Cell Specific Metabolomic and Fluxomic Responses to Polylactic Acid Nanoplastics Exposure in Human Intestinal Cells

As the demand for sustainable alternatives to fossil-based plastics accelerates, polylactic acid (PLA) has emerged as one of the most widely used biodegradable polymers, particularly in food-contact applications. However, its degradation into micro- and nanoplastics (M/NPs) raises critical concerns regarding their potential impact on human health—especially through oral exposure and intestinal absorption.1 In the light of the above, the present study aims to investigate the metabolic fate and consequences of PLA-NPs in two distinct human colorectal adenocarcinoma cell lines: CaCo-2 and HT29. PLA-NPs (170±64 nm) were synthesized in-house using a microfluidic-assisted nanoprecipitation method (2). Cells were treated with 100 µg/mL PLA-NPs and sampled at 24, 48, and 72 hours. Metabolic profiling was performed on both pellet and supernatant via Gas-Chromatography Mass Spectrometry. In parallel, fluxomic experiments were conducted using fully ¹³C-labeled glucose media to trace the metabolic processing of PLA-derived carbon. Interestingly, while both cell lines demonstrated altered energy homeostasis, and disrupted amino acid and lipid metabolism, the magnitude and direction of these perturbations differed, suggesting divergent adaptive mechanisms to xenobiotic stress. Metabolic flux analysis revealed a striking early accumulation of unlabeled (¹²C) intracellular lactate, indicative of PLA hydrolysis into lactic acid monomers. This effect was most pronounced within 24 hours and diminished over time, concurrent with a rise in extracellular lactate levels, comprising both ¹²C and ¹³C isotopologues, averting cytosolic acidification under metabolic strain. The observed pattern suggests both continued endogenous glycolytic activity and xenobiotic processing of PLA-derived substrates. Overall, our findings underscore the capacity of PLA-NPs to traverse epithelial membranes and perturb core intracellular pathways exerting subtle but functionally relevant metabolic interference. This highlights a previously underappreciated avenue of interaction between biodegradable plastics and host physiology. Furthermore, the rapid rerouting and extrusion of PLA-derived carbon metabolites suggests the engagement of protective metabolic circuits aimed at preserving redox equilibrium and cellular pH. These observations call for deeper investigation into the consequences of prolonged exposure, particularly with regard to intestinal homeostasis, epithelial barrier function, and downstream immunometabolic signaling using in vivo models.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.