Ferritin-loaded and melanin-coupled nano-particles as well as preparation method and application thereof

Abstract

The invention discloses a ferritin-coupled melanin nano-particle and a preparation method and application thereof, and relates to the technical field of biomedicine, the ferritin-coupled melanin nano-particle AFn-MNP comprises ferritin and a melanin nano-particle, the ferritin and the melanin nano-particle are covalently linked to form a core-shell structure, and the ferritin-coupled melanin nano-particle has excellent iron ion chelating ability and antioxidant activity. The AFn-MNP can effectively remove reactive oxygen species (ROS), inhibit ferroptosis of photoreceptor cells and reverse oxidative damage of retina. In a dry age-related macular degeneration (dAMD) model, single intravitreal injection of AFn-MNP significantly improves the visual function and protects the retina structural integrity. The invention provides a novel nano treatment strategy for treating dAMD and other retinal degenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a ferritin-coupled melanin nanoparticle, its preparation method, and its application. Background Technology

[0002] Dry age-related macular degeneration (dAMD) is a chronic degenerative retinal disease that primarily affects the macula and is a leading cause of irreversible vision loss in older adults worldwide. With the aging of the global population, the prevalence of dAMD is increasing annually, placing a heavy burden on individuals and society. Statistics show that the prevalence of dAMD exceeds 30% in people over 75 years of age, making it a significant public health issue. Unlike exudative AMD (wet AMD), dAMD has an insidious disease progression, manifesting as oxidative stress-induced damage to retinal pigment epithelium (RPE) cells, drusen deposition, and progressive photoreceptor degeneration, ultimately leading to geographic atrophy and retinal structural destruction.

[0003] The pathogenesis of dAMD is complex, involving multiple pathophysiological processes. Oxidative stress is one of the core driving factors. As a high-oxygen-consuming tissue, the retina, when chronically exposed to light and a hyperoxic environment, is prone to producing excessive reactive oxygen species (ROS), leading to oxidative damage to lipids, proteins, and DNA. Studies have shown that ROS levels are significantly elevated in the retinas of dAMD patients, while the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX) are reduced. Furthermore, the role of iron homeostasis dysregulation in dAMD is receiving increasing attention. Iron is a cofactor for many enzymes, but excessive iron generates hydroxyl radicals through the Fenton reaction, exacerbating oxidative damage. Research indicates that iron content in the RPE cells of dAMD patients is significantly increased and positively correlated with disease severity.

[0004] In recent years, ferroptosis, as a form of iron-dependent programmed cell death, has been shown to be involved in the pathogenesis of dAMD. Ferroptosis is characterized by iron overload, lipid peroxide accumulation, and GPX4 system inactivation. In dAMD models, ferroptosis features are observed in both photoreceptor cells and RPE cells, such as upregulated ACSL4 expression and decreased GPX4 activity. Ferroptosis forms a positive feedback loop with mitochondrial dysfunction, endoplasmic reticulum stress, and inflammatory responses, accelerating retinal degeneration. However, there are currently no specific treatments for ferroptosis in clinical practice. Existing treatments for dAMD are limited; for example, antioxidant supplementation (AREDS2 formulation) can only delay disease progression but cannot reverse degeneration. Anti-vascular endothelial growth factor (anti-VEGF) drugs are effective for wet AMD but ineffective for dAMD. Therefore, developing multifunctional agents that can simultaneously target oxidative stress and ferroptosis is a current research focus. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a ferritin-coupled melanin nanoparticle, its preparation method, and its applications, aiming to systematically solve the key challenge of synergistic regulation of oxidative stress and ferroptosis in the treatment of dry age-related macular degeneration (dAMD). Ferritin-coupled melanin nanoparticles (AFn-MNPs) are prepared by covalently linking ferritin (AFn) with melanin nanoparticles (MNPs) to form a core-shell structure. AFn-MNPs integrate four core functions: reactive oxygen species (ROS) scavenging, iron ion chelation, anti-inflammatory regulation, and photoreceptor protection. Furthermore, the invention clarifies the large-scale preparation method and application directions of AFn-MNPs.

