1. Multi-walled Carbon Nanotube In Vitro Bundling of F-Actin
Taylor Ferebee,1
Dr. Brian S. Gentry 2
1
Roanoke College
2
Hollins University
Introduction
Drug delivery using carbon nanotubes (CNTs) is a prevalent topic in
current medical research. Many cells readily incorporate CNTs,
making them an attractive option for targeting diseased cells. As a
result there have been numerous studies that investigate both the
process by which nanotubes are taken up by cells and the changes
they induce. One such change is bundling of the F-actin
cytoskeleton.
Figure 1: TEM image of MWNTs
Few investigations, however,
have focused on the bundling
morphologies of actin filaments
as a result of nanoparticle
interaction. Insight into
this behavior is crucial to the
understanding of how the cell
reacts to these particles. Herein, we examine in vitro bundling
morphologies that arise as a result of interactions between F-actin
and multi-walled carbon nanotubes (MWNTs). Using fluorescence
microscopy, we identify key morphologies that may provide insight
into the possible F-actin-MWCNT interactions.
Figure 2: Rhodamine-phalloidin treated F-actin exposed to SWNT in previous
investigation from ”Carbon Nanotubes Reorganize Actin Structures in Cells and
Ex Vivo,” B.D. Holt, 2010.
Materials & Methods
Dispersions of the Multi-walled Nanotubes
Nanotubes purchased from CheapTubes were dispersed in a 0.1%
solution of the co-block polymer Pluronic F-127. The solution was
stirred for 8 hours and then sonicated for 16 hours.Using globular
actin, we add rhodamine-phalloidin and polymerize and at room
temperature to get a solution of F-actin. Since the
rhodamine-phallodin is fluorescent, we use fluorescent microscopy to
view the behavior of the filaments. We then repeat this processes
looking at various mixtures of actin and nanotubes.
Figure 3: Dispersed Nanotubes. Varied processing times.
Phase Transition Conjecture
With the knowledge of the behavior of actin
filaments in the presence of multi-walled
carbon nanotubes, we aim to create a phase
space that describes the emergent behaviors
of the two materials. Ideally, this will become
an N-dimensional phase space that
corresponds to critical concentrations of both
actin and MWNTs. As a basis, we
hypothesize that there is an underlying phase
transition that is not dissimilar to this one.
These transitions are similar to the
transitions associated with the additions of
cross-linkers, such as α-actinin.
Phase Diagram
Initial Results
Bundle Formation and Multi-walled Carbon Nanotubes
Figure 4: Fluorescent Image of F-actin in the presence of MWNTs as compared with TEM image of MWNTs
We note the similarities between the bundle morphologies and the spatial organization of the nanotubes. This
gives insight into the possible interactions between the filaments and the nanotubes.
We identify key F-actin bundling morphologies that arise as a result of various conditions including temperature
and time of polymerization.
We identify behavior that gives evidence that there is a thermodynamic interaction between the F-actin and the
MWNTs.
Morphological Results
(a). Bundle Formation at 25C (b) Bundle formation at 4C (c) Emergent Bundle Formation at 4 C
Conclusions & Future Directions
We were able to simulate cellular F-actin bundling by carbon
nanotubes conditions by simple mixing. To make the multi-walled
nanotubes biocompatible, We dispersed them in the co-polymer
Pluronic F-127. We carried out these experiments with
rhodamine-phalloidin stained actin in order to utilize fluorescence
microscopy. Together with the reports from other investigations, we
believe that these properties point to a preferential binding
interaction between the MWNTs and actin filaments that is most
likely results from the anisotropic nature of these materials.
Furthermore, these findings suggest that the understanding of the
interactions between these materials be studied for reasons including
biological toxicity and environmental impact.
Future experiments will examine the mixing behavior of the two
materials in more detail. This will provide information about the
effects of MWNTs on the cytoskeletons of cells that take up the
nanotubes.
We will introduce polarization microscopy to view the MWNTs and
the actin simultaneously. This will provide more information about
the interactions between the nanotubes and F-actin.
References
[1] Brian D. Holt Carbon Nanotubes Reorganize Actin Structures in
Cells and Ex Vivo 2010.
[2] Gianni Ciofani Dispersion of Multiwalled Carbon Nanotubes in
Aqueous Pluronic F127 Solutions for Biological Applications 2009.
[3] Brian D. Holt Cellular Processing of Single Wall Carbon Nanotubes
2008.
Acknowledgments
The authors thank Dr. DorthyBelle Poli for the laboratory space and
Dr. Rama Balasubramanian for technical guidance. We also thank
the Dr. Leonard D. Pysh of the Roanoke College Biology
Department of Dr. David Taylor of the Roanoke College
Mathematics, Computer Science, and Physics Department for
supporting the additional costs. MWCNTs were purchased from
CheapTubes, 3992 Rte 121 East, Cambridgeport, VT 05141.
Roanoke College Department of Math, Computer Science, and Physics thferebee@mail.roanoke.edu