "source targeting" translates into superior mitochondrial protective efficacy. Mitochondria, as the cell's energy factories and the origin of oxidative stress, are crucial for maintaining homeostasis. Mito ROS staining shows that (…) This treatment targets the high-oxalate-induced oxidative burst. Figure 6 GA-Fe@TPP effectively suppressed mitochondrial-specific ROS levels to basal levels. This confirms that "transporting" antioxidants to the core region of ROS production for source-specific scavenging is more therapeutically efficient than non-specific cytoplasmic distribution. Benefiting from the relief of oxidative stress, JC-1 probe monitoring showed that GA-Fe@TPP effectively blocked the collapse of mitochondrial membrane potential (ΔΨm) induced by high oxalate, maintaining the polarization state required to maintain the cellular respiratory chain. Figure 6 F).

[0108] Given that mitochondrial calcium homeostasis imbalance is a key pathological event downstream of oxidative stress, further investigation was conducted on mitochondrial calcium levels. 2+ Changes in levels. Previous studies have shown that high oxalate stimulation can induce Ca²⁺ levels by activating the mitochondrial calcium transporter (MCU). + Explosive influx, while Ca² + Overload, in turn, amplifies mtROS production, triggering the apoptosis process. Our mitochondrial-specific Ca²⁺... + Fluorescence imaging and flow cytometry analysis showed that high oxalate treatment led to severe Ca²⁺ exposure in mitochondria. + load( Figure 7 (AD); GA and GA-Fe pretreatment only partially alleviated the symptoms, while the GA-Fe@TPP group reduced mitochondrial Ca²⁺. + The signal dropped significantly, almost returning to the normal control level. This result strongly confirms that GA-Fe@TPP can effectively block the vicious cycle of "ROS burst - calcium overload - ROS amplification".

[0109] Furthermore, the transformation of the aforementioned biochemical benefits into physical structural integrity was further confirmed at the ultrastructural level. This was achieved using biological transmission electron microscopy (TEM). Figure 7 E) and ultra-high resolution fluorescence microscopy STED ( Figure 7 We visually demonstrated the protective effect of GA-Fe@TPP on mitochondrial network topology. In normal NRK-52E cells, mitochondria exhibit a highly interconnected tubular or linear network. However, high oxalate stimulation leads to drastic morphological remodeling of mitochondria, transforming them from elongated strips to diffuse short rod-like or even globular structures (punctate / globular phenotype). This widespread fragmentation suggests hyperfission activation and impaired fusion, resulting in network continuity breakdown. In contrast, the GA-Fe pretreatment group only alleviated this fragmentation trend to some extent, with some mitochondria still exhibiting swelling and truncation characteristics. Notably, the mitochondrial morphology in the GA-Fe@TPP-treated group was highly similar to that of the normal control group, mainly existing as linear or branched structures, maintaining high network connectivity. This morphological evidence strongly suggests that GA-Fe@TPP can effectively inhibit excessive mitochondrial cleavage induced by high oxalate levels, preventing organelles from failing due to structural disintegration by maintaining fusion-fission kinetic homeostasis. Thanks to TPP-mediated precise delivery, GA-Fe@TPP can achieve subcellular targeting of ROS–ΔΨm–Ca²⁺. +"The comprehensive regulation of the axis and the maintenance of mitochondrial dynamics balance."

[0110] 2.5 GA-Fe@TPP alleviates high oxalate-induced cell damage First, the biocompatibility of GA-Fe@TPP was established. CCK-8 activity testing confirmed (…). Figure 8 GA-Fe@TPP showed no significant cytotoxicity to rat (NRK-52E), human (HK-2), and canine (MDCK) renal tubular epithelial cells across a wide therapeutic concentration range (≤ 200 μg / mL), indicating its excellent biocompatibility. Based on this, a high-oxalate (1 mM, 24 h)-induced NRK-52E cell damage model was established to evaluate the material's cytoprotective efficacy.

[0111] The results showed that high oxalate stimulation significantly reduced cell viability to 71.6%. However, pretreatment with both GA-Fe and GA-Fe@TPP dose-dependently improved cell viability. Notably, thanks to TPP-mediated precise mitochondrial targeting, the GA-Fe@TPP group exhibited superior protective effects compared to the non-targeted GA-Fe group; particularly at a concentration of 50 μg / mL, the GA-Fe@TPP group showed the most significant recovery of cell viability, reaching 93.8%. Figure 9 This essentially reversed the damage caused by oxalate.