[0006] In order to achieve the objective of this invention, the following solution is proposed: First aspect: The structure and functional properties of AFn-MNP nanocomposites The AFn-MNP nanocomposite was prepared by forming an MNP core through dopamine polymerization and then coupling it with ferritin via an EDC / NHS-mediated reaction, resulting in a stable nanosystem with a well-defined core-shell structure. In this composite, melanin nanoparticles (MNPs) serve as the antioxidant core, providing sustained free radical scavenging and metal ion chelating capabilities through phenolic hydroxyl groups; ferritin (AFn) acts as a surface functional armor, achieving specific iron ion binding and enhanced biocompatibility through its hollow spherical structure. The synergistic effect of melanin nanoparticles (MNPs) and ferritin (AFn) not only significantly improves the dispersion stability and targeting of the material but also achieves a multifunctional integration of antioxidant, iron homeostasis regulation, and inflammation suppression. The average aqueous dispersion particle size of AFn-MNP is 246.6 nm ± 22.1 nm, and the Zeta potential is -10.9 mV ± 1.03 mV, exhibiting excellent colloidal stability and cellular uptake efficiency.

[0007] The second aspect: Preparation method and key characterization of AFn-MNP The specific steps include: mixing dopamine hydrochloride with ammonia and deionized water, allowing it to self-polymerize in an alkaline environment to form MNPs. After centrifugation and purification, MNPs are coupled with activated ferritin under EDC / NHS conditions overnight. Unreacted substances are removed by dialysis, followed by freeze-drying to obtain solid AFn-MNPs. Transmission electron microscopy (TEM) confirmed that the morphology consists of uniform spherical nanoparticles. Dynamic light scattering (DLS) showed that the water-dispersed particle size was 246.6 nm ± 22.1 nm, and the Zeta potential was -10.9 mV ± 1.03 mV, demonstrating good monodispersity and surface stability. Fourier transform infrared spectroscopy (FTIR) showed the disappearance of the NH2 peak at 1618 cm⁻¹, confirming successful covalent bonding between AFn and MNPs. UV-Vis absorption spectroscopy showed that the characteristic peak of AFn (235 nm) redshifted to 260 nm, indicating the formation of a core-shell structure.

[0008] Thirdly: The application of AFn-MNP in the treatment of dAMD and related diseases The application of AFn-MNP in the preparation of drugs for treating dry age-related macular degeneration (dAMD), retinitis pigmentosa, and other eye diseases related to oxidative damage mainly includes the following mechanisms: 1. Inhibition of ferroptosis: By efficiently chelating excess Fe²⁺ in cells through ferritin components, the Fenton reaction is blocked, the accumulation of lipid peroxides (such as 4-HNE) is reduced, and the ferroptosis process in photoreceptor cells is reversed. 2. Regulate the immune microenvironment: Promote the polarization of microglia from the pro-inflammatory M1 phenotype (CD86⁺) to the anti-inflammatory M2 phenotype (CD206⁺), and reduce the release of inflammatory factors such as IL-1β, IL-6, and TNF-α; 3. Protect photoreceptor function: Clear ROS through melanin components, improve mitochondrial membrane potential (ΔΨm), and restore the amplitude of a and b waves in electroretinography (ERG).

[0009] The beneficial effects of this invention are as follows: 1. Nanomaterials possess unique advantages in drug delivery, such as targeting, sustained release, and multifunctional integration. Melanin nanoparticles (MNPs) are natural pigments with excellent metal ion chelation and ROS scavenging capabilities. Ferritin (AFn) is an endogenous iron storage protein that specifically binds iron ions. This invention innovatively combines MNPs with AFn to construct an AFn-MNP nanocomplex, synergistically treating dAMD through a dual mechanism. Experiments show that AFn-MNPs can effectively alleviate sodium iodate (NaIO3)-induced retinal oxidative damage and ferroptosis.

