SUMMARY Mitochondrial dysfunction in post-menopausal women due to loss of estradiol (E2) alone or in combination with age-induced accumulation of reactive oxygen species (ROS) may play a central role in development of metabolic dysfunction-associated steatotic liver disease (MASLD). Impaired oxidative phosphorylation (OXPHOS) and increased ROS production are linked to dysfunctional hepatic lipid metabolism and liver steatosis. Despite the impact of age and E2 loss on mitochondrial function in post-menopausal women, there is a lack of targeted therapies to treat menopause-associated hepatocellular mitochondrial dysfunction. Current therapies for menopause, which include hormone replacement therapy (HRT) or selective estrogen receptor modulators (SERMs), have adverse and off-target effects such as increased risk for gynecologic cancer and deep vein thrombosis and stroke. Moreover, none of these treatments specifically target mitochondrial function. New pharmaceuticals targeting hepatocellular mitochondrial function with or without E2 would reduce morbidity due to metabolic disease and improve the quality of life for post-menopausal women. The long-term goal of this research is to develop effective therapies to treat mitochondrial dysfunction in post-menopausal women. The objective of this proposal is to determine if two distinct, targeted nanoparticles can improve mitochondrial function in HepG2 cells cultured in MASLD-like conditions and in aged and E2-deficient female mice. The central hypothesis is that two unique nanoparticles – one that works via the nucleus and one that works directly at the mitochondria via E2 - can be delivered to improve mitochondrial function in aged and E2-deficient hepatocytes whose function is impacted by MASLD. Co-Investigator Dr. Gaharwar has developed a new class of molybdenum disulfide (MoS2) nanoflowers that scavenge ROS, increase transcription factor a mitochondria (TFAM) protein and mitochondrial biogenesis, and yield increased OXPHOS/ATP production. Based on preliminary data using poly(lactic-coglycolic acid)-poly(ethylene glycol)-triphenylphosphine (PGLA-PEG-TPP) nanoparticles covalently bonded to E2 (mito-E2) which demonstrates that mito-E2 colocalizes with mitochondrial (mt) estrogen receptor (ER) beta (β), it is predicted that mito-E2 will target mtERβ and mtER alpha (a) to improve 𝛽 oxidation and ATP production and decrease ROS. The hypothesis will be tested with two Aims: (1) Determine if MoS2 nanoflower and mito-E2 improve mitochondrial function in aged and E2- deficient MASLD-like conditions; (2) Determine the molecular mechanisms of mito-E2 at mtERa and mtER𝛽 in HepG2 cells cultured in MASLD-like conditions. It is expected that each of the nanoparticles will improve mitochondrial function in MASLD-like HepG2 cells and in mouse menopause models through different but complimentary mechanisms. This innovative proposal lays the foundation to develop nanoparticle therapeutics to treat post-menopausal MASLD....