This is a copy of the slides presented at the meeting but not formally written up for the volume.

The potential applications of nanomaterials in medicine are numerous and very promising. Translation of potential to practical value, however, requires demonstration not only of efficacy but also of safety. Assessing nanoparticles? potential toxicity to the immune system is an important step in building nanopartricles? safety profile. Unfortunately, immunotoxicological analysis of new molecular entities is not a straightforward process. There is no universal guide for immunotoxicity in general, as there is no formal guide specific for nanomaterials?. Our presentation will address key points for consideration during early preclinical studies on nanoparticles? effects to the immune system. We will also review methodological challenges in in vitro preclinical immunological testing of nanomaterials developed for biological applications. Funded by NCI Contract N01-CO-12400.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

Manipulation of the physicochemical properties of materials at the nanoscale has the potential to revolutionize electronic, diagnostic, and therapeutic applications. Because of the potential large-scale use of nanomaterials, it is important to determine if there is any unique toxicity of the nanoscale materials as compared to the bulk. It is essential for the purposes of interpreting results from cell culture and animal models that the nanomaterials are thoroughly characterized and that correlations are made between observed toxicological responses and the physicochemical characteristics of the materials. We hypothesize that nanomaterials by virtue of their size and surface activity can induce oxidative stress following exposures in cells or tissues and that they can move beyond the site of original deposition. To test this hypothesis, we use acellular assays (e.g. reactive oxygen species; dissolution; agglomeration analyses) to both identify potential benchmark nanomaterials and to predict responses in cells or tissues. Using in vitro (lung epithelial, endothelial cells) and in vivo assay systems, we have characterized responses, including cellular uptake and tissue distribution, of nanomaterials as a function of shape (using Pt), surface coating (using semiconductor quantum dots), size, and chemical composition. From these studies, it is clear that a dose metric other than mass is required to accurately predict response in acellular, in vitro, or in vivo systems and that surface area is a better predictor, at least within a given particle type. Other factors contribute to reactivity, though, including crystal phase, chemical composition, mode of synthesis, surface coating, and agglomeration state.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

Semiconductor, metal and ceramic nanocrystals have optical, magnetic and chemical properties that can be radically different from molecular or bulk systems. These unique and tunable features can be leveraged in biomedical applications. As an introduction we will give several examples in which such systems have been used to improve the diagnosis, imaging, and treatment of disease. Then we will discuss both the synthetic and analytical challenges with forming these materials for problems in medicine. We will first present general techniques for imparting water solubility to nanocrystals using amphilphilic co-polymers. Our strategy uses as a starting point high quality nanocrystals that are best formed in organic solvents. We then transfer the materials to water using copolymers generated using a maleic anhydride coupling scheme that permits the coupling of a wide variety of PEG polymers, both unfunctionalized and functionalized, to hydrophobic tails. The hydrodynamic size and concentration of the nanoparticle-polymer complexes requires new techniques, including size-exclusion chromatography, cryogenic transmission electron microscopy and mass spectroscopy. Ultimately, many of the key features of the nanoparticles are defined by their specific and non-specific interactions with biomolecules in complex biological fluids. Characterization of this full 'nanocrystal-polymer-biomolecule' complex requires additional methods drawn from both biochemistry and cell biology. In particular, we find analytical ultracentrifugation to be ideally suited for quantitative evaluation of these systems.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

Nanoparticles (NPs) are showing great promise in their utility towards biomedical applications in areas such as drug delivery, diagnostics and image contrast enhancement. The standardization of physical characterization protocols for NPs is critical for their eventual approval and use in clinical settings, and for the development of reliable nanosize reference materials. Field-flow fractionation (FFF) is emerging as a powerful tool to obtain information on the composition, size, and molecular weight of fractionated NP solutions. FFF is classified into several sub-techniques based on the applied "field", with the most common and broadly applicable being asymmetric-flow (A-FFF). A-FFF separates constituents according to their hydrodynamic size, and can be coupled with various detectors, such as UV-Vis, multi-angle light scattering (MALS), differential refractive index (DRI), photon correlation spectroscopy (PCS), fluorescence, and, more recently, inductively coupled plasma-mass spectrometry (ICP-MS). Depending on the different detector systems employed, further information such as the number and distribution of ligands or drug molecules attached to a multifunctional nanomaterial and the frequency of dimer, trimer and higher order aggregates can be obtained.In the present work we have employed commercial A-FFF systems customized with various detectors, including MALS, PCS, DRI and UV-Vis, to establish fundamental protocols for the characterization of gold nanoparticles and their bio- or dendridic-conjugates. These protocols are being applied in the development of new gold-based nanosize reference materials intended for the cancer research community. We optimized the experimental conditions by controlling various parameters, such carrier composition, membrane material, and ratios of channel-to-cross flow rates. Also, samples were separated by collection mode from A-FFF, to coincide with the MALS signal, and subsequently investigated for purity and accuracy by further supportive analysis of off-line UV-Vis and PCS measurements. We report on the results of these studies and their implications for biomedical research.NCL is funded by NCI Contract N01-CO-12400.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote biocompatibility of water-soluble quantum dots (QDs). Each hydrophilic ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring on the QD surface, and potential biological functional groups (biotin, amino, and carboxyl groups) at the other end of the PEG. Water-soluble QDs capped with one type or mixtures of the functional ligands were prepared via cap exchange with the native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and over broad pH range were prepared. Surface binding assay showed that DHLA-PEG-biotin-functionalized QDs strongly interacted with NeutrAvidin-modified surfaces. The new functional surface ligands studied here provide not only stable and highly water-soluble QDs but also simple and easy access to various biological entities.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

