This study aims to develop a hydrophilic nanoscale inhibitor with magnetic targeting capability to precisely inhibit the enzymatic activity of HAAO in prostate tissue, thereby blocking quinolinic acid (QA)-mediated abnormal proliferation of prostate epithelial cells, offering a novel therapeutic strategy for benign prostatic hyperplasia (BPH). Furthermore, through proteomic analysis, in vitro cellular experiments, and molecular docking simulations, we systematically elucidate the critical role of the HAAO-QA pathway in BPH pathogenesis and reveal the high-efficiency binding mechanism between the nanoscale inhibitor and HAAO (Kd = 5.479×10⁻⁸ M), providing theoretical foundations for understanding disease mechanisms and developing targeted therapies.

1.Nanoparticle Synthesis and Characterization: Hydrophilic Fe₃O₄ nanoparticles (average diameter: 10±5 nm) were synthesized via a co-precipitation method in an ethanol-water system. Their morphology, elemental composition (Fe/O ratio 3:4), and surface charge (ζ-potential: 30±0.5 mV) were validated using TEM, energy-dispersive X-ray spectroscopy (EDS), and Zeta potential analysis.
2.In Vitro Cellular Assays: BPH-1 cell line was treated with metal oxide nanoparticles (Fe₃O₄, Fe₂O₃, Al₂O₄, etc.). Cell proliferation was assessed via MTT assay, while flow cytometry analyzed cell cycle arrest (S-phase accumulation) and apoptosis (no significant induction).
3.Proteomic Profiling: Proteins from 18 BPH patients and 3 healthy prostate tissues were extracted and analyzed by data-independent acquisition (DIA) mass spectrometry. HAAO was identified as upregulated (fold change ≥2) in BPH. Pull-down assays and Western blot confirmed Fe₃O₄-HAAO interaction (Kd = 5.479×10⁻⁸ M).
4.Molecular Mechanism Exploration: AutoDockTools-1.5.6 simulated binding modes between Fe₃O₄ and HAAO. QA levels (ELISA) and 3-hydroxyanthranilic acid (HPLC) were quantified post-nanoparticle treatment, revealing inhibition of QA-driven proliferation.
5.In Vivo Validation: A testosterone-induced BPH mouse model (C57BL/6) received intravenous FITC-labeled Fe₃O₄ (5 mg/kg) under magnetic guidance. Biodistribution was tracked via bioluminescence imaging (AniView100) and TEM. Prostate weight, volume, hormone levels (DHT/testosterone), and epithelial thickness (H&E staining) were evaluated to confirm therapeutic efficacy.

1.In vitro inhibition: Fe₃O₄ nanoparticles (0.2 mg/mL) significantly suppressed BPH-1 cell proliferation via S-phase arrest without inducing apoptosis.
2.Target identification: Proteomic analysis revealed ≥2-fold upregulation of HAAO in BPH tissues. Its metabolite QA promoted cell proliferation at physiological concentrations (300–600 nmol/L, peak at 11.47%) but inhibited growth at higher levels (>750 nmol/L).
3.Molecular interaction: Fe₃O₄ exhibited high-affinity binding to HAAO (Kd=5.479×10⁻⁸ M), with molecular docking simulating active site occupation. Fe₃O₄ treatment reduced QA levels by 14%–21% and increased 3-HAA accumulation by 96% in vitro.
4.In vivo efficacy: In testosterone-induced BPH mice, magnet-guided Fe₃O₄ delivery decreased prostate volume, weight , and QA levels , alongside reductions in DHT  and testosterone . H&E staining confirmed a 56.8% reduction in epithelial thickness, validating therapeutic effects.

1.Pathological basis: Upregulated HAAO expression in BPH tissues drives excessive prostate epithelial proliferation via QA-mediated acceleration of DNA synthesis (increased S-phase proportion).
2.Targeted intervention: High-affinity binding of Fe₃O₄ to HAAO (Kd=5.479×10⁻⁸ M) blocks its enzymatic conversion of 3-HAA to QA, reducing QA levels  and accumulating 3-HAA , thereby arresting cell cycle progression.
3.Therapeutic implications: Magnetically guided Fe₃O₄ accumulation in prostate tissues significantly alleviated hyperplasia markers and hormonal dysregulation  in BPH models, establishing a novel therapeutic strategy through amino acid metabolism modulation.

This study demonstrates that hydrophilic Fe₃O₄ nanoparticles selectively target HAAO (Kd=5.479×10⁻⁸ M) to block QA biosynthesis, effectively suppressing prostate epithelial proliferation. Magnetically guided delivery enables precise metabolic reprogramming in the prostate microenvironment, significantly alleviating pathological markers in BPH models . This work pioneers a non-hormonal therapeutic paradigm by integrating nanomaterial-enabled enzyme targeting with tryptophan metabolism intervention, advancing nanoparticle applications in urological disorders. Future investigations should prioritize long-term biosafety evaluation and exploration of combinatorial therapeutic synergies.