Ferric gallate nanonetwork, its preparation method and application

Abstract

This invention relates to the field of pharmaceutical technology, and in particular to a ferric gallate nanonetwork, its preparation method, and its applications. The preparation method includes the steps of: preparing a PVP aqueous solution, stirring vigorously at room temperature, and then adding Fe dropwise. 3+ A saline solution was added dropwise; after incubation, the gallic acid aqueous solution was added to the above mixture, stirred, and dialyzed to obtain the gallic acid nanonetwork. This preparation method can synthesize ultra-small gallic acid iron nanonetworks with a particle size of less than 10 nm, which have good crystallization regulation and antioxidant functions, and can be used to prepare drugs for treating kidney stones.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an iron gallate nanonetwork, its preparation method, and its application. Background Technology

[0002] Nephrolithiasis is one of the most common diseases in urology, characterized by high incidence and recurrence rates. Due to its unclear pathogenesis and the lack of effective prevention and treatment drugs, it seriously endangers people's health. Currently, the main treatments for kidney stones include extracorporeal shock wave lithotripsy, open surgery, percutaneous nephrolithotomy (PCNL), and minimally invasive PCNL. In recent years, some progress has been made in understanding the pathogenesis and surgical techniques of kidney stones, but no new, highly effective, clinically applicable drug with minimal side effects has yet been discovered.

[0003] Natural polyphenols possess strong antioxidant properties, but their limited water solubility, low plasma levels, and metabolic instability restrict their practical applications. Metal-Phenolic Networks (MPNs) are a novel organic-inorganic hybrid network system that has gradually developed in recent years. They utilize the coordination between phenolic ligands and metal ions to combine the activities of both polyphenols and metal ions, exhibiting excellent multifunctional properties such as antioxidant, anti-inflammatory, and antibacterial activities.

[0004] The formation of kidney stones is closely related to CaOx crystallization and oxidative damage to renal cells. Furthermore, nanoparticles with a diameter exceeding 10 nm have difficulty crossing the glomerular filtration process to reach the renal tubules, thus hindering their role in stone prevention and treatment. Therefore, ultra-small nanonetworks with crystallization regulation and antioxidant functions have great potential for application in the prevention and treatment of kidney stones. Summary of the Invention

[0005] To address the above problems, this invention provides a method for synthesizing ultrasmall metal-polyphenol nanonetworks (ferric gallate nanonetworks) with a particle size of less than 10 nm, and provides the uses of such nanonetworks.

[0006] The first aspect of this invention provides a method for preparing an iron gallate nanonetwork, comprising the steps of:

[0007] Prepare an aqueous solution of PVP (polyvinyl pyrrolidone), stir vigorously at room temperature, and then add Fe dropwise. 3+ Salt solution; after the addition is complete, incubate, then add gallic acid (Ga, Gallic acid) aqueous solution to the above mixture, stir, and dialyze to obtain the iron-gallate nanonetworks.

[0008] Preferably, the concentration of the PVP aqueous solution is 5-10 mg / mL, such as 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, etc.; the Fe 3+ The concentration of the saline solution is 80-120 mg / mL, such as 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, and 120 mg / mL. The PVP solution and the Fe... 3+ The volume ratio of the saline solution is 40-50:1, such as 40:1, 44:1, 45:1, 47:1, 50:1, etc.; the concentration of the Ga aqueous solution is 8-12 mg / mL, such as 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, etc. The Ga aqueous solution, PVP solution, and Fe... 3+ The volume ratio of the mixed salt solution is 8-12:1, such as 8:1, 9:1, 10:1, 11:1, 12:1, etc.

[0009] Preferably, the Fe 3+ The salt is FeCl3 or Fe(NO3)3.

[0010] The molecular weight cutoff for dialysis is mainly determined based on the molecular weight of the free drug and reagent. Preferably, the dialysis membrane M used in the dialysis is... W CO = 25000.

[0011] A second aspect of the present invention also provides an iron gallate nanonetwork prepared by the above preparation method.

