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8/18/2019 Nano Tisue Culture

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NANOTECHNOLOGY MEETS PLANT BIOTECH-

NOLOGY: CARBON NANOTUBES DELIVER DNA

AND INCORPORATE INTO THE PLANT CELL

STRUCTURE

Maged Fouad1,2

, Noritada Kaji1,2

, Mohammad Jabasini1

,Manabu Tokeshi

1,2 and Yoshinobu Baba

1,2,3

1 Department of Applied Chemistry, Graduate School of Engineering

2 MEXT Innovative Research center for Preventive Medical Engineering,

Nagoya University, JAPAN3 Health Technology Research Center, AIST, JAPAN

ABSTRACT

Carbon nanotubes (CNT) can intracellulary traffic through different cellular bar-

riers and deliver biomolecules into living cells. However, their use in plants is lim-ited 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 functionaliza-

tion 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 cellulase-

modified 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

978-0-9798064-1-4/µTAS2008/$20©2008CBMS 227

Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA


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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-

glucopyranoside) as a paracellular permeability enhan-

cer.

RESULTS AND DISCUSSION

We first investigated the CSCNT-cellulase system

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

the cell nucleus in 3 out of 50 cells showing internal-

ized CSCNT-cellulase (Fig. 2C). This is the first ex-ample of plant cell transfection of dynamically en-

hanced CNT that have the ability to cross the plantcell wall, the cell membrane and the nuclear mem-

brane and localize inside the cell nucleus.

Confocal microscope images of AlexaFluor 488

and Qdot 655 labeled CSCNT-cellulase showed alter-

native appearance and disappearance of cellulaseAlexa

488 patches within CSCNTQdot 655 (Fig. 3). This obser-

vation led to a conclusion that nanotubes exhibit ul-

trafast random motion with respect to the attached cel-

lulase molecules, which act as either joints or

molecular springs within the CSCNT-cellulase mi-

croparticle. This kind of motion is believed to be dueto the heterogeneous distribution of the adsorbed OG

molecules onto the nanotube surface. Therefore, the

momentum exerted by the Brownian movement of

such small molecules is distributed heterogeneously

along the CSCNT axis. The dynamic behavior of

CSCNT with respect to the joint-cellulase moleculesin 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 opti-

mal 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-adsorbedCSCNT-cellulase (Fig.4). Standard polyethylene glycol (PEG)-mediated protoplast

transformation can achieve 40-90% transient transformation efficiency using 1-2 mg

of DNA per 10

6

cells. Here, transient transformation of 6% of cells could beachieved when 10

5 cells were incubated with more than 1,000 times less DNA, that

is, 750 ng DNA (coated on 10 g CSCNT-cellulase).

Fig. 1. AFM image of cellu-

lase modified CSCNT

(bar:100 nm).

Fig. 2. (A) Detection of

CSCNT fluorescence inside

cell (arrow), v: vacuole,

scale bar: 10 m. (B) Locali-

zation of CSCNT (arrows)

inside endocytosis vesicles,

scale bar: 10 m. (C) Locali-

zation of CSCNT (green

spots) inside nucleus (blue;

Stained by DAPI).

A B

C

Fig. 3. Successive single focal

plane snapshots (1 sec. inter-

vals) of CSCNT-cellulase

microparticles in a 10% OG

solution. Arrows indicate cellu-

lase (10 m).

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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 dif-

ferentiated from CSCNT-transfected mesophyll cells showed fluorescence heteroge-

neity, 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 ap- peared 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 meso-

phyll cells (Fig. 5D).

CONCLUSIONS

Current applications of carbon nanobiotechnology to biology have mainly fo-cused 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 ger-

mintion and root growth. Environ. Pollut. 150, pp 243-250 (2007).[3] Sayes, C. M. et al. Functionalization density dependence of single-walled car-

bon nanotubes cytotoxicity in vitro. Toxicol. Lett. 161, pp 135-142 (2006).

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 withCSCNT-cellulase. Three-dimensional reconstruction image (25 μm depth) of Arabidopsis

cell expressing GFP (green) and containing scattered carbon nanotubes (red) (bar: 5 μm).

A B

A B C

D

Fig 5. (A) Nanotubes aggregatesinside the cell. (B) Disassembly of

nanotubes’ aggregates after cell

transfection (10 μm). (C) 3D con-

focal images of tracheid, showing

CNT (red) [reconstituted from

60*0.5 μm single focal planes (20

μm). (D) Single focal plane imag-

ing of individual nanotubes in the structure of trachieds (arrow) (1 μm)].

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