Conductive polypyrrole nano-composite based on polydopamine modification, preparation method of conductive polypyrrole nano-composite and application of conductive polypyrrole nano-composite in preparation of retina ischemia-reperfusion injury treatment medicine

Abstract

The invention discloses a conductive polypyrrole nano-composite based on polydopamine modification, a preparation method of the conductive polypyrrole nano-composite and application of the conductive polypyrrole nano-composite in preparation of a medicine for treating retinal ischemia reperfusion injury, and relates to the technical field of biomedical nano-materials. The polydopamine-modified conductive polypyrrole nano-composite forms a core-shell structure through copolymerization of dopamine and pyrrole monomers, and has the properties of oxidation resistance, iron ion chelation, inflammation resistance and conductivity. The nanocomposite can effectively remove active oxygen, inhibit ferroptosis of retinal ganglion cells, regulate polarization of microglial cells to M2 anti-inflammatory phenotype, and recover the endogenous electrophysiological microenvironment of the retina. In a retina ischemia reperfusion injury model, retina structure injury and function loss are remarkably relieved by injecting P-PPy, and good biocompatibility is shown; and a comprehensive and reliable technical route and evaluation standard are provided for application of the nano-composite in preparation of the medicine for treating the retinal ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, and in particular to a polydopamine-modified conductive polypyrrole nanocomposite, its preparation method, and its application in the preparation of drugs for the treatment of retinal ischemia-reperfusion injury. Background Technology

[0002] Retinal ischemia-reperfusion injury (RIRI) is a core pathological mechanism of blinding eye diseases such as glaucoma, retinal vascular occlusion, and diabetic retinopathy, affecting hundreds of millions of patients worldwide, and its treatment faces significant challenges. This injury process involves a "double whammy" of ischemic and reperfusion phases: ischemia leads to hypoxia and energy metabolism collapse, while reperfusion triggers oxidative stress, neuroinflammation, and multiple cell death pathways (such as apoptosis, necroptosis, and ferroptosis). Retinal ganglion cells (RGCs), highly specialized neurons, undergo irreversible loss due to mitochondrial dysfunction, excitotoxicity, and iron homeostasis imbalance, ultimately leading to retinal structural damage and visual loss. Current clinical treatments (such as intraocular pressure-lowering drugs and anti-VEGF therapy) primarily target symptom relief and cannot effectively reverse the RGC death process. Traditional single-target drugs struggle to address the complex multi-pathway cross-network mechanisms of RIRI, particularly the vicious cycle of oxidative stress-ferroptosis-neuroinflammation.

[0003] Polypyrrole (PPy), as the core conductive polymer, possesses a unique π-conjugated backbone structure, enabling it to directly neutralize reactive oxygen species through reversible redox reactions, providing long-lasting and regenerable antioxidant protection. Its excellent electroactivity maintains the endogenous electrophysiological microenvironment of the retina, supporting the electro-signal transduction function of RGCs. However, unmodified PPy nanoparticles suffer from limitations such as strong hydrophobicity leading to easy aggregation and poor biocompatibility. To overcome these limitations, we introduced polydopamine (PDA) for surface modification, forming a core-shell structured P-PPy nanocomposite. This fully leverages the multiple advantages of PDA: its abundant catechol groups not only endow the nanoparticles with strong ROS scavenging ability but also specifically chelate overloaded ferrous ions (Fe2+). 2+ P-PPy inhibits the Fenton reaction, blocking the generation of hydroxyl radicals at its source. The biomimetic adhesion properties of PDA promote the specific accumulation and long-term retention of nanoparticles at retinal lesion sites. Simultaneously, it significantly improves the hydrophilicity and dispersion stability of PPy. The synergistic effect of PDA and PPy is achieved through intermolecular interactions such as π-π stacking and hydrogen bonding, forming a stable and functionally integrated nanoplatform. The comprehensive advantages of P-PPy are reflected in multiple dimensions: in terms of physicochemical properties, it possesses high electrical conductivity (34.86 S·m). -¹) and enhanced electrochemical activity, supporting the recovery of retinal electrophysiological function; in terms of antioxidation, theoretical calculations have confirmed its effectiveness against superoxide anions (O2). - It exhibits high affinity adsorption (binding energy -14.99 kcal / mol) and synergistically scavenges various ROS; in terms of iron homeostasis regulation, it acts as a "biological sponge," dose-dependently chelating Fe. 2+ (41.4% chelation rate at 20 µg / mL); In terms of immunomodulation, it significantly promoted the polarization of microglia to the M2 anti-inflammatory phenotype and inhibited the activation of inflammatory pathways such as NF-κB; in terms of biosafety, it demonstrated excellent in vitro and in vivo compatibility with no significant retinal toxicity. This multifunctional integrated strategy successfully overcomes the limitations of traditional nanomedicine therapy for RIRI, combining electrophysiological support functions with multi-target pathological regulation for the first time, providing insights for the preparation of drugs for neurodegenerative retinal diseases and possessing significant clinical translational value. Summary of the Invention

[0004] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide a conductive polypyrrole nanocomposite based on polydopamine modification, its preparation method, and its application in the preparation of drugs for the treatment of retinal ischemia-reperfusion injury. The present invention aims to systematically solve the key problem of synergistic regulation of multiple pathological mechanisms in the treatment of retinal ischemia-reperfusion injury (RIRI), and provides a novel multifunctional nanocomposite P-PPy (conductive polypyrrole nanocomposite modified with polydopamine). This nanocomposite integrates four core functions: reactive oxygen species (ROS) scavenging, ferroptosis inhibition, anti-inflammatory regulation, and electrophysiological support, and clarifies its large-scale preparation method and application direction.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a conductive polypyrrole nanocomposite modified with polydopamine includes the following steps: S1. Dopamine hydrochloride and pyrrole monomer are dissolved in Tris-HCl buffer, FeCl3·6H2O is added as a catalyst, and the polymerization reaction is carried out by stirring at 25°C in the dark to form a core-shell structure. S2. After the reaction is complete, the mixture is centrifuged at 15,000 rpm for 30 minutes at 4°C to collect the precipitate. The precipitate is repeatedly washed with ultrapure water to remove unreacted monomers and byproducts. Finally, the solid polydopamine-modified conductive polypyrrole nanocomposite, namely P-PPy, is obtained by freeze drying and stored at -80°C for later use.