[0012] Preferably, the average particle size of the iron gallate nanonetwork is 4-10 nm.

[0013] A third aspect of the present invention provides a pharmaceutical formulation or composition comprising the above-described ferric gallate nanonetwork.

[0014] Preferably, the pharmaceutical preparation further comprises at least one pharmaceutically acceptable excipient, the selection of which may be based on the dosage form of the pharmaceutical preparation.

[0015] A fourth aspect of the present invention provides the use of the above-described pharmaceutical formulation or composition in the preparation of a CaOx crystallization-regulating drug.

[0016] The fifth aspect of the present invention provides the use of the above-described pharmaceutical preparation or composition in the preparation of a medicament for treating kidney stones.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a method for preparing ferric gallate nanonetworks, which can synthesize ultra-small ferric gallate nanonetworks with a particle size of less than 10 nm. These nanonetworks have good crystallization regulation and antioxidant functions, and can be used to prepare drugs for treating kidney stones. Attached Figure Description

[0019] Figure 1 Characterization diagrams of Fe-Ga; A) Fe-Ga transmission electron microscopy (TEM) and high-resolution (HRTEM) images; B) Selected area electron diffraction (SAED) pattern; C) Particle size distribution; D) Fourier transform infrared spectroscopy (FT-IR) analysis; E) Mapping analysis;

[0020] Figure 2 To assess the stability of Fe-Ga in different solvents;

[0021] Figure 3 To observe the regulatory effect of different concentrations of Fe-Ga on CaOx crystals in vitro using a conventional microscope;

[0022] Figure 4 To observe the regulatory effect of different concentrations of Fe-Ga on CaOx crystals in vitro using scanning electron microscopy (SEM);

[0023] Figure 5 The change in Zeta potential before and after Fe-Ga regulation of CaOx crystals;

[0024] Figure 6 The in vitro antioxidant capacity of Fe-Ga was assessed; UV-Vis was used to detect the scavenging capacity of different concentrations of Fe-Ga against DPPH·(A), ABTS+·(B) and ·OH(C,D) free radicals.

[0025] Figure 7 The effects of different concentrations of Fe-Ga on the viability of different types of renal cells; (A) NRK-52E cells; (B) MDCK cells; (C) HK-2 cells;

[0026] Figure 8 The effect of different concentrations of Fe-Ga on the viability of NRK-52E cells damaged by Na2Ox;

[0027] Figure 9 For observation of cell morphology and CaOx crystals on the cell surface; (A) ordinary microscope image; (B) HE stained image;

[0028] Figure 10 Effect of Fe-Ga on intracellular ROS levels in NRK-52E cells before and after Na2Ox damage; (A) Fluorescence microscopy image; (B) Semi-quantitative fluorescence statistics;

[0029] Figure 11To observe the changes in the number of live / dead cells in NRK-52E cells after treatment using a fluorescence microscope;

[0030] Figure 12 Results of kidney crystallization and damage in mice after appropriate treatment in each group; (A) HE staining image; (B) Polarizing microscope image; (C) PAS staining image; (DF) Quantitative statistical results of the number, area and perimeter of crystals in the polarizing microscope image;

[0031] Figure 13 HE-stained images of tissue sections from the heart, liver, spleen, and lungs of mice in each group; (A) HE-stained image of intact organs and tissues; (B) 100× magnified image of HE-stained organs and tissues. Detailed Implementation

[0032] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the 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.

[0033] In the description of this invention, unless otherwise explicitly defined, terms such as heating, cleaning, weighing, and freezing should be interpreted broadly. Those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0034] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0036] Unless otherwise specified, all reagents, materials, and equipment used in the embodiments of this invention are commercially available; unless otherwise specified, all test methods are conventional test methods in the field.

