For scientists who develop nanomaterials, cancer has long been an attractive target. After all, nanoparticles—which can have a diameter one-thousandth the thickness of a sheet of paper—are the perfect size for slipping into tumors. Despite decades of research and billions of dollars invested, though, few nanotechnology-based cancer treatments are on the market. Pharmaceutical companies have been wary of putting in the big bucks required to commercialize these complex therapies.
That may be changing, though, thanks in part to the Nanotechnology Characterization Laboratory (NCL). The U.S. government facility, located in Frederick, Md., was established to accelerate the pace at which cancer-targeting nanomedicines get into clinical trials. And this past year, major firms, including AstraZeneca, invested in nanotherapeutics evaluated by NCL early in the development process.
The idea behind nanomedicines is to load the tiny particles with cancer drugs and decorate their outer shells with targeting molecules—compounds that will latch exclusively onto tumors while sparing regular tissue. By virtue of their size, charge, and coatings, these nanomaterials can also be designed to evade the body’s immune system and circulate for lengthy periods in a patient’s blood, thereby delivering plenty of drug to sites where it’s needed.
But the complexity of these tiny therapeutics has made it challenging to push their development forward. Not only do researchers have to worry about the drug’s purity and compatibility with the body during early stages of testing, but they also have to consider the nanoparticle’s properties. Is the particle stable? Are all the particles in a dose the same size and shape? Is the drug attached strongly enough to the particle to circulate in the bloodstream but weakly enough to release when it reaches a tumor?
Answering these questions hasn’t been easy. So far, only a handful of nanomedicines have gotten Food & Drug Administration approval for treating cancer.
Launched by the National Cancer Institute, NCL accepts about 12 nanomedicine hopefuls each year from academic teams, companies, and government labs in the U.S. for preclinical evaluation—free of charge. Among the battery of tests NCL runs on submitted particles are characterizations of their size, shape, and stability. Scientists there also determine whether the materials cause toxicity or incite immune reactions in healthy cells, and they assess how the candidates are processed in the bodies of rodents.
After nine years of running studies on some 280 nanomaterial formulations, the lab has helped put six medicines into clinical trials. In doing so, it’s accumulated tremendous behind-the-scenes knowledge about what works and what doesn’t for nanomedicines. “The lessons that we learn in developing these different kinds of technologies is something we share with the community,” says Anil K. Patri, deputy director of NCL. To disseminate its findings, the lab hosts workshops and publishes papers with titles such as “Common Pitfalls in Nanotechnology” (Integr. Biol. 2013, DOI: 10.1039/c2ib20117h).
To become one of NCL’s clients, a team—be it from industry, academia, or government—first has to apply online. The lab accepts submissions quarterly and judges potential nanomedicines on the basis of a number of criteria. Some of the obvious ones, says Jennifer Hall Grossman, a scientist at NCL, are that the medicine is actually “nano”—typically between 10 and 100 nm in diameter—and that it has efficacy against cancer. Beyond that, she says, NCL also determines whether to accept a candidate material by looking at how well the substance has been characterized by its maker and how the team plans to translate it to the clinic.
“Taking a nanomaterial concept from basic research into clinical trials is quite challenging,” Patri says. “We bridge that gap.”
Once a client is chosen, NCL will ask for samples to do some prescreening. “We try to fail things early if we think something’s not going to work,” Hall Grossman says. This means doing some basic sterility and size testing for starters.
Sterility, even though it seems like a simple test to pass, is frequently a sticking point for materials submitted to NCL. About 10% of the samples the lab has received over the years have been contaminated with bacteria, says Marina A. Dobrovolskaia, head of immunology at NCL. As many as 30% of samples have had unacceptable levels of endotoxin, a lipopolysaccharide from the outer membrane of gram-negative bacteria that incites inflammatory reactions in animal and human cells.
A lot of times, the scientists producing nanomaterials are academic chemists and materials scientists whose focus is on the proper fabrication of the particles rather than the microbial inhabitants of their lab benches, say experts at NCL. So this step in preclinical testing can be a rude awakening for some clients.
“With endotoxin, I ask our clients to itemize their synthesis steps for me,” Dobrovolskaia says. “Usually, I can go through the procedure and highlight in red all the places where endotoxin can get into the system,” such as glassware, stir bars, and rinse water.
When it’s not a financial burden, NCL will ask clients to return to the bench, clean their equipment, and synthesize new batches. For some, though, redoing a nanomedicine synthesis isn’t economically feasible. In these cases, Dobrovolskaia says, NCL works with scientists to develop ways of removing the contamination from existing batches without destroying the particles.
Running tests on a nanomedicine to determine its size is also a good way to head problems off early in a project, NCL scientists say.
One pitfall NCL commonly encounters in preclinical testing is the irreproducibility of clients’ samples. NCL sometimes receives nanomedicines—most often from academic labs—that vary in size or shape from batch to batch. This inconsistency makes it difficult to evaluate the efficacy of a material in later immunology or toxicology studies.
The variability, says NCL Deputy Director Patri, arises because standard characterization techniques haven’t really been established for nanomaterials. Organic chemists have at their fingertips a number of well-established methods, such as mass spectrometry and nuclear magnetic resonance spectroscopy, to analyze newly synthesized compounds, he adds, but nanotechnology specialists don’t.
This is one of the reasons, Patri says, that big pharma has been reluctant to invest in nanomedicines. “There’s no confidence because there are no standards.”
To address this problem, NCL works with the National Institute of Standards & Technology to develop nanoparticle reference materials. So far, they’ve made suspensions of gold nanoparticles measuring 10, 30, and 60 nm in diameter that scientists can use to calibrate particle-sizing equipment such as dynamic light-scattering instruments. Because some nanomedicines are covered in a layer of targeting or other molecules, NCL is also working on establishing coated versions.
