(7e) Single-Cell Level Study of Precise siRNA Delivery and Dynamic in Acute Myeloidleukemia Cells By Nanochannel Electroporation System

Single-Cell Level Study of Precise siRNA Delivery and Dynamic in Acute Myeloidleukemia Cells by Nanochannel Electroporation System

Keliang Gao^a, Xiaomeng Huang^a,b, Lei Li^a, Lingqian Chang^a, Xinmei Wang^a, Guido Marcucci^b, and L. James Lee^{a,c *}

^aNanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices

^b Department of Internal Medicine

^C Department of Chemical and Biomolecular Engineering

The Ohio State University, Columbus, Ohio 43210, USA

^*Corresponding Author’s E-mail: lee.31@osu.edu

A micro/nano-fabrication process of a nanochannel electroporation (NEP) biochip and its application for precise delivery of siRNA for non-viral gene transfection was described to study the dynamic of siRNA in leukemia cells. A novel dip coating process was optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4-butanediol diacrylate (1,4-BDDA), was adopted to convert the microridge-nanowire-microridge array into a microchannel-nanochannel-microchannel array. Secondary machining by the femtosecond laser ablation was applied to shorten one side of microchannels from 3000 to 50 µm to facilitate cell loading and unloading. The biochip was then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case could be opened for cell unloading after NEP to allow for the follow-up cell culture and analysis. To demonstrate its application, NEP was applied to deliver precise dosage of Mcl-1 siRNA to single acute myeloid leukemia cells. Various amount of siRNA was injected into the cells through different NEP conditions, and critical thereshold was established by checking cell viability. Mcl-1 mRNA was down-regulated by siRNA, shown by molecular beacon detection. Single cell PCR was adopted to illustrate intracytoplasmic siRNA copy number. Two different kinds of leukemia cells, KG1a as wild type FLT3 and MV4-11 as FLT3-ITD, were studied for comparison. The results established different thereshold for wild type and mutant cells due to apoptosis resistance, and showed that more siRNA were required for apoptosis of aggressive FLT3-ITD cells.  Dynamic analysis was revealed with different dosage of siRNA, and illustrated the intracytoplasmic mechanism of down-regulation and recovery.