TOXIC EFFECTS OF POLYLACTIC ACID NANOPLASTICS ON IN VITRO MODELS OF ASTROCYTES AND NEURONS

Marianna MARINO¹, Martina LOMBARDI^1-3, Raffaella D’AURIA¹, Erwin Pavel LAMPARELLI¹, Giovanna DELLA PORTA^1,4, Marta Anna SZYCHLINSKA⁵, Federica SCALIA⁶, Francesco CAPPELLO^6,7, Rosaria MECCARIELLO⁸, Andrea VIGGIANO^1,4, Antonietta SANTORO^1,4 | ¹Department of Medicine, Surgery and Dentistry, “Scuola Medica Salernitana”, University of Salerno, Italy; ²Theoreo Srl, Montecorvino Pugliano, SA, Italy; ³European Institute of Metabolomics - Fondazione EIM, Baronissi, SA, Italy; ⁴Interdepartmental Observatory for Scientific Health Initiatives in Environmental Legislation and Disease Prevention, SHIELD, University of Salerno, Italy; ⁵Department of Precision Medicine in Medical, Surgical and Critical Care (MEPRECC), University of Palermo, Italy; ⁶Department of Biomedicine, Neuroscience and Advanced Diagnostics, University of Palermo, Italy; ⁷Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy; ⁸Department of Physical Sciences and Wellness, University of Naples Parthenope, Italy

Micro and nanoplastics (MP/NPs), resulting from the fragmentation of plastics, are eliciting increasing concerns about their effects on human health. Evidence indicates that MPs/NPs can enter the human body through various exposure routes and may penetrate the brain, causing neurotoxicity. However, research in this field remains limited, especially regarding MPs/NPs derived from eco-friendly plastics like polylactic acid (PLA), which are increasingly being produced and used as alternatives to petroleum-based plastic polymers.^1,2 To this end, the present study aimed to fill this research gap by evaluating the potential effects of PLA MPs/NPs on in vitro models of the nervous system. Specifically, for the experimental setup were employed rat glioma C6 cells, as an astrocyte model, and rat adrenal pheochromocytoma PC12 cells, as a neuronal model stimulated to differentiate into neurons using Nerve Growth Factor (NGF). Both cell types were treated with PLA NPs (170±64 nm) at concentrations of 100 and 300 µg/mL for 24 h. Results indicate that once PLA-NPs are efficiently internalized by both cell models, they can impair normal astrocytic and neuronal physiology. Notably, in NGF-differentiated PC12 cells, PLA-NPs interfere with cell proliferation by inducing an S-phase cell cycle arrest. Treatment also promotes a reduction in the activation of key proteins involved in proliferation and neuronal differentiation, such as extracellular-signal-regulated kinases (ERK) and protein kinase B (pAKT). Furthermore, an increase in the pro-inflammatory rat cytokine CINC1, together with an enhanced level of the stress protein SIRT1, was observed. Analogously, C6 cells showed signs of activation following PLA-NPs exposure, as demonstrated by augmented expression of pAKT and glial fibrillary acidic protein (GFAP). ELISA immunoassay demonstrated a reduction in the vascular endothelial growth factor type alpha (VEGF-α), a protein whose downregulation correlates positively with increased intracellular ROS. To further investigate this issue, we evaluated Reactive-Oxygen Species (ROS) level, and in both cell models, a higher ROS production was evidenced, further corroborating that PLA-NPs treatment could promote the onset of an inflammatory state. These findings highlight the potential neurotoxic effects of PLA-NPs, emphasizing the need for further studies to fully understand the impact of PLA-NPs on the nervous system.

This work is under the support of the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.1, “Fund for the National Research Program and for Projects of National Interest (NRP)” by the Italian Ministry of University and Research(MUR), funded by the European Union – NextGenerationEU. Project title: “Plastic Contamination byPoly(Lactic Acid) (PLASTAMINATION): organ injuries and underlying molecular mechanisms”, MUR,PRIN-PNRR2022 CODE NUMBER: P2022AA47Y- CUP D53D23021910001

References

1. Yunn et al. Science of the Total Environment 924 (2024) 171681 
2. García-Rodríguez et al., Journal of Hazardous Materials 475 (2024) 134900