[0006] As a preferred embodiment: in step S1, the concentration of dopamine hydrochloride is 2 mg / mL, the concentration of the pyrrole monomer is 1.5 μL / mL, the pH of the Tris-HCl buffer is 8.5, and the concentration of the catalyst is 0.5 mg / mL.

[0007] A polydopamine-modified conductive polypyrrole nanocomposite, prepared by the aforementioned method, possesses ROS scavenging, iron ion chelation, anti-inflammatory, and conductive properties. This polydopamine-modified conductive polypyrrole nanocomposite has an average particle size of 98.47 ± 23.95 nm, a zeta potential of -33.67 ± 8.85 mV, and a conductivity of 34.86 S·m. - ¹.

[0008] The application of the aforementioned polydopamine-modified conductive polypyrrole nanocomposite in the preparation of a drug for treating retinal ischemia-reperfusion injury, wherein the polydopamine-modified conductive polypyrrole nanocomposite is used to prepare a drug for treating retinal ischemia-reperfusion injury.

[0009] As a preferred embodiment, the drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to inhibit ferroptosis of retinal ganglion cells (RGCs) in retinal ischemia-reperfusion injury.

[0010] As a preferred embodiment, the drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is capable of chelating excess Fe in cells. 2+ It blocks the Fenton reaction and reduces lipid peroxidation levels, thereby inhibiting ferroptosis in retinal ganglion cells.

[0011] As a preferred embodiment, the drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to modulate the immune microenvironment in retinal ischemia-reperfusion injury.

[0012] As a preferred embodiment, the drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite can promote the polarization of microglia from the pro-inflammatory M1 type to the anti-inflammatory M2 type, reduce the release of inflammatory factors such as TNF-α, IL-1β and IL-6, and regulate the immune microenvironment in retinal ischemia-reperfusion injury.

[0013] As a preferred embodiment, the drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to repair neuronal electrical activity in retinal ischemia-reperfusion injury.

[0014] As a preferred embodiment: the drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite supports the conduction of endogenous electrical signals in the retina through its conductive properties, improves mitochondrial membrane potential, and repairs neuronal electrical activity; the polydopamine-modified conductive polypyrrole nanocomposite is used to prepare a drug for treating retinal ischemia-reperfusion injury caused by glaucoma, diabetic retinopathy, or retinal vascular occlusion.

[0015] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as can be seen from the above technical solution, the polydopamine-modified conductive polypyrrole nanocomposite provided by this invention has polypyrrole (PPy) as a conductive core, providing continuous free radical scavenging ability and electrical conduction support; polydopamine (PDA) as a surface functional modifier, through its abundant catechol groups, achieves iron ion chelation and enhanced bioadhesion. The synergy of the two not only significantly improves the dispersion stability and biocompatibility of the material, but also achieves multifunctional integration of anti-oxidation, ion homeostasis regulation, and electrical signal support. The polydopamine-modified conductive polypyrrole nanocomposite (P-PPy) prepared by the preparation method of the polydopamine-modified conductive polypyrrole nanocomposite provided in this application has good colloidal stability and surface properties, and excellent charge storage and transport capabilities. The application of polydopamine-modified conductive polypyrrole nanocomposite (P-PPy) in the preparation of drugs for the treatment of retinal ischemia-reperfusion injury has good biosafety, providing an innovative nano-solution for the drug development of ischemic retinopathy.

[0016] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the neuroprotective mechanism of polydopamine-modified conductive polypyrrole (PDA-PPy) nanoparticles of the bio-sponge armor of the present invention in retinal ischemia-reperfusion injury (RIRI). Figure 2 This is a schematic diagram illustrating the characterization of PPy and P-PPy nanoparticles (NPs) of the present invention; Figure 3 The P-PPy method of this invention alleviates oxidative stress and O2 in R28 cells under the OGD / R model. - A theoretical schematic diagram of adsorption; Figure 4 This is a schematic diagram illustrating the inhibition of OGD / R-induced ferroptosis and mitochondrial-related cell death in R28 cells by P-PPy according to the present invention. Figure 5This is a schematic diagram illustrating the effect of P-PPy of the present invention on retinal degeneration induced by I / R in the mouse retina; Figure 6 This is a schematic diagram illustrating how P-PPy inhibits retinal inflammation in a retinal I / R model by promoting M2 microglia polarization, as described in this invention. Figure 7 This is a schematic diagram illustrating how the P-PPy therapeutic drug of the present invention effectively reduces retinal I / R damage by significantly reducing cell apoptosis and regulating the expression of key neurons and glial proteins. Figure 8 This is a schematic diagram illustrating the molecular mechanism of P-PPy protection revealed by retinal RNA sequencing in this invention. Figure 9 This is a schematic diagram illustrating the in vitro and in vivo biosafety assessment of the P-PPy therapeutic agent of the present invention. Detailed Implementation

[0018] The present invention is as follows Figure 1 As shown in Figure 9, a method for preparing a conductive polypyrrole nanocomposite modified with polydopamine includes the following steps: S1. Dopamine hydrochloride and pyrrole monomer are dissolved in Tris-HCl buffer, FeCl3·6H2O is added as a catalyst, and the polymerization reaction is carried out by stirring at 25°C in the dark to form a core-shell structure. S2. After the reaction is complete, the mixture is centrifuged at 15,000 rpm for 30 minutes at 4°C to collect the precipitate. The precipitate is repeatedly washed with ultrapure water to remove unreacted monomers and byproducts. Finally, the solid polydopamine-modified conductive polypyrrole nanocomposite, namely P-PPy, is obtained by freeze drying and stored at -80°C for later use.

[0019] In step S1, the concentration of dopamine hydrochloride is 2 mg / mL, the concentration of the pyrrole monomer is 1.5 μL / mL, the pH of the Tris-HCl buffer is 8.5, and the concentration of the catalyst is 0.5 mg / mL.

[0020] A polydopamine-modified conductive polypyrrole nanocomposite was prepared by a method for preparing a polydopamine-modified conductive polypyrrole nanocomposite. This method yielded a polydopamine-modified conductive polypyrrole nanocomposite possessing ROS scavenging, iron ion chelation, anti-inflammatory, and conductive properties. The average particle size of this polydopamine-modified conductive polypyrrole nanocomposite was 98.47 ± 23.95 nm, the zeta potential was -33.67 ± 8.85 mV, and the conductivity was 34.86 S·m. - ¹.

[0021] The application of the polydopamine-modified conductive polypyrrole nanocomposite in the preparation of drugs for treating retinal ischemia-reperfusion injury.

