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Posted: Aug 30, 2013
Nanoparticles enhance RNA interference
(Nanowerk News) Nanoparticles that deliver short strands of RNA offer a way to treat cancer and other diseases by shutting off malfunctioning genes. Although this approach has shown some promise, scientists are still not sure exactly what happens to the nanoparticles once they get inside their target cells. Understanding the mechanism of delivery is an important step to meet regulatory requirements along the drug approval process. A new study from researchers at the Massachusetts Institute of Technology (MIT) sheds light on the nanoparticles’ fate and suggests new ways to maximize delivery of the RNA strands they are carrying, known as short interfering RNA (siRNA). siRNA is capable of interfering with the natural processes of gene expression, effectively silencing the gene’s ability to produce a protein of interest.
“We’ve been able to develop nanoparticles that can deliver payloads into cells, but we didn’t really understand how they do it,” says Daniel Anderson, who led the MIT team. “Once you know how it works, there’s potential that you can tinker with the system and make it work better.” Dr. Anderson and his collaborators published their findings in the journal Nature Biotechnology ("Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling").
Research has shown that siRNA-carrying nanoparticles enter cells through a process called endocytosis, which cells use to engulf large molecules. The MIT team found that once the nanoparticles enter cells they become trapped in bubbles known as endocytic vesicles, or endosomes, where their contents either get recycled back out of the cell or degraded. This prevents most of the siRNA from reaching its target mRNA, which is located in the cell’s “cytosol,” the main body of the cell.
In the current study, Dr. Anderson and his colleagues at the David H Koch Institute for Integrative Cancer Research found that once cells endocytose lipid-RNA nanoparticles, they are broken down within about an hour and excreted from the cells. They also identified a protein called Niemann Pick type C1 (NPC1) as one of the major factors in the endosome-recycling process. Without this protein, the particles could not be excreted from the cells, giving the siRNA more time to diffuse from the endosome and reach its targets in the cytosol.
In studies of cells grown in the lab without NPC1, the researchers found that the level of gene silencing achieved with RNA interference was 10 to 15 times greater than that in normal cells. Notably, the lack of NPC1 also causes a rare lysosomal storage disorder that is usually fatal in childhood. The findings suggest that patients with this disorder might benefit greatly from potential RNA interference therapy delivered by this type of nanoparticle, the researchers say. They are now planning to study the effects of the absence of the NPC1 gene on siRNA delivery in animals, with an eye toward testing possible siRNA treatments for the disorder.
The researchers are also looking for other factors involved in endosome recycling that could make good targets for possibly slowing down or blocking the recycling process, which they believe could help make RNA interference drugs much more potent for treating cancer. Possible ways to do that could include giving a drug that interferes with endosome recycling, or creating nanoparticle materials that can more effectively evade the recycling process.
To create their multi-pronged therapy, Dr. Huang and his collaborators incorporated an siRNA agent designed to suppress production of vascular endothelial growth factor (VEGF), which helps trigger angiogenesis (i.e., new blood vessel growth that is essential to feed a growing tumor), and the anticancer drug gemcitabine, which inhibits DNA replication, into a single lipid/calcium/phosphate (LCP) nanoparticle. The calcium phosphate component promotes the releases the two drugs when taken up into cells by destabilizing the endosomal membrane. The researchers then attached a small molecule known as anisamide that binds to a receptor that is overexpressed on many human cancer cells as a means of targeting the nanoparticle, and coating for polyethylene glycol to make the particle soluble in blood. The researchers also prepared LCP nanoparticles containing only anti-VEGF siRNA or gemcitabine.
Tests in mice with tumors grown from human NSCLC cells showed that LCP’s loaded with both drugs effectively suppressed angiogenesis and increased apoptosis, or programmed cell death, resulting in almost complete reduction in tumor growth. Treatment with the LCP loaded with gemcitabine or anti-VEGF siRNA still produced significant suppression of tumor growth, less than the combination LCP treatment but still more efficaciously than free drug without a nanoparticle vehicle. The researchers also found that treatment with the dual-agent nanoparticle produced almost no toxic side effects. In particular, they noted the lack of liver toxicity that can limit the effectiveness of free gemcitabine therapy.