Integration and Invention: Frontiers of Nanotechnology and Biotechnology

Web Sites

Center for Study of Gene Structure and Function at Hunter College
A consortium based at Hunter College that brings together biologists, chemists, biopsychologists, biophysicists, and bioanthropologists working within the CUNY system. Additional information about this conference is available on their Web site: Frontiers of Nanotechnology & Biotechnology.

Dip-Pen Lithography Subgroup
This site by a branch of the Mirkin group outlines the process of Dip-Pen Nanolithography.

Nanosphere Chemistry
This Web site by the maker of the Verigene system, which uses nanospheres with oligonucleotides for diagnostics, explains some of the science behind the commercial system.

National Cancer Institute Alliance for Nanotechnology in Cancer
The NCI Alliance for Nanotechnology in Cancer is a comprehensive, systematized initiative encompassing the public and private sectors, designed to accelerate the application of the best capabilities of nanotechnology to cancer.

National Heart, Lung, and Blood Institute Program of Excellence in Nanotechnology
The National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH) has made four 5-year awards to initiate a unique, diverse, and nationwide Program of Excellence in Nanotechnology.

National Human Genome Research Institute DNA Sequencing Technology Program
Goals and awards made under this NHGRI program are described.

NIH Nanotechnology and Nanoscience Information
This page contains information on currently active NIH and Bioengineering Consortium (BECON) research and training opportunities and listings of funded grants for NIH and BECON program announcements related to nanotechnology and nanoscience.

NIH Nanomedicine Roadmap Initiative
This tran-NIH initiative, a component of the NIH Roadmap for Medical Research, funds centers that are developing a deep understanding of a fundamental biological nanoscale molecular complex or system and, in parallel, developing a research program to apply that knowledge to study a specific medical problem.

Journal Articles

The Oligonucleotide Nanoparticle Conjugate and the "Antisense Nanoparticle"

Giljohann DA, Seferos DS, Patel PC, et al. 2007. Oligonucleotide loading determines cellular uptake of DNA-modified gold nanoparticles. Nano Lett. 7: 3818-3821.

Park SY, Lytton-Jean AKR, Lee B, et al. 2008. DNA-programmable nanoparticle crystallization. Nature 451: 553-556.

Rosi NL, Giljohann DA, Thaxton CS, et al. 2006. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science 312: 1027-1030.

Seferos DS, Giljohann DA, Rosi NL, Mirkin CA. 2007. Locked nucleic acid-nanoparticle conjugates. Chembiochem. 8: 1230-1232.

Seferos DS, Giljohann DA, Hill HD, et al. 2007. Nano-flares: probes for transfection and mRNA detection in living cells. J. Am. Chem. Soc. 129: 15477-15479.

Taton TA, Mirkin CA, Letsinger RL. 2000. Scanometric DNA array detection with nanoparticle probes. Science 289: 1757-1760.

Novel Nano-Biomaterials via Biomineralization

Amemiya Y, Arakaki A, Staniland SS, et al. 2007. Controlled formation of magnetite crystal by partial oxidation of ferrous hydroxide in the presence of recombinant magnetotactic bacterial protein Mms6. Biomaterials 28: 5381-5389.

Matsunaga T, Okamura Y, Fukuda Y, et al. 2005. Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1. DNA Res. 12: 157-166. Full Text

Matsunaga T, Suzuki T, Tanaka M, Arakaki A. 2007. Molecular analysis of magnetotactic bacteria and development of functional bacterial magnetic particles for nano-biotechnology. Trends Biotechnol. 25: 182-188.

Nakagawa T, Maruyama K, Takeyama H, Matsunaga T. 2007. Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite. Biosens. Bioelectron. 22: 2276-2281.

Nakagawa T, Hashimoto R, Maruyama K, et al. 2006. Capture and release of DNA using aminosilane-modified bacterial magnetic particles for automated detection system of single nucleotide polymorphisms. Biotechnol. Bioeng. 94: 862-868.

Osaka T, Matsunaga T, Nakanishi T, et al. 2006. Synthesis of magnetic nanoparticles and their application to bioassays. Anal. Bioanal. Chem. 384: 593-600.

Suzuki T, Okamura Y, Calugay RJ, et al. 2006. Global gene expression analysis of iron-inducible genes in Magnetospirillum magneticum AMB-1. J. Bacteriol. 188: 2275-2279. Full Text

Yoshino T, Matsunaga T. 2006. Efficient and stable display of functional proteins on bacterial magnetic particles using mms13 as a novel anchor molecule. Appl. Environ. Microbiol. 72: 465-471. Full Text

Integrating Biotechnology and Nanotechnology for Frontier Sciences

Bai H, Xu K, Xu Y, Matsui H. 2007. Fabrication of Au nanowires of uniform length and diameter using a monodisperse and rigid biomolecular template: collagen-like triple helix. Angew. Chem. Int. Ed. Engl. 46: 3319-3322.

Gao X, Matsui H. 2005. Peptide-based nanotubes and their applications in bionanotechnology. Adv. Mater. 17: 2037-2050.

MacCuspie RI, Nuraje N, Lee SY, et al. 2008. Comparison of electrical properties of viruses studied by AC capacitance scanning probe microscopy. J. Am. Chem. Soc. 130: 887-891.

