Iridium-kaempferol complex as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to an iridium-kaempferol complex as well as a preparation method and application thereof. One Ir (Cp *) Cl2 structure in [Ir (Cp *) Cl2] 2 is substituted by one C15H10O6 to prepare the iridium-kaempferol complex, an Ir (III) center is combined with a kaempferol ligand for the first time, the metal organic complex Ir-Kae which not only utilizes the stability of the Ir (III) complex but also utilizes the pharmacological activity of kaempferol is developed, and through the synergistic effect of multiple mechanisms such as oxidation resistance, inflammation resistance and neuroprotection, the metal organic complex Ir-Kae can be used for preparing the metal organic complex. The spinal cord injury can be effectively treated at multiple target points. The Ir-Kae has a remarkable free radical scavenging capability, can effectively reduce the level of active oxygen in cells and organisms, realizes remarkable oxidation resistance and oxidative stress resistance, can improve the living and death degree of damaged hippocampal neurons, promotes neuronal repair, and has a remarkable anti-aging effect on hippocampal neurons. The nerve injury and the spinal cord injury are effectively treated, and a new direction is provided for assistant treatment of the spinal cord injury medicine.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to iridium-kaempferol complexes, their preparation methods, and their applications. Background Technology

[0002] Spinal cord injury (SCI) is a severe traumatic disease of the central nervous system, referring to a pathological state in which the structure and function of the spinal cord are altered due to external mechanical forces, resulting in temporary or permanent loss of motor, sensory, and autonomic nerve functions below the level of injury. Pathophysiologically, the progression of SCI can be divided into two stages: primary injury and secondary injury. Primary injury is caused by external force acting directly on the spine and spinal cord, often accompanied by mechanical damage such as vertebral fractures, dislocations, and intervertebral disc herniation, leading to instantaneous contusion, hemorrhage, or compression of the spinal cord tissue. The subsequent secondary injury involves a series of complex cellular and molecular cascade reactions, including local ischemia, inflammatory responses, ion imbalances, lipid peroxidation, and apoptosis, further expanding the extent and severity of spinal cord injury. Studies have shown that oxidative stress-related signaling pathways such as MAPK, NF-κB, and HIF-1 are significantly activated after SCI, and their mechanisms of action are closely related to cellular responses to hypoxia, reactive oxygen species metabolism, and the regulation of neuronal death. Therefore, effectively clearing the large number of oxygen free radicals generated in the acute phase of SCI, or inhibiting their excessive generation, and reshaping the redox balance has become an important treatment strategy to block the progression of secondary damage and promote nerve repair.

[0003] In the field of drug development, metal complexes have attracted attention due to their unique electronic structure and tunable chemical properties. Among them, iridium(III) (Ir(III)) complexes have been extensively studied in fields such as chemical sensors, biological probes, photocatalysis, and organic light-emitting diodes due to their high emission efficiency, long excited-state lifetime, excellent photothermal stability, and easily tunable emission wavelength. In recent years, some Ir(III) complexes have also been explored for biomedical applications. However, the mechanisms of action of Ir(III) complexes in disease treatment reported in existing technologies mainly focus on the generation of reactive oxygen species (ROS) using their photophysical properties. For example, various cationic Ir(III) complexes have been reported for use in photodynamic therapy (PDT) or chemokinetic therapy (CDT), with the mechanism of action being to act as photosensitizers or catalysts to promote the generation of ROS to kill tumor cells. This "pro-oxidation" strategy is completely opposite in pharmacological mechanism to the "antioxidant" strategy required for treating oxidative stress-related diseases. On the other hand, natural small molecule compounds are an important source of drug discovery. Kaempferol is a naturally occurring flavonoid compound widely found in various fruits, vegetables, and traditional Chinese medicines. Studies have shown that kaempferol possesses a variety of pharmacological activities, including antioxidant, anti-inflammatory, and antibacterial effects. Its antioxidant activity stems from the phenolic hydroxyl groups in its structure, which can directly scavenge free radicals or activate endogenous antioxidant pathways (such as the Nrf2 pathway). However, as a single component, kaempferol has limitations such as low bioavailability, rapid metabolism in vivo, and relatively dispersed target sites, which restricts its clinical efficacy.

[0004] In summary, it is highly significant to develop organometallic complexes capable of resisting oxidative stress and treating spinal cord injuries by combining the stability and modifiability of Ir(III) complexes with the well-established antioxidant activity of kaempferol. However, no relevant reports have been found to date. Summary of the Invention

[0005] Therefore, based on the above background, this invention provides an iridium-kaempferol complex, its preparation method, and its application. This invention is the first to combine the Ir(III) center with a kaempferol ligand to develop an organometallic complex, the iridium-kaempferol complex Ir-Kae, which can utilize both the stability of the Ir(III) complex and the multi-target pharmacological activity of kaempferol. It can achieve synergistic treatment of spinal cord injury through multiple mechanisms, including inhibiting oxidative stress, regulating the inflammatory microenvironment, and promoting the recovery of nerve function, thus providing a new direction for the drug treatment of spinal cord injury.

[0006] The technical solution provided by this invention is as follows:

[0007] The iridium-kaempferol complex has the following structural formula:

[0008]

[0010] Furthermore, its preparation includes the following steps:

[0011] S1: Dissolve [Ir(Cp×)Cl2]2 and kaempferol in a C1-C4 alcohol solvent, adjust the pH of the solution to 8-14, and react at 60-80℃ for no less than 1 hour;

[0012] S2: After the reaction is complete, remove the solvent and purify the residue.

[0013] Further, the molar ratio of [Ir(Cp×)Cl2]2 to kaempferol in step S1 is (2-3):(4-6).

[0014] Furthermore, in step S1, after adjusting the pH of the solution to 10, the reaction is carried out under heating conditions.

[0015] Furthermore, in step S2, the residue is purified by silica gel column chromatography.