[0022] The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to inhibit ferroptosis of retinal ganglion cells (RGCs) in retinal ischemia-reperfusion injury.

[0023] The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite can chelate excess Fe in cells. 2+ It blocks the Fenton reaction and reduces lipid peroxidation levels, thereby inhibiting ferroptosis in retinal ganglion cells.

[0024] The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to modulate the immune microenvironment in retinal ischemia-reperfusion injury.

[0025] The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite can promote the polarization of microglia from pro-inflammatory M1 type to anti-inflammatory M2 type, reduce the release of inflammatory factors such as TNF-α, IL-1β and IL-6, and regulate the immune microenvironment in retinal ischemia-reperfusion injury.

[0026] The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to repair neuronal electrical activity in retinal ischemia-reperfusion injury.

[0027] The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite supports the conduction of endogenous electrical signals in the retina through its conductive properties, improves mitochondrial membrane potential (ΔΨm), and repairs neuronal electrical activity; the polydopamine-modified conductive polypyrrole nanocomposite is used to prepare drugs for treating retinal ischemia-reperfusion injury caused by glaucoma, diabetic retinopathy, or retinal vascular occlusion.

[0028] Example 1: Preparation and characterization of a polydopamine-modified conductive polypyrrole nanocomposite (P-PPy) Preparation method: S1. Dopamine hydrochloride (2 mg / mL) and pyrrole monomer (1.5 μL / mL) were dissolved in 10 mM Tris-HCl buffer (pH 8.5), and FeCl3·6H2O (0.5 mg / mL) was added as a catalyst. The mixture was stirred at 25℃ in the dark for 24 hours. S2. After the reaction was completed, the mixture was centrifuged at 15,000 rpm for 30 minutes at 4℃ to collect the precipitate. The precipitate was washed three times with ultrapure water to remove unreacted monomers and byproducts. Finally, solid P-PPy nanoparticles were obtained by freeze-drying and stored at -80℃ for later use.

[0029] Characterization methods for polydopamine-modified conductive polypyrrole nanocomposites: The surface morphology of the material was observed using a field emission scanning electron microscope (SEM, Hitachi SU8000) at an accelerating voltage of 15 kV; the internal structure was analyzed using a high-resolution transmission electron microscope (HRTEM, JEOL JEM-2100) at an accelerating voltage of 200 kV; the hydration particle size and zeta potential were measured using a Malvern Zetasizer Nano ZS90 laser particle size analyzer, with each sample measured in triplicate and the average value taken; Fourier transform infrared spectroscopy (FTIR, Nicolet iS50) was performed in the range of 4000–400 cm⁻¹. -1 Chemical bond changes were analyzed within the wavenumber range; optical properties were characterized using ultraviolet-visible spectroscopy (UV-vis, Shimadzu UV-2600) in the 200-800 nm wavelength range; conductivity was measured using the four-probe method; and cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed using an electrochemical workstation (CHI760E).

[0030] Characterization results: Figure 2 In the diagram, A represents the synthesis of PPy and P-PPy NPs. Figure 2 Image B shows scanning electron microscope (SEM) and transmission electron microscope (TEM) images of PPy and P-PPy NPs (small image: SEM, scale bar: 100 nm; TEM, scale bar: 100 nm). Figure 2 C and Figure 2 In the middle D, the energy dispersive spectroscopy (EDS) analysis (C) and particle size distribution (D) histograms of PPy and P-PPy NPs are respectively. Figure 2 China E and Figure 2 The values ​​in the middle are the Zeta potential (E) and UV-Vis absorption spectra (F) of PPy and P-PPy NPs, respectively. Figure 2 G represents the conductivity measurement results of PPy and P-PPy nanoparticle tablets. Figure 2 H in the figure represents the cyclic voltammetry (CV) test curve. Figure 2 Middle I, Figure 2 J represents the electrochemical impedance spectroscopy (EIS) spectra of PPy and P-PPy nanoparticles: Nyquist plot (I) and Bode plot (J). All results are expressed as mean ± standard deviation of three independent experiments (n=3).

[0031] Figure 2 The results show: Figure 2 A indicates that P-PPy was successfully synthesized via oxidative polymerization, forming a core-shell structure; Figure 2The B-value indicates that the SEM and TEM images show that the nanoparticles are uniformly spherical with good monodispersity and an average diameter of 98.47 ± 23.95 nm. Figure 2 As shown in Figure C, EDS elemental analysis confirms the uniform distribution of C, N, O, and Fe elements. Figure 2 As shown in Figure D, the particle size distribution statistics show good monodispersity; Figure 2 As shown in Figure E, the Zeta potential is -33.67±8.85 mV, indicating that the surface modification was successful. Figure 2 As shown in Figure F, the UV-Vis absorption spectrum reveals characteristic absorption peaks, confirming the successful modification of PDA. Figure 2 As shown in Figure G, the conductivity measurement result is 34.86 S·m. -1 The value was significantly higher than that of unmodified PPy; Figure 3 As shown in Figure H, the cyclic voltammetry curves exhibit typical capacitance characteristics. Figure 3 Medium I- Figure 3 As shown in Figure J, electrochemical impedance spectroscopy indicates that P-PPy possesses excellent charge transport properties. Conclusion: P-PPy was successfully synthesized and possesses ideal physicochemical properties and electrochemical performance.

[0032] Example 2: In vitro cell experiments Cell culture and model establishment: R28 retinal progenitor cell line and BV2 microglia cell line were used and cultured in DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 incubator. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used in the experiments. An OGD / R model was established by seeding cells in 6-well plates (2 × 10⁶ cells / wells). 5 When the cells reached 80% confluence, the medium was replaced with sugar-free DMEM and cultured in a three-gas incubator (94% N2, 5% CO2, 1% O2) for 4 hours to simulate ischemic conditions. Then, the medium was replaced with normal complete medium and cultured for another 24 hours under normoxic conditions to simulate the reperfusion process.

[0033] Experimental grouping and treatment: The following groups were set up: control group, OGD / R model group, PPy treatment group (5, 10, 20, 50 μg / mL), and P-PPy treatment group (5, 10, 20, 50 μg / mL). Treatment time was 24 hours. Detection indicators and methods: Cell viability assay: CCK-8 assay, add 10 μL of CCK-8 solution to each well, incubate at 37°C for 2 hours, and measure absorbance at 450 nm.

