'Label-free' imaging tool tracks nanotubes in cells, blood for biomedical research

 Researchers have demonstrated a new imaging tool for tracking structures called carbon nanotubes in living cells and the bloodstream, which could aid efforts to perfect their use in biomedical research and clinical medicine.

The structures have potential applications in drug delivery to treat diseases and imaging for cancer research. Two types of nanotubes are created in the manufacturing process, metallic and semiconducting. Until now, however, there has been no technique to see both types in living cells and the bloodstream, said Ji-Xin Cheng, an associate professor of biomedical engineering and chemistry at Purdue University.

The imaging technique, called transient absorption, uses a pulsing near-infrared laser to deposit energy into the nanotubes, which then are probed by a second near-infrared laser.

The researchers have overcome key obstacles in using the imaging technology, detecting and monitoring the nanotubes in live cells and laboratory mice, Cheng said.

"Because we can do this at high speed, we can see what's happening in real time as the nanotubes are circulating in the bloodstream," he said.

Findings are detailed in a research paper posted online Dec. 4 in the journal Nature Nanotechnology.

The imaging technique is "label free," meaning it does not require that the nanotubes be marked with dyes, making it potentially practical for research and medicine, Cheng said.

"It's a fundamental tool for research that will provide information for the scientific community to learn how to perfect the use of nanotubes for biomedical and clinical applications," he said.

The conventional imaging method uses luminescence, which is limited because it detects the semiconducting nanotubes but not the metallic ones.

The nanotubes have a diameter of about 1 nanometer, or roughly the length of 10 hydrogen atoms strung together, making them far too small to be seen with a conventional light microscope. One challenge in using the transient absorption imaging system for living cells was to eliminate the interference caused by the background glow of red blood cells, which is brighter than the nanotubes.

The researchers solved this problem by separating the signals from red blood cells and nanotubes in two separate "channels." Light from the red blood cells is slightly delayed compared to light emitted by the nanotubes. The two types of signals are "phase separated" by restricting them to different channels based on this delay.

Researchers used the technique to see nanotubes circulating in the blood vessels of mice earlobes.

"This is important for drug delivery because you want to know how long nanotubes remain in blood vessels after they are injected," Cheng said. "So you need to visualize them in real time circulating in the bloodstream."

The structures, called single-wall carbon nanotubes, are formed by rolling up a one-atom-thick layer of graphite called graphene. The nanotubes are inherently hydrophobic, so some of the nanotubes used in the study were coated with DNA to make them water-soluble, which is required for them to be transported in the bloodstream and into cells.

The researchers also have taken images of nanotubes in the liver and other organs to study their distribution in mice, and they are using the imaging technique to study other nanomaterials such as graphene.

The paper was written by doctoral student Ling Tong; postdoctoral research associate Yuxiang Liu; doctoral students Bridget D. Dolash and Yookyung Jung; biomedical engineering research scientist Mikhail N. Slipchenko; Donald E. Bergstrom, the Walther Professor of Medicinal Chemistry; and Cheng.

The research is funded by the National Science Foundation.

Story Source:

The above story is reprinted from materials provided by Purdue University. The original article was written by Emil Venere.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

Journal Reference:

Ling Tong, Yuxiang Liu, Bridget D. Dolash, Yookyung Jung, Mikhail N. Slipchenko, Donald E. Bergstrom, Ji-Xin Cheng. Label-free imaging of semiconducting and metallic carbon nanotubes in cells and mice using transient absorption microscopy. Nature Nanotechnology, 2011; DOI: 10.1038/nnano.2011.210

Note: If no author is given, the source is cited instead.

Stopping malignancy in its tracks

 

An unusual chemical compound isolated from a mud-dwelling fungus found in a soil sample collected in Daejeon, South Korea, could lead to a new family of antitumor drugs. Discovered by teams led by Jong Seog Ahn at the Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, and  Hiroyuki Osada at the RIKEN Advanced Science Institute, Wako, the compound prevents cancerous cells from forming mobile colonies—the point at which cancers become malignant and spread through the body. The teams began collaborating after Yukihiro Asami from RIKEN joined KRIBB.

The researchers spotted the compound while searching extracts of the fungus for candidate drug compounds using a recently developed screen called a 3D epithelial culture system. To date, this kind of biological assay has rarely been used to search for natural products with novel bioactivity, says Ahn. It was during the 3D screen, which they spiked with cancerous cells, that the researchers realized that a compound produced by the fungus was inhibiting the cancer cells from clumping together to form colonies (Fig. 1). This type of screen is difficult using a conventional two-dimensional cell culture. 

