A Timely Release Of Gene-Silencing Molecules

Future treatments for diseases such as cancer or AIDS may use small interfering RNAs to silence disease-related genes. But scientists must first figure out how to safely and effectively deliver these molecules inside cells. Researchers have now developed a particle with low toxicity that escorts the nucleic acids into cells and then releases them to the cytosol (J. Am. Chem. Soc., DOI: 10.1021/ja300690j).

To transport small interfering RNAs (siRNAs) into cells, scientists must find a way to move the negatively charged molecules through the cells’ hydrophobic membranes. Previous strategies have taken advantage of endosomal uptake, a process in which the cell absorbs molecules and small particles by engulfing them inside vesicles called endosomes. But that’s only half the battle, says David Thompson of Purdue University. Once inside the cell, the siRNA must escape the endosome and enter the cytosol. Thompson wanted to develop what he calls bioresponsive materials that use biological cues, such as the acidic environment inside of endosomes, to free the therapeutic nucleic acids.

The delivery vehicle that Thompson and his team designed starts with a polyvinyl alcohol polymer backbone decorated with molecules of polyethylene glycol and cholesterol. The polyethylene glycol makes the material water soluble. Meanwhile, the cholesterol molecules allow the polymer to bind β-cyclodextrin molecules. These rings of seven sugars grab onto the cholesterol molecules through hydrophobic interactions. Once bound to the polymer, the β-cyclodextrin molecules use their positively charged amine groups to help the polymer snag a negatively charged siRNA molecule. The polymer wraps around the nucleic acid to form a particle.

The team designed the molecules linking the cholesterol molecules to the polymer backbone to break only in the acidic environment of an endosome. Thompson hypothesizes that the freed cholesterol could act like a detergent and “punch through the endosome,” releasing the cyclodextrin-bound siRNA into the cell.

The researchers tested their method on hamster cells expressing a gene for the green fluorescent protein. They mixed the cyclodextrin-bound polymer with a siRNA molecule that turns off the fluorescent protein gene. The resulting particles measured between 120 and 170 nm across, a size that cells readily take in, Thompson says. When the researchers watched how fast the particles moved through an electric field, they determined that the particles had almost neutral surface charge.

A day after the researchers added the siRNA packages to the hamster cells, the cells’ fluorescence dropped by 85%. Branched polyethylenimine, a commonly used siRNA delivery agent, had a similar effect. However, Thompson’s particles were less toxic to the cells than the branched polyethylenimine. The team incubated cells with 0.01 mM of the delivery agents and found that almost all of the cells treated with the new particles survived, while half of the cells treated with polyethylenimine died.

“This is an exciting approach,” says Joseph DeSimone of the University of North Carolina, Chapel Hill. He says that the particles’ near-neutral charge is a major advantage for researchers who would like to target siRNA to specific cell types, because charged particles can stick to cells non-specifically. But DeSimone would like to see more evidence that cholesterol’s release is responsible for the siRNAs’ escape from the endosomes. Thompson plans to study the delivery mechanism in more detail, as well as testing the approach in live animals.