[0016] Based on the same inventive concept, the present invention also provides the iridium-kaempferol complex, or the iridium-kaempferol complex prepared by the preparation method thereof, and its use in the preparation of drugs for the prevention and / or treatment of oxidative stress-related diseases.

[0017] Furthermore, the oxidative stress-related diseases include neurological damage-related diseases.

[0018] Furthermore, the neurological injury-related diseases include secondary spinal cord injury.

[0019] Furthermore, the iridium-kaempferol complex in the drug is the only active ingredient.

[0020] Based on the same inventive concept, the present invention also provides a medicament for treating and / or adjuvant treatment of nerve injury-related diseases, comprising an effective amount of the iridium-kaempferol complex and a medically acceptable adjuvant.

[0021] The beneficial effects achieved by this invention are as follows:

[0022] This invention is the first to combine the Ir(III) center with kaempferol ligand to develop an organometallic complex, iridium-kaempferol complex Ir-Kae, which can utilize both the stability of the Ir(III) complex and the pharmacological activity of kaempferol. It can achieve effective treatment of spinal cord injury through multiple targets by synergistic effects of multiple mechanisms such as anti-oxidation, anti-inflammation and neuroprotection.

[0023] Experiments have demonstrated that the Ir-Kae of this invention possesses significant free radical scavenging capabilities, effectively reducing intracellular and systemic reactive oxygen species levels, achieving significant antioxidant and stress-resistance abilities, and improving the survival rate of damaged hippocampal neurons, promoting neuronal repair, thus providing effective treatment for nerve and spinal cord injuries and offering a new direction for drug-assisted treatment of spinal cord injuries.

[0024] Instruction manual illustrations

[0025] Appendix Figure 1 The relevant characterization results of Ir-Kae prepared in the embodiments of the present invention are as follows: Figure 1 A represents Ir-Kae mass spectra; Figure 1 B represents the Ir-Kae carbon NMR spectrum; Figure 1 C represents the Ir-Kae 1H NMR spectrum; Figure 1 D represents the fluorescence spectra of different compounds; Figure 1 E represents the ultraviolet-visible absorption spectra of different substances.

[0026] Appendix Figure 2 The free radical scavenging results of Ir-Kae in this embodiment of the invention are shown below. Figure 2 A represents the ABTS free radical scavenging method; Figure 2 B represents the DPPH free radical scavenging method; Figure 2 C represents the reaction of O2• - Concentration-dependent clearance efficiency; Figure 2 D represents the concentration-dependent scavenging efficiency of H2O2; Figure 2 E represents the concentration-dependent scavenging efficiency of •OH; Figure 2 F is the efficiency graph of SOD simulated activity.

[0027] Appendix Figure 3 The results of the evaluation of the intracellular reactive oxygen species scavenging ability of Ir-Kae in the embodiments of the present invention are shown.

[0028] Appendix Figure 4 This invention relates to the toxicity results of Ir-Kae on hippocampal neurons in an embodiment of the invention.

[0029] Appendix Figure 5 This invention relates to the effects of Ir-Kae on hydrogen peroxide (H2O2)-induced oxidative stress damage in primary hippocampal neurons.

[0030] Appendix Figure 6 The results of the influence of Ir-Kae on the survival of hippocampal neurons in an embodiment of the present invention are shown.

[0031] Appendix Figure 7 The results of protein blotting and in vitro polarization experiments of macrophages are from embodiments of the present invention.

[0032] Appendix Figure 8 Behavioral and electrophysiological data of Ir-Kae in this embodiment of the invention (left: BMS Score, right: spinal cord electrophysiology).

[0033] Appendix Figure 9 The images show the mouse in vivo imaging results of this invention, where the left image is a mouse in vivo ROS imaging image; and the right image shows the fluorescence intensity of ROS between each group. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Preparation of the iridium-kaempferol complex Ir-Kae

[0036] [Ir(Cp×)Cl2]2 (100.0 mg; 0.13 mmol) was mixed with kaempferol (purchased from AmBeed, product number A272671) (64.3 mg; 0.25 mmol) and dissolved in anhydrous ethanol (10 mL). The pH of the system was adjusted to 10 using sodium hydroxide solution (0.01 mol / L), and the reaction mixture was heated to 70 °C for 2 hours. After the reaction was complete, the solvent was removed, and the residue was purified by silica gel column chromatography (CH2Cl2 / MeOH, volume ratio 10 / 1) to give a brown solid.

[0037] The molecular structure of [Ir(Cp×)Cl2]2 is shown below:

[0038]

[0040] The iridium-kaempferol complex Ir-Kae of the present invention is prepared by substituting one Ir(Cp×)Cl2 structure in [Ir(Cp×)Cl2]2 with one C15H10O6, and the specific reaction for its preparation is as follows:

[0041]

[0042] The brown solid prepared above was characterized as shown in the appendix. Figure 1 .

[0043] The specific characterization operations are as follows:

[0044] ① After the Ir-Kae complex is prepared, the reaction solution is rotary evaporated and lyophilized to obtain a solid product. For spectral and mass spectrometry testing, an appropriate amount of sample is dissolved in methanol (MeOH) or methanol / water (v / v = 1:1) system, sonicated for 5–10 min, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane before use. For NMR testing, the sample is dissolved in DMSO-d6 (or CD3OD, selected according to solubility), allowed to stand and clarify, and then transferred to a 5 mm NMR tube.

[0045] ② Electrospray ionization mass spectrometry (ESI-MS) was used to analyze the molecular ion characteristics of Ir-Kae. Samples were prepared in MeOH (or MeOH / H₂O) at approximately 10–50 μM (or 0.01–0.05 mg / mL), with 0.1% formic acid added if necessary to improve ionization efficiency. Spectra were acquired using a syringe pump (typically 5–10 μL / min) or direct injection mode of LC-MS. The mass spectrometry scan range was set to m / z 100–2000, and the main peak and possible adduct / aggregate ion signals were recorded to determine the bulk ion peak of the product and to assist in verifying complex formation.

