Effects of Polylactic Acid-nanoplastics on the Intestinal Barrier: in Vitro Analysis on Human Epithelial Cells

Bioplastics, during their degradation process, break down into progressively smaller particles known as microplastics (MPs) and nanoplastics (NPs). They can enter ecosystems and subsequently the human body through various exposure routes, including inhalation, ingestion, and dermal contact. The increasing use of bioplastics is raising concerns about their potential risks for human health.1 Among biodegradable polymers, polylactic acid (PLA) is one of the most widely used to replace conventional petroleum-based materials. However, its toxic effects in humans are still limited. This prompted us to investigate the potential toxicity of PLA-based NPs (PLA-NPs) evaluating their effects on human intestinal barrier in vitro. We used PLA-NPs conjugated with the fluorochrome Rhodamine, with an average size of 170±64 nm. To simulate intestinal barrier, an in vitro co-culture model using human colorectal adenocarcinoma cell lines Caco-2 and HT29, seeded at a 9:1 ratio respectively, was used. After 21 days of differentiation, each monolayer exhibits functional and morphological features comparable to native intestinal tissue, including mucus production and tight junction formation. PLA-NPs treatment (100 μg/mL) began on day 5 after cell seeding and continued throughout the 21-day differentiation period and for an additional 5 days post-differentiation (total 26 days). To assess the integrity and function of the epithelial barrier, transepithelial electrical resistance (TEER) was measured throughout the entire treatment period. TEER values were consistently higher compared to controls, suggesting a sustained enhancement of barrier integrity. Confocal microscopy following PLA-NPs treatment, showed that Caco-2 cells exhibited increased expression of the tight junction protein ZO-1, with a clear reticular pericellular localization, consistent with enhanced junctional assembly. In parallel, HT29 cells showed an increased number of intracellular MUC-2–positive granules, indicative of elevated mucin synthesis and secretory activity compared to the controls. These results suggest a cellular adaptation aimed at reinforcing both the physical and mucus components of the intestinal barrier. Complementary Western blot analyses of the tight Junction protein E-cadherin in Caco-2 cells also corroborated these results showing an activation of its phosphorylation. Taken together, these findings support the idea that intestinal epithelial cells respond to prolonged PLA-NPs exposure through structural and molecular adaptations that may lead to a chronic inflammation status.This work was supported by 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 1. Santoro A. et al., Curr Neuropharmacol. 2024;22:1870-1898. doi: 10.2174/1570159X22666240216085947.