NANO LETTERS
Carbon Nanosyringe Array as a Platform for Intracellular Delivery
2009 Vol. 9, No. 4 1325-1329
Sangjin Park,† Youn-Su Kim,‡ Won Bae Kim,*,‡ and Sangyong Jon*,† Cell Dynamics Research Center, Research Center for Biomolecular Nanotechnology, Department of Life Science, Department of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea Received September 29, 2008; Revised Manuscript Received January 12, 2009
ABSTRACT We report a novel platform for intracellular delivery of genetic material and nanoparticles, based on vertically aligned carbon nanosyringe arrays (CNSAs) of controllable height. Using this technology, we have shown that plasmid and quantum dots can be efficiently delivered to the cytoplasm of cancer cells and human mesenchymal stem cells. The CNSA platform holds great promise for a myriad of applications including cell-based therapy, imaging, and tracking in vivo, and in biological studies aimed at understanding cellular function.
With the growing interest in cell therapy using mesenchymal stem cells1-5 or immune cells,6-8 there is an increasing need for efficient devices and methods to deliver genetic material into cells9-11 and label cells with fluorescent or magnetic nanoparticles for tracking in vivo.12-16 Such devices should facilitate simultaneous, highly efficient cellular delivery of a variety of exogenous macromolecules or nanomaterials without the need for application of an external force and have no harmful effects on cell viability. Cell plasma membranes are a formidable barrier to the delivery of exogenous macromolecules in cellular engineering and labeling and cell therapy. Attempts have been made to breach this barrier, particularly using mechanical means. Delivery of genetic material has been successfully achieved using a microinjector method that is in common use,17-20 but there is concern about damage to the cell membrane caused by intrinsic invasiveness of the micro- or submicrosized needle used in these procedures. Bertozzi et al. recently reported that functionalized carbon nanotubes attached to an atomic force microscope tip could deliver quantum dots into cells without affecting cell viability.21 However, both the microinjector and the carbon nanotube nanoinjector system have limitations including limited delivery of cargo (e.g., genes or nanoparticles) into a single cell as a function of time (i.e., low throughput), and the need for complicated microscope instruments to control the injection position. Simultaneous gene delivery into a number of cells has * To whom correspondence should be addressed. E-mail: (S.P.)
[email protected]; (W.B.K.)
[email protected]. Phone: (+82) 62-970-2304. Fax: (+82) 62-970-2484. † Cell Dynamics Research Center, Research Center for Biomolecular Nanotechnology, Department of Life Science. ‡ Department of Materials Science and Engineering. 10.1021/nl802962t CCC: $40.75 Published on Web 03/02/2009
2009 American Chemical Society
recently been demonstrated using carbon nanofiber arrays22-24 or silicon nanowire arrays25 with a high aspect ratio, but several factors may limit their use in practice. First, an external gravitational force, achieved by centrifugation, is necessary to pierce the cells with the nanofibers (which vary in thickness and length), potentially causing severe cell damage. Second, only a small quantity of plasmid DNA, physically adsorbed or covalently bound on the surface of carbon fiber, is available for delivery into cells. Third, only the delivery of a limited variety of compounds, such as plasmid, is possible using this approach owing to absence of compartments that can load a variety of cargos having different natures and shapes. Consequently, there is need for a device that can facilitate simultaneous and highly efficient cellular delivery of a variety of exogenous macromolecules without the need to apply an external force, and without harmful effects on cell viability. Here, we report a simple intracellular delivery system based on novel patterned carbon nanosyringe arrays (CNSAs) comprised of vertically orientated nanodimensional syringes of controllable height.26 Using this array, we were able to demonstrate successful intracellular delivery of plasmid and quantum dots (QDs) into cancer cells and human mesenchymal stem cells. This is the first study of a cellular delivery process using a nanosyringe array platform with hollow nanotubes providing empty compartment for cargo loading and distinguishing it from carbon nanofiber and silicon nanowire arrays. The overall process for the cellular delivery of cargo using a CNSA platform is illustrated in Figure 1a. CNSA platforms were prepared by a series of chemical and physical steps, including (i) preparation of nanoporous anodized aluminum
Figure 1. (a) A schematic illustration of the construction of carbon nanosyringe arrays and their use in cellular delivery of cargo: (i) the thermal CVD method of carbon layer deposition within the well-ordered pore structure of a porous AAO template; (ii) removal of the uppermost carbon surface from the AAO template by ion milling, and subsequent chemical etching to expose the carbon nanosyringes; (iii) surface coating of the carbon nanosyringes with an amphiphilic polymer and loading cargo of interest; and (iv) intracellular delivery based on the carbon nanosyringe platform. (b) SEM images of CNSAs with exposed heights of 80 and 120 nm. (c) Chemical structure of the amphiphilic polymer used for coating carbon nanosyringes.
oxide (AAO); (ii) carbon deposition within the empty pores of the AAO; and (iii) ion milling and chemical etching to partially expose the carbon nanotube array. Typical scanning electron microscopy (SEM) images of the CNSAs with exposure lengths of ∼80 and ∼120 nm from the AAO layer are shown in Figure 1b. Each nanosyringe has an outer diameter of about 50 nm and an open portal in its tubular structure, as seen in the transmission electron microscopy (TEM) image of a single carbon nanosyringe (Figure S1, see Supporting Information). In the next step the hydrophobic surface of the nanosyringe is converted to a hydrophilic one by treatment of the CNSA with an amphiphilic polymer (1 wt % in water) previously shown to stably coat carbon nanotubes for dispersion in water.27 The chemical structure of the amphiphilic polymer is shown in Figure 1c. This surface engineering step is necessary to enable loading of cargo (by capillary action) into the hollow cores of the tubular CNSA syringes (the compartment space), because the cargos targeted for intracellular delivery are generally dissolved or dispersed in an aqueous solution. In the final step, cells seeded onto the CNSA get pierced in a spontaneous manner by cell’s own gravity, and intracellular delivery of cargo ensues. We examined the potential of CNSAs as an intracellular delivery platform for genetic material using enhanced green fluorescent protein (EGFP)-encoded plasmid (pEGFP) as a model cargo. A series of CNSAs set at different heights (40, 80, 120, and 160 nm) was used to assess the optimal height for intracellular delivery. A piece of the CNSA (∼1 cm × 1 cm) was treated with a drop of water containing pEGFP 1326
(100 µg/mL) to load the cargo, and any unloaded pEGFP was removed by gentle washing. NIH3T3 cells were seeded and grown on the pEGFP-loaded CNSAs. Lipofectamine 2000, one of the best commercially available nonviral gene delivery vectors, was used as a positive control. Figure 2a,b shows the typical fluorescence microscopy images and flowcytometry histogram profiles of NIH3T3 cells, respectively, after treatment with the CNSAs (80 and 120 nm in height) or with Lipofectamine 2000. Although CNSAs of 40, 80, and 160 nm in height also resulted in high levels of gene expression (∼24-30% relative to control, untreated cells in the flow-cytometry data), the CNSA at a height of 120 nm showed the highest gene expression efficiency (∼34%). Negligible green fluorescence (