[0034] Oxidative stress indicators: MDA assay kits were used to measure lipid peroxidation; GSH and SOD assay kits were used to determine antioxidant enzyme activity; and DCFH-DA fluorescent probes were used to detect ROS levels.

[0035] Ferrocyte-related markers: Phen Green™ FL iron ion fluorescent probe for detecting intracellular Fe 2+ Western blot was used to detect ACSL4 and GPX4 protein expression; the JC-1 kit was used to detect mitochondrial membrane potential. Inflammatory factor detection: ELISA was used to detect the concentrations of IL-1β, IL-6, and TNF-α in cell supernatant; immunofluorescence was used to detect the expression of CD86 and CD206.

[0036] In vitro experimental results and findings: Figure 3 A in the diagram is a schematic diagram of the OGD / R (oxygen-glucose deprivation / reperfusion) model. Figure 3 In Figure B, we show the effect of different concentrations of PPy and P-PPy on the survival rate of R28 cells in the OGD / R model (n=6 biologically independent replicates). Figure 3 C, Figure 3 The values ​​in C and D represent the effects of different concentrations of PPy and P-PPy on LDH release (C) and ATP levels (D) in R28 cells under OGD / R conditions (n=6 biological independent replicates). Figure 3 China E- Figure 3 G represents the effects of PPy and P-PPy on oxidative damage markers MDA (E), GSH (F), and SOD (G) under OGD / R conditions (n=3 biological independent replicates). Figure 3 H, Figure 3 The images (H) and (I) show the ability of PPy and P-PPy to scavenge excess ROS in the OGD / R model using the DCFH-DA fluorescent probes: fluorescence images (H) and quantitative statistical analysis (I) (n=3 biologically independent replicates). Scale bar = 50 μm. (J) P-PPy on O2 - The adsorption configuration diagram. Figure 3 K represents the P-PPy pair with O2 - Differential charge density distribution of the adsorption configuration. Figure 3 L- Figure 3 N represents the pair of P-PPy pairs with O2. - Visualization of interactions in adsorption configurations: IGMH isosurface plot (L), scatter plot (M), and color scale (N). Data are expressed as mean ± standard deviation.

[0037] Multiple comparisons were performed using one-way ANOVA combined with Tukey post-hoc tests, and no significant differences were found (ns); all tests were two-tailed (p < 0.05, p < 0.01, p < 0.001).

[0038] Figure 3 The results show: Figure 3 As shown in Figure A, the OGD / R model has been successfully established; Figure 3 As shown in Figure B, P-PPy treatment significantly improved cell viability to 97.8%, which was superior to the 80.1% in the PPy group. Figure 3 C and Figure 3 As shown in Figure D, LDH release decreases and ATP levels recover. Figure 3 China E- Figure 3 As shown in the figure, MDA levels decreased, while GSH and SOD activities significantly increased; Figure 4 H- Figure 4 As shown in Figure I, ROS levels were significantly reduced; Figure 4 J- Figure 4 As shown in Figure N, theoretical calculations confirm the interaction between P-PPy and O2. - The binding energy is -14.99 kcal / mol, and it mainly binds through π-π interactions and hydrogen bonds. Conclusion: P-PPy significantly improves OGD / R-induced cell damage by effectively scavenging ROS and regulating redox balance.

[0039] Figure 4 In Figure A, the iron chelating capacity of P-PPy was assessed (n=6 biological independent replicates). Figure 4 B, Figure 4 C represents the detection of intracellular Fe in R28 cells treated with OGD / R and supplemented with 20 μg / mL PPy or P-PPy using a ferrous ion staining kit. 2+ Level: Representative fluorescence images (B) and statistical analysis of fluorescence intensity (C) (n=3 biologically independent replicates). Scale bar = 50 μm; Figure 4 D represents the relative survival rate of R28 cells after 24 hours of exposure to 10 μM Erastin and different concentrations of P-PPy (n=6 biological independent replicates). Figure 4 E represents the expression level of ACSL4 in R28 cells of each group detected by Western Blot, with β-actin as an internal reference. Figure 4 Image F shows the ultrastructure of mitochondria in three groups of cells observed using transmission electron microscopy. Scale bar = 0.5 μm. Figure 4 China G, Figure 4In the figure, H represents the fluorescence intensity (H) of the JC-1 polymer (red) and monomer (green) of R28 cells, respectively, as assessed by the JC-1 fluorescent probe (ΔΨm) (G) and quantified using Image J software (n=3 biologically independent replicates). Scale bar = 50 μm. Figure 4 The diagram in the middle illustrates the mechanism by which P-PPy resists ferroptosis by inhibiting the Fenton reaction through iron chelation. Figure 4 J- Figure 4 The expression levels of IL-6 and TNF-α in each group were observed by immunofluorescence staining (J), and the fluorescence intensity of IL-6 (K) and TNF-α (L) was quantified (n=3 biological independent replicates). Figure 4 M, Figure 4 N represents the relative expression levels of Bax (M) and Bcl2 (N) gene mRNA in each group of cells detected by qRT-PCR (n=3 biological independent replicates). Figure 4 O, Figure 4 In the middle section, P represents the differentiation of live and dead cells in each group using Calcein-AM and PI staining (O), and cell viability (P) is calculated (n=3 biologically independent replicates). (Q) The expression levels of Cleaved Caspase3, p-NFκB, and NFκB proteins were detected by Western blotting, with β-actin as an internal control. Data are expressed as mean ± standard deviation. Multiple comparisons were performed using one-way ANOVA combined with Tukey's post-hoc test, and no significant differences were found (ns); all tests were two-tailed (p < 0.05, p < 0.01, p < 0.001).

[0040] Figure 4 The results show: Figure 4 As shown in Figure A, P-PPy exhibits a dose-dependent iron ion chelating capacity, reaching 41.4% at 20 μg / mL; Figure 4 China B- Figure 4 As shown in Figure C, intracellular Fe was significantly reduced. 2+ level; Figure 4 As shown in D, it effectively resists Erastin-induced ferroptosis; Figure 4 As shown in Figure E, Western blot analysis revealed downregulation of ACSL4 expression. Figure 4 As shown in Figure F, TEM observation revealed the restoration of mitochondrial morphology; Figure 4 G- Figure 4 As shown in Figure H, the mitochondrial membrane potential was significantly improved; Figure 5 The diagram in I represents the mechanism of action. Figure 5 J- Figure 5 As shown in Figure L, the expression of inflammatory factors IL-6 and TNF-α is decreased; Figure 5 M- Figure 5As shown in the N-mean, Bax expression is downregulated and Bcl2 expression is upregulated; Figure 5 O- Figure 5 As shown in Figure P, cell survival rate increased; Figure 5 As shown in Figure Q, the expression of apoptosis-related proteins was improved. Conclusion: P-PPy effectively inhibits ferroptosis and inflammatory responses by chelating iron ions, inhibiting ACSL4 expression, and protecting mitochondrial function.

