'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.

Quantum dots cast light on biomedical processes

The light emitted by quantum dots is both more intense and longer lasting than that produced by the fluorescent markers commonly used in medical and biological applications. Yet these nano-scale light sources still suffer from one major drawback: they do not dissolve in water. Researchers at the University of Twente's MESA+ Institute for Nanotechnology and at the A*STAR agency in Singapore have found a way to remedy this. They have developed a coating which allows quantum dots to be used inside the human body, even inside living cells. The researchers published details of their coating recipe in the October issue of Nature Protocols.

The new coating enables quantum dots, which are semiconductor nanocrystals, to literally cast light on biological processes. These dots are "nuggets," consisting of several hundred to several thousand atoms, that emit visible light when they are exposed to invisible UV radiation, for example. They range from a few nanometres to several tens of nanometres in size. The coating's benefits are not limited to improved solubility in water alone. Other molecules can "lock on" to its surface. This could make coated quantum dots sensitive to certain substances, for example, or allow them to bind to specific types of cells, such as tumour cells.

Better option

Scientists studying biological processes often use fluorescent tags that bind to biomolecules. This makes it relatively easy to track such molecules, even inside living cells. Quantum dots are a better option. They emit long-lasting, bright light, the colour of which depends on the size of the quantum dots used. For a number of reasons, including their toxicity, they were previously unsuitable for use in living organisms.

The researchers therefore developed an amphiphilic coating, i.e. one with both hydrophobic and hydrophilic properties. The "water hating" side of the polymer material attaches to the surface of the quantum dot. Its exposed hydrophilic side then makes the quantum dot/coating combination soluble in water. The coating builds up on the surface of the quantum dot through a process of self-assembly. The coating polymer has the added benefit that other molecules can be bound to it. Another important plus is that it does not adversely affect the quantum dot's light-emitting properties.

The study is a collaborative venture between the University of Twente's MESA+ Institute for Nanotechnology and the A*STAR agency's Institute of Materials Research and Engineering, in Singapore. It is headed by Professor Julius Vancso, Professor of Materials Science and Technology of Polymers at the University of Twente, who is also a visiting scientist at the Singapore institute.

Story Source:

The above story is reprinted from materials provided by University of Twente.

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

Journal Reference:

Dominik Jańczewski, Nikodem Tomczak, Ming-Yong Han, G Julius Vancso. Synthesis of functionalized amphiphilic polymers for coating quantum dots. Nature Protocols, 2011; 6 (10): 1546 DOI: 10.1038/nprot.2011.381

Improved method for capturing proteins holds promise for biomedical research

Antibodies are the backbone of the immune system—capable of targeting proteins associated with infection and disease. They are also vital tools for biomedical research, the development of diagnostic tests and for new therapeutic remedies.

Producing antibodies suitable for research however, has often been a difficult, costly and laborious undertaking.

Now, John Chaput and his colleagues at the Biodesign Institute at Arizona State University have developed a new way of producing antibody-like binding agents and rapidly optimizing their affinity for their target proteins. Such capture reagents are vital for revealing the subtleties of protein function, and may pave the way for improved methods of detecting and treating a broad range of diseases.

The team's results appear in today's issue of the journal ChemBioChem.

Antibodies are Y-shaped structures, capable of binding in two or more places with specific target proteins. Synthetic antibodies are much simpler forms that attempt to mimic this behavior. As Chaput explains, creating affinity reagents with strong binding properties can be accomplished by combining two weak affinity segments on a synthetic scaffold. The resulting affinity reagent, if properly constructed, can amplify the binding properties of the individual segments by two or three orders of magnitude.

"This dramatic change in affinity has the ability to transform ordinary molecules into a high affinity synthetic antibody," Chaput says. "Unfortunately, the chemistry used to make these reagents can be quite challenging and often requires a lot of trial-and-error. With NIH funding, my group has reduced the complexity of this problem to simple chemistry that is user friendly and easily amenable to high throughput automation. Such technology is absolutely necessary if we want to compete with traditional monoclonal antibody technology. "

Traditionally, antibodies for research have been extracted from animals induced to produce them in response to various protein antigens. While the technique has been invaluable to medical science, obtaining antibodies in this way is a cumbersome and costly endeavor. Instead, Chaput and his team produce synthetic antibodies that do not require cell culture, in vitro selection or the application of complex chemistry. They call their reagents DNA synbodies.

The new strategy—referred to as LINC (for Ligand Interaction by Nucleotide Conjugates) uses DNA as a programmable scaffold to determine the optimal distance needed to transform two weak affinity binding segments or ligands into a single high affinity protein capture reagent. The result is an artificial antibody, capable of binding to its antigen target with both high affinity and high specificity. The process is rapid and inexpensive. It also offers considerable flexibility, as the distance between the two ligand components bonded to the short, double-stranded DNA scaffold can be fine-tuned for optimum affinity.

In earlier work, the group identified ligand candidates by producing thousands of random sequence peptide chains—strings of amino acids, connected like pearls on a necklace. The peptide sequences were affixed to a glass microarray slide and screened against a target protein to pinpoint those that were capable of recognizing distinct protein binding sites. Two promising ligand candidates could then be combined to form a DNA synbody.

