By Neal Singer
Melding nanotechnology and medicine, research led by Sandia, the University of New Mexico, and the UNM Cancer Research and Treatment Center has produced an effective strategy to target a cancerous cell using nanoparticles that deliver a mélange of killer drugs into it.
A paper, selected as the cover article of the May issue of Nature Materials and available online April 17, describes silica nanoparticles about 150 nanometers in diameter as honeycombed with cavities that can store large amounts and varieties of drugs.
“The enormous capacity of the nanoporous core, with its high surface area, combined with the improved targeting of an encapsulating lipid bilayer [called a liposome], permit a single ‘protocell’ loaded with a drug cocktail to kill a drug-resistant cancer cell,” says Sandia researcher and UNM professor Jeff Brinker, principal investigator on the research. “That’s a millionfold increase in efficiency over comparable methods employing liposomes alone — without nanoparticles — as drug carriers.”
The nanoparticles, surrounded by cell-like membranes formed from liposomes, together become the combination referred to as a protocell: the membrane seals in the cargo and is modified with molecules (peptides) that bind specifically to receptors overexpressed on the cancer cell surface. The nanoparticles provide stability to the supported membrane and contain and release the therapeutic cargo within the cell.
A current Food and Drug Administration-approved nanoparticle delivery strategy is to use liposomes themselves to contain and deliver the cargo. In a head-to-head comparison of targeted liposomes and protocells with identical membrane and peptide compositions, the paper reports that the greater cargo capacity, stability, and targeting efficacy of protocells leads to orders-of-magnitude greater cytotoxicity directed specifically to human liver cancer cells.
Specialized loading strategies
Another advantage to protocells over liposomes alone, says lead author Carlee Ashley (8621), a Harry S. Truman Fellow at Sandia/California, is that liposomes require specialized loading strategies. “We’ve demonstrated we can simply soak nanoparticles to load them with unique drug combinations needed for personalized medicine. Protocells can also effectively encapsulate potent protein-based toxins, carrying them as well as siRNAs that silence expression of proteins that cancer cells need to survive.”
RNA, the biological messenger that tells cells which proteins to manufacture, is, in this case, used to silence the cellular factory, a way of causing apoptosis or cell death. “Si” is short for “silenced interfering.”
DEEP IMPACT-The figure on the left (Hep3B) shows a greenly fluoresced cancerous liver cell penetrated by protocells. The small red dots are lipid bilayer wrappings. Their cargo - drug-filled nanoparticles, their pores here filled with white fluorescent dyes for imaging purposes - penetrate the cancerous cell. (Penetration is more clearly seen in the second image, where the green fluorescence has been removed.) The normal cell on the right (hepatocyte) shows no penetration. (Images, courtesy Carlee Ashlee, were taken with a confocal microscope at the University of New Mexico cancer center's Fluorescence Microscopy Facility.)
The lipids also serve as a shield that prevents toxic chemotherapy drugs from leaking from the nanoparticle until the protocell binds to and becomes internalized within the cancer cell.
This means that few poisons leak into the system of the human host, should a malignant cell not be located. This cloaking mitigates toxic side effects usually expected from more conventional chemotherapy.Instead, the particles — crafted small enough to float under the radar of the liver and other cleansing organs — can circulate harmlessly for days or weeks, depending on their engineered size, seeking their prey.
A library of phages — viruses that attack bacteria — was created at UNM’s cancer center by collaborator David Peabody. This permitted researchers to expose the phages to a group of cancerous cells and normal cells, allowing identification of peptides that bind specifically to cancer cells but not normal cells.
“Protocells modified with a targeting peptide that binds to a particular type of cancer exhibit a 10,000-fold greater affinity for that cancer than for non-cancerous cells,” says Carlee.
Jeff adds, “A key feature of our protocell is that the fluid stable supported bilayer allows high-affinity binding with just a few of these peptides overall. This reduces nonspecific binding and immune response.”
The method is being tested on human cancer cells in culture and will shortly be tested in mice at UNM’s nationally accredited cancer center.
Preselecting particles for size
Work still ongoing includes engineering the size of the porous silica particle, which is formed by aerosolizing a precursor solution. The porous nanoparticle fabrication process — called evaporation-induced self-assembly and pioneered in the Brinker lab — produces particles from 50 nanometers to several micrometers in diameter. Particle sizes between 50 and 150 nanometers in diameter are ideal for maximizing circulation and uptake into cancer, so the particles are pre-selected for size before their formation into protocells.