Yang L, Nuraje N, Bai H, Matsui H. 2008. Crossbar assembly of antibody-functionalized peptide nanotubes via biomimetic molecular recognition. J. Pept. Sci. 14: 203-209.

Zhao Z , Matsui H. 2007. Accurate immobilization of antibody-functionalized peptide nanotubes on protein-patterned arrays by optimizing their ligand-receptor interactions. Small 3: 1390-1393.

Nanostructured Interfaces for Therapeutic Delivery

Popat KC, Eltgroth M, LaTempa TJ, et al. 2007. Titania nanotubes: a novel platform for drug-eluting coatings for medical implants? Small 3: 1878-1881.

Tao SL, Desai TA. 2005. Gastrointestinal patch systems for oral drug delivery. Drug Discov. Today 10: 909-915.

Tao S, Young C, Redenti S, et al. 2007. Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space. Lab Chip 7: 695.

Nanomechanics of Musculoskeletal and Exoskeletal Tissues

Dean D, Han L, Grodzinsky AJ, Ortiz C. 2006. Compressive nanomechanics of opposing aggrecan macromolecules. J. Biomech. 39: 2555-2565.

Dean D, Han L, Ortiz C, Grodzinsky AJ. 2005. Nanoscale conformation and compressibility of cartilage aggrecan using microcontact printing and atomic force microscopy. Macromolecules 38: 4047-4049.

Han L, Dean D, Mao P, et al. 2007. Nanoscale shear deformation mechanisms of opposing cartilage aggrecan macromolecules. Biophys J. 93: L23-25.

Ng L, Grodzinsky AJ, Patwari P, et al. 2003. Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy. J. Struct. Biol. 143: 242-257.

Ng L, Hung HH, Sprunt A, et al. 2007. Nanomechanical properties of individual chondrocytes and their developing growth factor-stimulated pericellular matrix. J. Biomech. 40: 1011-1023.

Bionanotechnology: an Air Force and DoD Perspective

Liu C. 2007. Micromachined biomimetic artificial haircell sensors. Bioinspir. Biomim. 2: S162-169.

Lynch SA, Desai SK, Sajja HK, Gallivan JP. 2007. A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. Chem. Biol. 14: 173-184. Full Text

Slocik JM, Naik RR. 2006. Biologically programmed synthesis of bimetallic nanostructures. Adv. Mater. 18: 1988-1992.

Slocik JM, Stone MO, Naik RR. 2005. Synthesis of gold nanoparticles using multifunctional peptides. Small 1: 1048-1052.

Tomczak MM, Glawe DD, Drummy LF, et al. 2005. Polypeptide-templated synthesis of hexagonal silica platelets. J. Am. Chem. Soc. 127: 12577-12582.

Nanotechnology, the manipulation of matter on a nanometer length scale to create new properties, has attracted tremendous attention among researchers in a variety of disciplines. The interface between nanotechnology and biology provides some of the most exciting opportunities in research today, including new ways to detect and diagnose disease, new ways to deliver therapy, and new ways to probe basic biological processes. On January 18, 2008, an array of stimulating speakers and members of Hunter College and the surrounding community convened to discuss opportunities at this interface, at the 21st Annual International Symposium of the college's Center for Study of Gene Structure and Function.

Biology and nanotechnology have much to offer each other.

As summarized by Hiroshi Matsui of Hunter College, biology and nanotechnology have much to offer each other. In one direction, biology offers an array of tools that can be exploited to manipulate materials on the finest length scales. Matsui, for example, described using the highly specific recognition between biomolecules to provide "assembly instructions" that could wire up integrated circuits. He also used specific biological patterns to nucleate mineral growth. Conversely, nanotechnology offers great promise in the development of new medical treatments and tools for basic research.

Like Matsui, Chad Mirkin of Northwestern University has used molecular recognition to assemble large structures (featured on the January 31 cover of Nature). In addition, however, Mirkin is actively pursuing the other side of bionanotechnology, in which fine-scale manipulation provides new tools for biology and medicine. He and his team attach large numbers of oligonucleotide chains to gold nanoparticles. In the process, they create a new entity with properties not possessed by either original component. These hybrid particles are already in commercial use for diagnostics, and the researchers have shown that they can also deliver nucleic acids directly into a variety of cells for potential therapeutic uses.

Tadashi Matsunaga of Tokyo University of Agriculture and Technology has explored the biological processes by which bacteria use protein seeds to grow magnetic nanocrystals. These tiny crystals are ideal for magnetic separation technology for biological research.

Tejal Desai of the University of California, San Francisco, is exploiting nanotechnology to deliver traditional drug-based therapies more effectively. By tailoring the nanoscale architecture of either oral drug carriers or the surfaces of implants, she hopes to deliver the drugs more steadily, with reduced side effects. In addition, nanoscale structuring often promotes intertwined growth of cells, whether on an artificial hip or a retinal implant.

Christine Ortiz of Massachusetts Institute of Technology has also explored the way that tissue macromolecules are synthesized in artificial scaffolds. She applies the tools and concepts of mechanical engineering at the nanometer scale to understand the mechanics of structural tissues like cartilage, and to reveal how the tissue properties reflect the underlying molecular structure and interactions.

