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

This presentation reviews existing ISO standards relating to the measurement of nanoscale materials and current ISO initiatives concerning best practices for preserving the occupational health and safety of those involved in the manufacture and use of nanotechnologies. The advent of the nanotechnology revolution has raised serious concerns regarding potential health and safety hazards associated with the production and use of engineered nanomaterials. These issues have prompted widespread calls that regulatory agencies evaluate and take appropriate actions to protect human health and safety, preserve the environment and enable international commerce in nanotechnologies. Both national and international regulatory agencies and standards organizations such as EPA, NIOSH, ANSI, BSI, APPIE, DIN, EEC, ISO and others are collaborating to develop a unified approach to standardization. There are a variety of existing international standards that address the measurement of nanoscale materials and the quantification of exposure to some types of airborne nano or ultrafine materials. Relevant national and ISO documents are often difficult to find due to the large number of technical committees, often linked to specific industrial sectors. Recently, a new ISO committee TC229 entitled “Nanotechnologies” was established to address issues specifically associated with engineered nanomaterials. It is the purpose of this committee to assemble international experts to evaluate current standards, compile relevant materials and develop a strategy for developing international standards relating to nanotechnology. Initially, three working groups will develop international standards and best practices in the areas of terminology, metrology, and occupational health and safety.

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

Magnetic nanoparticles have found utility in many biological applications, including imaging, cancer therapy, drug delivery, sensing and hyperthermia for tumor therapy. Hyperthermia is raising the tissue temperature between 41.5 - 46 degrees Celsius to kill cancerous cells while preserving the normal cells. Due to the fact that many robust synthetic strategies exist for iron oxides which results in high quality, monodisperse and crystalline nanoparticles, hyperthermia applications have traditionally used magnetic oxide nanoparticles. On the other hand, new materials for hyperthermia that combine the advantages of stability with those of magnetic behavior are desirable. We report the synthesis of MnZnFe, CoGdZn and ZnGdFe nanoparticle systems which are ideal for biological applications over magnetic oxides due to their conjugation chemistry, and surface chemistry. We present an AC magnetic heating studies of these nanoparticle systems which exhibit magnetic field heating. The frequency dependence of the heating follows general trends predicted by power loss equations and is similar to traditional materials. The heating pattern of Zn-Gd-Fe (20mg/ml) using alcohol thermometer at 961 kHz and 433 KHz and the heating pattern of Mn-Zn-Fe [Zn = 0.5 conc.](20mg/ml) using alcohol thermometer at 961 kHz and finally the heating pattern of Co-Gd Zn [Zn = 0.2 conc. Gd (1-X)] (20mg/ml) using alcohol thermometer at 961 kHz are reported. X Ray Diffraction studies and SQUID magnetic measurements and TEM and EDX particle size and constituents measurements are also included for the nanoparticle systems. In conclusion, high quality heating nanoparticle composites were developed for hyperthermia treatment of cancer. The composites generate sufficient heat at room temperature and stops heating at the Curie temperature Tc of the respective nanoparticle system.

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

One proposed approach to reduce the spread of HIV is to prevent transmission of the virus through the use of a topical microbicide. One microbicide strategy is a charge inhibition based approach using polyanionic compounds designed to interfere with the process of HIV-1 attachment to potential target cells. This strategy however, is predicated on further understanding the charge characteristics of whole virions and the relative contribution of viral and host-cell proteins to such charge. Electrokinetic methods (i.e. ζ-potential) provide information on the surface structure of biological cells without producing significant alteration of the cellular organization. Electrophoretic fingerprinting (EF) is obtained from 3D templates of the mean electrophoretic mobility (the raw data from which ζ-potential is calculated) of a given particle versus pH and solution conductivity at a fixed temperature. The EF thus represents a surface, described by isomobility lines, over all pertinent electrochemical conditions. These initial electrophoretic analyses have been performed using human CD4+ T cell lines. The cell lines are derived from human white blood cells which are the principle targets of the HIV-1 virus. Tissue culture work was carried out under Class II aseptic conditions. Cell types were maintained in RPMI growth medium supplemented with 10% heat inactivated foetal calf serum, 2mM glutamine, 100 i.u./ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 incubator. The cells were routinely passaged every 3-4 days in 75 cm3 fillter cap tissue culture flasks, by the addition of 4 mls of cells to 16 mls of fresh growth medium.Electrophoretic mobility (ζ-potential) was measured as a function of pH and ionic strength over a range chosen to cover that known for fluids found in the lower female reproductive tract, including vaginal fluid and semen. Measurements were made using a Malvern ZetaSizer NanoZS operating in the fast field reversal mode (PALS). Data was analyzed using SURFER™ software and the results validated from the covariance matrix of the linear fit. Challenges in the measurement and characterization of the cells include the difficulty of the cell preparation, cleanliness of the samples and sample handling required to maintain cell vitality.Overall, the EF’s analyzed under environments characteristic of physiological conditions for each CD4 T cell line resulted in similar zwitterionic surface charge features. These results suggest that the best candidate for a microbicide active needs, itself, to be zwitterionic so as to be able to mirror-image the shift in sign of surface charge as the pH of the vaginal tract changes. Current results suggest that HIV interaction with target cells is enhanced by physiological fluids. The data provides core information on the physico-chemical properties of model cellular targets for HIV-1 infection and pave the way for rational development of charge-based intervention strategies.