[0010] 2. Through in vitro experiments, it was verified that in the NaIO3-induced oxidative damage model, AFn-MNP increased the survival rate of 661W cells to 90.7%, reduced MDA levels by 40%, increased GSH and SOD activities by 2.9 times and 1.6 times, respectively, and reversed mitochondrial ultrastructural damage.

[0011] 3. In the dAMD mouse model, a single intravitreal injection of AFn-MNP (10 μg) significantly preserved the thickness of each retinal layer. ERG showed that visual function parameters recovered by nearly 80%. Immunofluorescence and Western blot confirmed the downregulation of ACSL4 expression and activation of the Nrf2 pathway.

[0012] 4. AFn-MNP achieves highly effective treatment of dAMD through synergistic effects of multiple mechanisms, while also exhibiting good biocompatibility, providing an innovative nano-solution for drug development of neurodegenerative retinopathy. Attached Figure Description

[0013] Figure 1 Figure 1 shows the preparation and characterization results of AFn-MNP. Among them, (A) is a schematic diagram of the construction of biomimetic melanosomes and its mechanism of regulating iron metabolism; (B) is a transmission electron microscope (TEM) image of MNP; (C) is a transmission electron microscope (TEM) image of AFn-MNP; (D) is a dynamic light scattering (DLS) analysis diagram of MNP; (E) is a dynamic light scattering (DLS) analysis diagram of AFn-MNP; (F) is a Zeta potential analysis diagram of MNP and AFn-MNP; (G) is a UV spectrum of AFn, MNP and AFn-MNP; and (H) is a Fourier transform infrared (FTIR) spectrum of AFn, MNP and AFn-MNP.

[0014] Figure 2 Figures showing the changes in retinal heterogeneity in mice under NaIO3-induced oxidative damage revealed by single-cell RNA sequencing; (A) shows the 11 retinal cell types; (B) shows the clustering strategy based on the normalized expression heatmap of each cluster; (C) shows the relative changes in the proportion of different cell clusters between the control group and the NaIO3 model group; (D) is a violin plot showing the proportion of 8 retinal cell clusters (n=2) between the control group and the NaIO3 model group, with P values ​​calculated using the Wilcoxon rank-sum test; (E) shows the normalized expression heatmap and violin plot of ferroptosis-related genes in cone cells, with P values ​​obtained using the hypergeometric test; (F) shows the normalized expression heatmap and violin plot of ferroptosis-related genes in rod cells, with P values ​​obtained using the hypergeometric test; all results are expressed as mean ± standard deviation, and P values ​​were calculated using one-way ANOVA unless otherwise specified.

[0015] Figure 3Figure 1 shows the experimental results of AFn-MNP alleviating NaIO3-induced ferroptosis and oxidative damage in 661W cells. (A) shows the cell viability of 661W cells treated with different concentrations of MNP and AFn-MNP (n=6 biological independent replicates); (B) shows the effect of MNP and AFn-MNP on GSH, an oxidative damage marker in 661W cells treated with NaIO3 (n=3 biological independent replicates); (C) shows the effect of MNP and AFn-MNP on MDA, an oxidative damage marker in 661W cells treated with NaIO3 (n=3 biological independent replicates); (D) shows the effect of MNP and AFn-MNP on SOD, an oxidative damage marker in 661W cells treated with NaIO3 (n=3 biological independent replicates); (E) shows the results of observation using transmission electron microscopy of 10 μg / mL MNP, AFn-MNP, and 30 mM... (F) Ultrastructure of mitochondria in 661W cells treated with NaIO3 for 24 hours (scale bar = 500 nm); (G) Immunofluorescence confocal microscopy image of FerroOrange cells (scale bar = 200 μm); (G) Quantitative fluorescence analysis (n = 3 biologically independent replicates); (H) Relative viability of 661W cells exposed to 10 μM Erastin and different concentrations of MNP and AFn-MNP for 24 hours (n = 6 biologically independent replicates); (I) Schematic diagram of intracellular signaling events of ferroptosis; Data are expressed as mean ± standard deviation. Statistical significance (not significant in ns, *p<0.05, **p<0.01, ***p<0.001) was assessed by two-way one-way ANOVA and Tukey post-hoc test.

