Ikuo Obataya

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

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 mum 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 approximately 50%, microinjection approximately 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.

Direct insertion of proteins into a living cell using an atomic force microscope with a nanoneedle

We have developed a tool for directly inserting proteins into living cells by using atomic force microscopy (AFM) and an ultrathin needle, termed a nanoneedle. The surface of the nanoneedle was modified with His-tagged proteins using nickel chelating nitrilotriaceticacid (NTA). The fluorescent proteins, DsRed2-His6 and EGFP-His6, could be attached to and detached from the surface of the nanoneedle. These results suggest that the Ni-NTA modified nanoneedle can successfully be used for specific delivery of proteins. The nanoneedle modified with DsRed2-His6 was able to penetrate the surface of a living HeLa cell, as confirmed by laser scanning fluorescence microscopy and monitoring an exerting force on the nanoneedle using AFM. Force curves using the nanoneedle indicated that the needle was able to penetrate at displacement speeds of 0.10–10 µm/s. These results suggest that this technique can be used to directly insert proteins into living cells and is applicable for modulation or regulation of single cell activity.

Force Analyses of the Interaction between the Surface of a Living Cell and Modified AFM Probes

We have developed a tool for performing surgical operations on living cells, known as cell surgery, using AFM and a modified AFM tip. The apparatus for cell surgery is designed to keep cells alive whilst enabling measurement of exerting forces on the AFM tip during surgery. The AFM tips are shaped into very thin needles using focused ion beam etching (FIB). Obtained force‐distance curves using these nano‐needles indicate the needles can penetrate cell membranes. This technology enables the extended application of AFM to analyses and therapy of living cells.

Development of a Gene Transfer Technique Using AFM

We report on a new low invasive technique for gene transfer of living cells using an ultra thin needle, termed a nano‐needle, comprising part of an etched atomic force microscope (AFM) tip. A silicon AFM tip was sharpened using focused ion beam etching to create an etched Si tip with a small diameter (100 to 200 nm). This small diameter ensured no damage was caused to the cell during needle insertion. In this study, we have demonstrated the cell penetration process using a nano‐needle and a DNA immobilized nano‐needle. This new technique could be applied for control of gene expression in a single cell.