Trojan Family

Little Big Science

It’s not as small as you think, but biomedical nanoscience may be just the right size to take on the biggest bully in human biology. We’re talking cancer with a capital C.
By Carl Marziali
Popular introductions to nanotechnology usually crow about the minuteness of it all. Nano means small, we are told: really, really small. Think “teeny” and keep going.

Like a bolted morsel triggering the cough reflex, such naοvetι forces an involuntary guffaw out of Timothy Triche, chief pathologist at Childrens Hospital Los Angeles and a member of the USC Provost’s Biomedical Nanoscience Initiative. Nanoscale objects (a nanometer is a billionth of a meter) are plenty big compared to, say, an atom. And that’s a good thing, explains the physician-researcher who once dreamed of becoming a particle physicist.

There is a reason no one has ever heard of quark-based medicine or lepton therapy: nanoparticles in medicine need to hit a sweet spot. Make them too big, above 100 nanometers, and they get trapped in the bloodstream, unable to reach their target cells. Too small, below 10 nm, and the particles vanish into the churning waste-treatment plants of the kidneys and liver. “These things are certainly not tiny like elementary particles,” says Triche of the nanomedicines his lab is currently developing. “These things at least have some discrete constitution.”

One might expect the nano scale to be an attractive research area – being so much more manageable than lab work at the atomic level. But science does not always progress by the most direct route. In the first half of the 20th century, Einstein’s famous formula (you know, that one) and quantum mechanics worked like a gold rush, pulling scientists from the East Coast of classical mechanics to the western ranges of high-energy physics. Overlooked in the physics frenzy was that nondescript land in the middle, a Nebraska of understated promise, an Omaha waiting for its sage.

The sage has been slow in coming.

So far, nanotechnology has excelled at producing clever tag lines. (“Nano: the next big thing.” “Size matters.” And Nobelist Richard Feynman’s early entry, in a famous 1959 lecture predicting the age of nano: “There’s plenty of room at the bottom.”)

The field also has scored impressive breakthroughs in funding. (“Nanotechnology,” an Oxford don reportedly sneered, comes from a Greek word meaning “grant.”)

What nanotechnology has not yet done, for the most part, is produce really major advances in basic science, engineering or medicine. A cynic, echoing the saw about nations stuck on the verge of greatness, might say that nano is the technology of the future, and always will be.

“But,” one might ask, “what about my nano-treated stain resistant khakis from Gap? Or my nanosilver-enhanced antibacterial athletic socks from Sharper Image, which cost four times as much as the regular kind?”

The marketing industry is not above exploiting nano for commercial gain. This is not to deny that many products contain nanoscale components, or that such components are useful – they do, and they are. But the English chemist Michael Faraday was already making gold particles a few nanometers across in a beaker back in the 1850s. Nano-sized specks of titanium dioxide, with their unique reflective properties, have been brightening white paint for decades – long before a popular paint manufacturer began touting its “nano guard” process.

The truth is that making bits of matter on a nanoscale, especially metals, can be surprisingly simple. With the cost of manufacturing coming down and the understanding of nanoscale matter’s useful properties going up, the use of nano in consumer products is taking off.

But actually making nanoscale structures, machines, engineered objects with a specific function – that is the hard part, says USC chemist Mark Thompson, who with Richard Cote of the Keck School of Medicine of USC co-directs the Biomedical Nanoscience Initiative announced by Provost C. L. Max Nikias in the fall of 2005.

Fittingly, for a major research university, the USC initiative is focused precisely on the hard part. Nanoscale biomedicine also happens to be the newest and most promising part.

“The excitement is that it’s a very open area,” says Thompson, who specializes in molecular materials and thin films. “There’s a lot yet to be learned, and a lot of techniques to be developed before we can exploit things on the nanometer scale. The biological aspect of nano is really quite recent.”

So recent, in fact, that Thompson knows of only one successful, truly nano-engineered biomedical product: Abraxane, a new formulation of the anti-cancer drug known as Taxol.

Taxol’s problem has been that it does not dissolve spontaneously in the bloodstream. This was corrected by mixing the drug with a kind of castor oil. But the additive made Taxol more toxic and caused allergic reactions in many patients.

The new drug, Abraxane, consists of Taxol enclosed in nanoparticles that travel throughout the body, but that bind preferentially to breast-cancer cells.

