Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Nanocomplexes Derived from Agriculture Waste with Activity Against Hepatic Diseases

Metabolic-Associated Fatty Liver Disease (MAFLD) and related fibrosis progression are driven by multiple pathological factors, including fibrogenic signaling, lipotoxic injury, and oxidative stress. Current therapeutic approaches aim to mitigate these processes but face limitations in bioavailability and specificity. The development of advanced nanomaterials for targeted drug delivery are needed. Naturally derived bioactive compounds are emerging as promising tools, offering a sustainable strategy to modulate liver disease progression and enhance the therapeutic efficacy of antifibrotic and metabolic interventions. Transforming growth factor beta1 (TGF-β1) stands out as a principal mediator of hepatic fibrosis by activating hepatic stellate cells (HSCs) and promoting excess extracellular matrix (ECM) deposition. In our first study, conducted in a rat model of CCl₄-induced liver fibrosis, we have demonstrated that the encapsulation of TGF-β1 receptor inhibitor Galunisertib (GLY-LY2157299) into polymeric nanomicelles significantly enhanced its bioavailability and therapeutic efficacy compared to the free form of the drug. This led to reduced collagen deposition, mitigated fatty acid-induced degeneration, and restoration of normal liver architecture. At the same time, alteration of lipid metabolisms and saturated fatty acids (FFAs) overload, such as palmitic acid (PA), induce lipotoxicity driving hepatocyte injury and steatosis. This highlights the need for targeted interventions not only for hepatic fibrogenic processes but also for associated metabolic dysfunction. To better understand these processes and explore targeted interventions, we optimized an in vitro system mimicking liver steatosis using a combination of PA and oleic acid (OA), the two most abundant dietary FFAs. Notably, PA and OA are metabolized differently in hepatic cells. PA is a potent activator of PPARα and induces endoplasmic reticulum (ER) stress, impairs autophagy, and promotes lipid accumulation, exacerbating metabolic dysfunction. Our findings highlight that PA increases cell death, downregulates enzymes crucial for protecting against FFA-induced toxicity, and disrupts lipid droplet formation, thereby intensifying lipotoxic stress. Following these insights, we further explored a sustainable, phytochemical-based approach for hepatoprotection and treatment of liver steatosis. Specifically, an olive leaf extract (OLE) obtained by water-based ultrasound-aided extraction was tested in our in vitro system of FFA-induced steatosis. OLE, rich in polyphenols such as oleuropein and hydroxytyrosol, exerted potent antioxidant activity and important hypolipidemic effects, reducing lipid accumulation, restoring antioxidant enzyme activity, stabilizing mitochondrial function, and inducing lipophagy in FFA-treated hepatocytes. Collectively, our data emphasize new potential cross-cutting therapeutic approaches for MAFLD, ranging from the use of nanostructured system to natural bioactive compounds. Targeted delivery via nanocarriers, combined with interventions that counteract lipotoxicity, such as "green" extracts like OLE, is our future goal. The integration of precision drug delivery with sustainable phytochemicals is offering a promising strategy for developing effective and environmentally conscious therapies for a broad spectrum of liver disorders. Keywords: Hepatic Fibrosis, TGF-β1, Galunisertib, Nanomicelles, Lipotoxicity, Palmitic Acid, Oleic Acid, Endoplasmic Reticulum Stress, lipid metabolism, Olive Leaf Extract, Oxidative Stress, Sustainable Therapy.