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

In the past four decades lipid vesicles (liposomes) have evolved from widely used biomembrane models into important drug and gene carriers. The phosphatidylcholine phospholipids PC used in the drug carriers are biocompatible and biodegradable but they function as a relatively inert shell and require the incorporation of cholesterol to maintain the drug encapsulated in the liposome; The PC are also incapable of associating with ligands and have very weak interactions with nucleic acids. Moreover, they are not particularly good for cytoplasmic delivery of the encapsulated cargo. Recently, we have devised three classes of new lipids and have improved the synthesis of a fourth class that enable the preparation of a bioresponsive targeted carrier with improved nucleic acid delivery. Class 1 are low pH sensitive and include a diortho ester PEG lipid or a di-orthoester PC. Class two are redox sensitive lipids and include thiocholesterol based and thio diacyl chain based lipids that can be used in a sequential assembly process to encapsulate nucleic acid drugs in a charge neutral or negatively charged nanolipid particle. Class 3 is a new family of lipids that provide increased in vivo bilayer stability without the need for crosslinking of the bilayer. Class 4 is an improved synthesis of a triNTA diacyl lipid. This lipid can be used to attach His-6 containing molecules to the bilayer vesicle after the liposomes have been prepared and loaded with drugs. These lipids form a tool kit that can be used to prepare a variety of targeted drug, protein and nucleic acid delivery vesicles with attached targeting ligands. The synthesis, characterization and use of these lipids in a variety of drug delivery applications will be described. Suported by NIH EB003008 & NIH GM061851.

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

The unique and diverse physico-chemical properties of nanoscale materials suggest that their toxicological properties may differ from materials of similar composition but larger size. Studies suggest that particle size, surface area and surface chemistry of engineered nanoscale materials can impact toxicity equally, if not more so, than chemical composition. The National Toxicology Program (NTP) (ntp.niehs.nih.gov) is a multi agency program headquartered at the NIEHS that coordinates toxicology research and testing programs within the federal government and conducts research to provide information about potentially toxic chemicals to health, regulatory, and research agencies, scientific and medical communities, and the public. The NTP is currently engaged in a research program that is evaluating the toxicological properties of current major nanoscale materials classes. These materials represent a cross-section of composition, size, surface coatings, and physico-chemical properties. The studies are designed to investigate fundamental questions concerning how nanoscale materials are absorbed and distributed in vivo and whether they can adversely impact biological systems. Some of these fundamental questions include: What are the appropriate methods for detection and quantification of nanoscale particles in tissues? How are nanoscale materials absorbed, distributed in the body and taken up by cells? Are there novel toxicological interactions? The NTP's nanotechnology safety initiative (http://ntp.niehs.nih.gov/go/nanotech) is focusing research with respect to specific types or groups of nanoscale materials: Non-medical, commercially relevant/available nanoscale materials to which humans are intentionally being exposed, e.g., cosmetics and sunscreens; Nanoscale materials representing specific classes (e.g., fullerenes and metal oxides) so that information can be extrapolated to other members of those classes; Subsets of nanomaterials to test specific hypotheses about a key physiochemical parameter (e.g., size, composition, shape, or surface chemistry) that might be related to biological activity. Ongoing research activities are addressing (1) the fate and distribution of nanoscale metal oxides and quantum dots in the body following their dermal application to rodents with attention given to the role of surface coating, size, polarity, vehicle, and skin condition on the ability of nanoscale TiO2 to penetrate the skin; (2) whether nanoscale TiO2 and ZnO applied dermally to mice in combination with UVA-containing light affects cell signaling, and (3) the potential for TiO2 applied dermally to haired and hairless mice in combination with UV-containing light to cause skin cancer.