“The nanoparticle is designed to actually interact with a receptor that is more highly expressed on cancer cells,” says Cote, who is a research pathologist and urologist based at the USC/Norris Comprehensive Cancer Center. The particle latches on to the receptor and gets absorbed by the cell, he explains, multiplying the drug’s specificity compared to regular Taxol.

How do you get the Taxol into the nanoparticles? Like a glass bottle blown around a model ship, the nanoparticles are grown in Taxol instead of in water or another solvent. Their insides can’t help but be full of the drug.

Even better would be a nanocapsule so specific that it binds only to the cancer cell. That is the goal of Triche’s laboratory in its work against Ewing’s sarcoma, a bone tumor found among children and young adults. (Two of the researchers in Triche’s group are themselves fighting Ewing’s.)

The laboratory has been encapsulating a standard chemotherapy drug, brand name Irinotican, with a very non-standard nanocapsule. To Triche’s knowledge, this is the first capsule designed to bind a receptor specific to a tumor cell.

“Total specificity,” in which only cancer cells are harmed, is the great quest of chemotherapy.

“If you keep your relatively toxic drug in a nanoparticle, and the nanoparticle goes to the tumor, and relatively little of it goes to the bone marrow,” says Triche, you achieve that specificity.

To keep the patient’s immune system from attacking the nanoparticles, they are coated with what Triche calls “stealthy liposomes” – sugars, essentially – that fool the immune system but do not interfere with the nanoparticle’s precision-guided targeting mechanism.

“The miracle to me is that it actually works,” he says.

Triche calls his method “targeted-targeted therapy” – targeted to find the tumor, and targeted to kill it. In the interest of full disclosure, he adds that the nanoparticle is not 100-percent specific: a small, non-essential number of immune cells share the target receptor, as do some cells in the testes. For the same reason, many other less-specific chemotherapy drugs are known to compromise fertility.

“It was pretty successful,” deadpans Siwen Hu, a post-doc in Triche’s lab who played a key role in developing the Irinotican-injected nanoparticles. Her remark is an understatement. “It” turns out to be not only a series of wildly successful experiments with nanoparticles carrying the cancer drug, but also a bold extension of that concept using silencing RNA (a 1998 discovery recently awarded the Nobel Prize in Medicine). Just as they carry chemotherapy drugs, nanoparticles also can smuggle siRNA into a tumor cell, turning off genes essential to tumor growth. The smuggler, a protein that loads iron into cells, works like a Trojan horse for the delivery of siRNA. Beware of nanoparticles bearing gifts.

In experiments on mice with induced Ewing’s cancer, Triche’s lab achieved complete remission in 80 percent of cases.

“We conclude that this novel delivery system is a powerful and simple method to induce gene silencing, with the potential to move to clinical trials,” Hu said at a 2005 meeting of the American Association for Cancer Research, which presented her with a scholar-in-training award. Among others, The Economist covered the breakthrough.

Impressed by the initial results, the Nanoparticle Identification Laboratory at the National Cancer Institute has commissioned huge batches of nanoparticles, intending to perform all the tests required by the Food and Drug Administration for a drug filing. It’s a win-win proposition for Triche and USC. If the nanoparticles pass muster, the university retains the right to submit the actual filing and to hold the patent.

The work at Triche’s lab demonstrates another striking fact about nanotechnology: for all the academic hype about interdisciplinary research, nano may be one area where cross-campus collaborations are not just useful, but indispensable. Triche works closely with Magnus Nordborg, associate professor in molecular and computational biology in the USC College of Letters, Arts and Sciences; his nanoparticle research group includes another computational biologist, a graduate student of USC math-biology-medicine polymath Simon Tavarι.

“This is the new face of biomedical research,” Triche says. “Statistics and mathematical modeling become incredibly important.”

In that sense, the Biomedical Nanoscience Initiative formalizes and supports what was already a university-wide movement.

“The initiative came along because when Max Nikias went into the provost’s office he already had an understanding that nano was important,” says Thompson, the chemist. (Nikias is an engineer and past dean of the USC Viterbi School.)

But instead of starting a generic nanoscience program, the likes of which exist elsewhere, Nikias searched for a synergy unique to USC. He found it in a marriage of the University Park campus, the Keck School and Childrens Hospital Los Angeles.

“If you really want to have a footprint, you have to pick and choose your areas,” says Cote. “And where would USC have an advantage over, for example, a Caltech, in nanosciences? What do we have that Caltech doesn’t have?

