XU RESEARCH GROUP REPORTS ADVANCES IN USING NANOPARTICLES FOR PROBES OF LIVING CELLS AND EMBRYOS

In two new articles published in national journals, the research group led by Old Dominion University chemist and biochemist Xiao-Hong Nancy Xu reports major advances in using nanoparticles as probes for the imaging of living cells and zebrafish embryos.

The articles describe the researchers' development of:
· Single nanoparticle optical biosensors that are about one-millionth the size of a pencil point and could aid in the creation of anticancer vaccines;
· Nanoparticle probes to investigate molecular transport and physical and chemical properties inside embryos; and
· An in vivo system to screen the biocompatibility and toxicity of nanomaterials.

Xu said her group is exploring the potential of nanoparticles to be in vivo imaging and therapeutic agents and hopes to develop an effective and inexpensive in vivo model system to screen biocompatibility and toxicity of nanomaterials. The latest articles report the group's initial successes in using nanoparticles for diagnostic and therapeutic imaging, while simultaneously evaluating the biocompatibility and toxicity of the nanoparticle probes.

Both articles focus on the silver nanoparticles that the Xu group produces and sends on missions, sometimes to explore the outer membranes of cells where protein receptors trigger cellular responses and sometimes to enter the embryos to report back on the effect of nanomaterials on the development of embryos.

These nanoparticles literally light up living cells and embryos, and allow scientists using a well-designed imaging system (named as SNOMS) developed in the Xu lab to spy on functioning cells and the effect of nanoparticles on the cells. Using inherent brightness and color of individual silver nanoparticles, Xu's imaging system is able to characterize nanometer sizes of individual nanoparticles in real-time, and to trace individual nanoparticles as they are transported through pores and canals into the embryo.

Single-nanoparticle photonics and the single nanoparticle imaging system developed by the Xu group enable the direct characterization of size and location of nanoparticles inside cells and embryos. These innovations helped Xu and her group to win a 2007 Nanotech Brief Nano 50th award from NASA.

Immediately after publication of the group's latest articles, summaries of the research findings began appearing on science news Web sites. "I knew that these two papers are important studies," Xu said, "but what I did not expect was the instantaneous positive response from so many colleagues around the world, and media sources hoping to feature this work."

Richard Gregory, chair of ODU's Department of Chemistry and Biochemistry, noted that an article about Xu's work was featured in mid-October on the Nanowerk.Com site and added, "Her work is getting a lot of attention, and deservedly so. She and her group are taking nanoparticle research to new and exciting places."

Beginning about eight years ago, Xu, an associate professor of chemistry and biochemistry at ODU, reported research findings that placed her in the vanguard of scientists using very tiny optical sensors to study living cells. She currently has grants totaling more than $2.5 million from the National Science Foundation and the National Institutes of Health to support her work.

Xu was named in a 2006 article prepared by the National Cancer Institute as a pioneering developer of nanotechnology that can be used in the war against cancer. The article, titled "Mission to the Inside of a Living Cell," noted the benefits of studying biochemical reactions inside live cells, rather than dead cells. Similar studies in the past were conducted with dead cells or purified biomolecules extracted from cells. The article also gave the Xu group high marks for producing silver nanoparticles that are exceedingly bright and do not photodecompose.

Now the Xu group has taken these imaging tools one step further and demonstrated the possibility of imaging living embryos.

Research by Xu's group reported on Sept. 28, 2007, in the American Chemical Society's journal, ACS NANO, revealed that silver nanoparticles showed potential therapeutic effects, as well as toxicity. The findings indicate that organisms in the wild might be affected by nanoparticles that got into rivers and other bodies of water.

The lead authors of the ACS NANO paper are Kerry J. Lee and Prakash D. Nallathamby; co-authors include Lauren M. Browning. All three are graduate students working toward their Ph.D. degree in biomedical sciences under the direction of Xu. Christopher J. Osgood, ODU associate professor of biological sciences, a collaborator of Xu, is also a co-author; Xu is the corresponding author of the paper. The title of the paper is "In Vivo Imaging of Transport and Biocompatibility of Single Silver Nanoparticles in Early Development of Zebra Fish Embryos."

