Rapamycin-Selenium Nanoparticles Reverse Vascular Aging via Mitophagy Activation

Vascular endothelial senescence is a central driver of age-related cardiovascular diseases including atherosclerosis, hypertension, and diabetes. Senescent endothelial cells accumulate in arterial walls, secreting pro-inflammatory cytokines (the senescence-associated secretory phenotype, SASP) and losing normal vascular functions such as permeability regulation and anti-thrombotic signaling. Oxidative stress is a primary trigger of this senescence, yet existing interventions — including rapamycin (RPM) and selenium supplementation — each face delivery and toxicity challenges when used alone. This study aimed to test whether a novel nanoparticle combining both agents could synergistically counteract vascular oxidative senescence.

The team synthesized RPM-coated selenium nanoparticles (RPM-SeNPs) by reducing sodium selenite with ascorbic acid in the presence of poloxamer 407 and RPM, yielding uniformly small particles with a mean diameter of 67.51 ± 2.07 nm and a PDI indicating excellent dispersity — significantly better than uncoated SeNPs. The RPM:Se molar ratio was 1:120. Physicochemical characterization via TEM, EDS, UV-Vis, FTIR, and dynamic light scattering confirmed successful RPM coating and stable colloidal properties. Coumarin-6 labeling confirmed cellular uptake of the nanoparticles into endothelial cells.

For in vivo studies, male C57BL/6J mice (n = 10 per group) received intraperitoneal paraquat (PQ, 7 mg/kg/day for 1 week) to induce systemic oxidative senescence of aortic endothelium. RPM-SeNPs (0.08 mg Se/kg), SeNPs (0.08 mg Se/kg), or free RPM (0.05 mg/kg) were administered intravenously every other day. RPM-SeNPs significantly outperformed both SeNPs and free RPM in reducing aortic SA-β-gal activity and lowering SASP markers including IL-1β, p16, p21, and p53 at both mRNA and protein levels. Oxidative stress indicators — MDA levels and intracellular/mitochondrial ROS — were markedly reduced, while GSH levels and GPX activity were restored. Endothelial dysfunction markers improved commensurately.

In vitro, mouse aortic endothelial cells (MAECs) were pre-treated with 200 mM H₂O₂ for 4 hours to induce oxidative senescence, then exposed to RPM-SeNPs (2 μM Se), SeNPs (2 μM Se), or free RPM (100 nM) for 24 hours. RPM-SeNPs rescued mitochondrial membrane potential (assessed by JC-1), restored ATP production, and reduced mitochondrial ROS (MitoSOX). Critically, RPM-SeNPs upregulated GPX4 — particularly the mitochondrial isoform (mtGPX4) — far more than either agent alone, providing direct antioxidant defense at the mitochondrial membrane. Mitophagy was confirmed to be activated by co-localization of MitoTracker and LysoTracker signals, alongside increased LC3B-II, decreased p62/SQSTM1, increased Beclin1, and reduced phospho-ULK1 at Ser757. Western blots showed suppression of PI3K, phospho-Akt (Ser473), and phospho-mTOR (Ser2448), mechanistically linking RPM-SeNPs to mTOR pathway inhibition and downstream mitophagy activation.

The dual mechanism is notable: RPM inhibits mTOR to disinhibit ULK1 and activate the autophagy initiation complex, while selenium drives selenoprotein synthesis (particularly GPX4) that shields mitochondria from lipid peroxidation and maintains the redox environment required for functional mitophagy machinery. Together, these actions clear dysfunctional mitochondria, preventing accumulation of mtDNA damage and SASP induction. The authors acknowledge the study is preclinical and relies on paraquat and H₂O₂ as oxidative stressors — models that may not fully replicate physiological aging. Human clinical translation, dosing optimization, and long-term safety profiling remain to be established. No conflicts of interest were declared.