Effects of Immune Cell Heterogeneity and Protein Corona on the Cellular Association and Cytotoxicity of Gold Nanoparticles: A Single-Cell-Based, High-Dimensional Mass Cytometry Study

[Image: see text] Understanding how nanoparticles (NPs) interact with biological systems is important in many biomedical research areas. However, the heterogeneous nature of biological systems, including the existence of numerous cell types and multitudes of key environmental factors, makes these interactions extremely challenging to investigate precisely. Here, using a single-cell-based, high-dimensional mass cytometry approach, we demonstrated that the presence of protein corona has significant influences on the cellular associations and cytotoxicity of gold NPs for human immune cells, and those effects vary significantly with the types of immune cells and their subsets. The altered surface functionality of protein corona reduced the cytotoxicity and cellular association of gold NPs in most cell types (e.g., monocytes, dendritic cells, B cells, natural killer (NK) cells, and T cells) and those immune cells selected different endocytosis pathways such as receptor-mediated endocytosis, phagocytosis, and micropinocytosis. However, even slight alterations in the major cell type (phagocytic cells and non-phagocytic cells) and T cell subsets (e.g., memory and naive T cells) resulted in significant protein corona-dependent variations in their cellular dose of gold NPs. Especially, naive T killer cells exhibited additional heterogeneity than memory T killer cells, with clusters exhibiting distinct cellular association patterns in single-cell contour plots. This multi-parametric analysis of mass cytometry data established a conceptual framework for a more holistic understanding of how the human immune system responds to external stimuli, paving the way for the application of precisely engineered NPs as promising tools of nanomedicine under various clinical settings, including targeted drug delivery and vaccine development.