Organelle Sorting and Proteomic Analysis to Identify Proteins Involved in the Uptake and Intracellular Trafficking of Nanoparticles.

Monitoring nanoparticle uptake and intracellular trafficking by cells is required to understand how nanomedicines are processed by cells. Many methods currently applied to achieve this test the involvement of known mechanisms and pathways after blocking them, or by measuring colocalization of nanoparticles with known intracellular compartments. However, unknown and non-canonical mechanisms are often involved in nanomedicine uptake and the organelles in which they are trafficked are not fully identified. Therefore novel methods to characterize how and where nanomedicines are internalized and trafficked in unperturbed cells are highly sought. Here a novel platform is presented to characterize uptake and intracellular trafficking of nanoparticles without requiring prior knowledge on the involved mechanisms and their intracellular location. After uptake and cell lysis, organelle sorting by fluorescence assisted cell sorting is used to isolate and purify the organelles in which nanoparticles are internalized and trafficked. Next, the composition of the recovered organelles with nanoparticles is determined by organelle proteomics. Using polystyrene nanoparticles to validate the method, it is shown that this workflow allows to discover novel receptors involved in nanoparticle uptake and proteins mediating nanoparticle trafficking toward the lysosomes. The knowledge gained is essential to test and improve novel formulations for nanomedicine applications.