“We have a huge biological and medical program. We have a medical school. And, in fact, that has been a major attraction. It has generated interest from the Caltech community and UC Berkeley.”

Cote’s own research program exemplifies the marriage foreseen by the provost. Cote, a urologist-pathologist from the Keck School of Medicine; Thompson, a chemist-materials scientist from the USC College; and Chongwu Zhou, an electrical engineer from the USC Viterbi School of Engineering, are co-investigators on what may be the initiative’s signature project: an effort to develop nano-sized sensors that would screen for many types of cancers at once, with just a drop of a patient’s blood, and in a single step.

To grasp the significance of such a sensor, it helps to understand the current state of cancer screening. So-called early detection strategies – mammograms, colonoscopies and such – detect tumor masses. Unfortunately, by the time a tumor mass forms, millions or billions of cells have already turned against the body. In the brave new nano world, early detection means something different.

What if you could detect the very genesis of a cancer at the cellular level? The difference between nanoscale cancer screening and today’s idea of “early detection” is the difference between forecasting a snow storm and waking up to four inches of the stuff.

One existing test approximates the nano ideal. Known as PSA, for “prostate specific antigen,” it detects a protein expressed by prostate tumors. Unfortunately, PSA is notorious for its high false-positive rate: many biopsies come back negative, and many of the tumors are so slow-growing they will never threaten the patient. The test also requires a relatively large blood sample, says Cote, followed by up to 20 expensive and time-consuming steps.

“And that’s just for one disease,” adds the Keck School researcher, whose specialty is in the detection of micrometastases in epithelial tumors.

“Now imagine that I can take a drop of blood, that I can put it on a chip that’s two-by-two centimeters in dimension, and that I can run hundreds or even thousands of reactions at once with that drop of blood.”

Great idea. But it requires sensing ability at the nano scale.

This is where Zhou’s nanowires and nanotubes come in. In his gleaming white, deceptively sparse laboratory in Tutor Hall, the USC Viterbi researcher leads visitors in hospital slippers on a tour of the tools of nanoscience.

On a table nearest the security doors is an atomic force microscope: essentially a very sharp needle, 10 nm in diameter at the tip, that is repelled by the atomic force of other matter. By tracing a sample – say a silicon wafer – with the needle and measuring the force pushing it away, the microscope produces a textured map of the sample’s surface.

Down the hall to the right, past graduate students deep in analysis at their workstations, Zhou points to his two workhorse instruments: tabletop metal boxes facing each other across a small room.

One is a laser ablation system for making nanowires. The laser heats the surface of a target, vaporizing some of the material. By supplying the appropriate catalyst, usually a cluster of gold atoms, Zhou and his students induce the vapor to reform into solid nano-sized wires or, under different conditions, two- or three-dimensional wire structures.

Across the room is Zhou’s homemade device for growing carbon nanotubes. Its name is descriptive to a fault: “carbon nanotube chemical vapor deposition.” By flowing carbon-containing gases into the device – providing a sapphire catalyst – and heating the chamber to around 900 degrees Celsius, Zhou and his team have found that the carbon self-assembles into nanotubes, and that these nanotubes are ideally oriented for conducting electricity. (Scientific American reported on the discovery earlier this year.)

Why do nanowires and nanotubes matter? In the semiconductor world, conventional silicon circuits are approaching their physical limit. If they could be mass-produced, nanoscale circuits would provide faster conduction, lower heat loss, smaller circuit size and greater computing power.

More importantly for medical research, the unique properties of nano structures seem tailor-made for sensor design. An effective sensor for a cancer marker needs to find the target protein, capture it and communicate its capture. Larger sensors can capture target proteins, but the molecules are like fleas on an elephant – too small to be noticed or to affect the mighty pachyderm’s gait.

As luck would have it, nanowires and nanotubes are similar in scale to proteins. Moreover, as objects get smaller, the ratio of their surface-area to their volume gets bigger and bigger. By analogy, a nanoscale man would be all skin, with little or no muscle or bone.

Since the skin of the nanosensor is the part that interacts with target proteins, even the binding of a single protein would feel less like a flea bite than a mastiff’s chomp.

“The properties of the nanowire are very strongly influenced by anything happening on the surface,” says Thompson, the chemist. “And so now you can get a substantial change in your transistor response by binding something to the surface of a nanowire, that you wouldn’t have gotten with a larger sensor.”

