Carbon nanotubes (CNT) can intracellulary traffic through different cellular ... have studied the toxicological impact and safety profile of carbon nanomaterial on.
NANOTECHNOLOGY MEETS PLANT BIOTECHNOLOGY: CARBON NANOTUBES DELIVER DNA AND INCORPORATE INTO THE PLANT CELL STRUCTURE 1
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Maged Fouad1,2, Noritada Kaji1,2, Mohammad Jabasini1, Manabu Tokeshi1,2 and Yoshinobu Baba1,2,3
Department of Applied Chemistry, Graduate School of Engineering MEXT Innovative Research center for Preventive Medical Engineering, Nagoya University, JAPAN 3 Health Technology Research Center, AIST, JAPAN
ABSTRACT Carbon nanotubes (CNT) can intracellulary traffic through different cellular barriers and deliver biomolecules into living cells. However, their use in plants is limited by the cellulosic wall surrounding the plant cell. Here we show that CNT with immobilized cellulase can serve as an efficient DNA delivery system for plant cells. Tracking the cellular fate of nanotubes revealed two novel phenomena: (1)A possible nuclear localization and (2)When the transfected cell decides to differentiate into tracheary cell (water conducting cell), nanotubes were observed to incorporate into cellular structure. Our work aims at methodological development that paves the way toward on-chip-nanoscale-gene delivery applications. KEYWORDS: Carbon nanotubes, Plant cell, Gene delivery, Tracheary cells INTRODUCTION Since, their discovery, carbon nanotubes (CNTs) have been eminent members of the nanomaterial family. Because of their unique physical, chemical and mechanical properties, they are widely predicted and regarded as new potential materials to bring enormous benefits in cell biology studies1. Also, an increasing number of reports have studied the toxicological impact and safety profile of carbon nanomaterial on both plant2 and mammalian cells, indicating that a high degree of CNT functionalization leads to a dramatic reduction in toxic effects3. THEORY In protoplast-based transfection methods, the entire plant cell wall is removed to make the DNA/DNA vector accessible to cell transcription machinery. Meanwhile, the viability of protoplasts and their capability of dividing are strongly reduced by chemicals applied to disorganize the cell wall. In our experiment, we used cellulasemodified Cup-stacked CNT (CSCNT-cellulase) to create nanoholes in the cell wall, through which CSCNT with adsorbed biomolecules can move intracellularly, hence circumventing complete cell wall removal. EXPERIMENTAL CSCNT have lengths between 1 m-100 m and the mean diameter is 60~100 nm. Cellulase was immobilized on fuctionalized CSCNT via a carbodiimide reaction Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 978-0-9798064-1-4/µTAS2008/$20©2008CBMS
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(Fig. 1). Arabidopsis thaliana ecotype Columbia Col-0 and Glycyrrhiza glabra were used as the model plants used in this study. One mL of cell suspension was mixed with 10 g of CSCNT-cellulase for 4h at 25°C in the presence of 10% OG (n-Octyl-ß-D- Fig. 1. AFM image of celluglucopyranoside) as a paracellular permeability enhan- lase modified CSCNT (bar:100 nm). cer. RESULTS AND DISCUSSION We first investigated the CSCNT-cellulase system A B on A. thaliana cells. In our experimental conditions, CSCNT-cellulase was uptaken by 20% of cells (fig. 2A), while uptaken by 15% of G. glabra cells (fig. 2B). After 8 h, CSCNT-cellulase was localized inside C the cell nucleus in 3 out of 50 cells showing internalized CSCNT-cellulase (Fig. 2C). This is the first example of plant cell transfection of dynamically enFig. 