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

Dendrimers are routinely synthesized as tuneable nanostructures that are designed and regulated as a function of their size, shape, surface chemistry and interior void space. They are obtained with structural control approaching that of traditional biomacromolecules such as DNA/RNA or proteins and are distinguishable by their precise nanoscale scaffolding and nano-container properties. This lecture will review progress on the use of these features for both targeted diagnostic and drug delivery applications. Recent efforts have focused on the synthesis and preclinical evaluation of a multi-purpose, STARBURST® poly(amidoamine) (PAMAM) dendrimer prototype that exhibits properties suitable for use as: (i) a targeted, diagnostic MRI contrast agent (ii) and/or for controlled delivery of cancer therapies. Special emphasis will be placed on the lead candidate, namely; (core: 1,4-diaminobutane; G=4.5); [dendri-PAMAM(CO2Na)64]. This dendritic nanostructure was selected based on a very favorable nanotoxicity profile*, the expectation that it will exhibit desirable kidney excretion properties and demonstrated targeting features.*The Nanotechnology Characterization Laboratory (NCL), an affiliate of the National Cancer Institute (NCI) has studied the lead compound and found it to be extraordinarily benign and highly biocompatible.

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

Description: This presentation reviews existing ISO standards relating to the measurement of nanoscale materials and current ISO initiatives concerning best practices for preserving the occupational health and safety of those involved in the manufacture and use of nanotechnologies. The advent of the nanotechnology revolution has raised serious concerns regarding potential health and safety hazards associated with the production and use of engineered nanomaterials. These issues have prompted widespread calls that regulatory agencies evaluate and take appropriate actions to protect human health and safety, preserve the environment and enable international commerce in nanotechnologies. Both national and international regulatory agencies and standards organizations such as EPA, NIOSH, ANSI, BSI, APPIE, DIN, EEC, ISO and others are collaborating to develop a unified approach to standardization. There are a variety of existing international standards that address the measurement of nanoscale materials and the quantification of exposure to some types of airborne nano or ultrafine materials. Relevant national and ISO documents are often difficult to find due to the large number of technical committees, often linked to specific industrial sectors. Recently, a new ISO committee TC229 entitled "Nanotechnologies" was established to address issues specifically associated with engineered nanomaterials. It is the purpose of this committee to assemble international experts to evaluate current standards, compile relevant materials and develop a strategy for developing international standards relating to nanotechnology. Initially, three working groups will develop international standards and best practices in the areas of terminology, metrology, and occupational health and safety.

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

Analytical ultracentrifugation (AUC) is a first-principle means to determine the molecular weight and size distribution of dissolved particles. Though primarily used for characterizing biomaterials, AUC also is used in polymer chemistry to determine size distributions and density distributions of latices, pigments, polymers and co-polymers. In fact, AUC can provide useful information for virtually any solute in a wide range of solvents. The methods are non-destructive, allowing subsequent analyses on the same sample. There are two different AUC methods-velocity and equilibrium. The principles of equilibrium and velocity sedimentation will be described, along with their advantages and disadvantages relative to one another and relative to other methods.

