Carbon Nanotube uptake by Alveolar Macrophages
Jonathan M. Malecki
University of Rochester
Institute of Optics
OPT407: Practical Electron Microscopy
Background and Introduction
|Experimental Procedure||Results and Discussion||
Conclusions and Acknowledgements
BACKGROUND AND INTRODUCTION
Figure 1: Carbon nanotubes are essentially a sheet a graphite rolled up into a tube.
Carbon nanotubes are long thin cylinders of carbon. They are essentially a sheet of graphite (a hexagonal lattice of carbon) that has been rolled up into a cylinder. The can have a single wall (SWNT) or multiple walls (MWNT) which are tubes inside tubes.
These tubes also have unique electrical properties such as being ballistic conductors according to Stefan Frank et al . Ballistic conduction is the unimpeded flow of charge over a relatively large distance which isn’t dominated by scattering events that is normally present. This kind of low resistance material can be very useful in nanotechnology. Ropes of SWNTs were thought to be able to achieve resistivity in the order of 10-4 ohms-cm at 300K. When measured by Thess et al , they were able to get readings of 0.34 x 10-4 ohms-cm which would make them the most highly conductive carbon fibers known.
Although layers of graphite can be easily peeled apart carbon fibers are shown to be very strong. In 1996 Michael Treacy, Ebbesen, and Murray Gibson  measured the Young's modulus, elastic strength, of multiwall nanotubes finding it to be about 1012 newtons per square meter which is about five times stronger than steel.
As with quantum dots nanotubes have more stable and brighter florescence than traditional biological dyes. When nanotubes are separated they will fluoresce at only one wavelength with different diameters emitting different wavelengths. This would allow doctors to provide a cocktail of different sized nanotubes that will detect different cells like tumors.
Not only could proteins be attached to the outside of the nanotubes and introduced to organisms, molecules could be placed inside the tubes. The tubes could then be enclosed allowing for the possibility of using the tube as a transport device to supply cells with molecules that would otherwise be broken down during the delivery process.
Although there are many possible uses for nanotubes, the detrimental effects are unknown. These tubes are made, handled and used without knowing how they would affect the human body. Probably the most likely means of exposure to nanotubes is inhalation. Therefore we would need to know what happens to the tubes after they taken up into the lungs. Will cells clean up nanotubes that find themselves in the lungs? Since macrophages are the cells that would target this kind of foreign particle we wanted to know if they would take up nanotubes and what kind of damage would result.
1. Macrophage prep
Rats were subject to inhalation of MWNT at concentrated amounts for 24 hours. They were then sacrificed and the lungs were lavaged. Alveolar macrophages were isolated from the lavage solution and fixed with 1% paraformaldehyde.
The macrophages were simply aliquoted onto carbon TEM grids. After they were allowed to settle to the grid the majority of the liquid was drawn off carefully with a kem wipe. The grid was then left to finish drying completely. The nanotubes alone were applied to the TEM grids in the same way after being mixed with either PBS or pure water. The PBS helped separate the tubes but caused salt crystals to form on the tubes and produce artifacts.
2. Microscope and EDS
Since the nanotubes are very delicate and thin, uncoated samples were tried first using the variable pressure setting on the Zeiss Supra 40VP SEM. These uncoated tubes were also analyzed with the AFM. The tubes as well as the macrophages were then sputter coated so that we could get a better micrograph in the SEM. The gold allows for much clearer micrographs which means very distinct nanotubes but probably does hide any delicate external features of the macrophages.
We also verified the makeup of the nanotubes by the EDS spectrum. Although the by definition the tubes consist of only carbon, some TEM images show darker spots which suggest there is some contamination as a result of the nanotubes formation process.
Although ideally the macrophages would be fixed, placed in an epoxy resin and sliced with a microtone, nanotubes inside the cells would not only destroy the cells as they were dragged through by the knife, they would also do a great deal of damage to the knife of the microtone. Therefore TEM samples were made up the same as they were for the SEM.
Results and Discussion
Micrographs of the tubes by themselves show the long thin structure as well as the flexibility of these tubes which is consistent with the theoretical shape of a carbon nanotube. These are MWNT which explain the variation in sizes as well as there large size. In this selection alone they vary from 122.38 nm to 24.4nm which is fairly consistent with this batch of tubes.