[0037] In this embodiment of the invention, SPSS 19.0 statistical software was used for analysis, and Graph Pad Prism software was used to visualize the experimental results. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0038] Example 1: Preparation of Ferric Gallate (Fe-Ga) Nanonetwork

[0039] Dissolve 66 mg of polyvinylpyrrolidone (PVP) in 8.8 mL of double-distilled water and stir vigorously at room temperature. Then, add dropwise 0.2 mL of ferric chloride (FeCl3) aqueous solution (100 mg / mL). After incubation for 1 h, add 1 mL of gallic acid (Ga) aqueous solution (10 mg / mL) to the above mixture and stir overnight. The final mixture is then dialyzed using a dialysis membrane (M... W The CO=25000) was dialyzed in deionized water for 24 hours and stored at 4℃ for later use.

[0040] Example 2: Structural Characterization of Fe-Ga Nanonetworks

[0041] 2.1. Transmission electron microscopy (TEM), selected area electron diffraction (SAED), and mapping analysis

[0042] Standard TEM samples were prepared by depositing diluted Fe-Ga nanonetwork suspension droplets onto a carbon-coated copper mesh. Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai F12 microscope at an accelerating voltage of 200 kV. Evaluation was performed using the accompanying selected area electron diffraction (SAED) and mapping analysis.

[0043] 2.2 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0044] Ga and Fe-Ga samples were mixed with KBr powder, pressed into 1 mm thin sheets, and analyzed using a Nicolet iZ-10 spectrometer at 4000 to 400 cm⁻¹. -1 FT-IR spectral analysis was performed within the specified range.

[0045] The characterization results of 2.1 and 2.2 are as follows: Figure 1 As shown. The results show that the average particle size of Fe-Ga prepared in Example 1 is approximately 6.7 ± 2.4 nm. Figure 1 C). Results from high-resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) ( Figure 1 In B), Fe-Ga has a polycrystalline structure. Figure 1B&1C). In the FT-IR spectral analysis results, Fe-Ga at 1290 cm⁻¹ -1 The infrared intensity of the HO-C stretching band is lower than that of Ga, indicating that the HO-C portion of Ga is similar to that of Fe. 3+ Coordination. At 2930cm -1 (CH stretching) and 1650cm -1 The absorption peak at (C=O) confirms the presence of PVP. Figure 1 D). Mapping analysis ( Figure 1 E) shows that C, Fe and O elements are uniformly distributed in Fe-Ga.

[0046] 2.3 Stability of Fe-Ga

[0047] Take an appropriate amount of Fe-Ga suspension and add it to double-distilled water (ddH2O), phosphate buffer (PBS), and culture medium (DMEM), respectively. After standing for 1 day, 3 days, and 7 days, observe whether there is turbidity or precipitation in the mixture and take pictures to record the situation.

[0048] Record the results as follows Figure 2 As shown, Fe-Ga placed in ddH2O, PBS, and DMEM culture medium showed no obvious turbidity or precipitation within one week, indicating that Fe-Ga has good stability.

[0049] Example 3: Determination of the Crystallization Regulation Ability of Fe-Ga on Calcium Oxalate (CaOx)

[0050] The effects of Fe-Ga on the morphology, size, and crystal form of CaOx crystals were investigated using in vitro chemical simulations. Specifically, 10 mM CaCl2 and 1 mM Na2Ox solutions were prepared using Tris-HCl buffer (containing 10 mM Tris and 90 mM NaCl, with pH adjusted to 7.4 by adding HCl dropwise). 50 mL of CaCl2 solutions containing different concentrations of Fe-Ga were added to beakers, followed by 50 mL of Na2Ox solution under rapid stirring, resulting in final CaCl2 and Na2Ox concentrations of 5 mM and 0.5 mM, respectively, and final Fe-Ga concentrations of 0, 50, 100, 150, and 200 μg / mL, respectively. After stirring for 1 h, the mixture was allowed to stand overnight at room temperature. The morphology and quantity changes of CaOx crystals were observed using an inverted microscope and scanning electron microscopy (SEM). The Zeta potential of the prepared samples was measured on a Zetasizer NanoZS (Malvern Instruments Ltd, UK). The results are as follows: Figure 3-5 As shown.

