查看完整版本: “量子点”助力RNA干扰技术

“量子点”助力RNA干扰技术

[b][color=Blue]【纳米科技世界论坛快讯】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.[/color][/b]

[attach]3582[/attach]
[color=DimGray]Each of these jars contains the same substance. The difference is the size of the particles. Quantum dots, suspended in liquid, absorb white light and then reemit it in a specific color that depends on the particle's size. Each quantum dot is about one ten-millionth of an inch in diameter and is composed of a few hundred atoms of material. Credit: Xiaohu Gao, University of Washington[/color]

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

[attach]3583[/attach]
[color=DimGray]A fluorescent image of the cell taken 15 minutes after introducing the quantum dot-siRNA complex. At this early stage the particles are in the cell membrane. Credit: University of Washington[/color]


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

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

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

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.

[attach]3584[/attach]
[color=DimGray]A fluorescent image of the cell taken four hours into the same experiment. At this time the quantum dot-siRNA complex is distributed throughout the cellular fluid. The dark region in the middle of the cell is the nucleus. Credit: University of Washington[/color]
The recent experiments used quantum dots, fluorescent balls of semiconductor material just six nanometers across (lining up 9,000 dots end to end would equal the width of a human hair). Quantum dots' unique optical properties cause them to emit light of different colors depending on their size. The dots are being developed for cellular imaging, solar cells and light-emitting diodes.

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

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 incoming material) and accumulates in the cellular fluid, 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, allowing 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. By contrast, the test protein was produced at 13 percent to 51 percent of normal levels when the siRNA was delivered with one of three commercial reagents, or reaction-causing substances, 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, while 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 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.


Source: University of Washington

[color=Blue]15年前，科学家发现了一种阻碍基因表达路径的方法——RNA干扰（简称RNAi）。这项荣膺2006年诺贝尔奖的发现承载着医学科学的迫切希望，它可以通过沉默基因来阻碍特定蛋白制造，从而达到疾病治疗的效果。不过到目前为止，RNA干扰技术很难在活体细胞中取得应用。[/color]

美国华盛顿大学和埃默里大学科学家的一项最新研究，首次成功利用“量子点”（quantum dots）技术解决了这一问题。研究证实，新技术向细胞内导入小分子干扰RNA（siRNA）的效率是现有方法的10至20倍。相关论文在线发表于《美国化学会志》（JACS）。

论文作者之一、华盛顿大学助理教授Xiaohu Gao说，“我们相信这会对siRNA输运领域产生重要影响。”另一位作者、佐治亚理工和埃默里大学教授聂书明也认为，“新的工作有助于攻克siRNA领域长期以来的一大障碍——如何在低毒性下高效地沉默基因。”

量子点即半导体荧光纳米球，具有特殊光学性质，它们能够按照尺寸发出不同颜色的光，因此被用于细胞成像、太阳能电池和发光二极管中。在最新研究中，科学家让直径6纳米的量子点与siRNA复合体结合，带正电荷的“质子海绵”（proton sponges）围绕在每个量子点的周围。而新方法的关键就是，研究人员可以调整量子点表层质子海绵的化学组成，从而精确控制量子点与siRNA的结合紧密度。

研究表明，在没有量子点的情况下，携带负电荷的siRNA无法进入细胞；而有量子点陪同时，带弱电的siRNA复合体能够穿过细胞膜，并且摆脱内涵体（包裹进入细胞物质的脂肪泡），从而在细胞液中积累，实现阻断蛋白制造的工作。

研究人员发现，实验中当通过量子点输运siRNA时，细胞内一种蛋白的生产可以降低到正常水平的2%。相比之下，用三种商业试剂或其他促反应物质来输运siRNA时，该蛋白的生产为正常水平

图[color=DimGray]片说明：导入量子点-siRNA复合体15分钟和1小时的细胞图像。可以看到，前者主要集中在细胞中，后者已经分散到整个细胞液中。（图片来源：University of Washington）[/color]

除了更高效地实现基因沉默，新技术的另一个重要方面就是荧光量子点可以让科学家观测siRNA在细胞内的运动。此前的示踪剂发光时间不超过一分钟，而用于细胞成像的量子点每次发光时间可达到一小时。在最新实验中，研究人员能够对siRNA路径进行多个小时的观测追踪。

此外，新方法对细胞的毒性也比现有化学物质弱10倍。不过，量子点为何比现有技术更加有效，其原因还是个谜。Xiaohu Gao表示，“我们认为这种改进是由逃脱内涵体以及量子点与siRNA分离的能力引起的。”

由于美国食品与药物管理局（FDA）没有批准量子点可以用于人体，研究人员正在试图将新技术“移植”到铁氧化物粒子上来。聂书明表示，“展望未来，这项工作对活体siRNA疗法具有重要意义——活体siRNA疗法将需要无毒的铁氧化物和生物降解聚合物运载体，而非量子点。”（科学网 任霄鹏/编译）

（《美国化学会志》（JACS），10.1021/ja800086u，Maksym V. Yezhelyev, Lifeng Qi, Ruth M. O’Regan, Shuming Nie, and Xiaohu Gao）