[0016] Figure 4Figure 1 shows the experimental results of AFn-MNP protecting against NaIO3-induced retinal damage by enhancing the antioxidant stress response. (A) shows the experimental timeline and design diagram; (B) shows representative micrographs of H&E-stained retinal sections from different groups, displaying retinal morphology (scale bar = 50 μm); (C) shows the total retinal thickness in H&E-stained sections of each group (n = 8 biologically independent mice per group); (D) shows the quantitative analysis of outer nuclear layer (ONL) thickness in each group (n = 8 biologically independent mice per group); (E) shows representative immunofluorescence staining images of retinal sections, showing the expression and localization of key markers 4-HNE, Nrf2, and ACSL4 (scale bar = 50 μm). (μm); (F) shows the number of 4-HNE positive cells in each group of retinal sections (n=8 biologically independent mice per group); (G) shows the mean fluorescence intensity of Nrf2 in each group of retinal sections (n=8 biologically independent mice per group); (H) shows the quantitative analysis of the mean fluorescence intensity of ACSL4 in each group of retinal sections (n=8 biologically independent mice per group); data are expressed as mean ± standard deviation, and statistical significance (no significance in ns, *p<0.05, **p<0.01, ***p<0.001) was assessed by two-way one-way ANOVA and Tukey post-hoc test.

[0017] Figure 5 The images show the experimental results of AFn-MNP inhibiting NaIO3-induced retinal atrophy and photoreceptor degeneration. (A) shows OCT images of different groups; (B) shows color fundus photography and fluorescence angiography (FFA) images of different groups; (C) shows immunofluorescence co-staining images of PKC-α and CtBP2 co-expression in retinal sections of mice from different groups (scale bar = 20 μm); (D) shows the quantitative analysis of PKC-α in different regions of retinal tissue in a specified group (n = 8 biologically independent mice per group); (E) shows the quantitative analysis of CtBP2 in different regions of retinal tissue in a specified group (n = 8 biologically independent mice per group); and (F) shows the immunofluorescence co-staining images of Iba-1 and IL-1β co-expression in retinal sections of mice from different groups (scale bar = 20 μm). (μm); (G) is a quantitative analysis map of Iba-1 in different regions of the retinal tissue of the specified group (n=8 biologically independent mice per group); (H) is a quantitative analysis map of IL-1β in different regions of the retinal tissue of the specified group (n=8 biologically independent mice per group); data are expressed as mean ± standard deviation, and statistical significance (no significance in ns, *p<0.05, **p<0.01, ***p<0.001) was assessed by two-way one-way ANOVA and Tukey post-hoc test.

[0018] Figure 6Figure 1 shows the experimental results of AFn-MNP protecting against NaIO3-induced retinal dysfunction; (A) is a full-field electroretinogram (ERG); (B) is an amplitude graph (n=8 biologically independent mice per group); (C) is a time-course graph (n=8 biologically independent mice per group); data are expressed as mean ± standard deviation, and statistical significance (not significant in ns, *p<0.05, **p<0.01, ***p<0.001) was assessed by two-way one-way ANOVA and Tukey post-hoc test.

[0019] Figure 7 Figure 1 shows the experimental results of the in vivo biocompatibility evaluation of AFn-MNP; (A) shows the intraocular pressure changes in mice on days 1, 3 and 7; (B) shows H&E stained sections of the retina-choroid-sclera complex (scale bar 200 μm); (C) shows the quantitative analysis of retinal thickness; (D) shows the results of retinal function detection by ERG after 7 days; (E) shows the quantitative statistical graph (n=3 biologically independent mice per group); data are expressed as mean ± standard deviation, and ns indicates no statistically significant difference. Detailed Implementation