[0041] Example 3: In vivo animal experiments Animal model establishment: Using 8-10 week old C57BL / 6J mice, weighing 20-25 g, retinal ischemia was induced by increasing intraocular pressure to 110 mmHg and maintaining it for 60 minutes through anterior chamber perfusion with balanced salt solution, followed by reperfusion to establish the RIRI model.

[0042] Dosage regimen: Animals were randomly divided into four groups: sham operation group, model group, PPy treatment group, and P-PPy treatment group, with 10 animals in each group. Twelve hours after the model was established, intravitreal injections were performed: the treatment group was injected with P-PPy (1 μL, 20 mg / mL), and the control group was injected with an equal volume of PBS.

[0043] Detection indicators and methods: Retinal function assessment: Full-field ERG recording of a wave, b wave and oscillatory potential under dark-adapted stimulation of 0.01, 3.0 and 10.0 cd·s / m².

[0044] Histological analysis: Eyeballs were fixed in 4% paraformaldehyde and paraffin sections were stained with H&E; immunofluorescence was used to detect the expression of β3-tubulin, GFAP, CtBP2, and PKC-α; and TUNEL assay was used to detect cell apoptosis.

[0045] Molecular mechanism study: Western blot was used to detect the expression of cleaved caspase-3, p-NF-κB p65 proteins; qRT-PCR was used to detect inflammation and ferroptosis-related genes; RNA sequencing was used to analyze differentially expressed genes and signaling pathways.

[0046] In vivo experimental results and findings: Figure 5 The diagram in Figure A illustrates the establishment of a mouse model of retinal ischemia-reperfusion injury and the subsequent experimental procedures, including intravitreal injection and data collection. Figure 5 The histological structures of the central, intermediate, and peripheral retinal regions of mice in each group were examined by hematoxylin and eosin (H&E) staining. Figure 5 C- Figure 5The diagrams in Figures C, D, and E show the quantitative measurement and intergroup comparison of the average thickness of each retinal layer in the central (C), mid-peripheral (D), and peripheral (E) regions (n=5 biologically independent mice per group). Figure 5 The dark-adapted full-field electroretinogram (ERG) waveforms under different stimulus intensities were recorded in the middle F. Figure 6 The amplitudes of specific ERG components (such as a-wave, b-wave, and oscillatory potential) were quantified under various stimulation conditions (n=5 biologically independent mice per group). Data are expressed as mean ± standard deviation. Multiple comparisons were performed using one-way ANOVA combined with Tukey's post-hoc test, and no significant differences were found (ns); all tests were two-tailed (p < 0.05, p < 0.01, p < 0.001).

[0047] Figure 6 The results show: Figure 6 A in the diagram represents the experimental procedure. Figure 6 As shown in Figure B, H&E staining revealed a significant improvement in the structural integrity of all retinal layers in the P-PPy treatment group. Figure 6 C- Figure 6 As shown in Figure E, quantitative analysis of the thickness of each retinal layer showed significant recovery in the thickness of the NFL / GCL, IPL, and INL layers; Figure 6 As shown in Figure F, the dark adaptation ERG waveform recording and display function has been improved; Figure 6 As shown in Figure G, the amplitude statistics of each component of the ERG show a significant increase in the amplitudes of the a-wave, b-wave, and OPs. Conclusion: P-PPy can effectively protect the structural and functional integrity of the retina.

[0048] Figure 6 Image A shows the changes in the expression levels of the M2 marker CD206 and the M1 marker CD86 in BV2 cells treated with PPy and P-PPy after LPS modeling, as indicated by immunofluorescence staining. Scale bar = 50 μm. Figure 6 B, Figure 6 (C) Statistical analysis of the average fluorescence intensity of CD206 (B) and CD86 (C) in each treatment group (n = 3 biological independent replicates). Figure 6 D represents the protein concentrations of inflammatory factors IL-1β, IL-6, and TNF-α in the cell culture supernatant of each group as detected by ELISA (n = 3 biologically independent replicates). Figure 6 The diagram in Figure E illustrates the mechanism by which P-PPy promotes the transformation of microglia from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype. Figure 6 In this study, the relative mRNA expression levels of M1 and M2 microglia phenotype-related marker genes in I / R retinal samples treated with P-PPy were detected by qRT-PCR (n = 3 biologically independent mice per group). Figure 6In the middle (G), retinal slices from the control group (PBS), IR group, IR+PPy group, and IR+P-PPy group were subjected to immunofluorescence staining with YM1 / 2 and IL-1β, covering the central, mid-peripheral, and peripheral areas. Scale bar = 50 μm. Figure 6 H, Figure 6 The values ​​in the table represent the quantitative analysis of the mean fluorescence intensity of YM1 / 2 (H) and IL-1β (I) in different regions of the retinal tissue of each treatment group (n = 5 biologically independent mice per group). Data are expressed as mean ± standard deviation. Multiple comparisons were performed using one-way ANOVA combined with Tukey's post-hoc test, and no significant differences were found (ns); all tests were two-tailed (p < 0.05, p < 0.01, p < 0.001).

[0049] Figure 6 The results show: Figure 7 As shown in Figure A, immunofluorescence reveals that P-PPy promotes the polarization of BV2 cells toward the M2 phenotype; Figure 7 China B- Figure 7 As shown in Figure C, CD206 expression is upregulated, while CD86 expression is downregulated; Figure 7 As shown in Figure D, ELISA detection revealed decreased secretion of inflammatory factors IL-1β, IL-6, and TNF-α; Figure 7 E in the diagram represents the mechanism of action. Figure 7 As shown in Figure F, qRT-PCR revealed upregulation of the M2 phenotypic markers Arg-1 and IL-10. Figure 7 As shown in Figure G, immunofluorescence of retinal sections showed upregulation of YM1 / 2 expression and downregulation of IL-1β expression; Figure 7 H- Figure 7 As shown in Figure I, quantitative fluorescence intensity confirmed the above changes. Conclusion: P-PPy improves the retinal inflammatory microenvironment by regulating microglia polarization.

