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MSEL MEL CSTL Metrology for the “Fate” of Nanoparticles in Biosystems Michael T. Postek, Andras Vladar, Thomas LeBrun and John Dagata, MEL John Small,

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Presentation on theme: "MSEL MEL CSTL Metrology for the “Fate” of Nanoparticles in Biosystems Michael T. Postek, Andras Vladar, Thomas LeBrun and John Dagata, MEL John Small,"— Presentation transcript:

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

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

3 MSEL MEL CSTL Metrology for the “Fate” of Nanoparticles Competence proposal is motivated by work stemming from:Competence proposal is motivated by work stemming from: –National Nanotechnology Initiative Subcommittee on Nanoscale Science, Engineering and Technology (NSET)Subcommittee on Nanoscale Science, Engineering and Technology (NSET) –Michael Postek –Alamgir Karim Nanotechnology Environmental and Health Implications Working Group (NEHI WG)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)Andrew Maynard (endorsement) –Environmental Protection Agency (EPA) Barbara KarnBarbara Karn Nora SavageNora Savage …. “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

4 MSEL MEL CSTL 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. 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. The collaboration will focus on developing standards for nanoscale devices …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 assays that could serve as a starting point for regulatory filings. Cancer Nanotechnology Plan, pg 24.

5 MSEL MEL CSTL Materials Today July/Aug 2003: 64 Why Now...Potential for Catastrophic Effects: Psychological and Economic X Flawed Science Big PRESS For Science, Nanotech Poses Big Unknowns By Rick Weiss Washington Post Staff Writer Sunday, February 1, 2004; Page A01 This approach will not reassure the public at large 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

6 MSEL MEL CSTL VALUE CREATION (IMPACT): Productivity / Market Access / Public Safety & Welfare S&T DEVELOPMENT OVER TIME New Invention Period Scientific Discovery March 2004 A new product development toolkit containing powerful new scientific and technical methods ……. is urgently needed to improve predictability and efficiency along the critical path from laboratory concept to commercial product. *Office of Cellular, Tissue, and Gene Therapies Why NIST…Lack of Measurement Infrastructure has Become a Critical Hurdle to Further Innovation Standards Development (Phase II) Measurement Methods & Tools (Phase I) Basic Science NIST’s Role Technology Improvement Period “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.

7 MSEL MEL CSTL It is this simple - what is the difference? Left Right Nanometer sized particles of Zinc Oxide Micrometer sized particles of Zinc Oxide Primary Particle size of 3-10 nm What is the effect on the body? 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

8 MSEL MEL CSTL 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 MSEL MEL CSTL Motivation Why Nanoparticle Metrology for Biological Systems? Nanoparticles are being produced and are a part of our environmentNanoparticles are being produced and are a part of our environment Nanoparticle measurements are challengingNanoparticle measurements are challenging Biological samples must be carefully preparedBiological samples must be carefully prepared Validation procedures are needed for standardization of measurementsValidation procedures are needed for standardization of measurements NIST needs to develop competence in this areaNIST needs to develop competence in this area NIST can make a differenceNIST can make a difference

10 MSEL MEL CSTL Measurement ChallengeMeasurement Challenge Technical StrategyTechnical Strategy Issues and BarriersIssues and Barriers Dissemination of ResultsDissemination of Results MilestonesMilestones Resources and LeveragesResources and Leverages Outline

11 MSEL MEL CSTL Measurement ChallengeMeasurement Challenge Technical StrategyTechnical Strategy Issues and BarriersIssues and Barriers Dissemination of ResultsDissemination of Results MilestonesMilestones Resources and LeveragesResources and Leverages Outline

12 MSEL MEL CSTL Underlying Basis for Measurement Need Exposure/UptakeBioactivityTransport Nano-particle Life-Cycle 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 Effect of size Control of mobility and distributionSurface chemistry effects

13 MSEL MEL CSTL Challenge: Biological Sample Preparation Biological samples require:Biological samples require: –Fixation –Dehydration –Embedding –Sectioning Dual beam FIBDual beam FIB CryomicroscopyCryomicroscopy CryomicrotomyCryomicrotomy Provides a new paradigm for NISTProvides a new paradigm for NIST –Physical metrology Biological sample preparation is not new to NIST, but not extensively practiced at NISTBiological 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.Biological samples require expertise that must be developed at NIST in order to assist our customers in metrology related situations.