“Their overall dimensions determine how widely they’ll be distributed in the bloodstream,” says Jeff. “We’re altering our synthesis to favor the smaller sizes.”
Also of importance to the circulation time of the particle are its electrical charge and hydrophobicity, which can improve or detract from its ability to remain free of unwanted molecular or energetic entanglements.
Protocells may be ready for testing in humans in as few as five years, researchers estimate.
Jeff is a Sandia Fellow and UNM Regent’s and Distinguished Professor of Chemical and Nuclear Engineering and member of the UNM cancer center.
Other institutions involved in the research include the University of California, Davis, and, in Canada, the University of Waterloo.
Funding was provided by the National Cancer Institute, the National Science Foundation, DOE’s Basic Energy Sciences program, the Air Force Office of Scientific Research, and Sandia LDRD. - Neal Singer
By Neal Singer
Easy-to-follow recipes for radioactive compounds like those found in nuclear fuel storage pools, liquid waste containment areas, and other contaminated aqueous environments have been developed by Sandia researchers.
“The need to understand the chemistry of these compounds has never been more urgent, and these recipes facilitate their study,” principal investigator May Nyman (6915) says of her group’s success in creating a method to self-assemble significant amounts of relevant compounds.
The trick to the recipes is choosing the right templates. These are atoms or molecules that direct growth much the way islands act as templates for coral reefs.
The synthesized materials are stable, pure, and can be studied in solution or as solids, making it easier to investigate their chemistry, transport properties, and related phases.
The compounds are bright yellow, soluble peroxides of uranium called uranyl peroxide. These and related compounds may be present in any liquid medium used in the nuclear fuel cycle. They also appear in the environment from natural or human causes.
Made with relatively inexpensive and safe depleted uranium, the recipes may be adapted to include more radioactive metals such as neptunium, whose effects are even more important to study, May says.
Cesium — an element of particular concern in its radioactive form — proved to be, chemically, an especially favored template for the compounds to self-assemble.
The work was done as part of the Actinide Materials Department of Energy (DOE) Energy Frontiers Research Center (EFRC) led by professor Peter Burns at Notre Dame University. Using the new method, researchers at the University of California-Davis are studying how materials behave in water and in different thermal environments, while researchers at DOE’s Savannah River Site study the analogous behavior of neptunium.
The research will be the cover article of the May 3 online European Journal of Inorganic Chemistry, to be published in print May 13. It currently is highlighted in preview in the online ChemViews Magazine. -- Neal Singer
By Patti Koning
More often than not, you can’t put a price tag on the rewards of scientific research — satisfaction at solving a tough problem, the respect of peers, knowing your work will have a larger impact on the world. But sometimes you can: just ask Kamlesh (Ken) Patel (8621). He recently won the Society for Laboratory Automation and Screening (SLAS) $10,000 Innovation Award for his outstanding podium presentation, “Preparation of Nucleic Acid Libraries for Ultra-High-Throughput Sequencing with a Digital Microfluidic Hub.”
“The SLAS Innovation Award was created specifically to recognize cutting-edge research and the individual behind the work, and Kamlesh’s exploration into nucleic acid libraries for ultra-high-throughput sequencing with a digital microfluidic hub will impact the scientific community for years to come,” says SLAS Innovation Award Committee Chair Jörg Kutter.
While Ken’s name is on the award, he’s quick to point out that his work is part of a much larger effort with contributions from a multidisciplinary team. Led by principal investigator Todd Lane (8623), the RapTOR (Rapid Threat Organism Recognition) Grand Challenge, part of the International, Homeland and Nuclear Security strategic management unit, has the ambitious goal of rapidly identifying and characterizing unknown pathogens. (Lab News, Aug. 26, 2010). In an outbreak scenario, whether the result of bioterrorism or a fast-moving, deadly virus like Ebola, RapTOR could greatly accelerate the response. Until you know what’s making people sick, treatment is like throwing darts.