[0046] ③ Nuclear magnetic resonance (NMR) 1 H NMR and 13 To confirm the changes in the chemical environment caused by coordination, ^1H NMR and ^13C NMR spectra of Ir-Kae were collected. ^1H NMR was typically measured on a 400 or 600 MHz NMR spectrometer, and ^13C NMR was measured at 100 or 150 MHz. Chemical shifts were measured using solvent residue peaks as internal standards (e.g., DMSO-d6: ^1H 2.50 ppm, ^13C 39.52 ppm; CD3OD: ^1H 3.31 ppm, ^13C 49.00 ppm). The influence of coordination on the electronic environment of the aromatic skeleton and functional groups was assessed by comparing the peak shifts, peak shape changes, and possible changes in hydroxyl-related signals between free kaempferol and Ir-Kae.

[0047] ④ Ultraviolet-Vis absorption spectroscopy (UV-Vis): The absorption spectra of Ir-Kae, free kaempferol, and Ir precursor / control samples were measured using a UV-Vis spectrophotometer. Samples were prepared with MeOH or MeOH / water at approximately 5–30 μM (absorbance controlled in the range of 0.1–1.0), using a 1 cm quartz cuvette, and scanned in the wavelength range of 200–600 nm (step size 1 nm or as per instrument settings). The absorption band shape, peak position, and intensity changes of Ir-Kae and control samples were compared to determine the spectral fingerprint changes caused by coordination and the possible contribution of charge transfer.

[0048] ⑤ Fluorescence spectroscopy

[0049] Emission spectra of Ir-Kae, free kaempferol, and Ir control were recorded using steady-state fluorescence spectroscopy. Sample solvents and concentrations were kept consistent with or appropriately reduced (typically 1–10 μM) compared to UV-Vis to avoid internal filtration effects. Suitable excitation wavelengths were selected (usually near the main absorption peak of their respective UV-Vis wavelengths, such as the 350–380 nm range, or determined based on preliminary experiments), and emission spectra were recorded at 390–650 nm. All samples were tested under the same slit width and gain conditions, and background was subtracted using blank solvent. The coordination-induced fluorescence quenching / energy or electron transfer effects were assessed by comparing the fluorescence intensity and spectral shape differences between Ir-Kae and free kaempferol, further supporting evidence of complex formation.

[0050] The above representation structure is shown in Figure 1 A to Figure 1 E, as shown in the figure, shows the systematic characterization of the prepared Ir-Kae: ESI-MS ( Figure 1 A) A significant main peak appears at m / z≈657.62, accompanied by a few weak addition / aggregation-related peaks, indicating that the product has a distinct main molecular ion characteristic; ^13C NMR and ^1H NMR ( Figure 1 B and Figure 1 The aromatic skeleton signal in C) is clearly discernible, and compared to free kaempferol, it shows a chemical shift change accompanied by a weakening / change in some hydroxyl-related signals, suggesting that coordination leads to a change in the electronic environment and deprotonation coordination; fluorescence spectrum ( Figure 1 D) shows that the luminescence intensity of Ir-Kae is significantly lower than that of kaempferol, exhibiting coordination-induced fluorescence quenching / energy or electron transfer characteristics; UV-Vis absorption ( Figure 1In E), Ir-Kae exhibits enhanced absorption and altered band shape / peak position compared to kaempferol, characterizing a new absorption fingerprint and indicating charge transfer contribution. These results corroborate each other, demonstrating that Ir and kaempferol form a stable coordination structure, and the resulting Ir-Kae is a metal complex product distinct from simple physical mixing.

[0051] Example 2: In vitro and in vivo related verification experiments

[0052] In vitro experiments

[0053] (1) Antioxidant activity experiment

[0054] 1) The Ir-Kae radical scavenging ability was determined using the ABTS and DPPH methods;

[0055] The specific experimental procedures are as follows:

[0056] ① Referring to existing literature, ABTS solution (5 mM) was mixed with potassium persulfate solution in a dark environment at room temperature to form ABTS·+ solution. Subsequently, different concentrations of Ir-Kae were added to the ABTS·+ solution and incubated in the dark for 120 minutes. The absorbance of the solution at 734 nm was then measured by UV-Vis spectroscopy to determine the degree of ABTS·+ scavenging.

[0057] ② Detection of DPPH free radical scavenging ability

[0058] Referring to previous literature, a DPPH working solution was prepared by mixing a DPPH stock solution with anhydrous ethanol (volume ratio 1:20). Subsequently, different concentrations of Ir-Kae were added to the DPPH working solution and incubated at 37°C for 120 minutes. The absorbance of the solution at 517 nm was measured using UV-Vis spectroscopy to determine the extent of DPPH scavenging.

[0059] ③ Detection of OH scavenging ability

[0060] 1 mM FeSO4 and 2 mM H2O2 were mixed in sodium carbonate buffer, and then different concentrations of Ir-Kae were added. After a 5-minute reaction, 3,3',5,5'-tetramethylbenzidine (TMB, T0440, Thermo Fisher Scientific) was added. Specifically, the mixture of 1 mM FeSO4 and 2 mM H2O2 in the sodium carbonate buffer generated ·OH. TMB, as a chromogenic substrate, was oxidized by the ·OH to generate a blue substrate with maximum absorbance at 652 nm. Due to the different concentrations of Ir-Kae, the ·OH in the system decreased accordingly, resulting in a decrease in absorbance. After a 5-minute reaction, the absorbance at 650 nm was measured, and the scavenging rate was calculated using the following formula to evaluate the ·OH scavenging ability of Ir-Kae. Where Sample represents the absorbance of the reaction mixture containing the sample and the free radical system, Blank represents the absorbance of the mixture containing only the sample but without the generation of free radicals, and Control represents the absorbance of the free radical system without any sample.

