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The ability to image viruses and proteins at the atomic level allows researchers to understand the structure and function of biological processes within the cell at the molecular level. (Source: Z. Hong Zhou) 

Although nanotechnology explores the very small, the field offers big opportunities in drug discovery and development. These opportunities include target selection and drug discovery, formulation, and delivery. Furthermore, these applications depend on a collection of advancing technologies, including electron microscopy and the use of nanoparticles.

When it comes to combining drugs with nanotechnology, many people imagine nano-size compounds. In fact, making drug particles smaller can make them easier to deliver, especially for injected compounds. “For intramuscular or subcutaneous delivery, you don’t want to use above 2 milliliters, because it gets painful,” says Gary Liversidge, PhD, chief technology officer at Elan Drug Technologies in King of Prussia, Pa. “With our NanoCrystal technology approach, we can get high drug loading—up to 900 milligrams per milliliter.” Despite loading so much compound in a small volume, Liversidge notes that “the viscosity is very low, so very thin needles can be used, which reduces the pain, as with the recently approved INVEGA SUSTENNA product we developed for Janssen.”

In addition, building drugs on nano-sized particles can often reduce the impact of food on orally delivered compounds. “Bioavailability of most poorly soluble drugs is influenced by food,” says Liversidge. “If you take an ordinary drug when fasted, bioavailability is low; if you take it when fed, the availability is higher.” He adds, “There’s a big range of variability, because it depends on fats in the food.” If a drug has a narrow therapeutic index—meaning that there’s a fine line between it being effective and safe—such differences in feeding-based bioavailability can, as Liversidge says, “be a big safety issue.” With Elan’s NanoCrystal technology, the surface-to-volume ratio can increase, allowing the “drug to dissolve much, much faster,” Liversidge explains. “In the fasted state, it dissolves quickly, and it dissolves quickly in the fed state, too.”

Moreover, incorporating a compound into a nanoparticle can improve the targeting of a drug. By getting the drug to the right place, efficacy can increase, while toxicity decreases. For example, researchers at Cerulean Pharma, Cambridge, Mass., are running Phase 2a trials on CRLX101—a nanopharmaceutical comprised of the anti-tumor agent camptothecin coupled to a polymer that self-assembles into a nanoparticle. This highly potent anti-cancer agent was too toxic when developed with traditional techniques, but that changed with Cerulean’s nanoparticle approach. “We have two years of encouraging safety data in patients,” says Oliver Fetzer, PhD, Cerulean’s president and CEO. Furthermore, both nanoparticles and released, free drug were found in a tumor biopsy taken from one patient two weeks after an injection of CRLX101, according to Fetzer.

Blasting with nanoparticles
Beyond using smaller needles to deliver drugs, some researchers hope to use nanotechnology to slip—or smash—compounds into individual cells. Perhaps surprisingly, that can be done with a laser and soot, which consists of nano-size particles of carbon black. According to Mark R. Prausnitz, PhD, professor of chemical engineering at the Georgia Institute of Technology, this approach relies on this equation:

C + H20 + energy ? CO + H2

So in the aqueous environment around a cell, a laser provides the energy that turns carbon black into carbon monoxide and hydrogen. These gases make a bubble that implodes, causing a tiny explosion that blows a hole in a nearby cell.

So far, Prausnitz doesn’t know how big the holes are. A similar approach that used ultrasound instead of a laser, however, made holes with diameters up to about 100 nanometers. “I think the holes we are making with the laser are smaller,” Prausnitz says. “We have much lower cell death.” In a recent article in Nature Nanotechnology, Prausnitz and his colleagues reported that this technique makes 90% of cells take up molecules, such as model drugs, and about 90% of the cells survive. Moreover, Prausnitz expects the survival rate to improve. “We got these results in cell culture, and other studies with similar methods found that viability goes up in vivo.”

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Nanofibers can be used to coat the bases of multiwell plates, where the double-headed arrow indicates the alignment of the fibers (A). Electron micrographs (B) reveal random (top) and aligned (bottom) fibers. Brain cells (C) cultured in ordinary Petri dishes (left) look flat in comparison to cells grown on aligned fibers (right). (Image: Mariano Viapiano) 

Imaging up close
Beyond using nano-size objects, nanotechnology encompasses looking at them. When asked what role nanotechnology plays in drug discovery and development, Robert Snyder, PhD, global marketing programs manager, life sciences, FEI Company, Hillsboro, Ore., replies: “For the first time, we can look at cellular operations at high resolution. The resolution of electron microscopy is approaching—even matching in some cases—the resolution of x-ray approaches.” Snyder envisions researchers watching the operation of cells, and then designing drugs that work within a specific system.

Reaching today’s resolution, according to Snyder, depended on vitrification, which freezes cells so fast that ice crystals don’t form. “Ice crystals are like knives that slice cells,” says Snyder. “Vitrification forms an amorphous ice, like glass, that is ideal for looking through.”

In addition, FEI’s Titan Krios transmission electron microscope automatically captures hundreds of thousands—even a million—images of a spot in a sample, and then averages the results. Z. Hong Zhou, PhD—director of the Electron Imaging Center for NanoMachines at UCLA—used this device to set the current resolution record of 3.3 angstroms, which was reported in the April 30, 2010, issue of Cell. At this resolution, even the smallest amino acid group—a methyl—appears as a bump in a protein chain. As Snyder says, “The next step is to bind a drug to a protein and see the conformational change in the electron density map.”

Build it like the brain
Nano-size objects might also reveal more about battling brain diseases. Using a biocompatible polymer called polycaprolactone (PCL), researchers at Ohio State University (OSU) create brain-like landscapes. “Using an electrospinning approach,” says Mariano Viapiano, PhD, an assistant professor at OSU’s Comprehensive Cancer Center, “we can cover a Petri dish—or a multiwell plate—with fibers that are nanometers in diameter.” The random mesh resembles the texture of the brain’s grey matter—largely cell bodies and short processes.

With an electric field, the researchers can orient the fibers, and that creates parallel lines of them that mimic the brain’s white matter, or myelinated axons. Viapiano and his colleagues then grow cells on these fibers. “The nanofibers provide topography and regulate the movement and orientation of the cells,” says Viapiano. “This is more representative of how cells move in the brain, compared with the rigid surface of a Petri dish.”

Viapiano and his colleagues, John Lannutti, PhD, and Jed Johnson, PhD, from the OSU College of Engineering, are perfecting a high-throughput model that uses these nanofibers to study brain-cancer cells. “We wanted to produce an in vitro ‘brain-like’ assay where we can grow brain-cancer cells and analyze their ability to move in conditions that approximate the brain texture and elasticity,” Viapiano says. “If we grow cancer cells conventionally, in a Petri dish, their shapes are flat, very different to what we see in a real tumor.”

Those cells don’t move like cancer cells, squeezing through brain tissue. On the nanofiber scaffolds, though, brain-cancer cells behave more like they would in the brain.

Viapiano and his colleagues can grow cancer cells on these nanofiber scaffolds and then bombard them with compounds to test changes in cell spreading and migration. “We think that compounds that disrupt the invasive motility of cancer cells in the fibers will have a high chance of being effective in vivo,” Viapiano says.

Nanotechnology can create many new tools for the pharmaceutical world. Moreover, big results will keep coming from these tiny technologies.

About the Author
Mike May is a publishing consultant for science and technology based in Houston, Texas.

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