Morley Stone of the Air Force Research Laboratory also discussed the use of specific peptide sequences to encourage the growth of chosen materials. Moreover, by tethering multiple sequences together, he and his colleagues have built customizable "surfactants" that modify the interface between any chosen materials. These techniques could allow the creation, for example, of multifunctional fabrics, with antimicrobial or electrical properties that complement their mechanical properties. The challenge, he observed, is knowing when to draw on nature only for general principles and when to copy it exactly.

Jeffery Schloss of the National Human Genome Research Institute endeavored to describe the vast range of bionanotechnology activities supported by the National Institutes of Health. Important recent efforts aim to characterize nanomaterials, as well as their health impacts, in both biomedical and industrial applications. In addition to the centrally formulated funding opportunities that specifically address needs of particular programs, however, he emphasized that most bionanotechnology research arises from the ideas of individual investigators.

The talks presented at Hunter illuminate many diverse aspects of the biology and nanotechnology. Although it is not always clear which applications will be the most important, it is clear that this will be an exciting interdisciplinary field for many years to come.

Speaker:
Chad Mirkin, Northwestern University

Highlights

• Oligonucleotides tethered to gold nanoparticles form a new entity with promise for diagnostics and therapeutics.
• Choosing the sequences on the nanoparticles and the complementary sequences on molecules that link them can promote self-assembly into the desired crystal structures.
• Complementary oligonucleotides pair cooperatively when bound to gold, enhancing both their sensitivity and selectivity.
• Oligonucleotide-covered nanoparticles rapidly enter a variety of cells, so they could be useful for delivering antisense DNA or other nucleic acid payloads.
• A single "nanoflare" particle both binds RNA and reports the binding through fluorescence.

Programming interactions with oligonucleotides

Chad Mirkin and his group at Northwestern University are pursuing a variety of approaches to control materials on a 1–100nm length scale. Their well known "dip-pen lithography" technique, for example, uses scanning-probe microscopes to spatially place molecules, including those, like proteins or oligonucleotides, that are too fragile for conventional lithgraphic methods. Recently they have extended this method from a single tip to as many as 55,000 cantilevers in parallel to create patterns over areas large enough to be measured in square-centimeters. They are also exploring supramolecular assembly and anisotropic nanostructures.

In his talk at Hunter, Mirkin focused on nanoparticles decorated with oligonucleotides. The promising and surprising properties of this combination earns it a unique new name, he said, opting for "oligonucleotide–nanoparticle conjugate." However awkward this nomenclature, these particles could be a powerful tool in both diagnostic and therapeutic bionanotechnology.

"Our view was that we would have to rely on inspiration from biology."

The researchers first developed these particles for biomolecule-directed assembly, exploiting the ability to routinely synthesize arbitrary nucleotide sequences. Base pairing between complementary sequences then provides the detailed assembly instructions referred to by Matsui. "Our view was, in the mid-90s, that we would have to rely on biological systems, or at least inspiration from biology."

Most of their work has used gold particles, Mirkin said, although the team has explored other noble metals as well as semiconductors, insulators, and magnetic nanoparticles. Gold has an advantage because it is usually prepared using a technique that leaves it covered in a shell of weakly bound ligands, so that oligonucleotides linked to thiols can displace them in large numbers—as many as 200 per particle.

In recent work, Mirkin and his colleagues have chosen sequences that promote the formation of crystallites with either a face-centered- (fcc) or body-centered-cubic (bcc) crystal structure. The researchers used the same oligonucleotide-decorated spheres in both cases, but changed the sequences used to bind them together. For the fcc structure, a single strand links the spheres, encouraging close packing, while for the bcc structure, two distinct strands make the connection.

Greater than the sum of the parts

Even when the nanoparticles don't form a regular crystal, their interaction can be seen "with the naked eye," Mirkin obvserved. Close proximity shifts the plasmon resonance of the gold spheres, changing the color from red to what Mirkin called "Northwestern purple." This color change sensitively monitors the binding of complementary oligonucleotides.

Densely attaching oligonucleotides to a gold nanoparticle creates a new entity with promise for both diagnostics and therapeutics.

A critical—and unexpected—observation was that oligonucleotides pair much more suddenly when they are densely attached to spheres than when they are floating freely. Instead of requiring a temperature decrease of about 25 degrees to get complete pairing, the transition for the nanoparticles system occurs in a single degree.

"We get particles that have properties that are very different from the inorganic core and the oligonucleotides from which they derive," Mirkin said. "That's one of the names of the game in nanoscience." One reason for the sharp transition is that there are multiple links between spheres. In addition, he suggested, the highly charged oligonucleotides attract oppositely charged counter ions which help to stabilize other oligonucleotides nearby. "It's a combination of these two effects that leads to these very narrow transitions."

In diagnostic assays, the sharper transition improves the selectivity of the assay, Mirkin said, to the extent that the binding change from a single point mutation can be detected. The narrower transition also improves the sensitivity, he observed, since weakly bound species completely unbind with no loss of the target species. A commercial tool based on this research uses the catalysis of silver reduction by the gold nanoparticles to achieve a 100,000-fold increase in sensitivity.

