Due to their special electrical, optical, and magnetic properties, materials less than 100 nm in size are very promising for biosensors, bio-separation, and drug delivery. [1][2][3] In recent years liposomes and polymers have been used as carriers for transfections. [4] Certain inorganic materials such as silica nanoparticles, carbon nanotubes, and silica nanotubes have been used as transporters with little toxicity in mammalian-cell transfections. [5][6][7] These zero-dimensional nanoparticles and one-dimensional nanotubes suggest that nanomaterials, if modified properly, can be used as carriers for transfections. However, the application of three-dimensional nanostructures as biomolecule carriers is less well-studied.Here, we report three-dimensional functionalized tetrapod-like ZnO nanostructures as novel carriers for mammalian cell transfections. In this work, silica-coated amino-modifed nanostructures were prepared. Through electrostatic interactions, ZnO tetrapods could be bound to plasmid DNA. When mixed with cells, the tetrapods attached to cell membranes. Just as phages stand on cells with six legs suitable for gene delivery, ZnO nanostructures stand on the cells with three needle-shaped legs for DNA delivery as a result of their tetrapodal shape. With three tips located on the cell surfaces, the opportunity of internalization of the tips by cells should be increased. In addition, the geometry of the tetrapods imply a much larger steric hindrance, which makes it difficult for the tetrapods to pass wholly through the cell membranes. Just as phages insert genes into cells without entering them, tetrapods delivered plasmid DNA into the cells while standing on the cell membrane. This result is helpful in decreasing any cytotoxic effects. These results demonstrate a novel application of tetrapod-like nanostructures for gene delivery.Three-dimensional ZnO nanostructures were synthesized by thermal evaporation at 900 8C. [8] The nanostructures consisted of four needle-shaped tetrahedrally arranged legs connected at the center, forming a tetrapod-like ZnO structure. The legs were single-crystalline and stable in air, with a mean diameter of % 80 nm and a length of 5-10 mm. As shown in Figure 1 A, one of the needle-shaped legs was perpendicular to the substrate while the other three legs remained in contact with the substrate. Utilizing their tetrapodal shape, the ZnO crystals were used as stable field emitters by our group or as atomic force microscopy probing tips by Tohda and co-workers. [9,10] Inspired by the use of microneedles for transdermal drug delivery, it was considered to use needle-shaped ZnO legs to penetrate cell membranes for plasmid DNA delivery. [11,12] For binding plasmid DNA, tetrapods were coated with silica and amino groups by using a sol-gel process, [6,13] where both tetraethyl orthosilicate (TEOS, precursor of silica) and aminopropyltriethoxysilane (APTES, aminomodification reagent) were added to the reaction solution. The process was simple, and the amount of amino groups on the tetrapod...