14 MSEL MEL CSTL SWNT DWNT MWNT Nanotubes (C, Au, Hybrid…)Quantum DotsDendrimers Challenge: Diversity of Nanoparticles Presents Numerous Characterization Challenges 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 MicellesLiposomes Functionalized Nanoparticles

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

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

17 MSEL MEL CSTL Challenge: High Measurement Precision of Surface Chemistry 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), , 2004 Variable surface chemistry induces 6 orders of magnitude difference in toxicity profile Small Changes Cause Enormous Differences in the Bioactivity

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

19 MSEL MEL CSTL Measurement ChallengeMeasurement Challenge Technical StrategyTechnical Strategy Issues and BarriersIssues and Barriers Dissemination of ResultsDissemination of Results MilestonesMilestones Resources and LeveragesResources and Leverages Outline

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

21 MSEL MEL CSTL A Multi-laboratory Effort Modeling Cell Culture Specimen Preparation Microscopy Analytical

22 MSEL MEL CSTL Initial Test Nanoparticles and Metrology Gold nanoshell Extracellular MetrologyIntracellular Metrology Cell CultureUltrastructure Particle Characterization TEMTEM (sectioned material) SEMSEM (sectioned material) FIB (sectioned material) Cryomicroscopy Dimensional MetrologySEM TEM DLS USAXS Particle ImagingSEM TEM Fluorescence AFM Transport Modelingxxx Particle PlacementOptical Tweezers Particle LocalizationFluorescence Particle RemovalOptical Tweezers Quantum Dot

23 MSEL MEL CSTL A Multi-laboratory Effort Modeling Cell Culture Specimen Preparation Microscopy Analytical Nanometrology Single Particle Analysis and Manipulation 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

24 MSEL MEL CSTL A Multi-laboratory Effort Modeling Cell Culture Specimen Preparation Microscopy Analytical Nanometrology Single Particle Analysis and Manipulation 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 STEM EDS maps with 50 nm resolution at 5mm WD CFe Overlay SiK O

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

26 MSEL MEL CSTL A Multi-laboratory Effort Modeling Cell Culture Specimen Preparation Microscopy Analytical Nanometrology X-ray Scattering FLOW-CELL SCHEMATIC Ultra-Small Angle X-ray Scattering (USAXS) Advantages of USAXS facility Probes dimensions from nm to µm scale with good statistics Small (adjustable) beam size (0.2 x 2 mm 2 ) permits µL samples Absolute scattering cross-section permits quantitative analysis X-ray energy (wavelength) tunability High beam flux at sample (>10 12 photons/s at 10 keV) USAXS SLIT-SMEARED GEOMETRY USAXS compliments the microscopy methods, providing statistically relevant data on populations of particles under in situ conditions

27 MSEL MEL CSTL A Multi-laboratory Effort Modeling Cell Culture Specimen Preparation Microscopy Analytical Nanometrology Dynamic Light Scattering D h = Hydrodynamic diameter of SCK Dynamic Light Scattering (DLS)

28 MSEL MEL CSTL Measurement ChallengeMeasurement Challenge Technical StrategyTechnical Strategy Issues and BarriersIssues and Barriers Dissemination of ResultsDissemination of Results MilestonesMilestones Resources and LeveragesResources and Leverages Outline

29 MSEL MEL CSTL 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) Microscopy Measurements

30 MSEL MEL CSTL 1)Endocytosis and the Encapsulation of Nanoparticles by Clathrin 2)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 Numerous proposed mechanisms and controversy in the scientific literature motivates intensive experimental and computational study of these types of assembly processes. Modeling 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. There is currently no systematic experimental or computational approach to predict how nanoparticles might affect normal biological processes. Issues/Barriers

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

32 MSEL MEL CSTL Measurement ChallengeMeasurement Challenge Technical StrategyTechnical Strategy Issues and BarriersIssues and Barriers Dissemination of ResultsDissemination of Results MilestonesMilestones Resources and LeveragesResources and Leverages Outline

33 MSEL MEL CSTL 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 Technology Transfer and Dissemination

34 MSEL MEL CSTL Measures of Success: Milestones Intercomparison of metrology techniques Test particle uptake in cell cultures 2006__ 2007__ 2008__ 2009__ 2010__ 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 MSEL MEL CSTL Summary 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 GOAL: Develop a 3D particle nanometrology infrastructure by 2010

36 MSEL MEL CSTL Measurement ChallengeMeasurement Challenge Technical StrategyTechnical Strategy Issues and BarriersIssues and Barriers Dissemination of ResultsDissemination of Results MilestonesMilestones Resources and LeveragesResources and Leverages Outline

37 MSEL MEL CSTL Resource Requirements

38 MSEL MEL CSTL 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

39 MSEL MEL CSTL Thank you

40 MSEL MEL CSTL Thank you


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