In the current study, the group instead makes use of pre-existing ligands with documented affinity for various disease-related proteins. The method involves the use of well-characterized ligands as building components for high quality DNA synbodies, eliminating the initial screening procedure and expanding the potential to tinker with the two-piece synbody in order to optimize affinity.

The peptides of choice for the study were those with high affinity for something called growth factor receptor bound protein 2 (Grb2). Grb2 has many cell-signaling functions and is an important focus of research due to its association with cellular pathways involved in tumor growth and metastasis.

By scouring the scientific literature, the group identified two peptides that recognize distinct sites on the surface of Grb2. Chaput points out, "this is a nice example where a few hours in the library can save you weeks in the lab."

The next step was to create an assortment of synbody constructs based on these peptides. To do this, one peptide was attached to the end of a short DNA strand, while the other peptide was attached to the complementary DNA strand further along its length (see figure 1).

The two peptide strands could be attached to the scaffold in either a forward or reverse direction and could be interchanged, with either occupying the terminal end of the first DNA strand. Further, the distance between peptide segments along the DNA strands could be adjusted to yield the best target affinity.

Experiments examined binding affinity for peptide chains separated by 3, 6, 9, 12, 15 and 18 base pairs along the DNA strand, (a distance range of 1.0-6.1 nm). Inspection revealed the best results for a synbody constructed of peptides separated by 12 base pairs at a distance of 4.1 nm, compared with the other 5 constructs.

The results for the best synbody in the study were impressive, demonstrating a binding affinity five- to ten-fold stronger than commercially available antibodies for Grb2, despite the synbody's comparatively primitive architecture. In further tests, the synbody was shown to exhibit high specificity—isolating Grb2 from other proteins in a complex biological mixture and selectively binding with its target.

The technique offers a new approach to producing high qualityaffinity reagents for disease research, diagnostic testing and the development of effective therapeutics.

Provided by Arizona State University (news : web)

Branch offices: New family of gold-based nanoparticles could serve as biomedical 'testbed'

 Gold nanoparticles are becoming the … well … gold standard for medical-use nanoparticles. A new paper by researchers from the National Institute of Standards and Technology (NIST) and the National Cancer Institute's Nanotechnology Characterization Laboratory (NCL) proposes not only a sort of gold nanoparticle "testbed" to explore how the tiny particles behave in biological systems, but also a paradigm for how to characterize nanoparticle formulations to determine just what you're working with.

Prospective uses of gold nanoparticles, says NIST chemist Vince Hackley, include high-precision drug-delivery systems and diagnostic image enhancers. Gold is nontoxic and can be fashioned into particles in a range of sizes and shapes. By itself, gold doesn't do much biologically, but it can be "functionalized" by attaching, for instance, protein-based drugs along with targeting molecules that cluster preferentially around cancer cells. The nanoparticles are generally coated as well, to prevent them from clumping together and to avoid rapid clearance by the body's immune system.

NCL's Anil Patri notes that the coating composition, density and stability have a profound impact on the nanomaterial safety, biocompatibility (how well the nanoparticles distribute in the body), and efficacy of the delivery system. "Understanding these parameters through thorough characterization would enable the research community to design and develop better nanomaterials," he says.

To facilitate such studies, the NIST/NCL team set out to create a nanoparticle testbed -- a uniform, controllable core-shell nanoparticle that could be made-to-order with precise shape and size, and to which could be attached nearly any potentially useful functionality. Researchers then could study how controlled variations fared in a biological system.

Their trial system is based on regularly shaped branching molecules called dendrons, a term derived from the Greek word for "tree." Dendron chemistry is fairly new, dating from the 1980s. They're excellent for this use, says NIST researcher Tae Joon Cho, because the individual dendrons are always the same size, unlike polymers, and can readily be modified to carry "payload" molecules. At the same time, the tip of the structure -- the "tree's" trunk -- is designed to bond easily to the surface of a gold nanoparticle.

The team made an exhaustive set of measurements so they could thoroughly describe their custom-made dendron-coated nanoparticles. "There aren't a lot of protocols around for characterizing these materials -- their physical and chemical properties, stability, et cetera," Hackley says, "so, one of the things that came out of the project is a basic series of measurement protocols that we can apply to any kind of gold-based nanoparticle."

Any single measurement technique, he says, is probably inadequate to describe a batch of nanoparticles, because it likely will be insensitive to some size ranges or confused by other factors -- particularly if the particles are in a biological fluid.

The new NIST/NCL paper provides the beginnings of a catalog of analysis techniques for getting a detailed lowdown on nanoparticles. These techniques include nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, dynamic light scattering, ultra-violet/visible spectroscopy and X-ray photoelectron spectroscopy. The dendron-coated nanoparticles also were tested for stability under "biologically relevant" conditions of temperature, acidity and some recognized forms of chemical attack that would take place in the bloodstream. In vitro biological tests are pending.

The work was funded in part by the National Cancer Institute, National Institutes of Health.

Story Source:

The above story is reprinted (with editorial adaptations ) from materials provided by National Institute of Standards and Technology (NIST).

Journal Reference:

Tae Joon Cho, Rebecca A. Zangmeister, Robert I. MacCuspie, Anil K. Patri, Vincent A. Hackley. Newkome-Type Dendron-Stabilized Gold Nanoparticles: Synthesis, Reactivity, and Stability. Chemistry of Materials, 2011; 23 (10): 2665 DOI: 10.1021/cm200591h