DNA introduction into living cells by water droplet impact with an electrospray process.

Electrospraying is performed by applying a high voltage to a capillary tip. A highly charged droplet at the capillary tip splits into microor nanoscale droplets, and a charged fineliquid aerosol is accelerated by a high-voltage electric field. This method has been used for soft ionization in the mass spectrometric analysis of macromolecules and biomaterials. Moreover, in desorption electrospray ionization (DESI) for mass spectrometry, the electrically charged droplets hit the surface molecules and are ionized under ambient conditions. Electrospraying is also used for the production of nanofibers and protein chips. Delivery of a foreign nucleic acid into a living cell is an important technique in molecular biology and the medical field. Previously developed transfection techniques, including methods that use a cationic polymer, lipofection, a gene gun, and electroporation, have been reported. Moreover, Pui et al. used electrospraying to increase the momentum of particles in the gene-gun method. However, these techniques sometimes cause severe damage to cells, especially mammalian cells, or require complex and expensive devices. We hypothesized that liquid droplet impact by electrospraying would transport a nucleic acid into a cell by making a transient channel through mild damage on the cell surface. Herein, we describe a technique for the introduction of a gene into eukaryotic (mammalian) and prokaryotic (bacterial) cells and avian embryonic tissue by spraying water droplets produced by an electrospray device without any cytotoxic reagents. Figure 1 shows a diagram of the electrospray equipment for gene delivery. Purified water is sprayed onto the cells as a charged aerosol generated by static electricity repulsion at the tip of the tube. Only water or phosphate-buffered saline (PBS) is used in this method, and the device is a simple structure basically consisting of a tube connected to a highvoltage power supply that is a constant potentiostat with the current limited to 100 mA. A stainless-steel capillary (caliber 0.1 mm, outside diameter 0.3 mm, length 40 mm) was impressed with a high voltage, and water was supplied at a flow rate of 100–200 mLmin . In this experiment, the stainless-steel tube was impressed from 7 to 18 kV. This voltage (over 7 kV) is higher than that used for mass spectrometry (1–3 kV), as an organic solvent, which is usually used in mass analysis for reduction of surface tension, cannot be applied to gene transfection because of its cytotoxicity. The sprayed dishes were placed on an electrically grounded square-plate electrode, and the inside of the culture dish was also grounded by attaching a small piece of metal foil to the plate. Adhesive Chinese hamster ovary (CHO) cells and HeLa cells were tested as models, and almost the same results were obtained. CHO cells were cultured in minimum essential medium (a-MEM, Gibco, USA) supplemented with 10% fetal bovine serum. Cells were plated in a 35-mm culture dish (Falcon, USA) at 2.0 ? 10 cells per dish and cultured at 37 8C under 5% CO2 in air. Three days later, the cells were used for electrospraying. The culture medium was removed from the dish and an aqueous solution (100 mL) of plasmid vector pEGFP-N1 (100 mgmL 1 in water, Clonetech, USA), as green fluorescence protein (GFP) encoding DNA, was added to the dish. Water was electrosprayed onto the cells from a height of 2 cm at 10 kV, and culture mediumwas directly added to the dish. After 24 h of cultivation, GFP-positive cells were counted with a hemocytometer under a fluorescence microscope (Olympus, Japan). We used nonmoving equipment in this experiment, which resulted in a limited spraying area on the dish. We evaluated the transfection rate of isolated GFP-positive cells to total cells, which included cells located in the external region of the sprayed zone, and obtained a transfection rate of 0.05 to 1.6%. The number of cells showing fluorescence increased with an increase in applied voltage, which indicates that the Figure 1. Diagram of the electrospray equipment for gene delivery. The plasmid DNAs located around the plasma membrane are introduced into cells by collision of water microdroplets.