Plant-Derived Nanovesicles from Ginkgo biloba Seeds Mitigate LPS-induced Endothelial Dysfunction and Promote Vascular Homeostasis

Maneea Moubarak¹Emese Szilágyi-Tolnai²Ani Lyudmilova Barbulova¹Immacolata Fiume¹Ildikó Kovács-Forgács²Judit Homoki²Georgina Pesti-Asbóth²Endre Szilágyi²Ramila Mammadova¹Matic Kisovec³Marjetka Podobnik³Dávid Papp⁴Gitta Schlosser⁴Judit Remenyik^2*Gabriella Pocsfalvi^1*

• ¹Institute of Bioscience and Bioresources, National Research Council (CNR), Napoli, Italy
• ²Debreceni Egyetem, Debrecen, Hungary
• ³Kemijski institut, Ljubljana, Slovenia
• ⁴Eotvos Lorand Tudomanyegyetem Kemiai Intezet, Budapest, Hungary

Background: Endothelial dysfunction is increasingly recognized as an early and central event in the onset of cardiovascular and neurodegenerative diseases. Ginkgo biloba extracts are known for their vascular protective properties, including enhancement of endothelial function, antioxidant and anti-inflammatory activity, nitric oxide preservation, and modulation of platelet aggregation. Plant-derived nanovesicles (PDNVs) are emerging as versatile bioactive carriers with demonstrated anti-inflammatory, anticancer, antimicrobial, regenerative, and microbiota-modulating effects. However, their vascular protective potential remains underexplored. This study aimed to assess the effects of PDNVs isolated from Ginkgo biloba seeds on endothelial responses under inflammatory stress. Methods: PDNVs were isolated from Ginkgo biloba seed homogenate using differential ultracentrifugation (DUC) followed by density gradient ultracentrifugation (DGUC) with linear and non-linear iodixanol gradients. Nanoparticle tracking analysis (NTA) and cryo-transmission electron microscopy (cryo-TEM) characterized vesicle size, concentration, and morphology. Untargeted mass spectrometry profiled the protein content of distinct PDNV fractions. Functional assays were conducted on human umbilical vein endothelial cells (HUVECs) exposed to lipopolysaccharide (LPS)-induced inflammatory stress. Results: Ginkgo PDNVs isolates formedcomprise a heterogeneous population, including vesicles with single-and double -bilayers vesicleslarger than >50 nm. Proteomics revealed seed storage proteins (legumin, ginnacin) and membrane-associated ATPases, HSP90, catalase, PEPC, and eEF1A. PDNVs were non-toxic up to 50 µg/mL; at 100 µg/mL, they enhanced mitochondrial activity but triggered early apoptosis and necrosis. PDNVs did not increase ROS production, even in the presence of H₂O₂. At 1 µg/mL, they significantly suppressed LPS-induced expression of IL-1β, TNF-α, IL-6, and IL-8 (mRNA and protein; p ≤ 0.05 to p ≤ 0.001). PDNVs preserved endothelial integrity by downregulating VCAM-1 and upregulating Occludin, maintained eNOS expression (p ≤ 0.01), and attenuated COX-1, COX-2, and PGIS induction. Thrombotic markers (TXB₂, vWF, PAI-1) remained unaffected. Conclusion: Ginkgo seed-derived PDNVs exhibit promising vascular protective and anti-inflammatory properties, supporting their potential as safe, multifunctional agents for endothelial modulation. Further studies are warranted to explore their therapeutic applications in vascular biology.