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

Gold nanoparticles have shown great promise in a variety of biological applications including the use in highly sensitive diagnostic assays 1,2, thermal ablation 3, radiotherapy enhancement 4, as well as drug and gene delivery 5. Such gold particles, however, suffer from losing or reducing sensitivity and selectivity due to aggregation under high ion strength of biological fluids and non-specific interaction with biomolecules, such as proteins or DNA. Poly ethylene glycol (PEG), which is known to lengthen the circulation time of biomedicines in the bloodstream, reducing the non-specific binding of proteins, and increasing efficacy and tolerability, is currently used as coating for different kind of nanoparticles to improve their stability and biocompatibility. The currently used strategy was to attach PEG moelcucles to gold nanoparticle through Au-SH chemical bonding. There is no available information, however, about how the length, conformation and attachment sites of PEG moiety affect the binding stability on gold nanoparticles, which play such critical role in retaining the solubility, while facilitating both selectivity and reactivity. In our present work we examined the stability of thiolated PEG with different length and multi-thiol anchors bound to gold nanoparticle by an assay of PEG displacement with different moities including di-thiolthreitol (DTT) and mercaptoethanol. Dynamic light scattering (DLS), Atomic force microscopy (AFM), multiangle-laser light scattering (MALS) incorporated with refractive index (RI), UV and DLS detectors were employed to characterize size, geometry, and packing density of PEG. This information will enable us to understand, optimize and control their efficacy and distribution of gold nanoparticle-based diagnostic or therapeutic agents under complex physiological environments. 1 Tkachenko, A.G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M.F.; Franzen, S.; Feldheim, D.L. J.Am. Chem. Soc.2003,125,4700.2 Nam, J.M.; Thxton, C.S.; Mirkin, C.A.; Science 2003, 301, 1884.3 Loo, C.; Lowery, A.; Halas, N.; West, J.; Drezek, R. Nano. Lett. 2005, 5, 709.4 Hainfeld, J.F.; Slatkin, D.N.; Smilowitz, H.M.; Phys. Med. Biol. 2004, 49, N309.5 Thomas, M.; Klibanov, A.; Proc. Natl. Acad. Sci. USA 2003, 100, 9138.

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

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

Nanoscale interfaces to proteins have been achieved for a variety of applications, in the form of electrodes to measure conductivity, for sensing on cantilevers or fluorescent quantum dots, or nanoparticles that can be used as reporters in receptor-ligand binding assays. One prevailing requirement is that the biological function of the protein is maintained when linked to nanoscale systems. Due to the structure-function relationship of proteins, the protein must maintain its folded structure. We covalently link cytochrome c and Ribonuclease S to Au or magnetic nanoparticles (NPs) and study the interface, with the goal of constructing design rules that govern the interaction. In both cases we devise methods to achieve linkage of a nanoparticle to the protein on a specific amino acid, in addition to chemical treatments that minimize non-specific adsorption. The protein linked to the nanoparticles is biophysically characterized. Protein secondary structure was quantified by circular dichroism spectroscopy (CD). From these measurements we determine that electrostatic forces dominate the NP-protein interaction and minimization of these results in folded proteins with minimal non-specific adsorption. For Ribonuclease S, these findings are integrated with measurements of enzymatic activity and binding constants KM to yield a picture of the how the protein interaction with the NP affects its binding to the substrate and activity. Experiments in which the NP labeling position, NP ligand, size, and material (Au, Fe3O4, CoFe2O4) are systematically varied will be discussed.

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

The field of nanomedicine is quickly evolving in response to achievements in genomics, proteomics, molecular biology, bioengineering, and the imaging sciences. New approaches to entrenched medical problems are being studied using a cadre of “nanotools”, one example of which is perfluorocarbon nanoparticles.Perfluorocarbon nanoparticles represent a platform technology with nominal sizes around 250nm, which can be modified to home to thrombi and the neovasculature in vivo after intravenous injection. They can be noninvasively imaged with ultrasound, magnetic resonance (MR, 1H and 19F), or SPECT/CT. In rabbit models, perfluorocarbon nanoparticles have been demonstrated to deliver drug payloads targeted to vascular tissues for anti-angiogenic and anti-restenotic applications and to noninvasively confirm and quantify delivery as well as to follow response to treatment. In canine studies these agents have been demonstrated to target and enhance the MR and ultrasound contrast of intravascular thrombi, and using ex vivo human carotid endarterectomy sections, these results have been extrapolated to human disease where the potential for sensitive detection of microthrombi in the fissures of ruptured plaques is clearly demonstrated. The development of emerging nanotechnology platforms, such as the perfluorocarbon nanoparticles, permits translation of immunohistology techniques from fixed tissue on a slide to live tissue in an animal. These new agents allow biochemical and physiological changes to be studied dynamically in vivo and permit the quest for site-directed therapy to be realized.