[0051] Bright-field images from an inverted microscope, such as Figure 3As shown, the control group had a large number of CaOx crystals, which showed aggregation and were mostly hexagonal rhomboid. After the addition of Fe-Ga, the number of crystals decreased in a concentration-dependent manner. The crystal morphology changed from hexagonal rhomboid to elliptical crystals with a concave center. Moreover, calcium oxalate dihydrate (COD) crystals with a tetragonal bipyramidal morphology were formed. The higher the Fe-Ga concentration, the more COD crystals were generated.

[0052] Scanning electron microscopy (SEM) results as follows Figure 4 As shown, the CaOx crystals generated in the control group were mainly hexagonal rhombic with a size of about 8 μm. After the addition of Fe-Ga, the morphology of the formed CaOx crystals changed significantly, gradually transforming from hexagonal rhombic to small-sized, lamellarly stacked, centrally concave elliptical crystals, with the size decreasing to about 4 μm.

[0053] Figure 5 This reflects that under the regulation of Fe-Ga, the formed CaOx crystal decreased from -16.5mV to -25.7mV, indicating that Fe-Ga improved the stability of the crystal and made it less prone to aggregation.

[0054] Example 4: Determination of the antioxidant capacity of Fe-Ga

[0055] The antioxidant capacity of the Fe-Ga nanonetwork in Example 1 was evaluated by detecting the scavenging efficiency of Fe-Ga against DPPH·, ABTS+· and ·OH free radicals.

[0056] 4.1 Determination of 2,2-bis(4-tert-octylphenyl)-1-picrylhydrazyl radical (DPPH·)

[0057] Different concentrations of Fe-Ga were mixed with equal volumes of 250 μM DPPH solution in ethanol and reacted for 30 min. The final Fe-Ga concentrations were 0, 25, 50, 100, 200, and 400 μg / mL, and the final DPPH concentration was 125 μM. The absorption curves of the reaction system in the range of 400-700 nm were detected using an ELISA reader.

[0058] 4.2 Determination of 2,2-adiazon-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS+·)

[0059] A mixture of 7 mM ABTS solution and 2.45 mM potassium persulfate solution was incubated at room temperature in the dark for 12–16 h to activate ABTS+· free radicals. Before testing, the mixture was diluted with PBS (pH = 7.4) to achieve an OD value of 0.7 ± 0.2 at 734 nm. Equal volumes of Fe-Ga solutions of different concentrations were mixed and reacted for 20 min. The final Fe-Ga concentrations were 0, 25, 50, 100, 200, and 400 μg / mL. The absorption curves of the reaction system in the 400–900 nm range were detected using a microplate reader.

[0060] 4.3 Methylene Blue (MB) Determination

[0061] Since the hydroxyl radicals (·OH) generated by the Fenton reaction can degrade strobilus (MB), the OD value of the remaining MB in the solution can reflect the ability of Fe-Ga to scavenge ·OH. Different concentrations of Fe-Ga were mixed with MB, and then Fe was added... 2+ / H2O2(Fe 2+ The reaction was carried out in Fenton's solution (1 mM for Fe-Ga and 10 mM for H2O2) for 15 min. The final concentrations of Fe-Ga were 0, 25, 50, 100, 200, and 400 μg / mL, and the final concentration of MB was 100 μM. The absorption curves of the reaction system in the range of 500-900 nm were detected using an ELISA reader.

[0062] 4.4 Determination of 3,3',5,5'-Tetramethylbenzidine (TMB)

[0063] Fe-Ga solutions of different concentrations were mixed with 125 μM TMB and Fenton solution (Fe... 2+ Equal amounts of Fe-Ga (1 mM; H2O2: 10 mM) were mixed in HAC / NaAC buffer (pH 4.5 acetate-sodium acetate buffer) to achieve final Fe-Ga concentrations of 0, 25, 50, 100, 200, and 400 μg / mL. After reacting for 5 min, the absorption curves of the reaction system in the 500-800 nm range were detected using a microplate reader.