[0061] • OH scavenging rate (%) = [1- (A_Sample-A_Blank.) / A_Control]×100%

[0062] ④ Detection of H2O2 scavenging ability

[0063] The ability of Ir-Kae to scavenge H₂O₂ was evaluated using the ammonium molybdate colorimetric method. H₂O₂ reacts with ammonium molybdate to form a stable yellow complex, exhibiting an absorption peak at 405 nm. Different concentrations of Ir-Kae were incubated with 2 mmol of H₂O₂ at 37°C for 120 min. After incubation, ammonium molybdate was added to the system to react and form a stable yellow complex. The absorbance of the system at 405 nm was then measured, and the scavenging rate was calculated using the following formula to evaluate the ·OH scavenging ability of Ir-Kae. Where Sample represents the absorbance of the reaction mixture containing the sample and the free radical system, Blank represents the absorbance of the mixture containing only the sample but without free radical generation, and Control represents the absorbance of the free radical system without any sample.

[0064] H2O2scavenging rate (%) = [1- (A_Sample-A_Blank.) / A_Control ]×100%

[0065] ⑤ Determination of O2•− scavenging ability

[0066] The scavenging ability of Ir-Kae for superoxide anion (O2•−) was determined using the nitroblue tetrazolium (NBT) reduction method (S0109, Beyotime). In this method, superoxide anion is generated in a non-enzymatic reaction system consisting of nicotinamide adenine dinucleotide (NADH), phenazine methanesulfonic acid (PMS), and NBT. Briefly, 1 mL of reaction mixture (containing 73 μmol NADH, 50 μmol NBT, and the test sample) was prepared in 0.1 mL / L phosphate buffer (pH 7.4). Subsequently, 15 μmol PMS was added to initiate the reaction, and the mixture was incubated at room temperature for 5 min. The superoxide anion reduces NBT to blue formazan. The scavenging ability of superoxide anion was quantified by measuring absorbance at 560 nm, and the scavenging rate was calculated using the following formula to evaluate the ·O2•− scavenging ability of Ir-Kae. Sample represents the absorbance of the reaction mixture containing the sample and the free radical system, Blank represents the absorbance of the mixture containing only the sample but without generating free radicals, and Control represents the absorbance of the free radical system without any sample.

[0067] O2•− scavenging rate (%) = [1- (A_Sample-A_Blank.) / A_Control]×100%

[0068] Assay of SOD enzyme activity

[0069] Materials and Methods: Total SOD Activity Assay

[0070] SOD Activity Assessment: The total superoxide dismutase (SOD) activity of the samples was determined using a total SOD activity assay kit (WST-8 method, Beyotime, S0101). This assay is based on a xanthine oxidase (XO) coupling reaction system.

[0071] The reaction principle is as follows: Xanthine generates superoxide anions (O2•) under XO catalysis. - The generated O2• - The colorimetric reagent WST-8 is reduced to produce a stable orange-yellow water-soluble formazan dye, which has the highest absorption peak at a wavelength of 450 nm.

[0072] When SOD is present in the sample, SOD can catalyze O2• - Disproportionation occurs to generate O2• - and O2• - This competitively inhibits WST-8 and O2• -The reaction reduces the amount of formazan dye produced. Therefore, the absorbance value at 450 nm is negatively correlated with the SOD activity in the sample.

[0073] Detailed operation steps:

[0074] According to the instructions, mix the sample to be tested with WST-8 / enzyme working solution.

[0075] After adding the reaction start-up working solution, incubate at 37°C for 30 minutes.

[0076] The absorbance of each well at 450 nm was measured using an ELISA reader.

[0077] The inhibition percentage is calculated using the following formula:

[0078] Inhibition percentage=1- (A_Sample-A_Blank.) / (A_Control-A_Blank ) ]×100%

[0079] Unit Definition: In the above detection system, when the inhibition percentage of superoxide anion is 50%, the SOD enzyme activity in the reaction system is defined as one enzyme activity unit (Unit). The final results are standardized based on the protein concentration (U / mg protein) or tissue weight of the sample.

[0080] See results Figure 2 ,in Figure 2 A and Figure 2 Figure B shows that ABTS and DPPH are gradually cleared with a dose-response effect as the concentration increases. Other figures show that the reactive oxygen species scavenging rate increases with the concentration of the complex, exhibiting SOD-like activity.

[0081] 2) Assessment of Ir-Kae's intracellular reactive oxygen species scavenging capacity

[0082] Intracellular reactive oxygen species (ROS) scavenging activity of Ir-Kae was detected using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime, catalog number S0033S) and ethidium dihydrofluorescein (DHE; Beyotime, catalog number S0063). PC12 cells were seeded in confocal culture dishes (NEST, catalog number 801001) and subjected to 6 hours of H2O2 exposure to induce oxidative stress. The culture medium was then replaced with conditioned medium containing control medium and Ir-Kae (40 μg / ml) for another 12 hours. After treatment, cells were labeled with DCFH-DA or DHE for 30 minutes, washed with PBS, and counterstained with DAPI in the dark for 5 minutes. Fluorescence images were acquired using a wide-field fluorescence microscope (Leica Dmi8 Thunder), and ROS levels were quantitatively analyzed using ImageJ software.

[0083] To detect intracellular reactive oxygen species (ROS) by flow cytometry, PC12 cells were seeded into 6-well plates and treated with hydrogen peroxide (H2O2) for 6 hours to induce oxidative stress. They were then cultured for another 12 hours in conditioned medium containing cerium ions (3.14 μg / mL), kaempferol (32.46 μg / mL), and Ce-Kaem NCs (40 μg / mL). Cells were collected by trypsin digestion, washed with PBS, and centrifuged at 5000 rpm for 5 minutes. The cell pellet was resuspended in PBS containing DCFH-DA (1:1000, v / v) and incubated in the dark at room temperature for 20 minutes. After incubation, the cells were washed with PBS and stored in the dark until analysis. Fluorescence signals were collected using a FACS Array Bioanalyzer (BDBiosciences) at an excitation wavelength of 488 nm, and the mean fluorescence intensity was quantified using FlowJo 7.6 software.