[0112] To elucidate its protective mechanism, the levels of intracellular oxidative stress and the degree of membrane damage were further quantified. Fluorescence microscopy observations were performed. Figure 10 A) and flow cytometry analysis ( Figure 10 (B) indicates that high oxalate induced the explosive production of intracellular ROS, while GA-Fe@TPP treatment significantly inhibited ROS fluorescence intensity, and its scavenging efficiency was significantly better than that of free GA and unmodified GA-Fe groups. Figure 10 (C). This powerful antioxidant capacity effectively breaks the downstream peroxidation damage chain.

[0113] Mitochondrial dysfunction and the accumulation of oxidative stress ultimately lead to cell death, and dead cell debris is an ideal breeding ground for crystal nucleation. Live / dead cell double staining and quantitative results show (…) Figure 11 In the oxalic acid-treated groups (A and C), the red fluorescence representing dead cells was significantly increased; in contrast, the proportion of green viable cells was dominant in the GA-Fe@TPP-treated groups, with very little red signal. Annexin V / PI double staining flow cytometry analysis further quantified this protective effect. Figure 11(B, D): High oxalate induced significant early and late apoptosis in NRK-52E cells, while GA-Fe@TPP significantly reduced the overall apoptosis rate. Apoptosis leads to the eversion of phosphatidylserine (PS), which can act as a calcium ion binding site, promoting crystal adhesion and aggregation. GA-Fe@TPP reduces the probability of microcrystal retention in the renal tubules from the source by inhibiting apoptosis.

[0114] 2.6 GA-Fe@TPP reduces crystal load in Drosophila crystallization model To further verify the intervention effect of GA-Fe@TPP on oxalate metabolism and crystal formation in vivo, a Drosophila strain was constructed. Drosophila melanogaster Malpighian tubule crystallization model. Malpighian tubules in Drosophila exhibit high evolutionary conservation with mammalian nephrons in ion transport, acid-base regulation, and metabolic excretion functions, and have recently been established as a powerful translational platform for studying the mechanisms of kidney stone formation and drug screening.

[0115] Wild-type fruit flies were continuously fed a diet containing 0.1% sodium oxalate (NaOx) for 7 days to induce model formation. Subsequently, Malpighian tubules were dissected and isolated for morphological evaluation. Figure 12 A, B). Bright-field and polarized light microscopy imaging shows ( Figure 12 In the model group (NaOx), the Margary tubes were filled with dense deposits; under polarized light, these deposits exhibited strong birefringence, confirming them as CaOx crystals. Excessive crystal accumulation led to significant obstructive dilation and distortion of the lumen, indicating severe excretory dysfunction. In contrast, co-feeding with GA-Fe@TPP significantly reversed this pathological process. Both the low-dose (L-GA-Fe@TPP) and high-dose (H-GA-Fe@TPP) treatment groups showed a dose-dependent reduction in crystal load. Particularly in the high-dose group, the Margary tube lumen became permeable again, with few bright crystal spots under polarized light, and the overall morphology almost returned to the normal control level. Image-based morphometric analysis further confirmed this observation: compared to the model group, the H-GA-Fe@TPP treatment group showed a significant decrease in crystal deposition area, crystal deposition count, and total perimeter. Figure 12 (D), which strongly confirms the excellent anti-crystallization efficacy of GA-Fe@TPP in vivo.

[0116] 2.7 Targeted enrichment characteristics of GA-Fe@TPP in mouse kidneys and its anti-calculous therapeutic efficacy Achieving efficient renal enrichment is a prerequisite for nanomedicines to exert their anti-crystalization effects. Due to the unique physiological structure of the glomerulus, only particles with a hydrodynamic diameter of less than 10 nanometers can pass through the kidney. This special structure limits the application of most nanomedicines in the kidney.

