102 | Polylactic acid nanoparticles as metabolically active entities: a metabolomics and fluxomics perspective

Martina Lombardi^1|2|3, Vera Abenante^2|3, Alessio Trotta^2|3, Erwin Pavel Lamparelli¹, Federica Montella¹, Giovanna Della Porta¹, Andrea Viggiano¹, Jacopo Troisi^1|2|3|4, Antonietta Santoro¹ | ¹Department of Medicine, Surgery and Dentistry, “Scuola Medica Salernitana”, University of Salerno, Italy; ²Theoreo Srl, Montecorvino Pugliano, Salerno, Italy; ³European Institute of Metabolomics - Fondazione EIM, Baronissi, Salerno, Italy; ⁴Department of Chemistry and Biology, “A Zambelli”, University of Salerno, Fisciano [SA], Italy.

The rapid incorporation of biodegradable polymers into daily-use and biomedical products has largely outpaced a comprehensive understanding of their biological effects and metabolic implications. Among the various, polylactic acid (PLA) represents one of the most extensively used, particularly in food-contact applications, and its fragmentation into micro- and nano-plastics (MPs/NPs) is raising increasing concerns for its potential implications for human health. Here, we present an integrated metabolomics and fluxomics investigation of the effects of PLA nanoparticles (PLA-NPs) combining in vitro models with an in vivo experimental model. PLA-NPs were synthesized in-house using a microfluidic-assisted nanoprecipitation approach and fluorescently labeled with rhodamine to enable intracellular localization. Human intestinal-derived HT-29 and Caco-2 cells were exposed to PLA-NPs (100 μg/mL) for 24, 48, and 72 hours, followed by untargeted metabolomics profiling by gas chromatography–mass spectrometry and high-resolution liquid chromatography-mass spectrometry. Metabolic analyses revealed non-overlapping, cell-specific responses to PLA-NPs exposure, involving coordinated perturbations in amino acid utilization, carbohydrate and lipid metabolism, and redox-related pathways. To disambiguate the intracellular fate of PLA-NPs, a fluxomic investigation was implemented by culturing cells in a fully 13C-labeled media. This approach enabled the discrimination of carbon fluxes derived from the polymeric material from those sustained by endogenous cellular metabolism. To extend these observations beyond in vitro systems, a parallel in vivo study was conducted in Wistar rats (n = 20), including 10 animals receiving PLA-NPs via drinking water and 10 matched controls over a 21-day exposure period. Untargeted and targeted metabolomics analyses of intestinal tissues and fecal samples are currently underway to evaluate metabolic adaptations associated with sustained PLA-NPs intake. Overall, these findings demonstrate that PLA-NPs is not metabolically inert, but can be actively integrated into cellular metabolic networks, where they elicit extensive metabolic rewiring. By demonstrating that PLA-derived nanoparticles are capable of actively modulating cellular metabolism, these findings provide a rationale for future investigations aimed at evaluating their effects on systemic metabolic regulation.
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 by Poly(Lactic Acid) (PLASTAMINATION): organ injuries and underlying molecular mechanisms”, MUR, PRIN-PNRR2022 CODE NUMBER: P2022AA47Y- CUP D53D23021910001