[0050] Figure 7 Image A shows TUNEL staining of retinal tissue sections from four treatment groups (control group, IR group, IR+PPy group, and IR+P-PPy group) to detect apoptosis, followed by co-localization imaging analysis with nuclear dyes. Representative staining images of the central, mid-peripheral, and peripheral regions are displayed. Scale bar = 50 μm. Figure 7 China B- Figure 7(B) and (C) represent quantitative analysis of the number of TUNEL-positive cells in the central (B), mid-peripheral (C), and peripheral (D) retinal regions of each treatment group (n = 5 biologically independent mice per group). (E) Immunofluorescence staining of β3-tubulin and glial fibrillary acidic protein (GFAP) was performed on retinal tissue samples from the control group (PBS), IR group, IR+PPy group, and IR+P-PPy group, covering the central, mid-peripheral, and peripheral regions. Scale bar = 50 μm. Figure 7 China F, Figure 7 G represents the quantitative analysis of the average fluorescence intensity of β3-tubulin (F) and GFAP (G) in different regions of the retinal tissue of each treatment group (n = 5 biologically independent mice per group). Figure 7 In the middle H, retinal slices from the control group (PBS), IR group, IR+PPy group, and IR+P-PPy group were subjected to immunofluorescence staining with CtBP2 and PKC-α, covering the central, mid-peripheral, and peripheral regions. Scale bar = 50 μm. Figure 7 Middle I, Figure 7 J represents the quantitative analysis of the mean fluorescence intensity of CtBP2 (I) and PKC-α (J) in different regions of the retinal tissue of each treatment group (n = 5 biologically independent mice per group). Data are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's post-hoc test was used for multiple comparisons, and no significant differences were found (ns); all tests were two-tailed (p < 0.05, p < 0.01, p < 0.001).

[0051] Figure 8 The results show: Figure 8 As shown in Figure A, TUNEL staining revealed that P-PPy significantly reduced apoptotic cells; Figure 8 China B- Figure 8 As shown in Figure D, the apoptotic cell count was significantly reduced in each region; Figure 8 As shown in Figure E, immunofluorescence of β3-tubulin and GFAP showed restored expression of neuronal markers and reduced glial cell activation; Figure 8 China F- Figure 8 As shown in G, the fluorescence intensity quantitatively confirmed the above changes; Figure 8 As shown in Figure H, CtBP2 and PKC-α immunofluorescence showed the restoration of expression of synaptic function-related proteins; Figure 8 Medium I- Figure 8 As shown in Figure J, fluorescence intensity statistics confirmed functional improvement. Conclusion: P-PPy exerts a neuroprotective effect by inhibiting apoptosis and protecting synaptic function.

[0052] Figure 8In Figure A, RNA sequencing analysis of retinal tissues from the IR group and the P-PPy treatment group was performed. Volcano plots were drawn using log2 (fold change) and p-value as parameters. The horizontal and vertical axes represent the degree of gene expression change and statistical significance, respectively (n=4 biologically independent mice in each group). Figure 8 In Figure B, the KEGG pathways that were significantly enriched were selected through the hypergeometric test. The top 20 pathways are displayed in a bubble diagram based on the enrichment factor and p-value, with the bubble size corresponding to the number of genes. Figure 8 In the middle, C represents the expression pattern of differentially expressed genes standardized using z-scores. A heatmap is generated through hierarchical clustering analysis, with colors ranging from blue to red indicating expression levels from low to high. Figure 8 The D-value represents the signaling pathways that were significantly altered in the retinal tissue of mice after P-PPy treatment, as shown by GSEA analysis. Figure 8 E represents the protein-protein interaction network of differentially expressed genes analyzed using the STRING database. The node size and color are adjusted according to the connectivity parameters. Figure 8 In the middle F section, retinal slices from the control group (PBS), IR group, IR+PPy group, and IR+P-PPy group were subjected to p-p65 immunofluorescence staining, with the staining area covering the central, mid-peripheral, and peripheral regions. Scale bar = 50 μm. Figure 8 G represents the quantitative analysis of the average fluorescence intensity of p-p65 in different regions of retinal tissue in each treatment group (n=5 biologically independent mice per group). Figure 9 The diagram in Figure H illustrates the cellular and molecular mechanisms by which P-PPy functions in the I / R model. Data are expressed as mean ± standard deviation. Multiple comparisons were performed using one-way ANOVA combined with Tukey's post-hoc test, and no significant differences were found (ns); all tests were two-tailed (p < 0.05, p < 0.01, p < 0.001).

[0053] Figure 9 The results show: Figure 9 In diagram A, the RNA sequencing volcano plot shows a large number of differentially expressed genes; Figure 9 As shown in Figure B, KEGG pathway enrichment analysis revealed significant enrichment of signaling pathways such as NF-κB and TNF. Figure 9 As shown in Figure C, the gene expression heatmap reveals changes in the expression of inflammation- and apoptosis-related genes. Figure 9 As shown in Figure D, GSEA analysis confirms the alteration of the signaling pathway; Figure 9 As shown in Figure E, the protein-protein interaction network reveals that NF-κB plays a central role; Figure 9 As shown in Figure F, p-p65 immunofluorescence revealed reduced nuclear translocation; Figure 9 As shown in G, quantitative fluorescence intensity confirmed the inhibition of NF-κB activation; Figure 9The diagram in H represents a summary of the mechanism. Conclusion: P-PPy regulates the expression of downstream inflammation and apoptosis-related genes by inhibiting the NF-κB signaling pathway.

[0054] This nanocomposite material was synthesized through the copolymerization of polydopamine and pyrrole monomers, forming a core-shell structure with enhanced antioxidant capacity. After intravitreal injection in RIRI model mice, PDA-PPy effectively scavenged reactive oxygen species (ROS), inhibited ferroptosis by maintaining iron homeostasis, and alleviated neuroinflammation by regulating microglia phenotype. These synergistic effects jointly reconstruct retinal redox-iron homeostasis, thereby protecting retinal structure and function. This integrated mechanism highlights the enhanced retinal neuroprotective effect of PDA-PPy on visual ischemia-reperfusion injury through a multi-target synergistic therapeutic strategy.

[0055] Example 4: Biosafety Evaluation Method In vitro toxicity testing: The cytotoxic effects of P-PPy on R28, BV2, hRMEC, and ARPE-19 cells were detected using the CCK-8 assay; the distribution of live and dead cells was observed using Calcein-AM / PI double staining.