The researchers isolated the bioactive compound and named it fusarisetin A. They then investigated its structure using an array of chemical characterization techniques, including nuclear magnetic resonance (NMR) and x-ray crystallography. They showed that fusarisetin A was a previously undescribed compound. Being able to grow crystals of the compound for x-ray studies was a breakthrough, says Osada. “It is very important for exact structural elucidation to get crystal analysis,” he says.

Having established that fusarisetin A is a new compound, the researchers probed its bioactivity in more detail. They showed that it simply blocks colony formation rather than killing cancer cells. They then compared the compound to others known to inhibit this process, and showed that it works differently to other compounds capable of blocking clumping. This suggests to the researchers that it could offer a new way to treat tumors.

The team is already working to discover how fusarisetin A inhibits the clumping of cancerous cells by looking for its molecular target. “We have already got candidate target proteins,” Osada adds.

Fusarisetin A itself is not bioactive enough to become a drug. However, it may be possible to fine-tune the structure to improve its activity, from which new drugs could be developed. “If we can get higher biological activity derivatives [of fusarisetin A], it may be possible,” says Ahn.

More information: Jang, J.-H., et al. Fusarisetin A, an acinar morphogenesis inhibitor from a soil fungus, Fusarium sp. FN080326. Journal of the American Chemical Society 133, 6865–6867 (2011).

Provided by RIKEN (news : web)

New technique tracks viral infections, aids development of antiviral drugs

 Scientists at the Naval Research Laboratory Center for Bio-Molecular Science and Engineering have developed a method to detect the presence of viruses in cells and to study their growth. Targeting a virus that has ribonucleic acid (RNA) as its genetic makeup, the new technique referred to as locked nucleic acid (LNA) flow cytometry-fluorescence in situ hybridization (flow-FISH), involves the binding of an LNA probe to viral RNA.

While individual parts of the technique have been developed previously, Drs. Kelly Robertson and Eddie Chang, in collaboration with researchers at the NRL Lab for Biosensors and Biomaterials, demonstrate for the first time that the combination of LNA probes with flow-FISH can be used to quantify viral RNA in infected cells. This also allows the scientists to monitor the changes in viral RNA accompanying antiviral drug treatment.

Once the probe is bound to the viral RNA inside mammalian cells, it is tagged with a fluorescent dye, then thousands of these tagged cells are measured rapidly by "flow cytometry" — a method for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus.

"The ability to rapidly measure thousands of cells for the presence of virus, sets this technique apart from currently used methods to monitor viral replication," said Robertson.

Traditionally, antibodies used to detect viruses must be produced and calibrated for each specific strain and are highly susceptible to viral mutations. Assays commonly used for quantifying viral loads and for drug development can be time consuming and rely on visible signs of cell damage, which is not produced in all viruses and can take long periods of time to occur.

Techniques such as quantitative reverse transcription-polymerase chain reaction (qRT-PCR), microarrays, and enzyme-linked immunosorbent assays (ELISAs), while highly sensitive, involve the lysis [the breaking down] of cells prior to measurement and are therefore unable to provide information about cellular viability, infected cell phenotypes, percentage of infected cells or the variation in infection among a cell population. The LNA probe differs from traditional nucleotide probes by binding more tightly to its target RNA.

LNA-flow FISH presents a fast and easy way to screen for compounds with antiviral activity and could be adapted for monitoring infections in the blood for vaccine therapy and development. This method adds a necessary tool for several emerging areas in cell biology that enables the use of high throughput measurements for entire populations and improves statistical analyses.

"This method can be expanded by adding more than one kind of LNA probe to enable multiple detection of different viral and host RNA," adds Robertson. "The multiplexing enhancement can be used to better understand infectious agents, allowing this technique to be used to aid in the development of antiviral drugs for a variety of viruses."

LNA flow-FISH offers an advantage over other techniques due to its simplicity and superiority. Methods involving genetic recombination of the virus to express a fluorescent protein as a means to mark the presence of virus can utilize flow cytometry for large-batch analysis of infected cells. However, an exception to this approach is viral strains that have not acquired genetic mutations, known as wild-type viruses (such as strains of Human Immunodeficiency Virus-HIV), which would require a large initial investment of labor for engineering each virus of interest.

Provided by Naval Research Laboratory (news : web)