Shock waves: a novel method for cytoplasmic delivery of antisense oligonucleotides

Intracytoplasmic delivery of oligonucleotides (ODN) can improve ODN-based s trategies such as the antisense approach and the use of immunostimulatory C pG dinucleotide containing ODN. Shock waves are established for the treatme nt of nephrolithiasis and other diseases. Here we describe the use of shock waves as a new physical method for the direct transport of antisense ODN i nto the cytoplasm and the nucleus of cells. Human peripheral blood mononucl ear cells together with antisense ODN were exposed to shock waves generated by an electrohydraulic lithotripter. ODN uptake was examined by flow cytom etry and fluorescence microscopy. By optimization of physical parameters we achieved the transfer of high amounts of ODN which were detected within le ss than 5 min after shock wave exposure, with viability of cells higher tha n 95%. Transfection of human peripheral blood mononuclear cells with an ant isense ODN directed against tumor necrosis factor (TNF) a resulted in a red uction in lipopolysaccharide-induced TNF production by 62% (n=5, P=0.006). Specificity of TNF suppression was confirmed with a four-mismatch oligonucl eotide. Positive atmospheric pressure abolished antisense-mediated inhibiti on of TNF synthesis by blocking shock wave-induced cavitation and formation of oscillating air bubbles. Electroporation was less effective. The use of shock waves is thus an efficient physical tool for ODN delivery to cells. Shock waves may allow the evaluation of target proteins in cell types diffi cult to transfect with other methods and thus may improve the antisense tec hnique for the analysis of unknown genes.