Some NCL clients don’t properly monitor the long-term stability of a material they’ve developed either. After six months on the shelf, a particle’s properties—size, shape, coating density—might have changed, Patri says. NCL once encountered a gold nanoparticle coated with polyethylene glycol (PEG) for which this was a particular problem. One batch of the proprietary material caused only mild inflammation in the lungs of rodents. But a second batch caused severe lesions on the animals’ lungs. After additional testing, the lab’s scientists realized that PEG, a molecule placed on particles to help them evade the immune system, was slowly coming off. That meant that older particles didn’t have enough camouflage to escape detection.
Another lesson NCL has learned over the years involves reagents used to manufacture nanomedicines. Typically, solvents or stabilizers needed in nanoparticle fabrication are toxic to living cells. Scientists are supposed to remove these substances from the final nanomedicine formulation by filtration and other techniques, but some are tough to get rid of. The surfactant and stabilizer cetyltrimethylammonium bromide (CTAB), for example, sometimes sticks around after the synthesis of gold nanorods, says Stephan T. Stern, head of pharmacology and toxicology at NCL. And because CTAB can kill cells, its presence confounds the results of preclinical toxicology studies of the materials.
As nanomedicines become more complex—carrying targeting compounds, drugs, camouflaging agents, and more—it’s also important to consider the biocompatibility of every single component, NCL says. The lab once received a client’s proprietary sample containing liposomes—hollow spheres made of lipids. The surfaces of these liposomes were modified with folic acid, a tumor-targeting compound for a wide variety of cancers. When administered to healthy cells, the liposomes caused an immune reaction as severe as that triggered by cobra venom factor. Several experiments later, the lab determined that the inflammatory culprit was the substance linking the folic acid to the liposome.
“NCL provides what I would call pharmaceutical mentorship,” says Lawrence D. Mayer, president and chief scientific officer of Celator Pharmaceuticals, in Vancouver, British Columbia. For academic groups without expertise in drug development, that means getting expert advice about what to do next with their particles. For small biotech companies, it often means getting down to the nitty-gritty of how a product works and building confidence in the material’s performance and safety.
According to Mayer, NCL helped Celator and its collaborators, Robert K. Prud’homme and colleagues at Princeton University, answer detailed questions about their nanomedicine, CPX-8. Tests NCL ran on the therapeutic candidate indicated how animals metabolize the material and how it interacts with the animals’ immune systems (J. Control. Release 2013, DOI: 10.1016/j.jconrel.2013.04.025).
CPX-8 is a polymeric nanoparticle containing a releasable form of the cancer drug docetaxel. Although not yet in clinical trials, it is a lot closer to that step than it would have been without NCL, Mayer says. Celator didn’t have sufficient staff or funds to run these tests back in 2009, when NCL accepted the firm’s application. Without the lab’s help, he adds, “our formulation would probably have languished.”
For small firms like Celator, buy-in from big pharma is almost essential to moving a nanomedicine candidate into expensive clinical trials. Until recently, though, large pharmaceutical firms haven’t exactly embraced the nano trend. Historically, it’s been unclear to pharma whether the tiny particles provide enough benefit to justify their costs, says Lawrence Tamarkin, chief executive officer at Rockville, Md.-based nanomedicine firm CytImmune Sciences. Testing from NCL, however, is helping to prove the tiny materials’ medicinal worth.
In 1995, FDA approved the first nanomedicine, a liposome made by Janssen Biotech called Doxil, to treat an AIDS-associated cancer. When administered to patients, Doxil induces fewer side effects than does its active chemotherapeutic ingredient, doxorubicin. But the nanomedicine doesn’t improve patients’ survival rates compared with doxorubicin.
These results have given the community some pause, given that in 2009 Doxil cost $5,594 per dose and doxorubicin cost far less, $62 to $162 per dose.
Still, recent positive results from more sophisticated nanomaterials that were preclinically characterized by NCL have sparked big pharma’s interest. These particles home in on tumor cells via targeting molecules on their surfaces.
Earlier this year, AstraZeneca, Amgen, and Pfizer each signed on to trap some of their own drugs inside the nanoparticles of Cambridge, Mass.-based Bind Therapeutics. Each deal could net the firm $47 million to $70 million in up-front and milestone payments.
Bind’s particles, which are composed of block copolymers linked to targeting compounds, encapsulate chemotherapeutics at their core. The firm’s own nanomedicine, called Bind-014, has a targeting molecule on its surface that binds to prostate cancer cells. When given to mice with grafted tumors, the particles shrank the diseased tissue to half its original weight. A nontargeting version of Bind-014, on the other hand, reduced the rate at which the rodents’ prostate tumors grew but didn’t shrink them (Clin. Cancer Res. 2012, DOI: 10.1158/1078-0432.ccr-11-2938).
Last December, AstraZeneca also invested an undisclosed amount in a nanomaterial made by CytImmune, Tamarkin’s firm. AstraZeneca will attach one of its proprietary cancer drugs to the Maryland company’s Cyt-6091 gold nanoparticles, which are coated with a two-in-one targeting molecule and drug called tumor necrosis factor.
NCL won’t get much of the glory if the nanomedicines from Bind and CytImmune make it to market. The work that NCL does, Tamarkin says, isn’t glamorous, but it’s critical in pushing the nanomedicine community forward. The lab, he adds, “brings a level of quality” to nanomaterial characterization that the pharmaceutical industry is just beginning to recognize.