Another paper published on Sept. 15 in the journal Analytical Chemistry, which also is an ACS publication, details the group's work with the single-nanoparticle optical biosensors that perform their investigations along the outer cell walls. Follow-up work, according to the paper, could aid in the development of effective anticancer vaccines.

The lead authors of this paper are Tao Huang, a postdoctoral researcher, and Nallathamby. Both are under the direction of Xu. A collaborator of Xu, Daniel Gillet of CEA, the French technological research organization, is a co-author, and Xu is the corresponding author of the paper. The title of the paper is "Design and Synthesis of Single-Nanoparticle Optical Biosensors for Imaging and Characterization of Single Receptor Molecules on Single Living Cells."

The researchers found that silver nanoparticles passively diffuse into zebra fish embryos through chorion pore channels that are responsible for molecular transport in and out of the embryos, according to the article in ACS NANO.

Zebra fish and humans have genetic and drug-target-site similarities that make the fish particularly useful in research concerning the treatment of human diseases. In addition, zebra fish are small, inexpensive and well-suited for whole animal studies.

Xu said another advantage for researchers is that the zebra fish's early embryonic development completes within 120 hours with well-characterized developmental stages. The embryos are transparent and develop outside of their mothers, permitting direct visual detection of pathological embryonic death, mal-development phenotypes and study of real-time transport and effects of nanoparticles in vivo.

Therefore, zebra fish embryos offer a unique opportunity to investigate the effects of nanoparticles upon intact cellular systems that communicate with each other to orchestrate the events of early embryonic development.

The researchers found that in relatively low concentrations the single-nanoparticle probes are not toxic to embryos, but that toxicity increases as concentrations increase. These findings are valuable in evaluating potential environmental harm that could come from nanoparticles getting into the wild. But they also point to ways that single nanoparticles might generate desired effects that may be useful for therapies.

An optical microscopy technique developed by the Xu group "exploits individual silver nanoparticles' inherent brightness and color to detect sizes of individual nanoparticles and trace them as they are transported through pores into the embryo," Xu said. The researchers are continuing to explore how chemical and surface properties of the nanoparticles-as well as their size and shape-affect their biocompatibility.

According to the ACS NANO paper, "This study…represents the first rigorous study and characterization of nanotoxity and nanobiocompatibility ever performed by investigating the effect of highly purified nanoparticles in vivo in real time and by considering the effect of possible trace chemicals from nanoparticle synthesis." The researchers also say that their study is the first of its kind "suggesting that the release of large amounts of (silver) nanoparticles into aquatic ecosystems may have drastic environmental consequences, should the sizes of nanomaterials remain unchanged during the environmental transport."

The second study reported in Analytical Chemistry found that the group's silver nanoparticles could be conjugated to proteins (immunoglobulin class IgG) to create biosensors that can detect and track the activity of individual receptors on the cells' outer membranes. These receptors bind with specific molecules to initiate the cellular physiological responses that drive cell development, tissue repair and immunity responses.

Scientists long have been intrigued by the ability of a protein molecule to induce a significant cellular response, but detecting interactions on such a small scale has been all but impossible. Conventional analytical tools are unable to effectively detect these vital molecules, and classical, mass-action kinetic theories cannot explain binding mechanisms at the single-molecule level.

The fluorescence probes suffer photobleaching, a problem that the Xu group says it has overcome to allow its nanoparticle optical biosensors to work for extended periods.

The researchers reported success in maintaining the biological activity of protein molecules that they attached to nanoparticles. They also describe techniques that allow them to "anchor functional receptors on the living cell surface, generating a new, clean model system for validation and calibration of tools for probing single receptor molecules on single living cells in real time."

In the summary of the article in Analytical Chemistry, the researchers wrote that they are using biosensors and detection schemes that they have developed to provide "effective characterization of anticancer vaccines and for better understanding of their biological functions."