Time-Resolved Proteomic Visualization of Dendrimer Cellular Entry and Trafficking

Our
understanding of the complex cell entry pathways would greatly benefit
from a comprehensive characterization of key proteins involved in
this dynamic process. Here we devise a novel proteomic strategy named
TITAN (Tracing Internalization and TrAfficking of Nanomaterials) to
reveal real-time protein–dendrimer interactions using a systems
biology approach. Dendrimers functionalized with photoreactive cross-linkers
were internalized by HeLa cells and irradiated at set time intervals,
then isolated and subjected to quantitative proteomics. In total,
809 interacting proteins cross-linked with dendrimers were determined
by TITAN in a detailed temporal manner during dendrimer internalization,
traceable to at least two major endocytic mechanisms, clathrin-mediated
and caveolar/raft-mediated endocytosis. The direct involvement of
the two pathways was further established by the inhibitory effect
of dynasore on dendrimer uptake and changes in temporal profiles of
key proteins.