[0117] To verify the kidney-targeting advantage of GA-Fe@TPP, a small animal in vivo imaging system was used to systematically compare the distribution and metabolic differences of non-targeted GA-Fe and mitochondrial-targeted GA-Fe@TPP (coumarin 6 labeled) in mice. In vivo fluorescence imaging results showed ( Figure 13 Both MPNs (A and D) rapidly distributed throughout the body after intravenous injection, mainly concentrating in the liver and kidneys in the abdominal region. The fluorescence signals of both GA-Fe@TPP and GA-Fe peaked approximately one hour after injection, then gradually weakened over time, and were essentially metabolized after 24 hours. This rapid clearance characteristic indicates that the ultra-small MPNs are fully metabolized in vivo, reducing the risk of systemic toxicity due to long-term accumulation. Comparison of in vitro organ imaging and quantitative analysis revealed (…). Figure 13 GA-Fe@TPP showed significantly higher enrichment levels in the kidneys than unmodified GA-Fe (B, E). This significant difference in enrichment is mainly attributed to the unique physiological and metabolic characteristics of the kidneys. As one of the organs with highly active energy demands, renal tubular epithelial cells are rich in mitochondria (second only to cardiomyocytes in volume fraction) to maintain continuous electrolyte transport and reabsorption. This provides a natural "high-density anchoring platform" for mitochondrial-targeted nanosystems, thereby significantly enhancing the selective accumulation of drugs in kidney tissue, especially the renal tubular region. As a lipophilic cation, TPP utilizes the electrostatic driving force generated by the mitochondrial transmembrane potential, enabling GA-Fe@TPP to be "efficiently captured" by the mitochondrially dense renal tissue. The stable binding of TPP to renal tubular mitochondria prolongs its renal retention time, avoiding rapid clearance, and enhances bioavailability through mitochondrial affinity enrichment, maintaining an effective drug concentration at the source of oxidative stress. Renal section fluorescence showed that GA-Fe@TPP exhibited a distinct tubular distribution pattern. Figure 13 (C) The fluorescence signal is mainly concentrated in the proximal tubule region.

[0118] In a mouse model of classical CaOx nephropathy induced by glyoxylate, the in vivo therapeutic efficacy of GA-Fe@TPP was further validated. Figure 14 HE staining results showed that the kidneys of the model group exhibited severe crystalline nephropathy characteristics: significant tubular dilation, vacuolar degeneration and necrosis of epithelial cells, and numerous casts obstructing the lumen accompanied by obvious inflammatory cell infiltration. Figure 14(B, C). In contrast, the GA-Fe@TPP treatment group showed significant relief of renal tissue lesions, with renal tubular structure becoming more intact and inflammatory damage significantly reduced, and the effect was superior to the GA or GA-Fe groups alone. PAS staining further confirmed that the brush border of the renal tubules in the model group was severely damaged and the integrity of the basement membrane was impaired; while GA-Fe@TPP treatment showed excellent protective effect, not only maintaining the regularity of the tubular structure, but also achieving effective repair of the basement membrane and brush border. Figure 14 (D, E). Observation by combined polarizing microscope and scanning electron microscope (SEM) ( Figure 14 (F, G), visually revealing the distribution of crystals within the kidney. Numerous CaOx crystals with strong birefringence were observed within the renal tubules of the model group; some crystals formed large clumps and adhered tightly to the surface of damaged epithelial cells. Quantitative statistical analysis (F, G) Figure 14 The H) results showed that the total perimeter, deposition area ratio, and crystal count of the model group were all at a high level; however, after GA-Fe@TPP intervention, all three indicators decreased significantly (P<0.0001), and its effect on inhibiting deposition was significantly better than that of the GA-Fe or free GA group. This indicates that GA-Fe@TPP can not only interfere with crystal nucleation and growth at the microscopic level, but also effectively block the pathological adhesion between crystals and cells. Biochemical tests showed ( Figure 15 In the model group, serum urea nitrogen (BUN), creatinine (Cr), and uric acid (UA) levels were abnormally elevated, indicating impaired renal function. GA-Fe@TPP treatment significantly reversed these biochemical abnormalities, restoring all indicators to levels close to the control group. Furthermore, histological observation of major organs such as the heart, liver, spleen, and lungs... Figure 16 (A, B) It was found that the GA, GA-Fe and GA-Fe@TPP treatment groups did not cause significant pathological changes in these organs, proving that the material has good biocompatibility.

[0119] 3. Conclusion This invention focuses on the core pathological link of "mitochondrial oxidative stress imbalance caused by pathological mineralization" in crystalline kidney injury. It constructs and systematically validates an ultrasmall metal-polyphenol nanomedicine, GA-Fe@TPP, with mitochondrial targeting capabilities. This nanosystem combines ultrasmall size, good physiological stability, and TPP-mediated precise mitochondrial delivery, enabling it to efficiently accumulate in the core lesion region of renal tubular epithelial cells' mitochondria. Functionally, GA-Fe@TPP not only exhibits broad-spectrum free radical scavenging ability and CAT / GPx-like multi-enzyme mimicry activity, but also significantly inhibits the nucleation, growth, and aggregation of CaOx crystals through multivalent coordination and steric hindrance effects, thus intervening in the kidney stone formation process at both the physical and chemical levels. In in vitro and in vivo multi-level models (cells, fruit flies, and mice), GA-Fe@TPP consistently demonstrates significant anti-crystallization, antioxidant, and renal function protective effects. In summary, this study proposes and validates a mitochondrial-targeted nanotherapy strategy that combines "source inhibition" and "downstream purification," providing new theoretical basis and potential technical pathways for the mechanistic intervention and precision nanomedicine treatment of kidney stones.

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A GA-Fe@TPP ultrasmall metal polyphenol network targeting mitochondria, characterized in that, The ultrasmall metal polyphenol network GA-Fe@TPP uses gentian acid as a ligand and Fe... 3+ It is assembled from nodes and its surface is modified with 4-carboxybutyltriphenylphosphine bromide.

2. The ultra-small metal polyphenol network GA-Fe@TPP according to claim 1, characterized in that, The ultrasmall metal polyphenol network GA-Fe@TPP has an amorphous structure.

3. The ultra-small metal polyphenol network GA-Fe@TPP according to claim 1, characterized in that, The particle size of the ultrasmall metal polyphenol network GA-Fe@TPP is less than 10 nm.

4. The ultra-small metal polyphenol network GA-Fe@TPP according to claim 1, characterized in that, The particle size of the ultrasmall metal polyphenol network GA-Fe@TPP is 7.06±1.78 nm.

5. A method for preparing an ultrasmall metal polyphenol network GA-Fe@TPP as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Synthesis of S1, GA-Fe MPNs Using gentic acid as an organic ligand, the metal ion Fe³⁺ + Using polyvinylpyrrolidone as an inorganic crosslinking center and as a protective polymer, the phenolic hydroxyl groups in gentian acid molecules react with the Fe³⁺ in the complex. + Fully coordinated crosslinking to construct metal-polyphenol nanonetworks GA-Fe MPNs; S2, TPP-PEG 2000 Synthesis of -NH2 4-Carboxybutyltriphenylphosphine bromide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole were dissolved in ultra-dry chloroform solvent and stirred until homogeneous, then activated; NH2-PEG was then added. 2000 -NH-Boc was dissolved in ultra-dry chloroform and slowly added dropwise to the above reaction solution. The reaction was carried out at room temperature, and the reaction solvent was removed by vacuum distillation. The crude product was dried, dissolved in deionized water, dialyzed, and lyophilized to obtain the product TPP-PEG. 2000 -NH-BOC; subsequent removal of the BOC protecting group yields the product TPP-PEG. 2000 -NH2; Synthesis of S3 and GA-Fe@TPP The GA-Fe MPNs were dispersed in deionized water and ultrasonically dispersed to form a uniform suspension. Under continuous stirring, EDC and NHS were added sequentially to the suspension, and the reaction was carried out at room temperature in a dark environment. Subsequently, TPP-PEG was added to the activated system. 2000 -NH2, continue stirring the reaction under light-protected conditions; after the reaction is complete, purify by dialyzing in deionized water, freeze-dry to obtain the ultrasmall metal polyphenol network GA-Fe@TPP.

6. The method according to claim 5, characterized in that, In step S1, the metal ion Fe³⁺ + It comes from FeCl3·6H2O, with a mass ratio of gentic acid to FeCl3·6H2O of 1:

1.

7. The method according to claim 5, characterized in that, In step S3, 4-carboxybutyltriphenylphosphine bromide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and NH2-PEG are used. 2000 The molar ratio of -NH-Boc is 5:5:5:

1.

8. The method according to claim 5, characterized in that, In step S3, GA-Fe MPNs are reacted with EDC, NHS, and TPP-PEG. 2000 The mass ratio of -NH2 is 1:1:1:

1.

9. The use of the ultrasmall metal polyphenol network GA-Fe@TPP as described in any one of claims 1 to 4 in the preparation of a medicament for the treatment and / or prevention of kidney stones.

10. The application according to claim 9, characterized in that, The ultrasmall metal polyphenol network GA-Fe@TPP achieves at least one of the following: a) Promotes the decomposition of hydrogen peroxide; b) Scavenging free radicals; c) Antioxidant; d) Inhibits the nucleation, growth, and aggregation of calcium oxalate crystals; e) Inhibit excessive mitochondrial cleavage and / or cell damage induced by high oxalate.

Images