Metrology for the “Fate” of Nanoparticles in Biosystems

1. Metrology for the “Fate” ofNanoparticles in Biosystems Michael T. Postek, Andras Vladar, Thomas LeBrun and John Dagata, MEL John Small, John Henry Scott, Scott Wight, Peter Barker, CSTL Alamgir Karim, Jack Douglas, Matthew Becker, Vincent Hackley, Andrew Allen, Stephanie Hooker, MSEL

2. MEL MSEL CSTL Metrology for the “Fate” of Nanoparticles Michael T. Postek, Champion Andras Vladar Thomas LeBrun John Dagata, Alamgir Karim, Champion Jack Douglas Matthew Becker Vincent Hackley Andrew Allen Stephanie Hooker John Small, Champion John Henry Scott Scott Wight, Peter Barker

3. …. “I would anticipate a highly successful project, and one that will support research into the impact of nanotechnology in a significant manner.” ……. Andrew Maynard, NIOSH Metrology for the “Fate” of Nanoparticles • Competence proposal is motivated by work stemming from: • National Nanotechnology Initiative • Subcommittee on Nanoscale Science, Engineering and Technology (NSET) • Michael Postek • Alamgir Karim • Nanotechnology Environmental and Health Implications Working Group (NEHI WG) • John Small • Michael Postek • The National Institute for Occupational Safety and Health (NIOSH) • Andrew Maynard (endorsement) • Environmental Protection Agency (EPA) • Barbara Karn • Nora Savage

4. Why Now…Impact on Society, Environment, and Commerce Without NIST’s contributions….. the lack of a common infrastructure for bioscience measurements, standards, & data will constrain innovation and stifle the commercialization of new bioscience-based applications in health care, agriculture and food, environmental protection, homeland security, and many other areas. NIST 2010 Strategic Plan, pg 11. NIST is the only organization that has the mission and the necessary combination of staff expertise, experience, industry contacts, industry respect, & worldwide recognition as the leading measurement and standards institution to address the high risk scientific and technological challenges associated with developing the measurement techniques and technical standards needed in this new field. National Nanotechnology Initiative, pg 119. …A joint collaboration with the National Institute of Standards and Technology (NIST) and the FDA is a high priority.The collaboration will focus on developing standards for nanoscale devices and both in vitro and in vivo characterization assaysthat could serve as a starting point for regulatory filings. Cancer Nanotechnology Plan, pg 24.

5. For Science, Nanotech Poses Big Unknowns By Rick WeissWashington Post Staff WriterSunday, February 1, 2004; Page A01 This approach will not reassure the public at large Materials Today July/Aug 2003: 64 • NANOTECHNOLOGY RESEARCH GRANTS INVESTIGATING ENVIRONMENTAL AND HUMAN HEALTH EFFECTS OF MANUFACTURED NANOMATERIALS: A JOINT RESEARCH SOLICITATION-EPA, NSF, NIOSH • Basically a toxicology study of nanoparticles • Need for known characterized particles • Need for standardized methodology X Flawed Science Big PRESS Why Now...Potential for Catastrophic Effects: Psychological and Economic

6. Standards Development (Phase II) NIST’s Role BasicScience “Instrumentation and metrology crosscut all the NNI Grand Challenges, and are vital to the success of the NNI. Advances in fundamental nanoscience, design of new nano-materials, and ultimately manufacturing of new nanoscale products will all depend to some degree on the capability to accurately and reproducibly measure properties and performance characteristics at the nanoscale. MeasurementMethods & Tools (Phase I) Why NIST…Lack of Measurement Infrastructure has Become a Critical Hurdle to Further Innovation VALUE CREATION (IMPACT): Productivity / Market Access / Public Safety & Welfare March 2004 A new product development toolkitcontaining powerfulnew scientific and technical methods ……. is urgently needed to improve predictability and efficiency along the critical path from laboratory concept to commercial product. New Invention Period Technology Improvement Period Scientific Discovery S&T DEVELOPMENT OVER TIME *Office of Cellular, Tissue, and Gene Therapies

7. Micrometersized particles of Zinc Oxide Left Primary Particle size of 3-10 nm Right Right Nanometer sized particles of Zinc Oxide It is this simple - what is the difference? FDA approval of zinc oxide nanoparticles is “grandfathered” in because of the previous approvals for micrometer-sized particles… … discussion held at the first NEHI meeting What is the effect on the body?