Leveraging DNA sequencing technology
Using the latest in DNA sequencing technology, RapTOR aims to transform slow, labor-intensive benchtop sample preparation methods to an automated microfluidic platform to create a fast, efficient, and flexible tool. “We’re taking advantage of DNA sequencing technology,” Ken says. “Reading the genetic code, the original building blocks, allows you to begin characterizing a pathogen at the most basic level.”
But getting at those building blocks is not easy — clinical samples are packed with information, most of which is not of use in characterizing an unknown pathogen. For example, more than 99 percent of the DNA in a blood sample is the human genome. DNA in a nasal swab is 90 percent human-derived and much of the rest is garden-variety bacteria. Suppressing all that background DNA is essential to get at the unknown pathogen.
DNA sequencing technology has evolved at a tremendous pace, even surpassing Moore’s Law, the 45-year-old prediction that computer processing power would double every two years. The pre-sequencing steps, however, have hardly changed since the bacteriophage genome was first sequenced in the mid-1970s.
Ken leads the Automated Molecular Biology (AMB) research to both scale down and automate traditional sample preparation methods such as normalization, ligation, digestion, and size-based separation — methods that traditionally require a skilled scientist and take days or even weeks. A critical component of RapTOR is bringing together the different sample prep steps to create a “one-stop shop” that connects to a DNA sequencer. Key to this is the digital microfluidic hub.
The hub is a Grand Central Station for samples, routing them from one step to the next with the flexibility to skip or repeat steps on the fly. But imagine a train station in which some trains are orders of magnitude larger than the others, and some travel at the speed of light and others at 60 mph. The digital microfluidic hub is designed to negotiate these differences, functioning like a train station that can shrink and enlarge trains as necessary and manipulate their speeds.
Instead of trains, droplets are the mode of transportation in this station and voltage serves as the engine. The sample is cargoed within a microliter-scale droplet that is spatially moved across the Teflon-coated surface of the hub when electrostatic forces are appropriately applied. The hub also manages the size of the sample, extracting the right amount for each process.
Reagents dispensed as needed
Size is only one variable that the microfluidic hub manages. Reagents and enzymes necessary for different manipulations are stored in reservoirs connected to the hub and dispensed as needed. “If we need to do a reaction at a set temperature, we can move the sample through a connector tube off the hub into a heated microreactor, perform the reaction at temperature with appropriate reagents, and then redispense the sample back onto the hub for the next processing step,” Ken says. “This is where AMB becomes very powerful — it allows you to connect multiple, different components together through a common flexible interface. All of the microreactors are replaceable, so contamination is not an issue.”
At the start of the Grand Challenge, the AMB team wasn’t sure how efficiently they could repeatedly move droplets on and off the hub. Turns out, the ability to move the droplets is one of the most powerful features of the device.
“We’ve concluded that this is one of the main contributions we’ve made to the field,” Ken says. “Interfacing to a droplet and other microfluidic chips is not just possible, it’s quite effective and a good path forward for processing samples.”
As the AMB team continues to refine the digital microfluidic hub, they are also working on a parallel project to culture cells within the droplets. “There are several exciting advantages to this approach — we can work with different microcultures of cells independently on the device. It is possible to study infections at the cellular level, working with small amounts of cells — thousands at best, not millions — in a hermitically sealed, safe environment,” Ken says. “The end goal is a device that could be used in Biosafety Level-3 containment, enabling safe diagnosis and research of infectious agents.”
Expanding the role of this technology, Ken was recently awarded additional funding from the US Army Criminal Investigations Laboratory to develop a microfluidic-based approach for genotyping in the field. Such a device would allow law enforcement to rapidly process forensic evidence at the crime scene for matching DNA, rather than sending a sample to the lab and waiting days for confirmation, generating immediate intelligence that can then be applied to the unfolding situation.
The digital microfluidic hub could have a wide range of applications — from crime scene investigators to first responders to a general practitioner’s office.
“An eventual goal might be to develop an all-in-one portable device, a sequencer with a sample prep front end. We have portable sensors, so why do not DNA sequencing in the field?” says Ken.“Going to the DNA level gives you so much definitive information, amazing characterization capabilities that we just don’t have today. It’s the new revolution, a really interesting and exciting way of doing research and solving clinical problems. It just makes sense for Sandia to be on that leading edge, applying this research to our national security missions in biodefense.” - Patti Koning