Delivering the goods

Although oligonucleotide–nanoparticle conjugates have great promise in these diagnostic applications, Mirkin spoke even more enthusiastically about their potential therapeutic use. In this context, the particles act in some ways like a customizable antisense RNA, but with important advantages, including tighter binding to their target because of the cooperativity.

The first challenge is to see whether the particles enter cells at all. Many other carriers have been used for this purpose, and "the lore in the literature is that you need positively charged entities," Mirkin said. In contrast, these conjugates are "some of the most negatively charged materials you can get," because of the oligonucleotides on the surface.

"We have yet to find a cell line where we don't get greater than 99.9% transfection."

Nonetheless, the particle complexes are very effectively taken up by a wide variety of cell lines. "We have yet to find a cell line where we don't get greater than 99.9% transfection," Mirkin said, "including nerve cells." They enter by endocytosis, he observed, but nonetheless retain their discrete character rather than being digested. Their resistance to degradation by nucleases may reflect their steric inaccessibility on the nanoparticle, Mirkin suggested. "That should translate into higher activity."

The uptake appears to be assisted by specific extracellular proteins, which tend to partially cancel the charge, the researchers found. Identifying these proteins could even avoid the need for the nanoparticles, Mirkin said, since they might act as a universal transfection agent when attached to "whatever it is you want to carry into the cell."

Mirkin's team confirmed that the nanoparticles can bind to messenger RNA in cells to reduce expression of green fluorescent protein in vitro. "We get about 60% knockdown, which is about as good as you can do under conditions where we have a lot of cell division," he said. Thus the oligonucleotide-nanoparticle conjugate shows great potential for delivering nucleic acids into cells.

Combining functions

The binding to complementary RNA can be made stronger, Mirkin noted, by using "locked nucleic acid," or LNA, chains, in place of the DNA oligonucleotides. (LNA is a bicyclic high affinity RNA analog in which the ribofuranose ring is locked in the 3′-endo conformation.) Team member Dwight Seferos used these LNA-modified particles to persistently knock down the activity of the survivin gene, which Mirkin said makes lung-cancer cells "effectively immortal."

Finally, Mirkin illustrated steps toward combining diagnostic and therapeutic activity in a single, multifunctional particle. His team created what they call a "nanoflare," in which a short complementary sequence weakly binds a fluorophore close to the gold nanoparticle, which quenches its fluorescence. When the longer target sequence binds to the complementary oligonucleotide, it also displaces the fluorophore, allowing it to emit light. "The beauty of this," Mirkin said, is that researchers can use nanotechnology to "build one kind of structure that gives you all of those capabilities."

Speaker:
Hiroshi Matsui, Hunter College

Highlights

• Nanotechnology lets researchers create powerful materials for biology and medicine, and biology can provide new ways to solve nanotechnology and engineering problems.
• To wire complex integrated circuits, the molecules must be told where to go.
• Antibody–antigen interactions can specify arrangements of labeled nanotubes, even arrangements in which they cross each other.
• Triple-chain peptides, like those found in collagen, form a template for crystals of prescribed diameter and length.
• The electrical capacitance of viral strains can be used to distinguish among them.

A two-way street

"Bionanotechnology is a great example for interdisciplinary research," said Hiroshi Matsui of Hunter College, who helped to organize the symposium. The flow of ideas goes in both directions between nanotechnology and biology, he said.

Drawing from his own research, Matsui illustrated bio-inspired assembly of nanowires. Such a process, he suggested, could help to extend integrated-circuit technology. Wiring together multiple microprocessors in a controlled way could extend the power of computers even when the individual chips can no longer be improved.

"You have to tell the atoms or molecules where to go."

Researchers have demonstrated self-assembly of simple, regular structures, but technology requires "much more complex structures with very high information content," Matsui said. "You have to find the information channel to convey the assembly instructions that tell the atoms or molecules where to go."

As a start toward communicating these assembly instructions, Matsui and his colleagues attach antigens such as biotin to separate gold pads on a substrate. When they decorate the ends of nanotubes with the complementary antibody, the nanotube naturally tends to connect between the pads.

The researchers extended the technique to attach two or three different kinds of wires in different places. For example, using mouse and human versions of the antigen/antibody pairs, they created a self-assembled arrangement in which the wires crossed over each other. This is a critical requirement for building complex circuits in which devices are not just connected to their neighbors. "Crossing is very important." Matsui said, "It's hard to do."

More conductive wires

The peptide nanotubes Matsui used to demonstrate this self-assembly are not practical for wiring, because they are electrically nonconductive. His team has explored several ways to make them more conductive after they are in place.

For example, by introducing into the peptide a motif from the zinc-finger-like matrix protein M1 from influenza virus, they nucleated growth of the semiconductor zinc sulfide. This technique produces ZnS with a wurtzite crystal structure at temperatures as low as room temperature. In related work, the team grew particles of zinc oxide and platinum of various sizes on their tubes, effectively tuning the resulting electrical properties. "The fine-tuning of the distance and particle size are crucial," Matsui said.

Control of the length of nanowires is "extremely difficult."

In recent work, the researchers have explored triple-chain-peptide templates, based on a motif found in the structural protein collagen. This strategy holds the promise of prescribing the length of nanowires directly, simply by adjusting the amino acid sequence. Although there are many ways to control nanowires' diameter, Matsui said, "control of the length is extremely difficult."