[0056] In vivo safety evaluation: Monitor intraocular pressure changes within 30 days after injection; assess retinal function using full-field ERG; perform histological analysis of retinal structure; and conduct blood biochemical tests to detect liver and kidney function indicators.

[0057] Safety Results and Findings: Figure 9 In Figure A, the survival rate of R28 cells after co-incubation with different concentrations of PBS (control group), PPy, or P-PPy for 24 hours was detected using the CCK-8 assay (n=6 biological independent replicates). Figure 9 China B- Figure 9 In the middle and D sections, BV2 cells (B), hRMECs (C), and ARPE-19 cells (D) were treated with different concentrations of PBS (control group), PPy, or P-PPy for 24 hours, and cell viability was determined by the CCK-8 assay (n=6 biologically independent replicates). Figure 9 In the middle E, R28 cells were treated with PBS (control group) and 100 μg / mL P-PPy. Live and dead cells were observed by Calcein-AM (green) and PI (red) fluorescence staining. Scale bar = 50 μm. Figure 9 In the middle F, the survival rate of R28 cells after incubation with 100 μg / mL P-PPy for 24 hours was quantitatively analyzed (n=3 biological independent replicates). Figure 9G represents the levels of IL-1β, IL-6 and TNF-α secreted by BV2 cells after treatment with 100 μg / mL P-PPy for 24 hours (n=3 biologically independent replicates). Figure 9 H, Figure 9 In the middle I, the retinal functional electrophysiological assessment was performed on mice in the PBS group and the P-PPy (1 μL, 20 mg / mL) treatment group (n=5 biologically independent mice in each group), and the amplitude (H) and implicit time (I) of specific waveform components were measured and compared between the two groups. Figure 9 In the middle J, intraocular pressure was monitored at multiple time points after intravitreal injection of P-PPy (1 μL, 20 mg / mL) (n=5 biologically independent mice in each group). Figure 9 In the middle (K), TUNEL staining was used to assess cell apoptosis in mouse retinal tissue, and DAPI counterstaining was used to label cell nuclei. Scale bar = 50 μm. (L) Tissue morphology was observed by H&E staining of retinal sections. Scale bar = 50 μm. (MO) The thickness changes of the nerve fiber / ganglionic cell layer (NFL / GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL) of the retina in the central (M), mid-peripheral (N), and peripheral (O) zones were measured (n = 5 biologically independent mice per group). Data are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's post-hoc test was used for multiple comparisons, and no significant differences were found (ns); all tests were two-tailed (p < 0.05, p < 0.01, p < 0.001).

[0058] Figure 9 The results show: Figure 9 As shown in Figure A, the R28 cytotoxicity assay showed that the cell viability rate remained at 92.6% after 24 hours of treatment with 200 μg / mL P-PPy. Figure 9 China B- Figure 9 As shown in Figure D, multiple cell line toxicity tests demonstrated good biocompatibility; Figure 9 China E- Figure 9 As shown in Figure F, staining of live and dead cells revealed no significant cell death. Figure 9 As shown in G, the detection of inflammatory factor secretion showed no stimulating effect; Figure 9 H- Figure 9 As shown in Figure I, ERG functional assessment showed no retinal toxicity; Figure 9 As shown in Figure J, intraocular pressure monitoring showed no significant changes; Figure 9 As shown in K, retinal apoptosis detection showed no abnormalities; Figure 9 As shown in the middle L, histological observation revealed that the structure was intact; Figure 9 M- Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 9As shown in Figure O, measurements of the thickness of each retinal layer showed no significant changes. Conclusion: P-PPy exhibits good biocompatibility under the experimental concentrations and conditions.

[0059] Data Analysis and Statistics: All experiments were independently repeated at least three times, and data are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 9.0. One-way ANOVA was used for comparisons among multiple groups, and Student's t-test was used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0060] Quality control measures: Experimental Environment Control: Cell experiments are conducted in a clean bench with regular microbial monitoring; animal experiments are conducted in an AAALAC-certified facility; molecular experiments are conducted in a dedicated PCR clean bench. Data Quality Control: Positive and negative controls are provided for each experiment; Western blot internal reference genes are validated; qPCR is used to detect primer specificity and amplification efficiency. Instrument Calibration: Microscopes, microplate readers, PCR instruments, and other instruments are calibrated regularly.

[0061] This application comprehensively validated the efficacy and safety of P-PPy nanocomposites in the preparation of therapeutic drugs for retinal ischemia-reperfusion injury through systematic in vitro and in vivo experiments. The results showed that P-PPy significantly improves retinal structure and function through multiple mechanisms, including ROS scavenging, iron homeostasis regulation, inflammatory response inhibition, and mitochondrial function protection, and exhibits good biocompatibility, providing a solid basis for clinical translation.

[0062] First: The structure and functional properties of polydopamine-modified conductive polypyrrole nanocomposites (P-PPy) The polydopamine-modified conductive polypyrrole nanocomposite provided by this invention uses Fe 3+ Using dopamine as a catalyst, a stable nanostructure with a well-defined core-shell structure was prepared via the oxidative copolymerization of dopamine and pyrrole monomers. In this composite, polypyrrole (PPy) serves as a conductive core, providing sustained free radical scavenging ability and electrical conduction support; polydopamine (PDA), as a surface functional modifier, achieves iron ion chelation and enhanced bioadhesion through its abundant catechol groups. The synergistic effect of these two components not only significantly improves the dispersion stability and biocompatibility of the material but also achieves a multifunctional integration of antioxidant, ion homeostasis regulation, and electrical signal support.

[0063] Second: Preparation method and key characterization of polydopamine-modified conductive polypyrrole nanocomposites (P-PPy) The polydopamine-modified conductive polypyrrole nanocomposite (P-PPy) prepared using the method provided in this application was confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to have a morphology of uniform spherical nanoparticles. Dynamic light scattering (DLS) showed that its hydrated particle size was approximately 98.47 nm, and its Zeta potential was -33.67 mV, demonstrating that the system possesses good colloidal stability and surface properties. Fourier transform infrared spectroscopy (FTIR) confirmed that PDA was successfully modified on the PPy surface, and ultraviolet-visible absorption spectroscopy showed that it had characteristic absorption in the near-infrared region. Electrochemical tests showed that the conductivity of P-PPy reached 34.86 S·m. -1 The value was significantly higher than that of unmodified PPy (30.06 S·m). -1 The cyclic voltammetry curves exhibit typical capacitive behavior, indicating that it has excellent charge storage and transport capabilities.

