Preparation of Size-Defined TPU Nano- and Microplastics and Their Cellular Uptake by RAW264.7 Macrophages

The increasing use of thermoplastic polyurethane (TPU)-based clear aligners has raised concerns regarding the potential release of nano- and microplastics during long-term intraoral use. However, systematic biological evaluation of aligner-related plastic particles remains limited, partly due to the lack of well-defined particle fractions suitable for toxicological investigations. In this study, commercial TPU powders were purchased and subjected to mechanical comminution followed by differential centrifugation to obtain particle fractions with distinct size distributions. The morphology and particle size characteristics of the resulting fractions were characterized using scanning electron microscopy (SEM) and dynamic light scattering (DLS), respectively. To investigate particle–cell interactions, TPU particles were incubated with RAW264.7 macrophages, and their localization relative to the cells was examined using confocal microscopy. SEM observations and DLS analyses confirmed the successful preparation of several TPU particle fractions spanning the micro- to nanoscale range, with stable isolation of particle populations down to approximately 200 nm. Confocal microscopy revealed distinct spatial distributions of TPU particles associated with RAW264.7 macrophages. Differences among particle fractions suggested size-dependent interactions between TPU particles and RAW264.7 macrophages. Microscopic observation demonstrated cellular uptake by RAW264.7 cells, while the WST-8 assay revealed a reduction in cell viability.These findings establish a practical size-fractionation platform for TPU-derived nano- and microplastics and provide a foundation for future studies investigating the biological responses and safety assessment of plastic particles associated with oral healthcare materials.