Unfortunately, under typical conditions for growing semiconductors, even this tough triple-chain motif is not stable, Matsui observed. His Hunter colleague Yujia Xu solved this problem by using recombinant technology, introducing a structure at the end of the wire to bind the strands firmly together. With this improvement, the team grew ZnO crystals on the triple-chain peptide even well below room temperature.

Distinguishing viruses

Matsui also described a project using tools of nanotechnology for biological applications. Specifically, the technique exploits the differing dielectric properties of the components of a virus, including the proteins of the envelope and capsid as well as the genetic core. "Since we have a sensitive measurement of capacitance, maybe we can distinguish viruses based on different structure and different content," he said.

Matsui showed what he called a proof of concept using atomic microscope with a conductive tip (C-AFM) to measure the capacitance when the tip was placed over a virus. The capacitance, clearly distinguished different strains of virus, as well as mutations that removed the glycoprotein from a virus.

Thus, Matsui demonstrated that the ability to work on the nanoscale has enabled researchers to borrow from biology and, at the same time, gain a better understanding of organisms from viruses to ourselves.

Speaker:
Tejal Desai, University of California, San Francisco

Highlights

• Synthetic nanoscale pores supply drugs more steadily in time than a traditional dissolving matrix does.
• The surfaces of standard medical implants can be modified to release drugs and promote integration with tissue.
• Oral ingestion of nanoengineered structures can deliver drugs in high, controlled concentration directly to epithelial cells.
• Endowing these carriers with nanoscale hairs makes them adhere to the intestinal walls.
• Drugs to treat retinal degeneration can be delivered using a structured plastic, which also serves as a scaffold for new cells.

Special delivery

Tejal Desai of the University of California at San Francisco uses the tools of microfabrication and nanotechnology to improve the delivery of therapeutic drugs. She emphasizes that different therapeutic interfaces have different requirements, so no single approach is likely to excel in all of them. Nonetheless, Desai, said, "there are some areas that have been challenges for decades that I think we finally have the tools to come closer to finding solutions for."

Nanotechnology can deliver drugs in areas that have been challenges for decades.

Applying nanotechnology to medicine doesn't necessarily require futuristic floating nano-robots, Desai said. "We're trying to get at how architecture at this scale can play a role in facilitating how cells interact with therapeutic molecules."

For example, Desai showed that the release of a drug from structures containing nanometer-sized channels can be very steady in time, because it is limited by transport through the channel. This steady release contrasts strongly with the traditional diffusion-limited release, for example from a pill, which starts quickly but rapidly decreases as the drug concentration in the body comes to equilibrium with that in the pill. With pills frequent dosing is usually required to maintain a drug concentration that is high enough to have an effect but is below the toxicity threshold. The steady release from nanoporous structures may get around this problem.

Desai and her team used silicon microfabrication techniques to create nanopore arrays that steadily release proteins like albumin and interferon. By using pores as small as seven nanometers in diameter, they even got the same constant release for the tiny molecules of glucose.

Modified implant surfaces

An inorganic nanoporous membrane can be incorporated into an implantable drug-release device, Desai said, releasing drugs for weeks. This fabrication strategy is relatively expensive, however.

She and her group have been exploring other ways to make well-controlled nanostructures that could be used with medical implants. They have used inorganic materials such as zirconium, titanium alloys, and tantalum, as well as organic materials. They employ both traditional fabrication techniques, like anodization and reactive-ion etching, as well as nanotemplating, which creates an inverted version of an existing structure.

You can introduce a controlled architecture at the interface while preserving the mechanical properties of an implant.

One particularly interesting structure consists of titania nanotubes. These nanotubes can be grown from bulk titanium alloy, such as that used in conventional implants like hip replacements or cardiovascular stents. "You can take the bulk material and simply modify the top several hundred nanometers," Desai observed, "to get this very controlled architecture at the interface."

For this application, using materials that are already compatible with—and approved for—long term use in the body is a major advantage. Polymeric coatings like those used in drug-eluting stents, in contrast, have had problems with degradation and consequent inflammation. "If one can embed a nanostructure within the material," Desai said, "one can avoid this use of multiple materials, and materials that degrade in the body."

Desai's team has demonstrated modified devices impregnated with antibiotic that "significantly reduce the colonization of bacteria." In addition, "the same nanostructures," Desai said, "serve as a template or an architectural cue for cells to differentiate and increase extracellular matrix deposition." Markers of matrix production are higher on the textured surfaces, she said, resulting over time in "really nice interdigitation of the native bone to the implant interface." By promoting this "osteointegration," nanotextured surfaces could improve the longevity of implants.

The oral route

Desai also described how nanotechnology can improve oral drug delivery. Although this route is familiar and easy for the patient, which can improve compliance, she said, "the oral route is one of the most complicated in terms of delivery." A delivery vehicle has to survive extreme acidity and digestive enzymes as well as mechanical agitation, and transfer its payload across a mucous layer and tight cellular junction whose purpose is to keep things like pathogens out.

A swallowed nanoengineered particle can adhere and controllably deliver drugs to cells that line the intestine.