Figure 2: Micrograph on the left is of MWNTs, the micrograph on the right is analyzed with Image J, the nanotube diameters are measured
2. Electron Flight Simulation
Electron Flight Simulation software was used to create a visual representation of how the electron beam interacts with nanotubes that are placed on top of the copper TEM grids. The red line represents the electron beam while the blue lines represent the paths that are taken by the electrons. The simulation represents a film of carbon on a substrate of copper with an acceleration voltage of 20kV. A count of 3000 electron trajectories were used.
Figure 3:Electron flight simulation of a carbon film on a copper substrate
3. Atomic Force Microscope
Although the SEM gives a depth to a micrograph than the TEM doesn’t, it still isn’t a 3D image. We were lucky enough to get the opportunity to get to try an Atomic Force microscope. AFM consists of a microscale cantilever with a probe at its end. The tip of this probe is brought into proximity of a samples surface. Deflections of the cantilever are caused by forces between the tip and the sample. With this instrument we were able to get a very defined 3 dimensional images as seen below. It is easy to make out the tubular shape with these 3 dimensional images.
Figure 4: 3 dimensional images by the atomic force microscope
Since the nanotubes formation process can inject contaminates if they are not made carefully we wanted to verify the chemical makeup of this batch of tubes. To do this we used energy dispersive x-ray spectrometry. The EDS detector is designed to detect x-rays and convert their energy into an electrical signal. This signal is processed so that it can identify the x-ray energies, and therefore the elemental source. When a spectrum of the MWNTs was taken it consisted of mostly carbon and copper as shown in figure 5. The most likely contaminate that the tubes would contain would be iron. The energy locations were marked on this figure, but it doesn’t show any significant amount iron.
Figure 5: Energy Dispersive X-ray spectrometry
Although the TEM shows that the nanotubes and macrophages are in close correlation with each other, it’s impossible to tell whether the tubes are in the macrophages or just laying on top as shown in figure 6. The TEM does give a very good image of the tubes themselves. To get a better idea of the placement of the nanotubes we used SEM.
Figure 6: TEM
Figure 7: Nanotube traversing the macrophage
It can be seen that the macrophages took up a large quantity of the MWNTs. This can be seen because they very distinctly stick out of the cells, some even traversing whole macrophages as seen in figure 7. This sample has a straight nanotube that has both ends outside the macrophage which suggests that the tubes ends aren’t just taken in, the cells are trying to completely engulf the tubes. Although no micrographs of this engulfing were obtained there were some that are possibly showing the beginning stages as in figure 8. Not all macrophages got away with taking up just one or two nanotubes; some were filled like in figure 9 with what seems like more than the cell would be able to take up on its own.
Figure 8: Nanotube uptake by macrophages
Figure 9: Energy Dispersive X-ray spectrometry
This project tells us two major points, first is that cells will take up nanotubes. This is one step closer to having nanotubes deliver protected molecules to cells. The second point is that nanotubes also have the ability breakdown cells, even those that are meant to scavenge garbage from the lungs like these macrophages. Therefore although MWNTs have many possible uses, care should be taken when handling them because of their destructive possibilities on body cells. Although most of the macrophages were still intact when they were viewed on the SEM, some had some serious damage to them, although it seems to take a great deal of tubes to destroy these cells.
Thank you to Brian McIntyre and Andreas Liapis for their light hearted instruction and patience, Karen Bentley for the time and help on her TEM as well as for the suggestions and ideas for this project. Thank you to the Oberdörster lab, especially Nancy Corson for the nanotubes and macrophage samples.
1. Stefan Frank et al., Science 280 1744 (1998)
2. Andreas Thess, Roland Lee, Pavel Nikolaev, Hongjie Dai, Pierre Petit, Jerome Robert, Chunhui Xu, Young Hee Lee, Seong Gon Kim, Andrew G. Rinzler, Daniel T. Colbert, Gustavo Scuseria, David Tománek, John E. Fischer, Richard E. Smalley "Crystalline Ropes of Metallic Carbon Nanotubes", Science, 273, 483 (1996).
3. M M J Treacy, T W Ebbesen and J M Gibson Exceptionally high Young's modulus observed for individual carbon nanotube Nature,381 67880 1996.
Jonathan M. Malecki, April 27, 2009