[0064] 4.5 Discussion of Results

[0065] The test results for 4.1-4.4 are as follows: Figure 6 As shown, DPPH· and ABTS+· were used as intrinsic reactive nitrogen radicals (RNS) to evaluate the antioxidant capacity of the Fe-Ga nanonetwork in this embodiment of the invention. After incubation with the antioxidant, the characteristic absorption peak of DPPH· at 517 nm gradually decreased. Notably, we found that the absorbance of DPPH· decreased significantly after mixing with different concentrations of Fe-Ga (25, 50, 100, 200, and 400 μg / mL). Figure 6A) indicates that Fe-Ga has a strong free radical scavenging ability, and ABTS+· detection also showed similar results ( Figure 6 B). The above results indicate that an appropriate concentration of Fe-Ga can remove most of DPPH· and ABTS+·.

[0066] •OH is considered a strong free radical and is involved in cell damage. MB and TMB were used to test the scavenging ability of Fe-Ga on •OH generated by the Fenton reaction solution. As expected, the Fenton reaction solution showed poor degradation of MB in the presence of Fe-Ga. Figure 6 C), indicating that Fe-Ga possesses highly efficient ROS scavenging ability. Similarly, in TMB determination, Fe-Ga at a concentration of 400 μg / mL almost completely scavenged ·OH (…). Figure 6 D) indicates that Fe-Ga has good ·OH scavenging activity.

[0067] Example 5 Cell Experiment

[0068] To test the safety of the Fe-Ga nanonetwork from Example 1 in different types of kidney cells, NRK-52E, MDCK, and HK-2 cells were treated with different concentrations (25-500 μg / mL) of Fe-Ga.

[0069] 5.1 Cell Culture and Grouping

[0070] Rat renal tubular ductal epithelial cells (NRK-52E cells), canine kidney cells (MDCK cells), and human renal cortical proximal tubular epithelial cells (HK-2 cells) were cultured in DMEM / F12 or DMEM medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin-100 μg / mL streptomycin antibiotic, under a humidified constant temperature environment of 37°C and 5% CO2. When the cell density reached 80%-90%, the cells were digested with trypsin and collected. After centrifugation to remove the supernatant, the cells were resuspended in fresh complete culture medium for later use.

[0071] Cells were divided into the following groups: a) Normal control group: fresh serum-free medium was added only, and the cells were cultured for 24 h; b) Fe-Ga control group: fresh serum-free medium containing 150 μg / mL Fe-Ga was added, and the cells were cultured for 24 h; c) Damage group: fresh serum-free medium containing 1 mM Na2Ox was added, and the cells were cultured for 24 h; d) Fe-Ga protection group: fresh serum-free medium containing different concentrations of Fe-Ga and 1 mM Na2Ox was added, and the cells were cultured for 24 h.

[0072] 5.2 Cell viability assay

[0073] 5.2.1 Cell safety testing of Fe-Ga

[0074] NRK-52E, MDCK, and HK-2 cells were used at 5 × 10⁻⁶ 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured overnight. 100 μL of fresh serum-free medium containing different concentrations (0, 25, 50, 75, 100, 125, 150, 175, 200, 500 μg / mL) of Fe-Ga was added to each well, and the plates were incubated for 24 h in a humidified constant-temperature environment at 37°C with 5% CO2. Then, the medium was replaced with fresh serum-free medium containing 10% CCK-8 and incubated at 37°C for 2 h. OD values ​​were measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader to calculate cell viability.

[0075] The results are as follows Figure 7 A-7C cells maintained viability above 100%, indicating that Fe-Ga has good safety at concentrations below 500 μg / mL.

