(504c) Using Nanoelectroporation to Understand Intracellular Trafficking of Nanoparticles In Gene Delivery

Most of bio-macromolecular agents and nanoparticles in conventional delivery methods are taken into cells through endocytosis process. Generally, bio-macromolecular agents and nanoparticles are targeted to cell receptors and internalized into endosome. Although the total amount of  drugs/genes can be specified, the delivered dose dependent on the kinetics of cellular attachment, internalization and endosome escape, which is random due to biological heterogeneity. 

To overcome this limitation, we recently developed a novel and yet simple nanoelectroporation (NEP) method for bio-macromolecular agents and nanoparticle delivery to individual cells with precise control of dosage. The delivery is achieved by the focused electric field through a nanochannel connecting two microscale channels with a single cell located at the outlet of the nanochannel in one microchannel and gene/drug/nanoparticles located in the other microchannel. The focused electric pulse porates the cell membrane to form a nanopore and also provides electrophoretic mobility of charged molecules/nanoparticles to move them directry into the cytosol (i.e. by-passing the endocytothic pathway). By comparing the conventional deliverly method and the NEP delivery method, we can better understand cellular uptaking and intracellular trafficking of bio-macromolecular agents and nanoparticles in drug/gene delivery because NEP delivery by-passed endocytosis and the consequent endosome escape.

In this work, QD605-amine or QD605-polycations and Cy5-oligonucelotides (Cy5-G3139) were chosen as the FRET pair for nanoparticle model.They were either encapsulated in lipoplex nanoparticles or form polyplex nanoparticles by the electrostatic interaction. The endosome of lung cancer cell (A549) was stained by transfection of plasmid EGFP-Rab7.The confocal microscopy was used to investigate the intracellular fate and unpacking of the nanoparticles in the lung cancer cells during/after NEP or conventional nanoparticle transfection. We demonstrated that NEP is able to deliver nanoparticles into cells without going through endocytosis and with minimum cell damage, which is not achieveabe in the conventional bulk electroporation delivery method. The in-situ FRET detection showed the time-dependent nanoparticle unpacking inside the cell. This technology allows us to quantify the effects of nanoparticle type and formulation on the kinetics of cellular uptaking, endocytosis, endosome escape, and nanoparticle unpacking in cancer cells.