[0084] In vitro uptake and colocalization imaging of Ir-Kae

[0085] To investigate cellular uptake of nanoparticles, Ir-Kae was first fluorescently labeled. Courmarin-6 (C6, MCE) was dissolved in DMSO to prepare a 1 mg / mL stock solution, which was then added to Ir-Kae suspension (40 μg / mL) at a mass ratio of 1:200 and incubated at room temperature for 2 h in the dark. Unbound dye was removed by centrifugation three times at 12,000 × g and washing with PBS to obtain C6-labeled Ir-Kae (C6–Ir-Kae). Cell membrane labeling was performed using DiI. DiI was diluted to 2–5 μg / mL and added to the cells to be observed. After incubation at 37 °C for 5–10 min, the cells were washed three times with PBS and incubated for 10 min in serum-containing medium. Subsequently, C6–Ir-Kae (40 μg / mL) was added to the cells, and incubation was performed for 6, 12, and 24 h. After treatment was terminated at each time point, the cells were washed three times with PBS. Cells were fixed in 4% paraformaldehyde at room temperature for 10–15 min and stained with DAPI (1 μg / mL). Fluorescence images were acquired using a fluorescence microscope (Leica Dmi8 Thunder), with the excitation / emission bands for Coumarin-6 set to 488 nm / 500–550 nm, and the DiI channels set to 549–561 nm / 565–620 nm. Image quantification was performed using ImageJ software, measuring the mean fluorescence intensity (MFI) of Coumarin-6 and its co-localization coefficient with DiI to assess nanoparticle uptake and cell membrane localization. All experiments were repeated at least three times, and data are expressed as mean ± SD.

[0086] See results Figure 3 , Figure 3 Figure A reflects the cellular uptake of labeled Ir-Kae at different times. As shown in the figure, with the extension of incubation time (6h, 12h, 24h), the green fluorescence of C6-Ir-Kae gradually increases and expands in range within the cell, indicating that Ir-Kae can be continuously taken up by the cell and the uptake efficiency is time-dependent. The DiI-labeled cell membrane (red) and the green fluorescence of C6-Ir-Kae have spatial overlap in the Merge figure, suggesting that the uptake process of Ir-Kae may interact with the cell membrane or eventually be located in the intracellular region near the membrane.

[0087] Figure 3 B and Figure 3C shows that the fluorescence signal of intracellular reactive oxygen species was weakened after Ir-Kae treatment: the fluorescence signal of the Control group (untreated) was weak, indicating a low level of intracellular superoxide anion; the fluorescence signal of the H2O2 group was significantly enhanced, indicating that H2O2 successfully induced the cells to produce a large amount of superoxide anion; the fluorescence signal of the Ir-Kae + H2O2 group was significantly weaker than that of the H2O2 group, suggesting that Ir-Kae can reduce the level of intracellular superoxide anion. Figure 3 As can be seen from D, the DCFH-DA fluorescence signal of cells treated with Ir-Kae was significantly reduced: the fluorescence peak in the Control group was concentrated in the low intensity region, representing the baseline ROS level of normal cells; the fluorescence peak in the H2O2 group shifted to the right, indicating a significant increase in the proportion of ROS-positive cells; the fluorescence peak in the Ir-Kae+ H2O2 group shifted to the left, indicating a significant decrease in the proportion of ROS-positive cells compared to the H2O2 group. This shows that Ir-Kae can significantly clear H2O2-induced intracellular ROS.

[0088] (2) The toxicity of Ir-Kae to neurons was verified by using Cell Counting Kit-8 and cell live / dead staining experiments;

[0089] Ir-Kae cytotoxicity was assessed using a cell counting kit-8 (CCK-8; C0037, Beyotime). Primary hippocampal neurons were counted at 1×10⁻⁶ cells / year. 4 Cells were seeded at a density of three replicates per well in 96-well plates. After an initial 12-hour incubation to allow cell adhesion, the medium was replaced with conditioned medium containing gradient concentrations of Ir-Kae. After 12 hours of exposure, CCK-8 was added to each well to assess cytotoxicity, and the cells were incubated at 37°C for 30 minutes. The absorbance was then measured at 450 nm using a microplate reader. Cytotoxicity was evaluated using the following formula: 𝐴Sample represents the absorbance of the treatment group (containing different concentrations of Ir-Kae), Control represents the absorbance of the untreated normal cell control group, and Blank represents the absorbance of the medium and CCK-8 reagent alone. Cell viability was calculated using the untreated group as a reference, and the highest concentration maintaining >90% viability was selected for subsequent experiments.

[0090] Cell Viability (%) = (A_Sample-A_Blank.) / (A_Control-A_Blank ) ]×100%

[0091] result Figure 4 It is evident that the cell survival rate was higher than 90% when the Ir-Kae concentration was 40 μg / ml, indicating that the Ir-Kae concentration at no higher than 40 μg / ml had no significant toxicity to neurons.

[0092] (3) Verify the protective effect of Ir-Kae against hydrogen peroxide (H2O2)-induced oxidative stress damage in primary hippocampal neurons.

