(1) Background: Zinc oxide (ZnO) particles are widely used as zinc (Zn) fortifiers, because Zn is essential for various cellular functions. Nanotechnology developments may lead to production of nano-sized ZnO, although nanoparticles (NPs) are not intended to be used as food additives. Current regulations do not specify the size distribution of NPs. Moreover, ZnO is easily dissolved into Zn ions under acidic conditions. However, the fate of ZnO in commercial foods or during intestinal transit is still poorly understood. (2) Methods: We established surfactant-based cloud point extraction (CPE) for ZnO NP detection as intact particle forms using pristine ZnO-NP-spiked powdered or liquid foods. The fate determination and dissolution characterization of ZnO were carried out in commercial foods and human intestinal cells using in vitro intestinal transport and ex vivo small intestine absorption models. (3) Results: The results demonstrated that the CPE can effectively separate ZnO particles and Zn ions in food matrices and cells. The major fate of ZnO in powdered foods was in particle form, in contrast to its ionic fate in liquid beverages. The fate of ZnO was closely related to the extent of its dissolution in food or biomatrices. ZnO NPs were internalized into cells in both particle and ion form, but dissolved into ions with time, probably forming a Zn–ligand complex. ZnO was transported through intestinal barriers and absorbed in the small intestine primarily as Zn ions, but a small amount of ZnO was absorbed as particles. (4) Conclusion: The fate of ZnO is highly dependent on food matrix type, showing particle and ionic fates in powdered foods and liquid beverages, respectively. The major intracellular and intestinal absorption fates of ZnO NPs were Zn ions, but a small portion of ZnO particle fate was also observed after intestinal transit. These findings suggest that the toxicity of ZnO is mainly related to the Zn ion, but potential toxicity resulting from ZnO particles cannot be completely excluded.
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