May 22, 2006
Nanoparticles Improve Antisense Delivery and Expression
In the fight against cancer, antisense drugs that could block cancer genes from producing malicious proteins have the potential to become a powerful new weapon that would complement drugs that work in a completely different manner. And though laboratory studies have been promising, the pace of development of these new drugs has been slow because of the difficulty in getting antisense drugs, which are made of short pieces of DNA or RNA, into cancer cells.
Now, however, a team of investigators at Northwestern University’s Center for Cancer Nanotechnology Excellence has successfully used gold nanoparticles to not only deliver antisense DNA molecules safely into cancer cells, but also improve the ability of the antisense DNA to bind to its target. This work appears in the journal Science.
"When mutations in the body's genetic material cause too many copies of certain proteins, cancer and other diseases can result," said Chad Mirkin, Ph.D., director of Northwestern's Center for Cancer Nanotechnology Excellence, who led the study. "Whereas typical drugs target the proteins, it is possible through antisense therapy or gene therapy to target the genetic material itself before it is ever made into copies of harmful proteins. One way to target the genetic material is to block the messenger RNA by using antisense DNA, which prevents the message from ever becoming a protein."
The promise of using antisense to control gene function in diseased cells was first recognized over 20 years ago, but the major challenge in translating promise into clinical utility has been a lack of a method for delivering antisense drugs to cells inside the body while avoiding their break down along the way. The Northwestern team shows that by attaching multiple strands of antisense DNA to the surface of a gold nanoparticle (forming an "antisense nanoparticle") the DNA becomes more stable and can bind to the target messenger RNA (mRNA) more effectively than DNA that is not attached to a nanoparticle surface.
When compared to antisense DNA complexed with commercial agents such as Lipofectamine 2000® and Cytofectin®, the antisense nanoparticles were more effective in decreasing gene expression and protein production, a process known as gene knockdown. Antisense DNA bound to gold nanoparticles were also less susceptible to degradation, resulting in longer lifetimes; exhibited vastly less toxicity compared to the commercial agents; and were more readily absorbed by cells, exhibiting greater than 99 percent uptake.
Once inside cells, the DNA-modified nanoparticles act as messenger RNA "sponges" that bind to their targets and prevent them from being converted into proteins. The investigators showed that the antisense DNA molecules, while functioning, remain attached to the gold nanoparticles even in the presence of intracellular molecules that normally cause nanoparticles to release their DNA cargo.
In their experiments the researchers targeted mRNA sequences that code for enhanced green fluorescent protein (EGFP) expressed in a mouse cell. The antisense sequence of the DNA attached to the nanoparticles was complementary to the mRNA for EGFP expression. When the nanoparticles were introduced to the cells the fluorescence dimmed -- a result of the nanoparticles binding to the mRNA and shutting down the protein's expression, or fluorescence. The investigators also found that they could tailor the degree of gene knockdown by controlling how many antisense DNA molecules they loaded onto each gold nanoparticle.
"In the future, this exciting new class of antisense material could be used for the treatment of cancer and other diseases that have a genetic basis," said Mirkin. His team is now working to optimize this system for maximum efficiency in delivering antisense agents to cancer cells.
This work, which was funded in part by the National Cancer Institute, is detailed in a paper titled, "Oligonucleotide-modified gold nanoparticles for intracellular regulation." An abstract of this paper is available though PubMed.