Our Science Initiatives – Nanotechnology
CCR investigators support NCI’s Alliance for Nanotechnology in Cancer through the Nanobiology Program and also work in close collaboration with the Nanotechnology Characterization Laboratory.
CCR’s Nanobiology Program (CCRNP) promotes multidisciplinary research that leads to the development of tools for nanoscale, biologically based strategies to prevent, diagnose, and treat cancer, AIDS, and biodefense-related viral diseases. The CCRNP works to understand the structure and function of biomolecules to aid in the design of nanodevices for in vivo imaging, diagnostics, and targeted drug delivery systems.
Principle investigators who are active in the program have several areas of interest, including membrane structure and function, protein interactions, biomedical image and database analysis, structural bioinformatics, structural glycobiology, molecular information theory, and computational RNA structure.
CCRNP investigators have formed collaborative partnerships with CCR investigators in other programs, labs, and branches, including the Molecular Imaging Program, the Radiation Oncology Branch, the Laboratory of Cell Biology, and the Laboratory of Medicinal Chemistry.
The Nanotechnology Characterization Laboratory (NCL) conducts preclinical efficacy and toxicity testing of nanoparticles intended for cancer therapeutics and diagnostics. The laboratory provides critical infrastructure support to NCI’s Alliance for Nanotechnology and is a partnership between the NCI, the U.S. Food and Drug Administration, and the National Institute of Standards and Technology. The NCL assists the bionanotech community in identifying structure-activity relationships related to nanoparticle safety and efficacy.
Research News
• CCR Nanobiology Program
• Collaborations
• Nanotechnology Characterization Laboratory
CCR Nanobiology Program News
Computational Approaches to the Construction of RNA-Based Nanodevices for Cancer Biology
CCR computational scientists are using their multidisciplinary expertise in algorithm design, RNA structure prediction and analysis, molecular modeling and high-performance computing to develop RNA-based nanodevices. Most of the recent work on the use of biomolecules for nanodevices has concentrated on DNA and proteins, but only in a few cases has taken into account the use of RNA molecules to construct such devices. An important property of protein-free RNA systems is that human immune response is typically low or undetectable. RNA-based systems are also very attractive for nanobiology because they are relatively easy to synthesize, and studies are finding RNA to be an equally, if not more, desirable material for designing functional nanostructures. RNA nanoconstructs can serve as building blocks and scaffolds to assemble more complex functional objects. In some cases, these shapes can self-assemble and can be of arbitrary sizes, while in other cases, they may be tailored for very specific purposes and must be within the correct size range (~20 nanometers) for the delivery of functional groups (RNA, peptides, or otherwise) to cells for controlling cell genetics, including cell death, or visualization of delivery pathways. Other potential applications include biosensors and crystallography substrates.
CCR scientists are investigating the structural properties of various RNA motifs and are working on computer applications for the interactive design of RNA nanostructures. It will be possible to visualize and manipulate three-dimensional (3D) representations of RNA interacting with other RNAs or other biomolecules. As part of this effort, an algorithm is being developed which generates nucleotide strands that form stem and loop motifs that trace a given target 3D structure. Current and future efforts involve the development of algorithms for automated sequence design, rigidity analysis, and assembly process optimization.
Hastings WA, Yingling YG, Chirikjian GS, and Shapiro BA. Structural and dynamical classification of RNA single-base bulges for nanostructure design. J Comput Theor Nanosci 3: in press, 2006.
Contact: Bruce A. Shapiro, Ph.D.