Improving oral delivery depends on two factors, Desai said. "If you can prolong the residence time of a drug or a drug carrier at the site of interest, and if you can improve the intimacy of contact between the drug-delivery device and your absorbing surface, then you can actually get better bioavailability." To accomplish this, she and her team envision a flat particle that would interdigitate with the epithelial surface and release drug asymmetrically, toward the intestinal wall.

Making the carrier flat dramatically improves the stability, Desai said. "Once it's bound, it stays bound." As an additional anchor, the team added a coating of nanowires, grown by chemical-vapor deposition. The wires, she said, allow the particle to strongly interdigitate with the finger-like villi that line the intestinal wall. This results in strong binding, both in lab cultures and in animal tests.

Desai and her colleagues have built carriers out of silicon as well as organic materials. By using their nanochannel controlled-release system, they can separately tune the delivery of multiple drugs directly toward the cells of the intestine.

Retinal treatment

Nanostructures can act as scaffolds for retinal progenitor-cell delivery.

Delivering drugs to the back of the eye is another challenging problem to which nanotechnology can contribute a solution. Although diseases such as macular degeneration frequently affect this region, it is difficult to access without disruption.

Desai and her team have been exploring what they call "nanostructure thin films," films of polymer, less than ten micrometers thick, containing nanowire morphologies. A useful feature is that they naturally wrap themselves into tight tubes, but unfurl when placed in the back of the eye.

The nanostructured films can deliver drugs, but they also act as scaffolds for retinal progenitor cells. The progenitor cells differentiate into mature photoreceptors on these films, which then degrade away, Desai said. Current research aims to understand how the nanostructure can orient the cells and help them to integrate with the host.

Speaker:
Christine Ortiz, Massachusetts Institute of Technology

Highlights

• The mechanical properties of tissue reflect its constituent molecules and their interactions, as well as their cellular and tissue-level arrangement.
• The stiffness of our joints relates directly to the bottle-brush arrangement of charged polymers that makes up cartilage.
• Age and other damage cause the polymers to get shorter and less stiff.
• When they are compressed beyond a certain point, both the interacting molecules and the tissue get dramatically stiffer.
• Interdigitation of different molecules affects the tissue properties.
• The study of single cells growing in a matrix can help researchers understand why artificial cartilage-like tissue lacks many of the properties of natural cartilage and guide steps to improve it.

A multi-level approach

Christine Ortiz of the Massachusetts Institute of Technology, in collaboration with Alan Grodzinsky (also of MIT), investigates the mechanical properties of materials at the nanometer scale. These nanomechanics methods are especially revealing for biological materials, which often contain structure at many different length scales.

"We combine observations at different length scales to understand biomechanical function."

Ortiz describes a quad-tiered approach for each material system. At the finest level, her team looks at individual molecules, directly imaging the configurations of macromolecules that make up complex tissues. To understand the interactions that lead these molecules to assemble in particular ways, they use artificial surfaces with similar but controlled surfaces. At a larger scale, they look at individual cells and the matrix that surrounds them. Finally, at the fourth level, the researchers characterize the small-length-scale nanomechanical properties at the level of intact tissues.

By looking at different length scales, Ortiz's team aims to relate the large-scale mechanical properties of tissues to their underlying structure. For example, the group studies the exoskeletons of various creatures with the hope of creating tough but lightweight materials that could serve a purpose such as body armor for soldiers.

A hierarchical structure

Ortiz described how nanomechanical investigation has illuminated the properties of cartilage, both in its normal state and as it degrades as a result of age.

Cartilage is "a fiber-reinforced composite," Ortiz said. The chondrocyte cells that build the tissue represent only a few percent of its volume. Through the remainder runs a matrix of highly charged polymers in a hierarchical, bottlebrush configuration, with glycosaminoglycans surrounding aggrecan chains, which in turn decorate a hyaluronan backbone.

"The stiffness in our joints is determined by polymer physics; it's based on macromolecular-level repulsion."

The high negative charge on these proteins attracts balancing positive ions from the surrounding solution. The microscopic repulsion between the chains provides about half of the elastic modulus of cartilage, Ortiz said. "The stiffness in our joints is determined primarily by polymer physics. Electrostatic double-layer repulsion between these polymer chains, in addition to nonelectrostatic components such as configurational entropy—that's what enables us to walk."

Ortiz's group has looked at individual matrix molecules, and found that the polymers are much shorter in older animals. "As you get older, enzymatic degradation contributes to the decrease in glycosaminoglycan chain length," she said. The decreased stiffness at the molecular level corresponds to a similar degradation at the tissue level.

Made to stick

The overall properties of cartilage are sensitive to the individual molecules, but also to the way they stick together. Ortiz and her team developed techniques to measure this stickiness directly, using an atomic force microscope tip covered with aggrecan to gently push and tug on a carpet of the same molecules on a surface.

"The molecular interactions are reflected in the whole tissue."

As the researchers push the materials together, the stiffness rises slowly. When the material is compressed by about 50%, though, the stiffness rises dramatically. "This is also a feature of the tissue," Ortiz said. "The molecular interactions are reflected in the whole tissue."

By comparing the response of molecules near a hard wall to those near a soft surface, the researchers were able to clarify a longstanding question relating to the nature of the intermolecular arrangement. "They're not just doing straight compression," Ortiz said, but our data suggests that there is actually some interdigitation with each other" that modifies their interaction.