2. (A) Detection of hanced CNT that have the ability to cross the plant CSCNT fluorescence inside cell wall, the cell membrane and the nuclear mem- cell (arrow), v: vacuole, brane and localize inside the cell nucleus. scale bar: 10m. (B) LocaliConfocal microscope images of AlexaFluor 488 zation of CSCNT (arrows) and Qdot 655 labeled CSCNT-cellulase showed alter- inside endocytosis vesicles, native appearance and disappearance of cellulaseAlexa scale bar: 10m. (C) Localization of CSCNT (green 488 patches within CSCNTQdot 655 (Fig. 3). This observation led to a conclusion that nanotubes exhibit ul- spots) inside nucleus (blue; trafast random motion with respect to the attached cel- Stained by DAPI). lulase molecules, which act as either joints or molecular springs within the CSCNT-cellulase microparticle. This kind of motion is believed to be due to the heterogeneous distribution of the adsorbed OG Fig. 3. Successive single focal molecules onto the nanotube surface. Therefore, the plane snapshots (1 sec. interof CSCNT-cellulase momentum exerted by the Brownian movement of vals) microparticles in a 10% OG such small molecules is distributed heterogeneously solution. Arrows indicate cellualong the CSCNT axis. The dynamic behavior of lase (10m). CSCNT with respect to the joint-cellulase molecules in OG solution was proposed to induce a physical force that synergistically adds to transfection efficiency. To prove that CSCNT-cellulase can function as a DNA delivery agent for plant cells, we used a plasmid containing green fluorescent protein (GFP) gene. The optimal ratio for DNA/CSCNT-cellulase was 1/7.5 (w/w). Transient GFP expression could be observed 48 h after A. thaliana cells were incubated with DNA-adsorbed CSCNT-cellulase (Fig.4). Standard polyethylene glycol (PEG)-mediated protoplast transformation can achieve 40-90% transient transformation efficiency using 1-2 mg of DNA per 106 cells. Here, transient transformation of 6% of cells could be achieved when 105 cells were incubated with more than 1,000 times less DNA, that is, 750 ng DNA (coated on 10 g CSCNT-cellulase).
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Fig 4. (A) Three snapshots showing CSCNT-cellulase aggregates penetrating the plant cell wall toward the interior of the cell. (B) Gene expression in Arabidopsis cell incubated with CSCNT-cellulase. Three-dimensional reconstruction image (25 μm depth) of Arabidopsis cell expressing GFP (green) and containing scattered carbon nanotubes (red) (bar: 5 μm).
After transfection of cellulase-immobilized CSCNT, A. thaliana mesophyll cells showed condensed masses of CNT that usually disassemble into scattered fragments and homogenously harbor the cells (Fig. 5A,B). Such disassembly possibly occurs due to the digestion of binding cellulase inside cell lysosomes. Tracheary cells differentiated from CSCNT-transfected mesophyll cells showed fluorescence heterogeneity, where an integer part of the tracheidal cell fluoresced in the AlexaFlour 488 red channel rather than the lignin-detection GFP-UV green channel. Both parts appeared complementary within the framework of the tracheary cell, suggesting the presence of an altered chemical composition (Fig. 5C). We anticipated that the transfected CNT were responsible for such fluorescence shift of some parts of TEs, where nanotubes deposit into their structure during lignin biosynthesis. Through optimization of detection conditions, individual nanotubes was detected in the structure of tracheids (Fig. 4). Such nanotubes arrangement was exclusively detected in tracheids formed from CNT-transfected A. thaliana mesophyll cells (Fig. 5D). Fig 5. (A) Nanotubes aggregates A C B inside the cell. (B) Disassembly of nanotubes’ aggregates after cell transfection (10 μm). (C) 3D confocal images of tracheid, showing D CNT (red) [reconstituted from 60*0.5μm single focal planes (20 μm). (D) Single focal plane imaging of individual nanotubes in the structure of trachieds (arrow) (1μm)].
CONCLUSIONS Current applications of carbon nanobiotechnology to biology have mainly focused on animal science and medical research. Here, we have demonstrated that their versatility can also be applied to plant science research to serve as a new and promising tool for plant DNA transfection and cell biology studies. REFERENCES [1] Bianco, A. Carbon nanotubes for the delivery of therapeutic molecules. Expert Opin. Drug Deliv. 1, pp 57-65 (2004). [2] Lin, D. and Xing, B. Phytotoxicity of nanoparticles: Inhibition of seed germintion and root growth. Environ. Pollut. 150, pp 243-250 (2007). [3] Sayes, C. M. et al. Functionalization density dependence of single-walled carbon nanotubes cytotoxicity in vitro. Toxicol. Lett. 161, pp 135-142 (2006).
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