Novel Human Monoclonal Antibodies to Components of the IGF System that Potently Inhibit the IGF-IR Signal Transduction Function
CCR researchers have developed a panel of novel human monoclonal antibodies against IGF-II, IGF-I, and IGF-IR that may potently inhibit the IGF-IR signaling function and IGF-II–mediated signaling through the insulin receptor. One of these antibodies, m610, bound with nM affinity to IGF-II and inhibited IGF-IR phosphorylation and phosphorylation of the downstream kinases Akt and MAPK as well as migration and proliferation of cancer cell lines. The researchers hypothesized that targeting IGF-II, in addition to blocking its interaction with the IGF-IR, would block that portion of the signal transduction through the insulin receptor due to its interaction with IGF-II. Lowering its level may not induce upregulation of its production as for IGF-I. Finally, targeting a diffusible ligand, such as IGF-II, may not require penetration of the antibody inside tumors but could shift the equilibrium to IGF-II complexed with antibody so the ligand concentration would decrease in the tumor environment without the need for the antibody to penetrate the tumor. These results indicate that an immunotherapeutic potential of IgG1 m10 is likely in combination with other antibodies and anti-cancer drugs, but only further experiments in animal models and clinical trials can evaluate this possibility. These new antibodies against the IGF-IR are currently being tested for incorporation into nanoliposomes for development of multifunctional nanoparticles that can specifically target cells expressing this receptor and help to image them in vivo.
Contact: Dimiter S. Dimitrov, Ph.D.
Selective Inactivation of Pathogenic Organisms, Viruses, and Tumor Cells for Vaccine Development by 5-Iodonaphthyl-1-Azide (INA) or Other Hydrophobic Reactive Probes
CCR researchers have discovered that labeling hydrophobic domains of viral proteins results in the inactivation of the virus without affecting its integrity or the conformation of envelope proteins. Researchers plan to further develop hydrophobic, cross-linking probes that will have the dual effect of inactivating the virus and making the treated virus resistant to detergent solubilization. This feature will allow elimination by detergent treatment of any possible residual infectious virus from the inactivated preparation, thus making it suitable for use in prophylactic vaccination. This new technology is broadly applicable for inactivating any organism with a membrane. It may be applied to a host of pathogens as well as whole-cell cancer vaccines. CCR researchers are also exploring the applications of fluorescently labeled inactivated viruses as nanoprobes in cancer research, such as activating cross-linking/photolabeling reagents at the tumor site.
Other research foci include the design and development of lipid-based nanoparticles for antibody-mediated, tumor-specific targeting and triggered release of anti-cancer drugs or genes to various tumor tissues; the use of viral proteins to build nanofusion machines that will directly deliver their cargo to cells’ cytoplasm; and the understanding of important nanoparticle-cell interactions that lead to specific recognition, imaging, and drug or gene delivery.
Raviv Y, Viard M, Bess JW, Jr, Chertova E, and Blumenthal R. Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope. J Virol 79: 12394–400, 2005.
Contact: Robert Blumenthal, Ph.D.
High-Speed Parallel Molecular Nucleic Acid Sequencing
CCR’s Molecular Information Theory Group specializes in using mathematical theory to produce future-technologies, such as designing molecular machines and developing nanotechnologies. This group has conceptualized several projects, including high-speed parallel molecular nucleic acid sequencing. Using this technique, the sequence of a single molecule of DNA or RNA can be read using a microscope to observe changes in the fluorescence of individual nucleic acid molecules being read by a DNA or RNA polymerase. The data are collected in a computer in parallel, allowing many sequences to be determined from a tiny volume. The technique could be used to read the sequences of mRNA directly from cells, bypassing current microarray technology. It may also be fast enough to allow individual laboratories to read entire genomes with a single device. This nanotechnology would have diagnostic uses, such as rapid identification of mutations that cause cancer and other genetic diseases. The Molecular Information Theory Group is also developing other future-technologies, such as a molecular computer and a molecular rotation engine.
Contact: Thomas D. Schneider, Ph.D.
Mutant Glycosyltransferases Assist in the Assembly of Glycoconjugates
CCR researchers have designed novel glycosyltransferases, based on their structural information, that have broader or requisite donor and acceptor specificities. Several mutant glycosyltransferases have been generated that can transfer a sugar residue with a chemically reactive functional group to N-acetylglucosamine, galactose, and xylose residues of glycoproteins, glycolipids, and proteoglycans (glycoconjugates). These glycoconjugates are cross-linked via modified glycan moieties and are assisting in the assembly of bionanoparticles that are useful for the development of the targeted-drug delivery system and contrast agents for MRI. The reengineered recombinant glycosyltransferases are also making it possible to synthesize oligosaccharides for vaccine development and remodel the oligosaccharide chains of glycoprotein drugs.