It is preferable for a number of reasons to resolve existing problems with atomic force microscopy (AFM) use instead of simply resorting to dynamic light scattering (DLS), optical or electron microscopy (SEM/TEM). Parameters involving direct measurements of the 3rd dimension, such as height, surface roughness and volume, are possible only with AFM. The range of examples of great scientific interest includes but not limited to visualization and quantitative measurements of single-wall carbon nanotubes, colloidal gold, polymer particles, quantum dots, oxides, liposomes and nanoemultions. Imaging can be performed in intermediate environment such as water, buffer solution or gas. A tip artifact or a tip dilation, the phenomenon specific to AFM, manifests itself in a broadening of the lateral dimensions of surface topography. Interestingly, the vertical resolution of AFM is not affected by the finite size of the probe. The false metrology parameters could be generated that reflect the convolution of the tip geometry and the geometry of the object being imaged, rather than the object morphology. We propose that the long-standing problem of tip deconvolution can be solved by scanning a pre-characterized nano-sphere prior to imaging unknown particles. Not only does particle characterization using AFM provide a resolution comparable to or greater than that of SEM/TEM/DLS, it also retains the capacity for both 3-D visualization and direct height and volume measurements. Because AFM individual particle characterization is more productive, non-intrusive and easier to use than electron microscopy, our solution to the problem of tip dilation represents an important contribution to several rapidly-growing fields of science and technology.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

National Cancer Institute is engaged in efforts to harness the power of nanotechnology to radically change the way we diagnose, image, and treat cancer. Novel and multi-functional nanodevices will be capable of detecting cancer at its earliest stages, pinpointing its location within the body, delivering anticancer drugs specifically to malignant cells, and determining if these drugs are effective. Functionalized nanoparticles would deliver multiple therapeutic agents to tumor sites in order to simultaneously attack multiple points in the pathways involved in cancer. Such nano-therapeutics are expected to increase the efficacy of drugs while dramatically reducing potential side effects. In vivo biosensors would have the capability of detecting tumors and metastatic lesions that are far smaller than those detectable using current, conventional technologies. Furthermore, they will provide rapid information on whether a given therapy is working as expected.In order to further these research goals, NCI Alliance for Nanotechnology in Cancer has been formed in 2004. The Alliance is investing $144.3 million over the next 5 years to pursue applied nanotechnologies for cancer detection, therapy, and prevention with an aim to achieve clinical translational stage of these technologies towards culmination of the program. The Alliance funds Centers of Cancer Nanotechnology Excellence, the development of nanotechnology platforms, and internal Nanotechnology Characterization Laboratory.This presentation will describe the details behind the organization and science and technology of the Alliance.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

The Nanotechnology Characterization Laboratory (NCL) conducts preclinical efficacy and toxicity testing of nanoparticles intended for cancer therapeutics and diagnostics. The NCL is a collaborating partnership between NCI, the U.S. Food and Drug Administration and the National Institute of Standards and Technology. As part of its assay cascade, NCL characterizes nanoparticles' physical attributes, their in vitro biological properties, and their in vivo compatibility using animal models. The Laboratory facilitates the rapid transition of basic nanoscale particles and devices into clinical applications by providing the critical infrastructure and characterization services to nanomaterial providers. It is a national resource available to investigators from academia, industry and government. The presentation will provide an overview of the NCL; discuss parameters that are critical to biocompatibility, and present assays used for preclinical characterization of nanoparticles.

This is a copy of the slides presented at the meeting but not formally written up for the volume.

Engineered nanoparticulate systems are anticipated to lead to advances in understanding biological processes at the molecular level and progress in the development of diagnostic tools and innovative therapies. Nanoparticle based imaging agents such as fluorescent dye-doped silica nanoparticles, quantum dots, gold nanoparticles, etc. have overcome many of the limitations of conventional contrast agents (organic dyes) such as poor photostability, low quantum yield and insufficient in vitro and in vivo stability. Additionally, the development of multifunctional nanoparticles, which can be detected simultaneously by multiple techniques e.g. Magnetic Resonance Imaging (MRI) and Optical Imaging integrate the advantages of high sensitivity (from optical method of detection, e.g., fluorescence) with the potential of true three dimensional imaging of biological structures and processes at cellular resolution (e.g. via MRI). Microemulsions have attracted considerable interest as potential delivery vehicles for drugs with poor aqueous solubility as well as for drug detoxification. Several favorable properties of microemulsions such as transparency, easy of preparation, sterilization and nanometer droplets size (providing a relatively high interfacial area and low interfacial energy) have made them suitable for drug detoxification applications. In this presentation, highlights of advances made in synthesis, characterization and performance evaluation of nanoengineered particulate systems in targeted areas such as imaging of biological specimens using photostable nanoparticle based imaging probes, pulmonary therapeutics, and microemulsion mediated detoxification of drugs will be discussed. The toxicological assessment of selected systems will also be presented.