Bye-bye clunky PSA test.

“If I functionalize this [nano]wire, and I bind something to it specifically, I might need to only do that in order to detect a specific reaction,” says Cote. “One step. We’ve in fact shown this.”

The team has already published some results and is awaiting word on a series of grants to start moving the technology from the lab to the doctor’s office.

The biggest remaining challenge, says Keck School doctoral student Henry Lin, is to make the sensor work in the protein stew that is a patient’s blood serum. “Non-specific binding,” in which a normal protein manages to latch on to the nanowire, is always a concern. The same challenge confronts a different nanosensor under development in Cote’s lab: a cantilever – essentially a springboard – treated to bind target proteins and to warp detectably from electromagnetic attraction or repulsion.

Once a cancer is found, nanotubes also might figure in its elimination. Cancer kills from within, so wouldn’t it be poetic justice for cancer’s killers to do the same?

Zhou is investigating the use of nanotubes as lethal vectors capable of infiltrating cancer cells but not healthy tissue. This approach relies on a common trait of cancer tumors: because they grow so fast, their structures develop gaps and cracks. If nanotubes could penetrate the tumor through these cracks, they could be heated with an infrared laser, says Zhou, destroying the cancer cells without the side effects and potential for drug resistance that plague chemotherapy.

All of these efforts share a common first step: Find a protein or other molecule that will bind, or latch onto, a receptor on the cancer cell. This is not always easy to achieve, especially if, to avoid false positives, the desired molecule must be incapable of binding to other proteins.

There are almost as many ways to identify binding molecules, or ligands, as there are cancers. All have their limitations. Richard Roberts, a chemist in USC College, has a few trillion reasons to tout his radical method.

Roberts specializes in “genetic evolutionary design.” The philosophy behind his approach might be summarized thus: “Let’s not restrict ourselves to molecules that exist today. Let’s try to find useful molecules that evolution has not yet created, or that it should create but might never get to.”

Roberts and his lab have built a library of trillions of molecules to literally throw at a cancer protein.

“We take the protein and we usually just use this library to target different sites on it,” he says. “We just mix the two together and see what sticks.

Though Roberts calls his approach “naοve,” it nevertheless delivers molecules that are frequently useful or “functional,” in the jargon of chemistry.

“It’s the brute force approach to being a locksmith,” says Roberts. “You have a pocket with a trillion keys in it. Our strategy lets you essentially test each key simultaneously.”

As a bonus, the approach also may deliver better formulations of common drugs. One method, mentioned earlier, is to enclose a drug such as Taxol in a protective nanocapsule. But there are other ways to skin a nanocat. Roberts and his lab believe they can use their synthetic library to make more durable versions of certain classes of drugs, such as peptides, that fall apart quickly in the bloodstream.

Synthetic approaches are hot in the fight against unnatural diseases, and Roberts’ “accelerated evolution” looks almost natural compared to the armies of nanorobots envisioned by computer scientist Aristides Requicha of the USC Viterbi School. Together with Zhou and Thompson, Requicha runs the Laboratory for Molecular Robotics, which is already working on components of nanorobots, such as tiny transistors and actuators.

Instead of putting blood into the sensor, Requicha wants to put the sensor in the blood. And not just one sensor, but a whole fleet – a “sensor-actuator network” that could provide real-time monitoring of the body’s health and carry out repair jobs.

While such nanorobots are a few years away – in Requicha’s most optimistic estimate – his laboratory already has nano pseudo-bots that could perform useful sensing and monitoring tasks in test tubes, perhaps to study how cells interact or how healthy cells differ from cancerous ones.

But the actual nanobot-in-body enterprise has what Requicha calls “a big power problem.” Nobody has figured out how nanobots in the bloodstream would power themselves or communicate wirelessly, both among themselves and with an external computer.

Mark Humayun and Anupam Madhukar do not pretend to have solved that problem, but their research on a different problem is revealing surprising insights on how to harness a cell’s own power.

Humayun, professor of ophthalmology, biomedical engineering and cell neurobiology in the Keck School, is best known to date for his retinal implant – a micro-scale device that has returned a degree of sight to some totally blind patients. Madhukar, professor of engineering at USC Viterbi, is an expert in optical electronics at the nanoscale. One of the earliest advocates of nano at USC, he proposed a program within engineering in 1992.