Contact: Pradman K. Qasba, Ph.D.
Self-Assembly of Biological Building Block Motifs for Nanoscale Cancer Biology Applications
CCR computational biologists are applying their expertise in the principles of molecular structures and the mechanisms of their formation to nanobiology. They employ biological (particularly protein) building block motifs in a self-assembly process to engineer molecular-sized components for particular functional designs. The key is to be able to control and manipulate the self-assembly of the building blocks to obtain diverse shapes, sizes, chemistry of surfaces, and dynamics. The database of protein structures is populated by a vast collection of protein and building block geometries and chemical properties. Their synthesis is fast, and they are expected to be nontoxic. Hence, they are very attractive as starting points for nanodesign. The challenge is to develop predictive, mechanistic schemes that allow controlling the assembly of the intermediate conformational states. The strategy is to choose potential building blocks and enhance their population times through introduction of native and non-native residues. The building blocks are then assembled, and their stabilities are estimated through high-performance computing. Proteins are also used in strategies that mimic naturally occurring functional constructs. It is expected that such advanced computational approaches will lead to a considerable acceleration of nanodesign.
Contact: Ruth Nussinov, Ph.D.
Collaborative News
Bridging the Imaging Gap in Nanobiology with 3D Electron Microscopy
Emerging methods in 3D biological electron microscopy provide powerful tools and great promise to bridge a critical gap in imaging in the biomedical size spectrum. This gap comprises a size range of great interest in biology and medicine that includes cellular protein machines, giant protein and nucleic acid assemblies, small subcellular organelles, and small bacteria. These objects are generally too large or too heterogeneous to be investigated by high-resolution X-ray and NMR methods, but the level of detail afforded by conventional light and electron microscopy is often not adequate to describe their structures at resolutions high enough to be useful in understanding the chemical basis of biological function.
CCR investigators are using electron microscopic imaging to discover and analyze biological complexity within the size gap with linear dimensions of about 50–1000 nm. Ultimately, the understanding of cellular architecture gained at this level will be crucial in designing effective strategies for disease prevention and treatment. A key mission is to quantitatively describe the spatial and temporal architecture of key molecular machines that fall into this “nano gap”. Areas of current interest include: 1) the development and application of novel technologies for 3D electron microscopy of specimens ranging in size from small molecules to tissues, including automated approaches to analyze the molecular structure and sub-cellular location of a variety of nanoparticles, 2) determination of the dynamic spatial and temporal architectures of cellular structures and molecular machines involved in fundamental processes such as energy transduction, cell division, and chemotaxis, and 3) determination of molecular mechanisms underlying the neutralization and cellular entry of HIV.
Subramaniam S and Milne JLS. Three-dimensional electron microscopy at molecular resolution. Annu Rev Biophys Biomol Struct 33: 141-55, 2004.
Subramaniam S. Bridging the imaging gap: Visualizing subcellular architecture with electron tomography. Curr Opin Microbiol 8: 316-22, 2005.
Contact: Sriram Subramaniam, Ph.D.
Development and Characterization of Affibody®-Based Bioconjugates for Molecular Imaging and Targeted Therapy of HER2-Positive Cancers
CCR researchers are developing an innovative strategy for individualized treatment of HER2-positive cancers by combining a non-invasive method for monitoring of HER2 in vivo and HER2-specific delivery of therapeutic agents. Affibody molecules obtained from our CRADA partner in Sweden (http://www.affibody.com) are used as the targeting agent. These very stable and highly soluble a-helical proteins are relatively small (8.3 kDa) and can be readily expressed in bacterial systems or produced by peptide synthesis. The His6-Zher2:324 binds to HER2 receptors with high affinity (22 pM) and is available with cysteine at the carboxy-terminal to facilitate conjugation. For imaging purposes, these molecules will be labeled with radionuclides. For therapy, the His6-Zher2:324 will be conjugated with thermo- or radio-sensitive liposomes when labeled with beacons for in vivo imaging and loaded with therapeutic agents (e.g., toxins, radiosensitizers, or kinase inhibitors) that will allow local drug release defined by real-time monitoring of their distribution. These nanoparticles will provide means for HER2-specific delivery of a variety of tumoricidal agents, including those whose application is currently limited due to their hydrophobicity and that, thereby, complement current therapeutic strategies. This approach, involving assessment of target presence and distribution in an individual patient followed by optimized, target-specific drug delivery, should significantly improve the efficacy of cancer treatment while reducing side effects.