Starting over

Naturally, explaining the properties of diseased and healthy cartilage is less satisfying than it would be to replace damaged tissue. One approach to cartilage repair is to grow new cartilage on a scaffold. So far, the resulting material is generally inferior, Ortiz said.

Using nanomechanics, Ortiz and her colleagues are exploring the properties of individual cartilage cells, chondrocytes, as they secrete a matrix around themselves. Surprisingly, in the early stages of this process, the combination of the cell and matrix is much softer than the cell alone. Only as the extracellular matrix matures does the complete system gain its traditional stiffness.

These techniques hold the promise of identifying the different contributions to the special properties of the artificial cartilage grown, for example, from mesenchymal stem cells. By comparing the nanomechanical properties as researchers vary the cell type, the matrix, the growth factors, and other culture conditions, they may be able to learn how to make a tissue that can relieve the sometimes debilitating discomfort of millions of people with osteoarthritis.

Speaker:
Morley Stone, Air Force Research Laboratory

Highlights

• Bionanotechnology offers many nonmedical opportunities for military applications, including optics, catalysis, and sensing.
• A library of peptide sequences from phages can nucleate the biomineralization of a variety of inorganic materials.
• Tethering different blocks together creates a customized surfactant that promotes interaction between specific materials.
• Although many manmade sensors rival their natural counterparts, synthetic flow sensors are much less sensitive.
• Synthetic versions of riboswitches offer a way to build self-replicating chemical detectors.

A bottom up approach

Morley Stone described some of the ways in which the Department of Defense, and his team at the Air Force Research Labs, are exploring bionanotechnology for non-medical uses. He cautioned that building complex systems—analogous to today's integrated circuits—will require researchers to face up to the need for defect tolerance. "When you start to build things from the bottom up, you can't help but start to run into incredibly high defect rates," he said, adding that "tolerance is something that biology does quite well."

"When you start to build things from the bottom up, you can't help but start to run into incredibly high defect rates."

Like others, Stone and his colleagues have used "phage display" to screen a library of peptide sequences from phages. Although this technique is well known for identifying protein interactions, it can also be used for inorganic materials to find "what kind of interaction with a given surface would be important if you want to nucleate and grow that material," he observed.

The team has assembled a library of peptide sequences useful for growing, or self-assembling, various materials, ranging from carbon nanotubes to noble metals, as well as a variety of oxides, Stone said. In most cases, the nature of the interaction that leads to nucleation and growth is poorly understood.

Combining functions

Stone described a variety of applications in which peptide blocks with distinct affinities were linked to form what he called "bifunctional biotemplates." These hybrid entities can help to assemble desired structures by positioning blocks in one location rather than another, for example.

In another application, one sequence promotes the formation of a nanoparticle, while the other acts to recognize, for example, a mercury ion. The agglomeration of the particles causes a detectable spectral shift, similar to—but in this case more sensitive than—those described by Chad Mirkin.

The researchers also combined a block that binds silk fibers with others that promote the formation of materials with the properties they desired to incorporate into what eventually would become a cloth product. They nucleated the growth of semiconducting zinc oxide or conductive gold nanoparticles, for example, or lysozymes containing antibiotic. "This is one step toward that vision of a self-decontaminating suit," Stone observed.

More generally, tethering peptides with different properties can create a controllable interaction between materials. "The peptides are really great in helping to mediate that interface," Stone said. In addition to promoting the nucleation and growth of specific materials or phases, they provide a "unique surfactant."

Going for the flow

When it comes to sensing applications, "we are very amateur nanotechnologists," Stone asserted. By contrast, "the insect world is filled with some very unusual sensory structures" that we might do well to understand. In particular, he said, "biological organisms can sense flow—and changes in that flow—orders of magnitude more sensitively than we can with traditional synthetic flow sensors, reportedly detecting energy as small as 10⁻¹⁹ joules."

Stone briefly described research by Chang Liu, now at Northwestern University, creating a flow sensor out of a silicon cantilever coupled to an elastomeric hair. Surprisingly, emulating the gelatinous "cupula" surrounding natural hair-bundle sensors dramatically improves the coupling of vibrations to artificial sensors as well. "Biological analogs," Stone concluded, "can give you very non-obvious insight into why you would want to do certain things."

Harnessing riboswitches

A riboswitch is a way to build self-replicating circuits.

Stone ended by describing a cell-based fluorescence assay that amplifies an incoming chemical signal. The assay uses a synthetic riboswitch to modulate the catalytic activity of a protease in the bacterium Escherichia coli. The riboswitch is based on modular aptamer domain in a messenger RNA. When the target chemical is present, the mRNA undergoes a conformational change that allows translation into the TEV (tobacco etch virus) protease.

To detect the protease, the researchers also introduced green and blue fluorescent proteins, linked by an 18-amino-acid segment that includes the TEV cleavage site. Ordinarily, excitation of the blue fluorophore transfers to the nearby green fluorophore. The presence of theophylline reconfigures the aptamer to allow protease translation, which cleaves the molecule and quenches the energy transfer. "This is not a fast assay," Stone admits, but "sometimes that's OK," and the biological specificity of the technique could be very powerful. Although the first demonstration responded to the caffeine analog theophylline, it could also be adapted for explosives-related molecules like 2,4-dinitrotoluene (DNT) by changing the aptamer domain. The promise of the method, Stone said, is that "it's a way to build self-replicating circuits."