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

Targeting potent anti-cancer therapeutics to solid tumors is best accomplished by first avoiding recognition and uptake by the immune system and second by limiting the biodistribution of the drug to the tumor. We have achieved these objectives by binding tumor necrosis factor alpha (TNF) to the surface of 30 nm pegylated colloidal gold particles. Pegylation of the gold nanoparticles is accomplished by binding thiolated polyethylene glycol in between the TNF molecules on the surface of the gold nanoparticles. This formulation is termed CYT-6091 (Aurimune). The liver or spleen (the RES) take-up little or no drug 6 hr after CYT-6091 is injected into mice, and TNF levels in the tumors increase over this time period (in contrast to decreasing levels of TNF seen in healthy tissues). Electron micrographs also show gold nanoparticles in the tumors, but few or no particles in healthy tissue. By getting more TNF to the tumors, CYT-6091 is both safer and more effective in causing tumor regression in mice. CYT-6091 has also been given to dogs with naturally occurring cancers. Most notably in both dogs with cancer and in healthy rabbits, CYT-6091 caused fever, but did not induce hypotension. Historically, hypotension has been the dose-limiting toxicity for TNF and the primary reason for its failure in human clinical trials. For human testing, the manufacturing of CYT-6091 was successfully scaled-up and produced under cGMP. CYT-6091 is currently being tested in end-stage disease cancer patients in an NCI sponsored Phase I clinical trial.

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

Cell surface carbohydrates play unique roles in a host of biologically relevant events such as cell differentiation, adhesion and development. They also mediate many undesired processes such as pathogenic infection and tumor metastasis. A common feature of all tumor cells is the altered expression and presentation of surface glycans that serve as “non-self” structures that can be recognized by the immune system. For many years, these tumor-associated carbohydrate antigens (TACA’s) have been employed in the development of tumor vaccines with varying degrees of success. In addition, these sugars may mediate tumor cell adhesion during the metastatic cascade. We have previously succeeded in preparing both gold nanoparticles and quantum dot nanocrystals that were coated with specific TACA’s. We have concentrated on the Thomsen Friedenreich (TF) antigen disaccharide (Galβ1-3GalNAcα-O-Ser/Thr) and have shown that gold particles coated with TF antigen can either promote or inhibit metastasis in vivo. Here we describe our progress toward the synthesis of a number of these multivalent nanoscaffolds and report on our characterization efforts and how the size and surface chemistry of the particles relates to their biological activities.

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

Characterization of nanobioparticles is a demanding task. This problem is particularly evident in the case of biomedical applications of nanoparticles where toxicological indices and ADME parameters are the result of complex interactions of the nanoparticle at the molecular, cellular and tissue levels. Furthermore, particles of this size frequently behave in ways that are intrinsically different than those at meso and sub-nano scale. The success of the biological application of nanoparticles depends, however, to a large extent, on our ability to characterize, and eventually predict and control, the properties and behavior of nanoscale particles in realistic biological environments. To help this process, the development of computer-aided nanoparticle characterization approaches is highly desirable. Nanobioparticles include a large array of dissimilar materials under a common name, making the definition of common microscopic criteria matching the modeled molecular properties with the macroscopically observed ones, a daunting task. In this presentation we will review our efforts at devising strategies that, from in-silico simulations of nanoparticles, will help us infer their behavior in complex environments. The approaches presented rely on the application of sensitivity analysis techniques that probe the intrinsic stability of the particle. Particles will be suitable candidates for biological use only if they show low sensitivity to those challenges. The nature of the parameters explored and the possible generalization of this approach will be discussed by presenting our results using metal-loaded fullerenes, gold particles and dendrimers. This work has been funded in part with funds from the NCI-NIH (Contract No. NO1-CO-12400). The contents of this publication do not necessarily reflect the views or policies of the DHHS, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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

Plasmonic nanoparticles have been successfully demonstrated as chemically functional nanosensors, based on the controllable electromagnetic properties at their surfaces. Surface enhanced spectroscopies such as surface enhanced Raman scattering (SERS) can be utilized to provide measurable signals that monitor properties local to the nanoparticle location, such as pH, with precision adequate for diagnostic purposes where applicable. A variety of different molecular layers can be developed that exploit this SERS-based sensing modality, to monitor specific chemical binding events. Plasmonic nanoparticles can also be utilized to specifically and selectively enhance the fluorescence of molecular markers in their direct vicinity. We will discuss the underlying physical principles of both SERS and fluorescence enhancement by plasmonic nanoparticles, and the role of the plasmon energy in both SERS and fluorescence ehancements, as well as the local substrate geometry, in these processes. Combining chemically functional monitors with therapeutic modalities such as photothermal cell ablation will expand the utility of this therapeutics approach.