Acoustically augmented probiotic extracellular vesicles for oral fucoxanthin nanotherapy in NAFLD: Hepatoprotection and macrophage immunometabolic modulation

Ultrasound engineering increased probiotic EV yield while preserving immunoregulatory bioactivity. • Microfluidic loading enabled oral 300 W-EV@Fx with enhanced gastrointestinal stability and hepatic exposure. • 300 W-EV@Fx relieved hepatocyte lipotoxicity, oxidative injury, and metabolic dysfunction. • 300 W-EV@Fx improved hepatic macrophage balance and inflammatory homeostasis in HFD-fed mice. • EV depletion/add-back revealed hepatocyte EV–macrophage crosstalk in vitro . Nonalcoholic fatty liver disease (NAFLD) progression involves hepatocellular lipotoxicity together with amplification of the hepatic inflammatory microenvironment. Therapeutic strategies that simultaneously alleviate hepatocyte injury and hepatic immune dysregulation remain underexplored. Here, we generated acoustically augmented probiotic extracellular vesicles from Lacticaseibacillus casei . Among them, vesicles generated under 300 W ultrasound stimulation (300 W-EV) showed an optimal balance of enhanced yield, structural integrity, and immunomodulatory activity. Fucoxanthin (Fx) was then encapsulated using microfluidic technology to construct an oral nanodelivery platform, 300 W-EV@Fx. In vivo , orally administered 300 W-EV@Fx showed enhanced hepatic exposure within 8 h, alleviated hepatic lipid deposition and oxidative damage in high-fat diet-fed mice, restored mitochondrial respiration, and ameliorated immune dysregulation characterized by Treg loss, macrophage infiltration, and M1-like/M2-like imbalance. In complementary mechanistic studies performed in vitro , 300 W-EV@Fx relieved lipotoxic hepatocyte injury. Coculture and EV depletion/add-back experiments further showed that hepatocyte-derived EVs functionally contributed to macrophage inflammatory responses. EVs from lipotoxic hepatocytes promoted macrophage ROS accumulation, inflammatory polarization, and glycolytic activation, whereas EVs from 300 W-EV@Fx-treated hepatocytes drove macrophage immunometabolic reprogramming toward an anti-inflammatory phenotype supported by mitochondrial oxidative metabolism. Overall, this study presents an oral delivery strategy that integrates efficient drug delivery with microenvironmental regulation, attenuating NAFLD progression through the coordinated relief of hepatocellular lipotoxicity and inflammatory microenvironmental imbalance.