[0093] Acquisition of hippocampal neurons:

[0094] Hippocampal tissue was isolated from neonatal SD rat sucklings (0-3 days after birth). Digestion was performed at 37°C for 25 minutes using 0.125% trypsin (25200-072, Gibco). Digestion was terminated by adding Durbeco Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12, 11320033, Gibco) containing 10% fetal bovine serum (FBS, A3161002C, Gibco). After undigested tissue fragments settled in the suspension, the supernatant was removed, and the tissue was resuspended in fresh culture medium. Neurons were gently dispersed using a Pasteur glass pipette. The final cell suspension was prepared at approximately 1 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of [number] cells / cm² in poly-D-lysine-coated (PDL, P6407, Gibco) glass-bottom confocal culture dishes (801001, NEST). Four hours after cell adhesion, the medium was replaced with Neurobasal-A medium (10888-022, Gibco) supplemented with 2% B27 (16954044, Gibco) for continuous culture. To maintain optimal neuronal activity, half of the medium was replaced every three days. The total in vitro culture time was adjusted according to the requirements of subsequent experimental protocols.

[0095] Primary hippocampal neurons were fixed, permeabilized, and blocked. Axons were labeled with anti-Tuj1 (1:200, ab49822, Abcam), followed by incubation with Alexa Fluor 555 (1:500, ab150118, Abcam) or Alexa Fluor 647 (1:500, ab150115, Abcam) secondary antibodies at room temperature for 1 h in the dark. DAPI (1 μg / mL) was used for nuclear staining. After acquiring fluorescence images, ImageJ was used to quantify parameters such as total axon length and the number of axonal branches to further evaluate the impact of Ce–Kaem NCs on the axonal development and regeneration capacity of primary hippocampal neurons.

[0096] like Figure 5 As shown in the figure, the control group had the highest number of neurons and the longest axon length. After hydrogen peroxide treatment, the axons shrank and the number of neurons decreased. After treatment with Ir-Kae complex, the axon length and the number of neurons recovered significantly.

[0097] Primary hippocampal neurons were seeded at a density of 1 × 10^6 cells per well in 6-well plates and allowed to adhere overnight. Ir-Kae (40 μg / mL) was then added to each well for 1 hour as a pretreatment. Cells were then stimulated with 100 μM H2O2 for 24 hours. Cells were stained with calcein-AM (for live cells) and propidium iodide (for dead cells), and washed with PBS after 20 minutes of incubation. Fluorescence images were acquired using a wide-field fluorescence microscope (Dmi8 Thunder, Leica). The excitation / emission wavelengths of calcein for live cells were 488 / 515 nm, and for dead cells, the PI wavelengths were 535 / 617 nm. The live / dead cell ratio was quantitatively analyzed using ImageJ software. Cell viability (%) (Survival) was calculated using the following formula: Survival (%) = Calcein-AM + Cell count / (Calcein-AM) + Cell count + PI + Cell count) × 100%, in addition, live cells (Calcein-AM) were counted separately. + ) and dead cells (PI) + The proportion of cells in the total cell count (excluding those without) serves as an indicator of cell survival and death. Treatment significantly improved cell survival.

[0098] Figure 6The leftmost column of images in group A represents the Sham group. The top image, from the Calcein channel, shows almost uniform, bright green fluorescence filling the field of view, indicating a very high percentage of live cells and good cell viability. The middle image, from the PI channel, shows no obvious red fluorescence, only a dark background, suggesting a very small number of dead cells (cells with almost no membrane damage). The bottom image, from the merged channel, shows green fluorescence dominating the field of view, with almost no red fluorescence, indicating a high number of live cells and a low number of dead cells. The middle column of images represents the H2O2 group. The top image, from the Calcein channel, shows significantly weakened and sparse green fluorescence, indicating a significant decrease in the number of live cells. The middle image, from the PI channel, shows dense red fluorescent spots / areas, indicating a sharp increase in the number of dead cells. The bottom image, from the merged channel, shows a significantly increased proportion of red fluorescence and diluted green fluorescence, visually demonstrating a small percentage of live cells and a large percentage of dead cells in this group. The rightmost column of images represents the Ir-Kae+ group. The top image of the H2O2 group, showing the Calcein channel, reveals a significant increase in green fluorescence density, approaching the brightness and distribution density of the Sham group, indicating a significant recovery in the number of viable cells. The middle image, showing the PI channel, shows significantly weaker red fluorescence intensity compared to the H2O2 group, with only a small amount or no obvious red signal remaining, suggesting a substantial reduction in the number of dead cells. The bottom image, showing the merged channel, shows green fluorescence dominating the field of view again, with sparse or even invisible red fluorescence, exhibiting a phenotype highly similar to the Sham group. This demonstrates that Ir-Kae has a significant protective effect against hydrogen peroxide (H2O2)-induced oxidative stress damage in primary hippocampal neurons. Figure 6 B also quantified cell survival rate. Ir-Kae effectively improved neuronal cell survival under the intervention of hydrogen peroxide. Although there was still a gap compared with the sham group, it showed that Ir-Kae had a significant protective effect against hydrogen peroxide (H2O2) induced oxidative stress damage in primary hippocampal neurons.

[0099] (4) The protein expression levels of IL-1β, TNF-α, IL-6, iNOS, and Arg-1 in Raw264.7 cells were detected by Western blotting. Cultured neuronal cells or spinal cord tissue samples were placed in RIPA lysis buffer (P0013B, Beyotime) containing protease and phosphatase inhibitors and lysed on ice for 30 min. The lysis buffer was centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant was collected for subsequent experiments. Protein concentration was quantified using the BCA method (A55865, Thermo Fisher Scientific). An equal volume of total protein (20–40 μg) was mixed with 5× SDS loading buffer and denatured in a metal bath at 95 °C for 5 min. The protein samples were then separated on 10–12% SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore) by semi-dry transfer. After blocking the membrane in TBST (containing 0.1% Tween-20) with 5% skim milk powder at room temperature for 2 h, it was incubated overnight at 4 °C with the following primary antibodies: IL-1β (1:1000, ab283818, Abcam), TNF-α (1:1000, ab215188, Abcam), IL-6 (1:1000, ab9324, Abcam), iNOS (1:1000, ab283655, Abcam), and Arg-1 (1:1000, 16001-1-AP, Proteintech). β-Tubulin (1:2000, ab314069, Abcam) was used as an internal control for signal normalization. After washing, the membrane was incubated with HRP-labeled secondary antibody (1:5000, Cell Signaling Technology) at room temperature for 1 h. Enhanced chemiluminescence (ECL) kit (P0018S, Beyotime) was used for color development, and band signals were acquired using the ChemiDoc XRS+ imaging system (Bio-Rad). ImageJ software was used for quantitative analysis of band intensity, with β-tubulin as a normalized reference for target protein expression levels.