[0020] This embodiment provides a method for preparing AFn-MNP, as detailed below: 5 mL of dopamine hydrochloride (50 mg / mL) solution was added to a mixture of anhydrous ethanol (20 mL), ammonia (10 mL), and deionized water (45 mL). The mixture was stirred and reacted at 25 °C in the dark for 24 hours to obtain MNP solution. Subsequently, ferritin (AFn) was diluted to a concentration of 250 μg / mL, and the pH was adjusted to 8.4 using 1 mol / L NaHCO3 solution. After activating the carboxyl groups on the surface of ferritin with 200 μL EDC (2.5 mg / mL) and 300 μL NHS (2.5 mg / mL), 1 mL of MNP solution (200 μg / mL) was added and stirred overnight at room temperature. After the reaction was completed, the mixture was dialyzed in PBS (pH=7.4) for two days (molecular weight cutoff: 7000 kDa). Finally, AFn-MNP was obtained by freeze drying and stored at -80 °C for later use.

[0021] The characterization method for AFn-MNP is as follows: The internal structure was analyzed using a high-resolution transmission electron microscope (HRTEM, JEOL JEM-2100) at an accelerating voltage of 200 kV; the hydrated 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; chemical bond changes were analyzed using Fourier transform infrared spectroscopy (FTIR, Nicolet iS50) in the wavenumber range of 4000 cm⁻¹–400 cm⁻¹; and optical properties were characterized using ultraviolet-visible spectroscopy (UV-vis, Shimadzu UV-2600) in the wavelength range of 200 nm–800 nm.

[0022] Characterization results and findings: Figure 1 The results showed that: (A) AFn-MNP was successfully synthesized via the template method, forming a core-shell structure; (B) TEM images showed that the particle size of MNP was approximately 50 nm, exhibiting a uniform spherical shape; (C) TEM images showed that the particle size of AFn-MNP was approximately 100 nm, exhibiting a uniform spherical shape; (D) Dynamic light scattering analysis showed that the hydrodynamic radius of MNP was 149.65 nm ± 5.65 nm; (E) Dynamic light scattering analysis showed that the hydrodynamic radius of AFn-MNP was 246.6 nm ± 22.1 nm; (F) Zeta potential measurements showed that MNP and A The surface charges of Fn-MNP are -23.5mV±0.71mV and -10.9mV±1.03mV, respectively; (G) UV-Vis absorption spectroscopy shows that AFn has a characteristic absorption peak at 235nm, and this peak is red-shifted to 260nm in AFn-MNP, confirming that AFn and MNP interact successfully; (H) FTIR spectroscopy shows the stretching vibration peaks of OH, NH and NH2 chemical bonds at 3500cm⁻¹-3200cm⁻¹, and the NH2 asymmetric stretching vibration peak of AFn at 1618cm⁻¹ disappears, confirming that the covalent connection is successful.

[0023] Experiment 1: In vitro cell experiments, as detailed below: (1) Cell culture and model establishment: The 661W mouse photoreceptor cell line (derived from immortalized mouse photoreceptor cells) was 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. A NaIO3-induced oxidative damage model was established by seeding cells in 6-well plates (2 × 10⁻⁶ cells / well). 5 Once the cells reached 80% confluence, the medium was replaced with a medium containing 30 mM NaIO3 for 24 hours to simulate the oxidative stress conditions of dry age-related macular degeneration (dAMD).

[0024] (2) Experimental grouping and treatment: The following groups were set up: control group (PBS), NaIO3 model group (30 mM), MNP treatment group (5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL), and AFn-MNP treatment group (5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL). The treatment time was 24 hours. All treatments were performed 1 hour before NaIO3 exposure to ensure the effect of pretreatment.

[0025] (3) Detection indicators and methods: ① Cell viability assay: CCK-8 assay, add 10 μL of CCK-8 solution to each well, incubate at 37°C for 2 hours, measure absorbance at 450 nm, and calculate cell viability.

[0026] ② Oxidative stress indicators: Commercial kits were used to detect malondialdehyde (MDA) levels to assess lipid peroxidation; glutathione (GSH) and superoxide dismutase (SOD) kits were used to determine antioxidant enzyme activity; and the DCFH-DA fluorescent probe was used to detect reactive oxygen species (ROS) levels.

