4.8 Article

Multispectral Fingerprinting Resolves Dynamics of Nanomaterial Trafficking in Primary Endothelial Cells

Journal

ACS NANO
Volume 15, Issue 7, Pages 12388-12404

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acsnano.1c04500

Keywords

intracellular trafficking; endosomal maturation; confocal Raman microscopy; hyperspectral imaging; near-infrared fluorescence; machine learning; immunofluorescence colocalization

Funding

  1. National Science Foundation CAREER Award [1844536]
  2. RI Consortium for Nanoscience and Nanotechnology - National Science Foundation EPSCoR [OIA-1655221]
  3. Div Of Chem, Bioeng, Env, & Transp Sys
  4. Directorate For Engineering [1844536] Funding Source: National Science Foundation

Ask authors/readers for more resources

This study introduced a novel approach to investigate the intracellular trafficking processes of nanomaterials, revealing key physicochemical properties and mechanisms. By utilizing single-walled carbon nanotubes in combination with fluorescence and Raman spectroscopy techniques, the progression of intracellular processing events was successfully monitored, providing valuable insights into the cellular uptake and processing of engineered nanomaterials.
Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials (ENMs) which gain cellular entry via active endocytic processes. A recent emphasis on routes of ENM 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 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 exceptional optical properties exhibited by noncovalent 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. 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 submicron spatial resolution. This approach uncovered correlations between DNA-SWCNT concentration, dielectric modulation, and irreversible aggregation within single intracellular vesicles. An immunofluorescence assay was designed to directly observe the DNA-SWCNTs in labeled endosomal vesicles, revealing a distinct relationship between the physical state of organelle-bound DNA-SWCNTs and the dynamic luminal conditions during endosomal maturation processes. Finally, we trained a machine learning algorithm to predict endosome type using the Raman spectra of the vesicle-bound DNA-SWCNTs, enabling major components in the endocytic pathway to be simultaneously visualized using a single intracellular reporter.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.8
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available