[0076] 5.2.2 Detection of the Cell Protective Efficacy of Fe-Ga

[0077] To investigate the protective effect of Fe-Ga on damaged NRK-52E cells, a cell model was established by treating cells with 1 mM Na2Ox for 24 h. Specifically, NRK-52E cells were injected at a rate of 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of / wells in 96-well plates and cultured overnight. Experimental groups were as follows: a) Normal control group: only fresh serum-free medium was added; b) Damage group: fresh serum-free medium containing 1 mM Na₂Ox was added; c) Fe-Ga protection group: fresh serum-free medium containing different concentrations (50, 75, 100, 125, 150, 175, 200, 500 μg / mL) of Fe-Ga and 1 mM Na₂Ox was added. After incubation for 24 h in a humidified constant temperature environment of 37°C with 5% CO₂, the medium was replaced with fresh serum-free medium containing 10% CCK-8 and incubated at 37°C for 2 h. OD values ​​were measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) labeler, and cell viability was calculated for each group.

[0078] The results are as follows Figure 8 As shown, cell damage was effectively inhibited under different concentrations of Fe-Ga protection. The cell viability of the Fe-Ga protection group with a concentration of 150 μg / mL recovered from 57.8% to 82%, indicating that Fe-Ga has a good protective effect on renal cells even at low concentrations.

[0079] 5.2.3 Observation of cell morphology and crystal changes

[0080] Observation under a conventional microscope: NRK-52E cells were seeded in 12-well plates at a density of 3 × 10⁻⁶ cells / well. 4 / well, cultured overnight. Experimental groups were as follows: a) Normal control group: fresh serum-free medium only; b) Fe-Ga control group: fresh serum-free medium containing 150 μg / mL Fe-Ga; c) Damage group: fresh serum-free medium containing 1 mM Na2Ox; d) Fe-Ga protection group: fresh serum-free medium containing a mixture of 50 or 150 μg / mL Fe-Ga and 1 mM Na2Ox. After incubation in a humidified constant temperature environment of 37°C and 5% CO2 for 24 h, the samples were observed under an inverted microscope.

[0081] The results are as follows Figure 9 As shown in Figure A, under bright-field mode, the cells in the normal control group and the Fe-Ga control group exhibited plump morphology and dense growth. However, after damage with 1 mM Na2Ox, the cell number decreased sharply, the cells shrank, and their morphology varied, with a large number of calcium oxalate monohydrate (COM) crystals forming on the cell surface. In contrast, the Fe-Ga protection group showed a significant increase in cell number, a significant improvement in the disordered cell morphology, a significant reduction in the amount of crystals, and a transformation from hexagonal rhombic COM to tetragonal bipyramidal COD, indicating that Fe-Ga inhibited Na2Ox-induced cell damage. The comparison between the two concentrations also shows that the high-concentration Fe-Ga group had a better protective effect.

[0082] HE staining observation: ① Remove the supernatant and wash 3 times with PBS; ② Add 4% paraformaldehyde and fix at room temperature for 20 min, then wash 3 times with PBS; ③ Stain with eosin for 5 min, then rinse with tap water for 3 min; ④ Wash twice with ddH2O, add an appropriate amount of fresh serum-free culture medium, observe the changes in cell morphology and crystals in different groups under bright field mode of an inverted microscope, and take pictures to record the results.

[0083] The results are as follows Figure 9 As shown in Figure B, cells in both the normal control group and the Fe-Ga control group grew well, while the cell density in the damaged group was significantly reduced, and a large number of CaOx crystals were formed. The morphology of the damaged cells in the Fe-Ga protected group improved, and the density was close to that of the normal control group.

[0084] 5.2.4 Detection of intracellular reactive oxygen species (ROS) levels

[0085] DCFH-DA labeling was used, and intracellular ROS changes were observed using fluorescence microscopy. Specifically, NRK-52E was seeded in 12-well plates at a density of 3 × 10⁻⁶. 4 / well, cultured overnight. Experimental groups are the same as above. Perform the procedure according to the ROS detection instructions: ① Aspirate the supernatant and wash 3 times with PBS; ② Add 500 μL of fresh serum-free medium containing 10 μM DCFH-DA to each well and incubate at 37°C in the dark for 30 min; ③ Wash 3 times with fresh serum-free medium to remove unloaded DCFH-DA, add an appropriate amount of fresh serum-free medium, and observe under an inverted fluorescence microscope.

[0086] The results are as follows Figure 10 As shown, the ROS expression level in the damaged group was significantly higher than that in the normal control group. Under the protection of Fe-Ga, the intracellular ROS level decreased significantly, and the higher the Fe-Ga concentration, the more obvious the decrease in fluorescence intensity.

[0087] 5.2.5 Staining of live and dead cells

[0088] Cell death was observed using Calcein AM / PI double staining. Specifically, NRK-52E cells were seeded in 12-well plates at a density of 3 × 10⁶ cells / wells, as described above. 4 / well, culture overnight. Experimental groups are the same as above. Then, according to the Calcein / PI cell viability instructions, perform the following operations: ① Aspirate the supernatant and wash once with PBS; ② Add 500uL of Calcein AM / PI detection working solution to each well and incubate at 37℃ in the dark for 30 min; ③ Observe under an inverted fluorescence microscope.

[0089] The results are as follows Figure 11 As shown, in the normal control group, the cell outline morphology was good, and the main fluorescence was strong green. In the damaged group, the cell number was significantly reduced, the cells shrank and became rounded, and the red fluorescence increased, indicating that Na2Ox caused a large number of cell deaths, and the surviving cells were in poor condition. In the Fe-Ga protection group, the cell condition was significantly improved, and the red fluorescence was significantly reduced. Under Fe-Ga protection, the viability of NRK-52E cells was significantly improved.

[0090] Example 6 Animal Experiment

[0091] A mouse CaOx crystal kidney model was established by intraperitoneal injection of glyoxylic acid to investigate the inhibitory effect of the Fe-Ga nanonetwork of Example 1 on CaOx crystal deposition and tissue damage.

[0092] 6.1 Fluorescent labeling of Fe-Ga

[0093] Take 4 mL of the prepared Fe-Ga solution and mix it with indocyanine green (ICG) at a mass ratio of approximately 10:1. Stir the mixture overnight at room temperature in the dark. After dialysis, the Fe-Ga-ICG complex is obtained and stored at 4°C in the dark for later use.

[0094] 6.2 In vivo therapeutic evaluation of Fe-Ga

[0095] Male BALB / c mice aged 6-8 weeks, weighing approximately 18±2g, were purchased from the Guangdong Provincial Medical Laboratory Animal Center. Mice were randomly divided into 5 groups: ① Normal control group: fed normal diet and given free access to sterile water for 6 consecutive days; ② Model group: fed normal diet and water plus 70mg / kg / day glyoxylic acid intravenously for 6 consecutive days; ③ Ga (20mg / kg) control group: same as the model group plus 20mg / kg Ga intravenously for 6 consecutive days; ④ Low-dose Fe-Ga (10mg / kg) treatment group: same as the model group plus 10mg / kg Fe-Ga intravenously for 6 consecutive days; ⑤ High-dose Fe-Ga (20mg / kg) treatment group: same as the model group plus 20mg / kg Fe-Ga intravenously for 6 consecutive days. All mice were allowed free access to their regular diet and were kept at 25℃ under light-dark cycles during the experiment. Mice were allowed 3 days to acclimatize to the surrounding environment before the start of the animal experiments. All procedures were performed in accordance with the animal management regulations of the Ministry of Health of the People's Republic of China and approved by the Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University.

[0096] 6.3 H&E and PAS staining of mouse kidney tissue

[0097] The kidneys of mice in each group were isolated, fixed in 4% paraformaldehyde, and then embedded in paraffin. The largest section was selected for slide preparation. Staining was performed with hematoxylin-eosin (H&E) and periodic acid-Scheffler (PAS) dyes, followed by analysis using PathScope. TM The mice were observed using a 4S scanner (DigiPath, USA). The extent of renal tubular damage was assessed, with tubules exhibiting the following histopathological changes considered damaged: tubular dilation, cast formation, tubular cell shedding, atrophy, and thickening.