[0100] Macrophage in vitro polarization experiment

[0101] To assess the effect of Ir-Kae on macrophage polarization, immunofluorescence staining was used to detect different phenotypes in RAW264.7 cells. After treatment with LPS (Lipopolysaccharide), IL-4, and Ir-Kae, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were permeabilized with 0.1% Triton X-100 PBS solution for 10 minutes, followed by blocking with 5% bovine serum albumin (BSA) for 1 hour to reduce non-specific binding. For M1 macrophage phenotype identification, cells were incubated overnight at 4°C with CD68 antibody (1:200, ab303565, Abcam) and inducible nitric oxide synthase (iNOS, 1:200, ab283655, Abcam) primary antibody. M2 phenotype identification was performed using a combination of CD68 and arginase-1 (Arg-1, 1:200, 16001-1-AP, Proteintech) antibody. After incubation with the primary antibody, cells were washed and then incubated with secondary antibodies labeled with Alexa Fluor 488 (1:500, ab150077, Abcam) or Alexa Fluor 555 (1:500, ab150118, Abcam) at room temperature in the dark for 1 hour. Finally, nuclei were stained with DAPI (1 μg / mL) for 5 minutes. Fluorescence images were acquired using a fluorescence microscope, and the colocalization of CD68 with iNOS or Arg-1 was quantitatively analyzed using ImageJ software. The colocalization of M1-type (CD68) cells was also calculated. + / iNOS + ) and M2 type (CD68) + / Arg-1 + The proportion of cells was used to assess the polarization status of macrophages.

[0102] See results Figure 7 ,Depend on Figure 7 As shown in Figure A, compared with the Control group, the number of CD68+ and iNOS colocalized cells (Merge purple) was significantly increased in the LPS group, suggesting that LPS can induce Raw264.7 cells to polarize towards the M1 type. Compared with the LPS group, the CD68+ / iNOS ratio was significantly higher in the LPS+Ir-Kae group. + The reduction in colocalized cells indicates that Ir-Kae can inhibit LPS-induced M1 polarization. Figure 7 B. As can be seen, compared with the Control group, the IL-4 group showed a significant increase in CD68+ and Arg-1 co-localized cells, suggesting that IL-4 can induce Raw264.7 cells to polarize towards the M2 type. Figure 7As shown in Figure C, compared with the Control group, the iNOS band gray value was higher in the LPS group, indicating that LPS induces upregulation of iNOS protein expression. Compared with the LPS group, the iNOS band gray value was lower in the LPS+Ir-Kae group, confirming that Ir-Kae can downregulate LPS-induced iNOS expression, thus verifying its inhibitory effect on M1 polarization at the protein level. Figure 7 As shown in Figure D, compared with the Control group, the Arg-1 band gray value was higher in the IL-4 group, indicating that IL-4 induces upregulation of Arg-1 protein expression. Compared with the IL-4 group, the increased Arg-1 band gray value in the IL-4+Ir-Kae group suggests that Ir-Kae can enhance IL-4-induced Arg-1 expression and promote M2 polarization; a decrease indicates inhibition. Combined with the M1 inhibition results, this shows that Ir-Kae can regulate polarization balance by inhibiting M1 polarization and promoting M2 polarization. Figure 7 As can be seen from E, compared with the control group, the gray values ​​of IL-1β and TNF-α bands in the LPS group were higher, indicating that LPS induced a large release of pro-inflammatory factors. Compared with the LPS group, the gray values ​​of pro-inflammatory factor bands in the LPS+Ir-Kae group were lower, which is consistent with the iNOS results and further confirms that Ir-Kae exerts an anti-inflammatory effect by inhibiting M1 polarization and reducing the secretion of pro-inflammatory factors.

[0103] The results above show that Ir-Kae can exert its effects by bidirectionally regulating macrophage polarization. For LPS-induced M1 polarization, Ir-Kae inhibits iNOS expression and reduces the release of pro-inflammatory factors (IL-1β, TNF-α), thus weakening its pro-inflammatory capacity. For IL-4-induced M2 polarization, Ir-Kae promotes Arg-1 expression and enhances its anti-inflammatory and repair potential.

[0104] (5) Related animal experiments

[0105] Eight-week-old female C57BL / 6 mice were used and fed normally. The mice were purchased from the Animal Center of Xi'an Jiaotong University.

[0106] Construction of a mouse model of spinal cord injury:

[0107] (1) Remove the hair from the back of the mouse to expose the skin on the back of the mouse;

[0108] (2) Anesthesia: Anesthesia was performed by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). The anesthesia was successful when breathing was stable, muscle strength in the limbs was significantly weakened, and pain reflex and corneal reflex disappeared.

[0109] (3) Exposure of the spinal cord: Wipe the skin twice with iodine-soaked cotton balls to disinfect it; make an incision along the midline of the spine, bluntly dissect the paraspinal muscles, remove the spinous process and lamina with bone forceps, and thoroughly expose the T8 segment of the spinal cord. During the operation, be careful to avoid additional tissue damage. Then use a fixator to open and fix the spinal cord to fully expose it.

[0110] (4) A spinal cord clipping injury model was established in mice by clamping the T8 segment of the spinal cord with a microvascular clip for 30 seconds.