[0064] Third: Application of a polydopamine-modified conductive polypyrrole nanocomposite (P-PPy) in the preparation of drugs for the treatment of retinal ischemia-reperfusion injury. In vitro experiments validated that in the oxygen-glucose deprivation / reperfusion (OGD / R) model, P-PPy treatment significantly increased R28 cell survival to 97.8% (vs. model group <60%), reduced ROS production and MDA content, restored GSH and SOD activity, and effectively reversed mitochondrial membrane potential collapse. In the RIRI mouse model, intravitreal injection of P-PPy significantly preserved the thickness of all retinal layers (especially the ganglion cell layer and nuclear layer), and full-field electroretinography (ERG) showed significant recovery of a-wave and b-wave amplitudes and oscillatory potentials (OPs), while optomotor response (OMR) confirmed improved visual function. Simultaneously, immunofluorescence and Western blot results indicated that P-PPy effectively inhibited ACSL4 expression, reduced cleaved caspase-3 production, and downregulated NF-κB pathway activation.

[0065] In summary, P-PPy, through its synergistic effects across multiple mechanisms, is applied to RIRI therapeutics while exhibiting good biocompatibility, providing an innovative nanotechnology solution for drug development in ischemic retinopathy.

[0066] The key design focus of this invention is the provision of a polydopamine-modified conductive polypyrrole nanocomposite. In this composite, polypyrrole (PPy) serves as the conductive core, providing sustained free radical scavenging capabilities and electrical conduction support; polydopamine (PDA), as a surface functional modifier, achieves iron ion chelation and enhanced bioadhesion through its abundant catechol groups. The synergistic effect of these two components significantly improves the dispersion stability and biocompatibility of the material, and also achieves multifunctional integration of antioxidant, ion homeostasis regulation, and electrical signal support. The polydopamine-modified conductive polypyrrole nanocomposite (P-PPy) prepared using the method provided in this application exhibits good colloidal stability and surface properties, and possesses excellent charge storage and transport capabilities. The application of the polydopamine-modified conductive polypyrrole nanocomposite (P-PPy) in the preparation of drugs for the treatment of retinal ischemia-reperfusion injury demonstrates good biocompatibility, providing an innovative nano-solution for drug development in ischemic retinopathy.

[0067] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a conductive polypyrrole nanocomposite modified with polydopamine, characterized in that: Includes the following steps: S1. Dopamine hydrochloride and pyrrole monomer are dissolved in Tris-HCl buffer, FeCl3·6H2O is added as a catalyst, and the polymerization reaction is carried out by stirring at 25°C in the dark to form a core-shell structure. S2. After the reaction is complete, the mixture is centrifuged at 15,000 rpm for 30 minutes at 4°C to collect the precipitate. The precipitate is repeatedly washed with ultrapure water to remove unreacted monomers and byproducts. Finally, the solid polydopamine-modified conductive polypyrrole nanocomposite, namely P-PPy, is obtained by freeze drying and stored at -80°C for later use.

2. The method for preparing a conductive polypyrrole nanocomposite based on polydopamine modification according to claim 1, characterized in that: In step S1, the concentration of dopamine hydrochloride is 2 mg / mL, the concentration of the pyrrole monomer is 1.5 μL / mL, the pH of the Tris-HCl buffer is 8.5, and the concentration of the catalyst is 0.5 mg / mL.

3. A polydopamine-modified conductive polypyrrole nanocomposite prepared by the method for preparing a polydopamine-modified conductive polypyrrole nanocomposite according to any one of claims 1-2, characterized in that: A conductive polypyrrole nanocomposite modified with polydopamine was prepared using the aforementioned method. This nanocomposite exhibits ROS scavenging, iron ion chelation, anti-inflammatory, and conductive properties. The average particle size of this conductive polypyrrole nanocomposite is 98.47 ± 23.95 nm, the zeta potential is -33.67 ± 8.85 mV, and the conductivity is 34.86 S·m. - ¹.

4. The application of the polydopamine-modified conductive polypyrrole nanocomposite as described in claim 3 in the preparation of a therapeutic drug for retinal ischemia-reperfusion injury, characterized in that: The polydopamine-modified conductive polypyrrole nanocomposite is used to prepare a drug for treating retinal ischemia-reperfusion injury.

5. The application of the polydopamine-modified conductive polypyrrole nanocomposite according to claim 4 in the preparation of a therapeutic drug for retinal ischemia-reperfusion injury, characterized in that: The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to inhibit ferroptosis of retinal ganglion cells (RGCs) in retinal ischemia-reperfusion injury.

6. The application of the polydopamine-modified conductive polypyrrole nanocomposite according to claim 5 in the preparation of a therapeutic drug for retinal ischemia-reperfusion injury, characterized in that: The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite can chelate excess Fe in cells. 2+ It blocks the Fenton reaction and reduces lipid peroxidation levels, thereby inhibiting ferroptosis in retinal ganglion cells.

7. The application of the polydopamine-modified conductive polypyrrole nanocomposite according to claim 4 in the preparation of a therapeutic drug for retinal ischemia-reperfusion injury, characterized in that: The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to modulate the immune microenvironment in retinal ischemia-reperfusion injury.

8. The application of the polydopamine-modified conductive polypyrrole nanocomposite according to claim 7 in the preparation of a therapeutic drug for retinal ischemia-reperfusion injury, characterized in that: The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite can promote the polarization of microglia from pro-inflammatory M1 type to anti-inflammatory M2 type, reduce the release of inflammatory factors such as TNF-α, IL-1β and IL-6, and regulate the immune microenvironment in retinal ischemia-reperfusion injury.

9. The application of the polydopamine-modified conductive polypyrrole nanocomposite according to claim 4 in the preparation of a therapeutic drug for retinal ischemia-reperfusion injury, characterized in that: The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite is used to repair neuronal electrical activity in retinal ischemia-reperfusion injury.

10. The application of the polydopamine-modified conductive polypyrrole nanocomposite according to claim 9 in the preparation of a therapeutic drug for retinal ischemia-reperfusion injury, characterized in that: The drug prepared from the polydopamine-modified conductive polypyrrole nanocomposite supports the conduction of endogenous electrical signals in the retina through its conductive properties, improves mitochondrial membrane potential, and repairs neuronal electrical activity; the polydopamine-modified conductive polypyrrole nanocomposite is used to prepare drugs for treating retinal ischemia-reperfusion injury caused by glaucoma, diabetic retinopathy, or retinal vascular occlusion.

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