8. Competence Goal • What we want to do: • Develop an innovative toolbox for metrology of nanoparticles in biological systems that currently does not exist • Focused Ion Beam sectioning • Cryo TEM/SEM/FIB stage integration • Cryomicrotomy • ….. • Form a strong group of experts in biological nanoparticle analysis • Develop an unbiased source of expertise • Provide better technical support for other agencies • What we are not doing: • Attempting to become NIH • Losing our metrology focus

9. Why Nanoparticle Metrology for Biological Systems? Motivation • Nanoparticles are being produced and are a part of our environment • Nanoparticle measurements are challenging • Biological samples must be carefully prepared • Validation procedures are needed for standardization of measurements • NIST needs to develop competence in this area • NIST can make a difference

10. Measurement Challenge Technical Strategy Issues and Barriers Dissemination of Results Milestones Resources and Leverages Outline

11. Measurement Challenge Technical Strategy Issues and Barriers Dissemination of Results Milestones Resources and Leverages Outline

12. Exposure/Uptake Nano-particle Life-Cycle Transport Bioactivity Underlying Basis for Measurement Need Effect of size Depending upon the system, increased particle size can have either negative or positive consequences……. Particle size therefore can be indicator of expected activity. …. Mattison et al., 2001 “….An essential aspect of engineered nanomaterials is how their properties depend on physical and chemical structure. Quantitative assessments of the risk associated with engineered nanomaterials can not therefore proceed without techniques capable of providing biologically-relevant physiochemical information with nanometer resolution.” – Andrew Maynard, NIOSH Control of mobility and distribution Surface chemistry effects

13. Challenge: Biological Sample Preparation • Biological samples require: • Fixation • Dehydration • Embedding • Sectioning • Dual beam FIB • Cryomicroscopy • Cryomicrotomy • Provides a new paradigm for NIST • Physical metrology • Biological sample preparation is not new to NIST, but not extensively practiced at NIST • Biological samples require expertise that must be developed at NIST in order to assist our customers in metrology related situations.

14. Challenge: Diversity of Nanoparticles Presents Numerous Characterization Challenges SWNT DWNT MWNT Functionalized Nanoparticles Micelles Liposomes These materials will be the first “in the pipeline” as new products but, new self-assembled soft material constructs with increasing difficult characterization challenges must be addressed Dendrimers Quantum Dots Nanotubes (C, Au, Hybrid…)

15. Challenge: Numerous Measurement Parameters of Nanoparticles particle diameter core diameter Core composition & stiffness shell thickness Molecular mass & distribution Surface chemistry & Extent of bio-functionality Aggregation number (# molecules per particle) Dispersion Properties Impacted by Measurable Parameters pH susceptibility Bioactivity Diffusion coefficient Anisotropic transport Membrane permeability Degradation mechanisms and byproducts

16. Challenge: Increasing Complexity Fullerene decorated Dendrimer AW Jensen, BS Maru, X Zhang, DK Mohanty, BD Fahlman, DR Swanson, and DA Tomalia Nano Letters, 5(6), 1171 -1173, 2005 Dendrimer decorated Polymer Brush Antibody labeled Gold Nanoshell CC Lee, M Yoshida, JMJ Frechet, EE Dy, and FC Szoka Bioconjugate Chem., 16(3), 535 -541, 2005 C Loo, A Lowery, N Halas, J West, and R Drezek Nano Letters, 5 (4), 709 -711, 2005

17. Challenge: High Measurement Precision of Surface Chemistry Small Changes Cause Enormous Differences in the Bioactivity Variable surface chemistry induces 6 orders of magnitude difference in toxicity profile CM Sayes, JD Fortner, W Guo, D Lyon, AM Boyd, KD Ausman, YJ Tao, B Sitharaman, LJ Wilson, JB Hughes, JL West, & VL Colvin “The Differential Cytotoxicity of Water-Soluble Fullerenes” Nano Letters, 4 (10), 1881 -1887, 2004

18. Challenge: Multiple mechanisms of particle translocation Modeling is Imperative Computational approaches can help elucidate potential mechanisms of translocation

19. Measurement Challenge Technical Strategy Issues and Barriers Dissemination of Results Milestones Resources and Leverages Outline

20. Transport Properties Extra & Intra Cellular Nanoparticle Characterization Cell Culture Bare Nanoparticle Characterization Sample Preparation Flocculation Particle Tracking Simulations Modeling Strategic Technical Approach

21. Modeling Microscopy A Multi-laboratory Effort Cell Culture Specimen Preparation Analytical

22. Gold nanoshell Initial Test Nanoparticles and Metrology Quantum Dot

23. Transmission Electron Microscopy • NEW Aberration Corrected TEM • Potential New NIST competence • Scanning Electron Microscopy • NEW Environmental SEM • Potential New NIST competence • Optical Tweezers • Single nanoparticle manipulation • in biological materials • Potential New NIST competence • Atomic Force Microscopy of • biological materials 3-D reconstruction of cells and particles Modeling Microscopy Single Particle Analysis and Manipulation A Multi-laboratory Effort Nanometrology Cell Culture Specimen Preparation Analytical

24. C Fe STEM EDS maps with 50 nm resolution at 5mm WD • Transmission Electron Microscopy • NEW Aberration Corrected TEM • Potential New NIST competence • Scanning Electron Microscopy • NEW Environmental SEM • Potential New NIST competence • Optical Tweezers • Single nanoparticle manipulation • in biological materials • Potential New NIST competence • Atomic Force Microscopy of • biological materials K Si Overlay O 3-D reconstruction of cells and particles Modeling Microscopy Single Particle Analysis and Manipulation A Multi-laboratory Effort Nanometrology Cell Culture Specimen Preparation Analytical