Contact: Jacek Capala, Ph.D.
Immunotherapeutic Cocktail of Nanoparticles to Attack Tumor Cells
Researchers in the Laboratory of Medicinal Chemistry (LMC) synthesize gold nanoparticles coated with tumor-associated carbohydrate antigens (TACAs)—in particular, the Thomsen-Friedenreich (TF) antigen disaccharide. This sugar is present on over 90% of carcinoma cells but rarely displayed on normal tissue. Researchers have shown that the carbohydrate was functional on the particle and that, when injected into mice with implanted breast tumors, these particles inhibited metastasis to the lungs. In addition, researchers are preparing nanoparticles that are coated with glycopeptides that contain the TF antigen as precursors to a possible anti-cancer vaccine. The goal is to prepare multifunctional particles as part of a cocktail for immunotherapy targeted against tumor cell surface carbohydrate epitopes and their surrounding environment. Researchers also have used CCR’s synthetic precursors to prepare the first quantum dots coated with the TF TACA. Quantum dots are highly useful semiconductor nanocrystals that have unique photoluminescent properties. Researchers were successful in accomplishing the first de novo synthesis of carbohydrate-coated quantum dots. From there, they proceeded to enhance their luminescent properties by making hybrid particles with small organic acids. They were able to synthesize very robust particles with high quantum yields and biofunctional sugars on the surface. Researchers were able to selectively label lung metastasis cells with these particles and are proceeding to tackle many other cell-labeling applications with the LMC’s newly developed protocol for quantum dot synthesis.
Contact: Joseph J. Barchi, Jr., Ph.D.
Labeled Nanoparticles Reveal Tumor Details
Conventional magnetic resonance imaging (MRI) contrast agents or dyes are widely used in hospitals today, but they have a number of limitations. Their small molecular size means that they can easily escape from vessels and increase signal or “enhance” non-specifically in the body. They are also notoriously difficult to target to specific tissues, including cancerous tissues. NCI researchers have recently designed a new nano-sized MRI contrast agent based on a family of macromolecules known as dendrimers. These nanoparticles are composed of branching molecules (dendron in Greek means tree) that allow multiple atoms of gadolinium, the paramagnetic agent that enhances MRI, to attach to tissues. The slower molecular tumbling rate of nanoparticles enhances their relaxivity and, therefore, their effect on the image. In addition, the large number of potential binding sites on the surface of dendrimers allows them to be dual or triple labeled with other imaging probes, such as radionuclide or optical probes, as well as with ligands, which target surface receptors found mainly on cancer cells.
This research is being performed by Martin W. Brechbiel, Ph.D., Head, Radioimmune Inorganic Chemistry Section, Radiation Oncology Branch, and Peter L. Choyke, M.D., Director, Molecular Imaging Program.
Nanotechnology Characterization Laboratory News
Nanoparticles Characterized
The Nanotechnology Characterization Laboratory (NCL) is now actively characterizing nanoparticles intended for clinical applications. At the close of fiscal year 2005, the NCL had accepted 26 particles for characterization, exceeding the 24 particles forecasted in the NCL business plan. Efforts are now focused on continuing to solicit nanoparticles, subjecting the particles to the NCL’s three-phase assay cascade, and identifying parameters that influence biocompatibility.
Contact: Scott E. McNeill, Ph.D.