[0027] ③ Ferroplasm-related indicators: Intracellular Fe²⁺ concentration was detected by FerroOrange fluorescent probe; ACSL4 and GPX4 protein expression was detected by Western blot; and mitochondrial membrane potential (ΔΨm) was detected by JC-1 kit.

[0028] ④ Detection of inflammatory factors: 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 (M1 phenotypic marker) and CD206 (M2 phenotypic marker).

[0029] ⑤ Morphological observation: Transmission electron microscopy (TEM) was used to observe the ultrastructure of mitochondria; immunofluorescence images were recorded using fluorescence microscopy.

[0030] (4) Results and findings of in vitro cell experiments: Figure 2A single-cell RNA sequencing diagram was used to reveal changes in retinal heterogeneity in mice under NaIO3-induced oxidative damage. The results showed that: (A, B) a clustering strategy based on standard marker expression heatmaps identified 11 retinal cell types; (C) compared with the control group, the proportions of retinal cell subsets changed significantly in the NaIO3 model group, with a significant decrease in the number of rod cells (ROD), cone cells (CONE), amacrine cells (AC), retinal ganglion cells (RGC), and retinal pigment epithelial cells (RPE), while the number of rod bipolar cells (RBC), Miller glial cells (MG), and immune cells increased; (D) a violin plot showed the differences in the proportions of 8 cell clusters between groups (n=2), and the p-values ​​were calculated using the Wilcoxon rank-sum test; (E) the expression of ferroptosis-related genes (such as Acsl4, GPX4, and Fth1) in cone cells was upregulated; (F) similar gene expression changes were observed in rod cells, and the p-values ​​were obtained using the hypergeometric test. Conclusion: Single-cell RNA sequencing revealed heterogeneous changes in retinal cell subsets under NaIO3-induced oxidative damage, with ferroptosis being the core driver of photoreceptor cell degeneration.

[0031] Figure 3 Figure 1 shows the experimental results of AFn-MNP alleviating NaIO3-induced ferroptosis and oxidative damage in 661W cells. The results show that: (A) CCK-8 assay showed that after 24 hours of treatment with AFn-MNP at a concentration of 200 μg / mL, the cell viability was still 90.7%, which was significantly higher than that of the MNP group (86.1%), showing good biocompatibility; (B, C, D) AFn-MNP treatment significantly reversed the NaIO3-induced increase in MDA, and the levels of GSH and SOD increased by 2.9-fold and 1.6-fold, respectively; (E) TEM observation showed that mitochondrial swelling and cristae disappearance were significantly improved in the AFn-MNP group; (FG) FerroOrange fluorescence staining showed that AFn-MNP reduced the intracellular Fe²⁺ level; (H) In the Erastin-induced ferroptosis model, AFn-MNP concentration-dependently increased cell viability; (I) Schematic diagram of the ferroptosis signaling pathway. In summary, AFn-MNP exerts a protective effect through iron chelation and ROS scavenging.

[0032] Conclusion: AFn-MNP comprehensively alleviates NaIO3-induced photoreceptor cell damage by enhancing antioxidant capacity, inhibiting ferroptosis, and protecting mitochondrial function.

[0033] Experiment 2: In vivo animal experiments, as detailed below: (1) Establishment of animal models: A dry age-related macular degeneration (dAMD) model was established using 8-10 week old male C57BL / 6J mice, weighing 20-25g, via tail vein injection of sodium iodate (NaIO3, 35mg / kg, dissolved in 1% DPBS solution). This model mimics the oxidative damage characteristics of human dAMD, inducing degeneration of retinal pigment epithelium (RPE) cells and loss of photoreceptor cells. Mice were fasted for 12 hours prior to injection but had free access to water. After injection, mice were kept in standard feeding conditions until the experimental endpoint. Successful model assessments included thinning of the outer retinal layer, accumulation of lipid peroxidation products, and visual function decline, which peaked 7 days post-injection.

