Revisiting the cellular toxicity of benzo[

Benzo[a]pyrene (Bap) is one of the main organic pollutants in the atmospheric haze that is rich in fine water drops and particulate matters. The understanding of the Bap's form in water is of great importance to unveil its real biological effects toward the respiratory system. To date, various reports have documented its toxicological effects in the molecular form. Herein, we found that Bap existed as self-aggregated nanoclusters of tunable sizes rather than as dissolved molecules in water and different sized nanoclusters illustrated varied cytotoxicity. These findings indicated that the size, which has been ignored in previous studies, is also a dominant parameter similar to the molecular concentration for determining Bap's cytotoxicity. Polystyrene (PS) nanoparticles, as a model for nanoplastics, could adsorb Bap nanoclusters and serve as carriers that enter the cells. The combination effect interestingly altered the cytotoxicity distinction of Bap of different sizes. The intracellular fate of the nanoparticles and subcellular organelle damages were studied to unveil the mechanisms of cytotoxic distinction. Small Bap nanoclusters entered cells faster than their large counterparts. The Bap of the PS@Bap complex was stably adsorbed on PS at the early stages of endocytosis until it was detached during the lysosomal transport and maturation process. The dissociated Bap may bypass the lysosome pathway and be released into the cytosol with a nanocluster structure or relocate into the endoplasmic reticulum. On the other hand, the detached PS preferred to bind to the mitochondria or be excreted out of the cell via the lysosomal pathway. Moreover, the PS@Bap complex resulted in a significant loss of the mitochondrial membrane potential and induced apoptosis through the mitochondria-involved apoptosis pathway. This study provides a new perspective towards the toxicological mechanism of insoluble hydrophobic organic compounds and reveals the environmental significance of nanoplastics for regulating the biological effects of conventional pollutants.