Gene silencer and quantum dots reduce protein production to a whisper

More than 15 years ago, scientists discovered a way to stop a particular gene in its tracks. The Nobel Prize-winning finding holds tantalizing promise for medical science, but so far it has been difficult to apply the technique, known as RNA interference, in living cells.

Now scientists at the University of Washington in Seattle and Emory University in Atlanta have succeeded in using quantum dots to address this problem. Their technique, which was reported in the Journal of the American Chemical Society, is 10 to 20 times more effective than existing methods for injecting the gene-silencing tools, known as siRNA, into cells.

"We believe this is going to make a very important impact to the field of siRNA delivery," said Xiaohu Gao, Ph.D., of the University of Washington. Gao collaborated with Shuming Nie, Ph.D., of Emory University and co-principal investigator of the Emory-Georgia Tech Nanotechnology Center for Personalized and Predictive Oncology, one of eight Centers of Cancer Nanotechnology Excellence funded by the National Cancer Institute.

Short pieces of RNA, the working copy of DNA, can disable production of a protein by silencing, or deactivating, a stretch of genetic code. Research laboratories regularly use the technique to figure out what a particular gene does. In the body, RNA interference could be used to treat conditions ranging from breast cancer to deteriorating eyesight.

The recent experiments used quantum dots, fluorescent balls of semiconductor material just six nanometers across. The unique optical properties of quantum dots cause them to emit light of different colors according to their size. In this work, each quantum dot was surrounded by a proton sponge that carried a positive charge. Without any quantum dots attached, the siRNA's negative charge would prevent it from penetrating a cell's wall. With the quantum-dot chaperone, the more weakly charged siRNA complex crosses the cellular wall, escapes from the endosome (a fatty bubble that surrounds material coming into a cell), and accumulates in the cell's cytoplasm, where it can do its work disrupting protein manufacture.

Key to the newly published approach is that researchers can adjust the chemical makeup of the quantum dot's proton-sponge coating. This ability allowed the scientists to precisely control how tightly the dots attach to the siRNA.

Quantum dots were dramatically better than existing techniques at stopping gene activity. In experiments, a cell's production of a test protein dropped to 2 percent when siRNA was delivered with quantum dots. In contrast, the test protein was produced at 13 to 51 percent of normal levels when the siRNA was delivered with one of three commercial reagents for siRNA delivery now commonly used in laboratories.

Central to the finding is that fluorescent quantum dots allow scientists to watch the siRNA's movements. Previous siRNA trackers gave off light for less than a minute, whereas quantum dots, developed for imaging, emit light for hours at a time. In the experiments the authors were able to watch the process for many hours to track the gene silencer's path. The new approach is also five to 10 times less toxic to the cell than existing chemicals, meaning that the quantum dot chaperones are less likely to harm cells. The ideal delivery vehicle would have no effect; the only biological change would be siRNA blocking cells' production of an unwanted protein.

The exact reason that the quantum dots were more effective than previous techniques is, however, still a mystery. "We believe the improvement is caused by the endosome escape, and the ability of the quantum dots to separate from the siRNA," Gao said.

Quantum dots are not yet approved for use in humans. The authors are now transferring their techniques to particles of iron oxide, several types of which have been approved by the Food and Drug Administration for use in humans. They are also working to target cancer cells by attaching to specific markers on the cells' surface. "Looking forward, this work will have important implications in in-vivo siRNA therapeutics, which will require the use of nontoxic iron oxide and biodegradable polymeric carriers rather than quantum dots," Nie said.

This work, which was supported in part by the National Cancer Institute's Alliance for Nanotechnology in Cancer, is detailed in the paper "Proton-Sponge Coated Quantum Dots for siRNA Delivery and Intracellular Imaging." An abstract of this paper is available through PubMed. View abstract