25. NOVA Nanolab - dynamic • sectioning of biological • material for • nanoparticle analysis • and metrology • PotentialNew NIST competence • Biological sample • preparation • for electron microscopy • PotentialNew NIST competence Modeling Microscopy A Multi-laboratory Effort Nanometrology • Focused Ion Beam • NOVA Nanolab • Biological Sample Preparation • UMd Cell Culture Specimen Preparation Analytical

26. Ultra-Small Angle X-ray Scattering (USAXS) USAXS compliments the microscopy methods, providing statistically relevant data on populations of particles under in situ conditions USAXS SLIT-SMEARED GEOMETRY FLOW-CELL SCHEMATIC Advantages of USAXS facility • Probes dimensions from nm to µm scale with good statistics • Small (adjustable) beam size (0.2 x 2 mm2) permits µL samples • Absolute scattering cross-section permits quantitative analysis • X-ray energy (wavelength) tunability • High beam flux at sample (>1012 photons/s at 10 keV) Modeling Microscopy A Multi-laboratory Effort Nanometrology Cell Culture Specimen Preparation Analytical X-ray Scattering

27. Dynamic Light Scattering (DLS) Dh = Hydrodynamic diameter of SCK Modeling Microscopy A Multi-laboratory Effort Nanometrology Cell Culture Specimen Preparation Analytical Dynamic Light Scattering

28. Measurement Challenge Technical Strategy Issues and Barriers Dissemination of Results Milestones Resources and Leverages Outline

29. Microscopy Measurements Issues/Barriers • Dynamic Light Scattering (DLS) of particles results in the hydrodynamic diameter which is highly influenced by particle shape and solvation • Sample preparation for complimentary electron beam techniques generally requires drying and good dispersal • Images are highly influenced by electron beam interaction and charging effects • Comparisons of DLS measurements with SEM, TEM and other techniques must be done • But will result in different values. • Resolution of these measurement differences is imperative • Important part of this competence effort 5 nm size Pt particles in their original environment (fuel cell)

30. Issues/Barriers Modeling Numerous proposed mechanisms and controversy in the scientific literature motivates intensive experimental and computational study of these types of assembly processes. • Endocytosis and the Encapsulation of Nanoparticles by Clathrin • Computational of Transport and Configurational Properties of Nanoparticles • 3) Influence of Particle-Substrate Interactions on the Rate of Diffusion • 4) Matrix-Driven Translocation of Nanoparticles There is currently no systematic experimental or computational approach to predict how nanoparticles might affect normal biological processes. Nanoparticle are unique in that they are commensurate in size with proteins & other biopolymers. This simple fact accounts for their high degree of bio-activity.

31. Fig. 6 Manipulation of cells and liposome using optical tweezers. Issues/Barriers Can we actually manipulate the nanoparticles and place them where we want them? Manipulation of cells and liposome using optical tweezers

32. Measurement Challenge Technical Strategy Issues and Barriers Dissemination of Results Milestones Resources and Leverages Outline

33. Technology Transfer and Dissemination • Direct interactions with manufacturers such as FEI Co., Gatan and others • Alliances with FDA and NIH • Nanotechnology Characterization Laboratory • Addresses several of the NNI Program Component Areas • Reported through the NNI • Publications and presentations • Workshops and conferences • Web-based tools for modeling and computation

34. 2006__ 2007__ 2008__ 2009__ 2010__ Measures of Success: Milestones • Intercomparison of metrology techniques • Test particle uptake in cell cultures Demonstration of dual beam sectioning Initial nanoparticle transport model • Demonstration of cryomicroscopy capabilities • Demonstration of a robust measure of dispersion • Demonstration of 3D reconstruction for biomedical applications • 3D chemical and dimensional metrology of test nanoparticles • Development of a 3D particle nanometrology infrastructure

35. Summary GOAL: Develop a 3D particle nanometrology infrastructure by 2010 This Competence at NIST will provide: • Development of an innovative toolbox for metrology of nanoparticles in biological systems • Currently does not exist • A strong group of experts in biological sample preparation • Unbiased source of expertise • Unique technical support for other agencies • Make NIST THE place where other agencies come FIRST

36. Measurement Challenge Technical Strategy Issues and Barriers Dissemination of Results Milestones Resources and Leverages Outline

37. Resource Requirements

38. Leverages • Nanotechnology Characterization Laboratory collaboration in particle characterization and standardization • Postek, Vladar, MEL • Scott, Small, CSTL • Hackley, MSEL • Food and Drug Administration collaboration in nanoparticle coatings • Karim, MSEL • ATP Intramural Project • Small, Postek • Postek, Small • 3-D Chemical Imaging Competence • Scott, Small • University of Maryland Laboratory for Ultrastructure Research • FEI Company • Hitachi High Technologies

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