[0034] (2) Dosing regimen: Animals were randomly divided into four groups: a blank control group (PBS), a NaIO3 model group (NaIO3), an MNP treatment group (MNP), and an AFn-MNP treatment group (AFn-MNP), with 10 animals in each group. Thirty minutes after NaIO3 injection, intravitreal injection was administered: the treatment group received AFn-MNP (1 μL, 10 mg / mL), while the control group received an equal volume of PBS or MNP solution. A 33-gauge blunt needle was used for injection, and the injection was administered slowly to avoid a sudden increase in intraocular pressure. Levofloxacin gel was applied post-operatively to prevent infection. Animals were housed under standard conditions (12-hour light / dark cycle, free access to food) until the experimental endpoint (usually 7 days).

[0035] (3) Detection indicators and methods: ①Retinal function assessment: Full-field ERG recording of a wave, b wave and oscillatory potential under dark-adapted stimulation of 0.01 cd·s / m², 3.0 cd·s / m² and 10.0 cd·s / m².

[0036] ② Histological analysis: After eyeball collection, the eyes were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and then 5μm thick sections were prepared. Hematoxylin-eosin (H&E) staining was performed to observe the structure of each layer of the retina. Immunofluorescence staining was used to detect the expression of β3-tubulin (neuronal marker), glial fibrillary acidic protein (GFAP, glial cell activation marker), CtBP2 (synaptic band protein), and PKC-α (bipolar cell marker). TUNEL assay was used to detect cell apoptosis, and DAPI counterstaining was used to locate cell nuclei.

[0037] ③ Molecular mechanism study: detection of ferroptosis and inflammation-related protein expression; qRT-PCR detection of inflammation and ferroptosis-related genes; RNA sequencing (scRNA-seq) to construct retinal single-cell atlas, and KEGG enrichment analysis of differentially expressed genes and signaling pathways (such as oxidative stress and ferroptosis pathway).

[0038] (4) Results and findings of in vivo animal experiments: Figure 4The experimental results show that AFn-MNP protects against NaIO3-induced retinal damage by enhancing the antioxidant stress response. The results are as follows: (A) A schematic timeline of the experiment shows the modeling, drug administration, and detection process; (B) H&E staining shows a significant improvement in the structural integrity of all retinal layers in the AFn-MNP treatment group; (C, D) Quantitative analysis of total retinal thickness and outer nuclear layer (ONL) thickness indicates that AFn-MNP effectively restores retinal thickness; (E) Immunofluorescence shows that AFn-MNP reduces the number of 4-HNE-positive cells, restores Nrf2 expression, and inhibits ACSL4 upregulation; (F, G, H) Quantitative analysis of the number of 4-HNE-positive cells, Nrf2, and ACSL4 fluorescence intensity confirms these changes. Conclusion: AFn-MNP significantly protects retinal structure by reducing lipid peroxidation, enhancing endogenous antioxidant defense (Nrf2), and inhibiting the key protein of ferroptosis (ACSL4).

[0039] Figure 5 The experimental results of AFn-MNP inhibiting NaIO3-induced retinal atrophy and photoreceptor degeneration are shown in the figure. The results show that: (A) OCT images show that AFn-MNP prevents retinal thinning; (B) Fundus photography and fluorescein angiography (FFA) show that AFn-MNP reduces retinal atrophy and vascular leakage; (C) Immunofluorescence co-staining of PKC-α and CtBP2 shows that AFn-MNP maintains synaptic protein expression; (D, E) Quantitative fluorescence intensity of PKC-α and CtBP2 confirms synaptic function protection; (F) Immunofluorescence co-staining of Iba-1 and IL-1β shows that AFn-MNP reduces microglial infiltration and inflammatory factor expression; (G, H) Quantitative fluorescence intensity of Iba-1 and IL-1β indicates an improved inflammatory microenvironment. Conclusion: AFn-MNP effectively alleviates retinal atrophy and photoreceptor degeneration by protecting synaptic structure and inhibiting neuroinflammation.

