100 | Tracking polystyrene nanoplastics by raman microscopy reveals intestinal and retinal toxicity

Valentina Iezzi¹, Roberta Maria Ferri¹, Annamaria Iannetta², Ludovica Sulcanese², Tommaso Silvestrini², Vanessa D’Altilia², Giovanni Angelozzi², Federico Perrella³, Carino Ferrante⁴, Giulia Fioravanti³, Monia Perugini², Elisabetta Benedetti¹ | ¹Department of Life, Health and Environmental Sciences, University of L’Aquila, Italy; ²Department of Bioscience and Agro-Food and Environmental Technology, University of Teramo, Italy; ³Department of Physical and Chemical Sciences University of L’Aquila, Italy; ⁴CNR-SPIN, c/o department of Physical and Chemical Sciences University of L’Aquila, Italy.

Micro- and nanoplastic pollution represents an increasingly serious threat to the environment and to the organisms that inhabit it, including humans. In this study, we evaluated the toxicity of 150-nm polystyrene (PS) nanoparticles using both the zebrafish animal model and the human intestinal epithelial Caco-2 cell line. In Caco-2 cells, PS nanoplastics (16 and 25 µg/mL) induced a dose- and time-dependent reduction in cellular protein content, as measured by the SRB assay, preceding overt cell loss. This effect was accompanied by the activation of inflammatory markers (IL1β, NOS) and dysregulation of OCEL indicating impairment of epithelial barrier integrity. Raman imaging revealed intracellular accumulation of PS nanoparticles at 15 and 24 h, followed by a marked reduction of intracellular particles at 48 h, suggesting selective loss of PS-load cells, potentially contributing to a leaky-gut phenotype. In vivo, Raman microscopy revealed nanoplastic localization in the ocular region, confirmed by the presence of characteristic polystyrene spectral peaks together with alterations in the amide I and amide III bands, indicative of oxidative stress and inflammation. These findings were supported by the increased expression of genes involved in inflammatory and oxidative stress responses (il1b, cat, sod1, gstm) and by a significant increase in neutrophil recruitment in the retina, assessed using the transgenic neutrophil reporter line Tg(mpx: GFP). Retinal homeostasis was further compromised, as demonstrated by marked overexpression of rpe65c, a retinal pigment epithelium–specific gene essential for visual cycle function, together with induction of sox2 and pax6a, key regulators of eye development and regeneration. This expression profile suggests activation of a stress-induced compensatory response. Finally, lipid metabolism was altered, with Oil Red O staining revealing neutral lipid accumulation in the head region and strong induction of pparγ, a master regulator of lipid storage. Collectively, these findings reveal Raman microscopy to be a reliable technique to detect PS in both in vitro and in vivo models and demonstrate, in vitro, that PS impair epithelial barrier function, enabling systemic inflammation, whereas in vivo they trigger inflammation, oxidative stress, metabolic reprogramming, and retinal gene dysregulation, posing a previously unrecognized risk to visual system development in aquatic organisms.