(98b) Enhanced Microfluidic Electroporation for Oligonucleotides Delivery

Electroporation provides an easy way for intracellular delivery of biomolecules in most cell-related research. However, current electroporation instruments often require a tedious trial-and-error search process to identify a good protocol and many protocols recommended limited cell population (105?106). It would be very valuable if electroporation could be carried out in a continuous mode, under a mild, uniform electric field that is effective with every porated cell.

We have recently developed a Converging Flow Electroporation (CFE) device for in vitro biomolecules uptake. Cells mixed with biomolecules will continuously flow through a converging microchannel where a low AC/DC electric field is applied. When sequentially passing the throat region, each cell experiences an identical but highly focused electric pulse. In this way, it simplifies or even eliminates the optimization process for a new cell types and proper electroporation protocols can be quickly established. Using pWizGFP plasmid and K562 & mES cells as models, CFE showed significant improvement on transgene expression (~ 15%) compared to the Bio-Rad Gene Pulser XcellTM Electroporation System. Cell lysis during poration was mostly avoided with no observable cell debris and quantitative result by MTS assay confirmed our observation of cell conditions with the cell viability more than 50% or even higher. Similar results were also observed with pWizGFP transfection in (CCE).

With its continuous-mode operation, many DNA away from cells may be wasted, or have to be recycled afterwards. The relatively low concentration of DNA around cell also limits the further improvement of its working efficiency. To further enhance CFE efficiency and to reduce oligonucleotides consumptions, multi-functional nanoparticles which carry DNA and targeting molecules are used in further study. Through receptors on the cell surface, nanoparticles are conjugated onto cells and flow together through the electroporation zone. This largely overcomes barriers in the uptake of DNA when cell membranes are upset. We demonstrated this concept using transferring (TfR)-targeted lipoplex nanoparticle and found better transfection of eGFP plasmids and ODN in K562 cells. The cell proliferation assays also show better cell viability.