Now the two are collaborating on a project that, if successful, would make the retinal implant a true nanoscale device. And more broadly, it would harness the cell as a power source for all kinds of nano applications.

Humayun’s group has already identified a molecule naturally occurring in spinach that makes nerve cells light-sensitive. The implications are potentially staggering. Humayun’s retinal implant bypasses dead retinal cells to electronically stimulate intact receptors in the eye. But from a nano perspective, the implant is still unwieldy and primitive. The molecule Humayun discovered might be able to do the same job as the implant, without the awkward electronics and external power source.

But, he says, “we have to get this into the cell membrane.”

Madhukar and graduate student Siyuan Lu think they know how to use nanotechnology to create protein-sized devices that can be embedded in cells to restore or endow new function. Scientists already attach fluorescent tags known as quantum dots to specific ligands, which then bind to desired proteins on the cell surface. Humayun, Lu and Madhukar propose replacing the fluorescent tag with an “active” device that will perform the desired function. Making the nerve cell light-sensitive, as for the retina, is just one of many possible applications.

“We can use these functional active nano devices, or FANS, by putting them directly at appropriate places in the cell,” says Madhukar, who holds faculty appointments in materials science, biomedical and chemical engineering and physics.

Details are still confidential – the researchers are filing for a patent – but Humayun can barely contain his enthusiasm. “Once you have control over the cell with a receptor, now you got ’em. You can drive the energy harnessed in the cell. It’s very powerful.”

It is a short hop from Madhukar’s active nano devices to Requicha’s robotic legions to Zhou’s nanowires and carbon nanotubes. The existence of such closely related projects in one university, with synergies yet unexplored, argues for a structure to bring them together. The Biomedical Nanoscience Initiative is an obvious start, but the provost also is considering the establishment of a biomedical nanoscience institute, as yet unnamed and unfunded, to coordinate projects, encourage collaborations and just gather USC nano scholars under one roof for a few hours a month.

In addition, USC may have room for a few more outstanding scientists.

“We would like to bring in, not just one or two, but a group of individuals who can really push the university forward in this field,” says Randolph Hall, vice provost for research advancement.

Meanwhile the university is already busy with major upgrades to its nanotechnology capability: millions of dollars for “core labs” (shared by all interested researchers) to acquire the latest fabrication equipment, microfluidic devices, electron microscopes and other basic tools of nano.

In the late 19th century, universities around the world upgraded their laboratories with stunningly powerful optical tools that afforded glimpses of matter at the micro scale – hence the name “microscope.”

Is nanotechnology the destination or a way station? Will mankind someday need even smaller tools to fix the nanomachines that fix the human body?

“In the Reign of Harad IV” – a short story by noted American fiction writer Steven Millhauser – tells of a miniature maker so accomplished that he begins to work in the invisible realm, confidently assembling minuscule masterpieces by feel even as his students and patrons write him off as insane. At the story’s conclusion, two students bestow exaggerated praise on his latest creation, an invisible kingdom.

Millhauser writes:

The maker of miniatures, knowing that [the students] had seen nothing, that their words were hollow, and that they would never visit him again, returned with some impatience to his work; and as he sank below the crust of the visible world, into his dazzling kingdom, he understood that he had traveled a long way from the early days, that he still had far to go, and that, from now on, his life would be difficult and without forgiveness.

Particle physics may yet return to haunt medicine’s miniature makers.

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Nano Cluster In his Tutor Hall lab, electrical engineer Chongwu Zhou (left) confers with chemist Mark Thompson and pathologist Richard Cote, co-directors of the USC Biomedical Nanoscience Initiative.

Photographs by Mark Berndt

Trojan Horse Breeders Siwen Hu, a postdoctoral fellow at USC-affiliated Childrens Hospital Los Angeles, reviews the progress of tumors in lab rats receiving chemotherapy via a revolutionary nanocapsule that she helped develop in the lab of CHLA chief pathologist Timothy Triche (left). Their new delivery system may be on the market soon; tests needed for an FDA filing have been commissioned by the National Cancer Institute.

At the Forge Chemical engineer Ted Lee (right) fires up a laser light and detector capable of measuring particles in a solution. Lee teaches a new undergraduate class in “nano-blacksmithing” offered through the USC Viterbi School. With him (from left) are journeymen nanosmiths Panteha Mirarefi and Anne-Laure Leny, both doctoral students in chemical engineering, and junior Jonathan Lo, an aspiring apprentice from Lee’s class.