Multiomic Profiling and Neuroprotective Bioactivity of Salvia Hairy Root-Derived Extracellular Vesicles in a Cellular Model of Parkinson’s Disease

Abstract

Vincenzo Vestuto,1,* Marisa Conte,1,* Mariapia Vietri,1,* Francesca Mensitieri,2 Valentina Santoro,1,3 Anna Di Muro,1 Mariaevelina Alfieri,4 Maria Moros,5,6 Maria Rosaria Miranda,1,3 Chiara Amante,1 Matteo Delli Carri,1 Pietro Campiglia,1 Fabrizio Dal Piaz,2,7 Pasquale Del Gaudio,1 Nunziatina De Tommasi,1,3 Antonietta Leone,1 Ornella Moltedo,1 Giacomo Pepe,1,3 Elisa Cappetta,1 Alfredo Ambrosone1 1Department of Pharmacy, University of Salerno, Fisciano, 84084, Italy; 2Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Baronissi, 84081, Italy; 3National Biodiversity Future Center (NBFC), Palermo, 90133, Italy; 4Clinical Pathology, Santobono-Pausilipon Children’s Hospital, AORN, Naples, 80122, Italy; 5Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC-Universidad de Zaragoza, Zaragoza, Spain; 6Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain; 7Operative Unit of Clinical Pharmacology, University Hospital “San Giovanni di Dio e Ruggi d’Aragona”, Salerno, 84131, Italy*These authors contributed equally to this workCorrespondence: Alfredo Ambrosone; Elisa Cappetta, Department of Pharmacy, University of Salerno, Fisciano, 84084, Italy, Email [email protected]; [email protected]: Extracellular vesicles (EVs) are promising tools for nanomedicine and nanobiotechnology. The purification of mammalian-derived EVs involves intensive processes, and their therapeutic application raises multiple safety and regulatory issues. Plants have the potential to serve as nonconventional sources of therapeutically relevant EVs. In this context, we recently identified hairy roots (HRs) of medicinal plants as a novel biotechnological platform to produce EVs for human health.Methods: Herein, we report the purification, omics profiling, and bioactivity of EVs isolated from HRs of the medicinal plants S. sclarea and S. dominica. EVs were isolated from conditioned media of HR cultures using differential ultracentrifugation (dUC) and size exclusion chromatography (SEC). The isolated EVs were characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The proteomic and metabolomic profiles of the EVs were determined using mass spectrometry. Uptake studies and bioactivity assays, including confocal microscopy, MTT, flow cytometry, ROS quantification, and untargeted metabolomics analyses, were conducted in SH-SY5Y cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA) to evaluate the therapeutic potential of EVs in an in vitro model of Parkinson’s disease.Results: S. sclarea HRs released nanosized round-shaped EVs with a distinctive molecular signature. HR EVs from S. sclarea and S. dominica revealed conserved cargo of secondary metabolites, predominantly triterpenoids, which are known for their antioxidant properties. We showed that HR EVs are safe, enter the cells, and strongly inhibit apoptosis in a cellular model of Parkinson’s disease. Cellular metabolomics revealed that EVs preserved metabolic homeostasis and mitigated cellular oxidative stress when co-administered with 6-OHDA. Mechanistically, HR EVs inhibited 6-OHDA autoxidation and substantially reduced the accumulation of its oxidative products, which are responsible for 6-OHDA-induced toxicity.Conclusion: Collectively, our findings provide compelling evidence that EVs isolated from the hairy roots of Salvia species are promising, non-mammalian alternative for the design of novel therapies targeting neurological disorders. Keywords: non-mammalian EV source, Salvia extracellular vesicles, hairy roots, nanomedicine, neuroprotection, Parkinson’s disease

Keywords

• non-mammalian ev source
• salvia extracellular vesicles
• hairy roots
• nanomedicine
• neuroprotection
• parkinson’s disease