High-efficiency DNA injection into a single human mesenchymal stem cell using a nanoneedle and atomic force microscopy

Abstract 

We describe a low-invasive gene delivery method that uses an etched atomic force microscopy (AFM) tip or nanoneedle that can be inserted into a cell nucleus without causing cellular damage. The nanoneedle is 200 nm in diameter and 6 μm in length and is operated using an AFM system. The probabilities of insertion of the nanoneedle into human mesenchymal stem cells (MSCs) and human embryonic kidney cells (HEK293) were higher than those of typical microinjection capillaries. A plasmid containing the green fluorescent protein (GFP) gene was adsorbed on a poly-l-lysine–modified nanoneedle surface, which was then inserted into primary cultured single human MSCs. A highly efficient gene delivery of over 70% was achieved in human MSCs, which compared very favorably with other major nonviral gene delivery methods (lipofection ~50%, microinjection ~10 %). The single cells expressing GFP were collected and the amount of delivered DNA in each cell was analyzed. The highest rate of expressed GFP per delivered DNA was achieved using the nanoneedle, because the nanoneedle could be inserted into the nucleus directly without causing significant cell damage.

Key words: Gene transfer, Gene expression, Atomic force microscopy (AFM), Mesenchymal stem cells, Confocal microscopy, Nanoindentation