Speaker:
Jeffery Schloss, National Human Genome Research Institute, NIH

Highlights

• From the earliest stages of the federal nanotechnology thrust, the National Institutes of Health (NIH) have encouraged applications in diagnosis, therapy, and fundamental biology.
• The National Cancer Institute and other institutes have established centers for exploring medical uses of nanotechnology.
• Most NIH research funds for bionanotechnology come through investigator-initiated grants.
• Recently expanded activities at NIH aim to establish principles for evaluating potential risks of nanomaterials in both medicine and manufacturing.
• Researchers are exploring the possibility of sequencing an individual DNA molecule by threading it through a nanometer-sized pore.

A continuing commitment

The National Institutes of Health have actively encouraged bionanotechnology research since the advent of the National Nanotechnology Initiative in the late 1990s, says Jeffery Schloss. Schloss, program director at the National Human Genome Research Institute (NHGRI), is also cochair of the Trans-NIH Nanotechnology Task Force. The early goals, he said, have held up well: high-sensitivity assays and diagnostics for early detection of disease, as well as targeted and multi-functional delivery of molecular therapies, and the use of nanotechnology tools to understand biology.

"Most research support for nanotechnology comes through investigator-initiated grants."

Some of this support comes through networks of centers focused on specific challenges, such as the Nanotechnology Alliance of the National Cancer Institute (NCI). One such center, for example, supports research by Chad Mirkin. Schloss noted that the network aims to be comprehensive, encompassing early detection, imaging, and therapy in cancer. He highlighted the notion of functional imaging, in which investigators are "not looking at the tumor mass per se, but for biochemical processes going on in the tumor area." Overall, Schloss said, the NCI program provides roughly one quarter of the $215 million annual investment in nanotechnology at NIH.

Despite the success of these centers, Schloss emphasized, "the majority of the research support for nanotechnology—and most other things at NIH—comes not through specific programs, but through investigator-initiated grants." These grants, which are spread throughout the NIH, include not just traditional hypothesis-driven research, but also discovery-driven, developmental, and design-directed activities.

Predicting risk

One working group of the task force has helped the National Institute of Environmental and Health Safety (NIEHS) to establish the NanoHealth Enterprise Initiative. With continued cooperation from the Environmental Protection Agency (EPA), the National Science Foundation (NSF), and the National Institute for Occupational Safety and Health (NIOSH), and through outreach to non-federal agency entities, this initiative aims to "examine the fundamental physical and chemical interaction of engineered nanomaterials with biological systems, at the molecular, cellular, and organ level," Schloss said. "We want to be able to understand and actually predict, if you made a [nano]material ... how it will interact with biological material," including issues of biocompatibility and toxicity.

In a related effort, NCI supports a partnership with FDA and the National Institute of Standards and Technology (NIST) called the Nanotechnology Characterization Laboratory, which aims to facilitate assay development toward regulatory approval for various types of nanomaterials. This is critical since, one hopes, these materials will have completely different properties than bulk materials. "You have to have all of that coming together with the applications research, if you'll ever get this stuff into the clinic," Schloss said.

Nanopore sequencing

In light of the sweeping scope of NIH-supported research, Schloss could only give brief glimpses of particular research projects. He emphasized projects not mentioned by other speakers at the symposium, such as the versatile dendrimer platform for combining multiple functions in synthetic molecular assemblies. He also highlighted the potential of arrays of carbon nanotube bundles for electrically probing brain function.

Can you use a nanopore, thread DNA through it, and read off the sequence?

In his role at NHGRI, Schloss is looking for cheaper ways to sequence DNA. He described one bold strategy, which he said is "a good approach, it just happens to be nano," that several teams are pursuing. The idea is to thread DNA segments through a nanoscale pore—either a naturally occurring or an artificial one—and read the sequence as the molecule passes through.

One idea, for example, involves monitoring the ion current through the pore as single-stranded DNA passes through it, if it's possible to engineer pores so the four bases will interrupt the ion flow differently. Positing that one needs to make the structures more robust, some teams work with nanomachined pores, 1–2nm in diameter, in a silicon-based membrane.

In addition to creating the pores, sequencing may require incorporating electrical detection circuitry or other sensors directly in the pore. "It's very tough," Schloss admitted. In another approach, some researchers are trying to exploit the natural base discrimination of DNA polymerase for the detection.

These examples only hint at the promise of bionanotechnology for medicine, in viewing biological systems as consisting of interchangeable parts, Schloss said. "Evolution has done it. Can we learn how to do it in the laboratory?"

How can the nanotechnology community develop tools and procedures to design reliable complex systems?

Can bio-inspired designs allow manmade sensors to match the sensitivity of their biological counterparts?

How can researchers grow artificial cartilage that will work as well as the real thing?

What features of nanometer-scale scaffolds will best encourage cell growth?

Can self-assembled wiring be complex enough and conductive enough to solve problems for integrated circuits?

How can the health, environmental, and safety risks of nanotechnology be responsibly addressed?

Can specific proteins act as universal transfection agents without involving nanoparticles?