Gene Silencer and Quantum Dots Reduce Protein Production

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Now scientists at the University of Washington in Seattle and EmoryUniversity in Atlanta have succeeded in using nanotechnology knownas quantum dots to address this problem. Their technique is 10 to20 times more effective than existing methods for injecting thegene-silencing tools, known as siRNA, into cells.

"We believe this is going to make a very important impact tothe field of siRNA delivery," said Xiaohu Gao, a UW assistantprofessor of bioengineering and co-author of a study publishedonline this week in the Journal of the American Chemical Society.

"This work helps to overcome the longstanding barrier in thesiRNA field: How to achieve high silencing efficiency with lowtoxicity, said co-author Shuming Nie, a professor in the WallaceH. Coulter Department of Biomedical Engineering, jointly affiliatedwith the Georgia Institute of Technology and Emory University.

Other co-authors are Maksym Yezhelyev and Ruth O'Regan at Emory andLifeng Qi at the UW.

Short pieces of RNA, the working copy of DNA, can disableproduction of a protein by silencing, or deactivating, a stretch ofgenetic code. Research laboratories regularly use the technique tofigure out what a particular gene does. In the body, RNAinterference could be used to treat conditions ranging from breastcancer to deteriorating eyesight.

The recent experiments used quantum dots, fluorescent balls ofsemiconductor material just six nanometers across (lining up 9,000dots end to end would equal the width of a human hair). Quantumdots' unique optical properties cause them to emit light ofdifferent colors depending on their size. The dots are beingdeveloped for cellular imaging, solar cells and light-emittingdiodes.

This paper describes one of the first applications of quantum dotsto drug delivery.

Each quantum dot was surrounded by a proton sponge that carried apositive charge. Without any quantum dots attached, the siRNA'snegative charge would prevent it from penetrating a cell's wall.With the quantum-dot chaperone, the more weakly charged siRNAcomplex crosses the cellular wall, escapes from the endosome (afatty bubble that surrounds incoming material) and accumulates inthe cellular fluid, where it can do its work disrupting proteinmanufacture.

Key to the newly published approach is that researchers can adjustthe chemical makeup of the quantum dot's proton-sponge coating,allowing the scientists to precisely control how tightly the dotsattach to the siRNA.

Quantum dots were dramatically better than existing techniques atstopping gene activity. In experiments, a cell's production of atest protein dropped to 2 percent when siRNA was delivered withquantum dots. By contrast, the test protein was produced at 13percent to 51 percent of normal levels when the siRNA was deliveredwith one of three commercial reagents, or reaction-causingsubstances, now commonly used in laboratories.

Central to the finding is that fluorescent quantum dots allowscientists to watch the siRNA's movements. Previous siRNA trackersgave off light for less than a minute, while quantum dots,developed for imaging, emit light for hours at a time. In theexperiments the authors were able to watch the process for manyhours to track the gene-silencer's path.

The new approach is also five to 10 times less toxic to the cellthan existing chemicals, meaning the quantum dot chaperones areless likely to harm cells. The ideal delivery vehicle would have noeffect; the only biological change would be siRNA blocking cells'production of an unwanted protein.

The exact reason that the quantum dots were more effective thanprevious 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 thesiRNA," Gao said.

Quantum dots are not yet approved for use in humans. The authorsare now transferring their techniques to particles of iron oxide,several types of which have been approved by the Food and DrugAdministration for use in humans.
They are also working to target cancer cells by attaching tospecific markers on the cells' surface.

Looking forward, this work will have important implications inin-vivo siRNA therapeutics, which will require the use of nontoxiciron oxide and biodegradable polymeric carriers rather than quantumdots, Nie said.

The research was funded by grants from the National Institutes ofHealth, the National Science Foundation and the Georgia CancerCoalition.