[0040] Figure 6 The experimental results of AFn-MNP protecting against NaIO3-induced retinal functional impairment are shown in the figure. Results include: (A) Full-field electroretinography (ERG) waveform recordings showing recovery of a-wave, b-wave, and oscillatory potential amplitudes in the AFn-MNP treatment group; (B) Amplitude statistics indicating that AFn-MNP significantly improved the amplitudes of each ERG component; and (C) Time-course statistics showing sustained functional improvement. Conclusion: AFn-MNP significantly improves NaIO3-induced functional impairment by maintaining retinal electrophysiological activity, with its advantages stemming from enhanced bioavailability and anti-ferroptosis capabilities.

[0041] Experiment 3: In vivo biosafety evaluation, details are as follows: Monitor intraocular pressure changes within 7 days after injection; assess retinal function using full-field ERG; and perform histological analysis of retinal structure.

[0042] Figure 7 The figure shows the experimental results of the in vivo biocompatibility evaluation of AFn-MNP. The results show that: (A) In wild-type mice without NaIO3-induced oxidative damage, PBS and AFn-MNP were injected into the vitreous cavity, and intraocular pressure changes were measured on days 1, 3, and 7 after injection using a handheld tonometer. It was observed that there was no significant change in intraocular pressure within 7 days after intraocular injection of AFn-MNP; (B) H&E staining showed that the retina-choroid-sclera complex structure was intact; (C) Quantitative analysis of retinal thickness showed no significant difference; (D, E) ERG detection showed that retinal electrophysiological function was not affected. Conclusion: AFn-MNP exhibits good biocompatibility in vivo, with no intraocular pressure fluctuations, retinal structural damage, or functional abnormalities, and is suitable for further clinical translation.

[0043] 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.

[0044] Quality control measures: ① Experimental environment control: Cell experiments are conducted in a clean bench with regular microbial testing; animal experiments are conducted in an AAALAC-certified facility; molecular experiments are conducted in a dedicated PCR clean bench. ② Data quality control: Positive and negative controls are set up for each experiment; Western blot internal reference genes are validated; qPCR is used to detect primer specificity and amplification efficiency. ③ Instrument calibration: Microscopes, ELISA readers, PCR instruments, and other instruments are calibrated regularly.

[0045] This experimental protocol comprehensively validated the efficacy and safety of AFn-MNP nanocomposite in treating dAMD through systematic in vitro and in vivo experiments. The results showed that AFn-MNP significantly improved retinal structure and function through multiple mechanisms, including ROS scavenging, iron homeostasis regulation, inflammatory response inhibition, and mitochondrial function protection, and exhibited good biocompatibility, providing solid experimental evidence for clinical translation.

[0046] The above embodiments are only used to illustrate the technical concept and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the protection scope of the present invention.

Claims

1. A type of iron-loaded protein-coupled melanin nanoparticle, characterized in that, It includes ferritin and melanin nanoparticles, which are covalently linked to form a core-shell structure.

2. The iron-loaded protein-coupled melanin nanoparticles according to claim 1, characterized in that, The average water-dispersed particle size was 246.6 nm ± 22.1 nm, and the Zeta potential was -10.9 mV ± 1.03 mV.

3. The method for preparing the iron-loaded protein-coupled melanin nanoparticles according to claim 1, characterized in that, Includes the following steps: Ferric protein solution and melanin nanoparticle solution were mixed and covalently linked under EDC / NHS catalysis, and then purified by dialysis to obtain AFn-MNP.

4. The application of the iron-loaded protein-coupled melanin nanoparticles according to claim 1 or 2, characterized in that, Including any one of the following applications: in the preparation of drugs that inhibit ferroptosis in photoreceptor cells; in the preparation of drugs that scavenge reactive oxygen species in the retina; in the preparation of drugs that protect retinal structures.

5. The application of the iron-loaded protein-coupled melanin nanoparticles according to claim 1 or 2, characterized in that, Application in the preparation of drugs for treating retinal diseases associated with oxidative damage.

6. The application according to claim 5, characterized in that, Retinal diseases associated with oxidative damage include one or more of dry age-related macular degeneration and retinitis pigmentosa.

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