Search for: All records

Blood is an attractive carrier for plasmid and RNA based medicine in cell therapy. Electroporation serves as its favorable delivery tool for simple operation, quick internalization, minimum cell culture involvement, and low contamination risk. However, the delivery outcomes of electroporation heavily depend on the treated cells such as their type, size, and orientation to the electric field, not ideal for highly heterogeneous blood samples. Herein a new electroporation system was developed towards effective transfection to cells in blood regardless their large diversity. By coupling replica molding and infiltration coating processes, we successfully configured a three-dimensional electrode comprised of a polymer micropillar array on which carbon nanotubes (CNTs) are partially embedded. During electroporation, cells sag between micropillars and deform to form conformal contact with their top and side surface. The implanted CNTs not only provide a robust conductive coating for the polymer micropattern, but also have their protruded ends face the cell membrane vertically everywhere with maximum transmembrane potential. Regardless their largely varied sizes and random dispersion, both individual blood cell type and whole blood samples were effectively transfected with plasmid DNA (85% after 24 hrs and 95% after 72 hrs, or 2.5-3.0 folds enhancement). High-dose RNA probes were also introduced which regulate better the expression levels of exogenous and endogenous genes in blood cells. Besides its promising performance on non-viral delivery route to cell-related studies and therapy, the invovled new fabrication method also provides a convenient and effective way to construct flexible electronics with stable micro/nanofeatures on the surface. 

Abstract Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400–800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.