(498d) Microfluidic Electroporation for Delivery of Cell-Penetrating Peptide Conjugates of Peptide Nucleic Acids (PNA) for Antisense Inhibition of Intracellular Bacteria

Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. PNAs, gene-specific artificial oligonucleotide analogs with peptide backbone, are designed to silence selected gene and inhibit bacteria. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents.

In this report, we show that microfluidic electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages which is typical in animals and humans. Electroporation (i.e. the exposure of cells to an external electric field with a higher-than-threshold intensity for a short duration) breaches cell membranes by creating nanoscale pores and destabilizing the membrane structures. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V/cm and 5 ms pulse was a factor of 14 higher than that without electroporation (in an experiment with a multiplicity of infection of 10:1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.