Size-dependent internalization of polystyrene microplastics as a key factor in macrophages and systemic toxicity.

Microplastics are emerging pollutants with a wide range of ecological and biological effects, including the ability to accumulate in organisms and induce toxicity. Although numerous studies have investigated the distribution and toxicity of microplastics in murine models and cell lines, the conclusions are inconsistent owing to variations in experimental designs, particle sizes, exposure methods, and dose quantifications. To address these gaps, we systematically evaluated the size-dependent internalization and toxicity of polystyrene microplastics (PS-MPs) using in vitro and in vivo models. Fluorescently labeled PS-MPs were used to confirm the negligible toxicity of fluorophores on macrophages, demonstrating their suitability for tracking particle accumulation. In vitro experiments using RAW 264.7 cell lines and primary peritoneal macrophages revealed size-dependent phagocytosis and cytotoxicity, with smaller particles (0.5 µm) demonstrating higher internalization and causing greater mitochondrial depolarization, reactive oxygen species generation, and apoptosis compared to that with larger particles (5 µm). Acute in vivo experiments comparing oral administration and tail-vein injection revealed that the absorbed dose and toxicity were significantly influenced by particle size, with smaller PS-MPs showing higher organ retention and alterations in hematological and metabolic parameters. Additionally, a 28-day subacute oral exposure study highlighted systemic toxicity, including weight loss, disrupted food intake, elevated oxidative stress markers, and reduced antioxidant enzyme activity. By integrating multiple exposure routes, macrophage models, and fluorescence toxicity evaluations, this study provided a comprehensive and realistic assessment of microplastic toxicity, offering valuable insights for advancing toxicological evaluations and regulatory frameworks. However, this study did not address the influence of other plastic types, shapes, or environmental factors on toxicity. Future studies are thus needed to explore these variables and the long-term implications of real-world microplastic exposure.