TY  - JOUR
TI  - Determination of Cellular Uptake and Endocytic Pathways
AU  - Gong, Jing 
AU  - Wang, Hong-Xia 
AU  - Leong, Kam  W
VL  - 9
IS  - 4
PY  - 2019
DA  - 2019/02/20
SP  - e3169
C1  - Bio-protocol 2019;9:e3169
DO  - 10.21769/BioProtoc.3169
UR  - https://doi.org/10.21769/BioProtoc.3169
AB  - Efficiency of drug and gene delivery via nonviral vehicles is contingent on proper cellular uptake and intracellular release. Further, various cargos, such as nucleases for gene editing or inhibitors for endosomal receptors, require transport to specific compartments of the cell. Hence, characterization of cellular uptake and endocytic pathways is crucial for the optimization of any nanoparticle-mediated intracellular delivery system. Previous work on endocytic pathways looks at the effect of various pathway inhibitors on the uptake efficiency of nanoparticles carrying fluorescently-labeled cargo. While this helps attribute particle uptake to specific pathways like caveolae-mediated or clathrin-mediated endocytosis, this does not provide a holistic picture of the delivery process. Here, we provide a general protocol that combines systematic studies of inhibitor effects on efficiency with quantification of particle-induced cell membrane permeability. By applying this methodology to a nucleic acid delivery system, for example a helical polypeptide-based nanoparticle for plasmid and guide RNA delivery, we gain understanding of the endocytic mechanisms and cell uptake for intelligent design of intracellular delivery.
KW  - Endocytosis
KW  - Cell uptake
KW  - Gene editing
KW  - Gene delivery
KW  - Cell-penetrating peptide
KW  - Intracellular trafficking
KW  - Macropinocytosis
KW  - Uptake mechanisms
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False