[0111] (5) Close the incision and administer 2000 U of gentamicin subcutaneously once a day for 3 consecutive days. Empty the artificial bladder every 8 hours.

[0112] Twelve mice with successfully constructed spinal cord injury models were randomly selected and randomly divided into the SCI group and the Ir-Kae group, with six mice in each group.

[0113] Six mice that underwent only laminectomy without spinal cord injury were selected as the blank control group, the Sham group.

[0114] Mice in the SCI group were injected with 10 μL of PBS immediately after laminectomy and spinal cord injury; mice in the Ir-Kae group were injected locally with 10 μL of Ir-Kae solution (Ir-Kae solution with a concentration of 40 μg / ml prepared with physiological saline) immediately after laminectomy and spinal cord injury.

[0115] ① Postoperatively, after the mice in each group acclimatized to their environment, they were allowed to move freely in an open area for 3 minutes, and their hind limb motor function was assessed using the BassoMouseScale (BMS) scoring system. The BMS score was mainly used to evaluate the frequency, range, gait quality, and forelimb-hindlimb coordination of hind limb movements, with a total score range of 0–9 points. BMS scoring was performed independently by two professionally trained assessors who were blinded to the experimental group assignments on postoperative days 0, 3, 7, 14, 21, and 28. Each observation lasted 2 minutes, and the scoring results were recorded every three days.

[0116] Four weeks post-spinal cord injury surgery, cortical motor evoked potentials (CMEP) were performed to assess the functional recovery of the cortical-spinal cord conduction pathway. Mice were anesthetized with 1% sodium pentobarbital (50 mg / kg) via intraperitoneal injection and fixed in a stereotaxic apparatus. Evoked potential signals were recorded using a MadLab-4C data acquisition and analysis system (Zhongshi Technology, China). A pair of tungsten microelectrodes (85 mm long, 0.5 MΩ impedance, KeDouBC) were used as bipolar stimulation electrodes, with an electrode spacing of 1 mm, inserted into the T6 segment of the spinal cord at a depth of approximately 300–500 μm. The stimulation parameters were monophasic square wave pulses (0.1 ms) with a pulse interval of 3 s. A 1 mm diameter silver ball electrode was placed in the T10 segment to record evoked potentials. All signals were bandpass filtered from 20–1000 Hz and subsequently analyzed on the MATLAB platform. Electrophysiological parameters such as amplitude and phase rhythm were quantitatively analyzed under blind conditions by technicians whose experimental groups were unknown. Results are shown below. Figure 5 .

[0117] Depend on Figure 8 It is evident that the BMS score, induced motor potential, and refractory period of mice in the Ir-Kae group were significantly improved after Ir-Kae treatment.

[0118] ② In vivo ROS chemiluminescence imaging

[0119] To assess the level of reactive oxygen species (ROS) generation in vivo after spinal cord injury, in vivo imaging analysis was performed using the chemiluminescent probe L-012 on postoperative day 3. Mice were fasted for 4 hours before imaging (with free access to water) and maintained stable condition through continuous inhalation anesthesia with 1.5–2.0% isoflurane throughout the imaging process. L-012 was prepared as a working solution with sterile saline and administered intraperitoneally at a dose of 25 mg / kg. Immediately after injection, mice were placed in an AniView 600 small animal in vivo imaging system (BLT, China) for chemiluminescence imaging, and signals were acquired using open-filter mode; the chemiluminescence of L-012 was mainly concentrated in the 470–480 nm blue light band. All imaging parameters were kept consistent across groups, and the obtained signal intensity was quantified by ROI using the system's accompanying analysis software to assess the in vivo ROS level in the spinal cord injury area. The results are shown in [Figure 1]. Figure 6 .Depend on Figure 6 It is evident that the local ROS levels in mice were significantly reduced after Ir-Kae treatment, indicating that Ir-Kae can effectively improve oxidative stress.

[0120] The embodiments shown are merely one implementation of the present invention, and the actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this and design similar structures and embodiments without departing from the spirit of the invention, they should all fall within the protection scope of the present invention.

Claims

1. An iridium-kaempferol complex, characterized in that, Its structural formula is shown below: 。 2. The method for preparing the iridium-kaempferol complex according to claim 1, characterized in that, Includes the following steps: S1: Dissolve [Ir(Cp×)Cl2]2 and kaempferol in a C1-C4 alcohol solvent, adjust the pH of the solution to 8-14, and react at 60-80℃ for no less than 1 hour; S2: After the reaction is complete, remove the solvent and purify the residue.

3. The method for preparing the iridium-kaempferol complex according to claim 2, characterized in that, The molar ratio of [Ir(Cp×)Cl2]2 to kaempferol in step S1 is (2-3):(4-6).

4. The method for preparing the iridium-kaempferol complex according to claim 2, characterized in that, In step S1, the pH of the solution is adjusted to 10, and the reaction is carried out under heating conditions.

5. The method for preparing the iridium-kaempferol complex according to claim 2, characterized in that, In step S2, the residue was purified by silica gel column chromatography.

6. The use of the iridium-kaempferol complex according to claim 1, or the iridium-kaempferol complex prepared by the method according to any one of claims 2 to 6, in the preparation of medicaments for the prevention and / or treatment of oxidative stress-related diseases.

7. The application according to claim 6, characterized in that, The oxidative stress-related diseases include neurological injury-related diseases, including secondary spinal cord injury.

8. The application according to claim 6 or 7, characterized in that, The iridium-kaempferol complex in the drug is the only active ingredient.

9. A medicament for treating and / or adjunctive treating neurological injury-related diseases, characterized in that, Includes an effective amount of the iridium-kaempferol complex of claim 1 and a medically acceptable adjuvant.

10. A medicament for treating and / or adjunctive treating neurological injury-related diseases according to claim 9, characterized in that, The iridium-kaempferol complex is the only active ingredient.

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