Multispectral Fingerprinting Resolves Dynamics of Nanomaterial Trafficking in Primary Endothelial Cells

Abstract

Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials which gain cellular entry via active endocytic processes. A recent emphasis on routes of nanomaterial uptake has established key physicochemical properties which direct certain mechanisms, yet relatively few studies have identified their effect on intracellular trafficking processes past entry and initial subcellular localization. Here, we developed and applied an approach where single-walled carbon nanotubes (SWCNTs) play a dual role - that of an engineered nanomaterial (ENM) undergoing intracellular processing, in addition to functioning as the signal transduction element reporting these events in individual cells with single organelle resolution. We used the unique optical properties exhibited by non-covalent hybrids of single-stranded DNA and SWCNTs (DNA-SWCNTs) to report the progression of intracellular processing events via two orthogonal hyperspectral imaging approaches of near-infrared (NIR) fluorescence and resonance Raman scattering. A positive correlation between fluorescence and G-band intensities was uncovered within single cells, while exciton energy transfer and eventual aggregation of DNA-SWCNTs were observed to scale with increasing time after internalization. These were confirmed to be consequences of intracellular processes using pharmacological inhibitors of endosomal maturation, which suppressed spectral changes through two distinct mechanisms. An analysis pipeline was developed to colocalize and deconvolute the fluorescence and Raman spectra of subcellular regions of interest (ROIs), allowing for single-chirality component spectra to be obtained with sub-micron spatial resolution. This approach uncovered a complex relationship between DNA-SWCNT concentration, fluorescence intensity, environmental transformations, and irreversible aggregation resulting from the temporal evolution of trafficking events. Finally, a spectral clustering analysis was applied to delineate the dynamic sequence of processes into four distinct populations, allowing stages of the intracellular trafficking process to be identified by the multispectral fingerprint of encapsulated DNA-SWCNTs. TOC Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY