Xu Group Reports Advance in Probes of Intracellular Function
The nanobiotechnology research group of Old Dominion University chemist and biochemist X. Nancy Xu has discovered a new means of studying a poorly understood type of cellular function that figures significantly in human disease.
Cells of all organisms contain proteins that serve as efflux pumps, pulling specks of nourishment and other substances into the cells and pushing unwanted substances out. These pumps can be a boon to good health, but they also can cause resistance to drugs that treat highly infectious diseases or cancer. Malfunctions of some of these types of transporters cause diseases, such as cystic fibrosis.
Researchers have identified these pumps - called ABC membrane transporters - but they have not yet understood exactly how the process works. Xu and her colleagues have been studying the pumps for more than 10 years, and their research article published online last month in the journal, Analytical and Bioanalytical Chemistry, is the latest in a series of high-profile articles they have produced about their probes of live cells.
In this latest research, the Xu group was able to design and construct fusion genes, express them in the cells, and generate two new strains of bacteria. Inside these specially created cells are specks of enhanced green fluorescence protein (EGFP). Under fluorescence microscopy, the EGFP lights up intracellular operations and the efflux pump can be studied over time.
The researchers report extensive experiments to ensure that the efflux pump process was not altered by the presence of EGFP. Tests with dye molecules showed that the efflux function of the new strains was efficient.
As a sidelight, the article notes that this latest study also shows that glucose adversely affects the fluorescence study of efflux function, "suggesting that efflux kinetics of ABC membrane transporters in live cells must be characterized in the absence of glucose."
Authors of the article, "Design and Probing of Efflux Functions of EGFP Fused ABC Membrane Transporters in Live Cells Using Fluorescence Spectroscopy," include Xu's doctoral students Feng Ding and Kerry J. Lee, research scientist Tao Huang, and postdoctoral researcher Ardeschir Vahedi-Faridi.
Over the past decade, the Xu research group in nanobiotechnology has looked into a possible "stealth" quality for single-nanoparticle probes of living cells or for similar nanoparticle vehicles that can deliver medicine into the cells. In other words, they have been studying means by which nanoparticles can penetrate cells and accomplish their mission without harming the cell or being ejected by the efflux pumping mechanism. This mechanism naturally targets foreign objects for ejection from cells.
The group's broader mission has been to create and use flecks of precious metals no larger than one-millionth of a meter in length as reliable probes of living cells and embryos. The researchers have found ways to synthesize and purify silver and gold nanoparticles that will stay stable - one size, or monodisperse - over an extended period. They have also reported breakthroughs in the way they image and characterize nanoparticles using dark-field optical microscopy and spectroscopy.
The small size of the nanoparticles that have been created enables them to penetrate living organisms, but the surface area is large relative to the overall size, and this allows the particles to perform better in optical sensing and to carry a larger payload of drugs. The rainbow colors of these nanoparticles also contributes to their usefulness as probes and sensors.
Not only has the group's research advanced techniques for nanoparticle delivery of drugs, but it also has found that the nanoparticles alone, without a drug payload - particularly the silver nanoparticles - can have a toxic effect on cellular functions. This means that the nanoparticles themselves could be used as medicines, say, to kill cancer cells. But this also raises questions about the potential toxic impacts of nanoparticles that escape into the natural environment.