[0098] The results are as follows Figure 12 As shown, A) HE-stained images of kidney tissue sections; B) Polarized light microscope images; C) PAS-stained images; D) Statistical results of the number, area, and perimeter of crystals in polarized light microscope images.

[0099] Among them, HE staining results ( Figure 12 A) shows that the kidneys of the normal control group mice were of good size and shape, with tightly and neatly arranged renal tubules and normal renal tubular cell morphology; kidney sections of the model group ( Figure 12 B) showed abundant, clustered, and interconnected crystals in the renal tubules, with varying degrees of cell swelling and structural damage. Interstitial vascular congestion and inflammatory cells were visible. In the Fe-Ga treatment group, the amount of CaOx crystal deposition in the kidneys and the extent of tubular damage significantly decreased with increasing drug concentration. PAS staining results ( Figure 12C) This also confirmed that the glomerular and tubular structures of the CaOx crystal model mice were severely damaged. Compared with the model group, the kidney damage in the Fe-Ga treatment group was improved, the cell structure was close to normal, and the renal tubular necrosis was significantly reduced. Fe-Ga inhibited the formation and aggregation of CaOx crystals in mice, effectively alleviated kidney damage caused by high oxalate, and reduced the risk of stone formation.

[0100] 6.4 In vivo safety testing

[0101] Heart, liver, spleen, and lung tissues from mice in each group were isolated, fixed in 4% paraformaldehyde, and then embedded in paraffin. The largest section was selected for slide preparation. The tissues were stained with hematoxylin and eosin (H&E) dye and then analyzed using PathScope. TM The 4S scanner (DigiPath, USA) was used for observation.

[0102] The results are as follows Figure 13 As shown, the histopathological sections of Fe-Ga treated mouse organs stained with hematoxylin and eosin (HE) exhibited the same characteristics as organs collected from the normal control group. The livers of mice in the Fe-Ga treatment group showed normal structure, with clear central and portal veins and hepatocytes arranged in normal sinusoidal intervals. The size of the heart chambers and the thickness of the atrial and ventricular walls were normal, with no significant damage to cardiomyocytes. The spleen maintained the ideal histological structure of the white and red pulp. The lung tissue showed an intact reticular structure without damage, and the alveoli, bronchi, and blood vessels were clearly visible.

[0103] The above embodiments synthesized an ultra-small iron gallate nanonetwork with a particle size of less than 10 nm using the preparation method of the present invention. Relevant tests characterized that the nanonetwork has stable performance, good biosafety, and good crystallization regulation ability for CaOx crystallization. It can be used to prepare CaOx crystallization regulation drugs. In addition, the nanonetwork also has good oxygen free radical scavenging activity and good antioxidant properties. Animal experiments also showed that it inhibited CaOx crystal deposition and tissue damage in mouse kidneys. Therefore, it can be used to prepare drugs for the treatment of kidney stones.

[0104] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a ferric gallate nanonetwork in the preparation of a drug for treating kidney stones, characterized in that, The iron gallate nanonetwork was prepared through the following steps: Prepare an aqueous solution of polyvinylpyrrolidone (PVP), stir vigorously at room temperature, and then add Fe dropwise. 3+ Salt solution; after the addition is complete, incubate, then add gallic acid (Ga) solution to the above mixture, stir, and use M... W The ferric gallate nanonetwork was obtained by dialysis with a CO=25000 dialysis membrane; the average particle size of the ferric gallate nanonetwork was 6.7±2.4 nm. The concentration of the PVP aqueous solution was 10 mg / mL; The Fe 3+ The salt solution concentration is 100 mg / mL, and the PVP solution and the Fe 3+ The volume ratio of the salt solution to the aqueous solution is 44:1; The concentration of the Ga aqueous solution is 10 mg / mL, and the volume ratio of the Ga aqueous solution to the mixed solution is 10:

1. The Fe 3+ The salt is FeCl3; PVP: Fe 3+ GA mass ratio = 66:20:10; The concentration of the iron gallate nanonetwork is between 500 μg / mL and 50 μg / mL.

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