Quantitative Analysis of Plasmid DNA Distribution and Transgene Expression Following Cell Squeezing.

Microfluidic cell squeezing is a promising mechanoporation strategy that has been successfully applied to deliver various macromolecules, including proteins and nucleic acids. However, its success is limited when mechanoporation is used alone to facilitate gene delivery. The goal of the current study is to develop a gene delivery strategy that integrates electrotransfection and mechanoporation.We designed and fabricated a microfluidic device for controlled cell squeezing. Mammalian cells were first electrotransfected with pDNA, followed by mechanical squeezing to promote nuclear entry of the delivered DNA. Intracellular pDNA transport and transgene expression were quantitatively evaluated in squeezed and un-squeezed cells.Cell squeezing increased cellular uptake and nuclear accumulation of pDNA by more than eightfold compared with un-squeezed controls. Despite this substantial enhancement in DNA delivery, transgene mRNA levels remained unchanged, and protein production was consistently reduced relative to controls. The lack of increased transgene expression might be partially attributable to squeezing-induced chromatin condensation.These findings indicate that chromatin condensation limits transgene expression following mechanoporation. Pharmacological inhibition of chromatin condensation may therefore represent a promising strategy to improve the efficiency of mechanoporation-facilitated gene delivery.

Duke Scholars

Author Fan Yuan Biomedical Engineering