MEL

CSTL MSEL

Metrology for the “Fate” of

Nanoparticles 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
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Metrology for the “Fate” of Nanoparticles

CSTL MSEL MEL

Michael T. Postek, Champion
Andras Vladar
Thomas LeBrun
John Dagata,

CSTL MSEL
John Small, Champion Alamgir Karim, Champion
John Henry Scott Jack Douglas
Scott Wight, Matthew Becker
Peter Barker Vincent Hackley
Andrew Allen
Stephanie Hooker
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Metrology for the “Fate” of Nanoparticles

CSTL MSEL

• Competence proposal is motivated by work stemming

from:
– National Nanotechnology Initiative
• Subcommittee on Nanoscale Science,
Engineering and Technology (NSET)
– Michael Postek
– Alamgir Karim
…. “I would anticipate a highly successful project,
• Nanotechnology Environmental and Health
and one that will support research into the impact
Implications Working Group (NEHI WG)
of nanotechnology in a significant manner.”
– John Small
……. Andrew Maynard, NIOSH
– Michael Postek
– The National Institute for Occupational Safety and
Health (NIOSH)
• Andrew Maynard (endorsement)
– Environmental Protection Agency (EPA)
• Barbara Karn
• Nora Savage
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Why Now…Impact on Society, Environment,
and Commerce

CSTL MSEL

Without
NIST is the only organization NIST’s
that has the contributions…..
mission and the necessary combination of
…A joint collaboration with the National Institute of Standards and Technology (NIST) and the FDA is a high priority.
the
stafflack of a common
expertise, infrastructure
experience, industry for bioscience
contacts, measurements,
industry standards,recognition
respect, & worldwide & data will
The collaboration
as the leadingconstrain innovation
measurement
will focus
and stifle the
and standards
on developing
commercialization
institution
standards
of new
to address the
for nanoscale
highbioscience-based
devices
risk scientific and applications
technological challenges
and
in both
healthin care,
associated vitro
with and in vivo and
agriculture
developing characterization
the food, assays
environmental
measurement that could
protection,
techniques serve as astandards
homeland
and technical starting
security,point
and for regulatory
many
needed other
in this filings.
areas.
new field.
Cancer Nanotechnology
NISTNanotechnology
National Plan,pgpg11.
2010 Strategic Initiative,
Plan, 24.119.
pg
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Why Now...Potential for Catastrophic Effects:
Psychological and Economic

X
CSTL MSEL

Flawed Science Big PRESS

For Science, Nanotech Poses Big Unknowns

By Rick Weiss
Washington Post Staff Writer
Sunday, February 1, 2004; Page A01

Materials Today
This approach will not reassure the public at 2003:
July/Aug large64
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Why NIST…Lack of Measurement Infrastructure has
Become a Critical Hurdle to Further Innovation

Productivity / Market Access / Public Safety & Welfare

CSTL MSEL

Standards
Development
(Phase II)
VALUE CREATION (IMPACT):

NIST’s
Role
Basic A new product development toolkit
“Instrumentation
Science and metrology crosscut all the NNI Grand
containing powerful new scientific and
Challenges, and are vital to the success of the NNI.
Advances in fundamental nanoscience, design of new technical methods ……. is urgently needed
nano-materials, and ultimately
Scientific manufacturing
New of new
Technology to improve predictability and efficiency along
nanoscale Discovery
products will allInvention
depend
Period
to some degree on
Improvement
the critical path from laboratory concept to
Period
the capability Measurement
to accurately and reproducibly measure commercial product.
properties and performance characteristics at the
Methods & Tools S&T DEVELOPMENT OVER TIME
(Phase I)
nanoscale. *Office of Cellular, Tissue, and Gene Therapies
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It is this simple - what is the difference?

CSTL MSEL
Micrometer sized particles
of Zinc Oxide
Left

FDA approval of zinc oxide nanoparticles

is “grandfathered” in because of the
Whatapprovals
previous is the effect
foron the body?
micrometer-sized
particles…
… discussion held at the first NEHI meeting

Right
Right sized particles
Nanometer
of Zinc Oxide
Primary Particle size of 3-10 nm
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Competence Goal

CSTL MSEL

• 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
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Motivation

CSTL MSEL

Why Nanoparticle Metrology for Biological

Systems?
• 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
 MEL

Outline

CSTL MSEL

• Measurement Challenge
• Technical Strategy
• Issues and Barriers
• Dissemination of Results
• Milestones
• Resources and Leverages
 MEL

Outline

CSTL MSEL

• Measurement Challenge
• Technical Strategy
• Issues and Barriers
• Dissemination of Results
• Milestones
• Resources and Leverages
 MEL

Underlying Basis for Measurement Need

CSTL MSEL Effect of size

Exposure/Uptake
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

Nano-particle
properties depend on physical and chemical structure. Quantitative
assessments of the risk associated with engineered nanomaterials
Life-Cycle
can not therefore proceed without techniques capable of providing
biologically-relevant physiochemical information with nanometer
resolution.” – Andrew Maynard, NIOSH

Transport Bioactivity
Control of mobility and distribution Surface chemistry effects
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Challenge: Biological Sample Preparation

CSTL MSEL

• 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.
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Challenge: Diversity of Nanoparticles Presents Numerous
Characterization Challenges
Dendrimers Quantum Dots Nanotubes (C, Au, Hybrid…)
CSTL MSEL

SWNT

DWNT

MWNT

Functionalized
Liposomes Micelles Nanoparticles

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
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Challenge: Numerous Measurement
Parameters of Nanoparticles
particle diameter
CSTL MSEL
core diameter Core composition
&
shell thickness
stiffness
Molecular mass
& distribution
Surface chemistry
Aggregation number &
(# molecules per particle) Extent of bio-functionality
Dispersion

Properties Impacted by Measurable Parameters

pH susceptibility
Bioactivity
Diffusion coefficient
Anisotropic transport
Membrane permeability
Degradation mechanisms and byproducts
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Challenge: Increasing Complexity

CSTL MSEL

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
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Challenge: High Measurement Precision of Surface Chemistry

CSTL MSEL

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
 MEL

Challenge: Multiple mechanisms of particle translocation

CSTL MSEL

Modeling is Imperative

Computational approaches can help elucidate potential

mechanisms of translocation
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Outline

CSTL MSEL

• Measurement Challenge
• Technical Strategy
• Issues and Barriers
• Dissemination of Results
• Milestones
• Resources and Leverages
 MEL

Strategic Technical Approach

CSTL MSEL
Transport Properties

Extra & Intra Cellular

Nanoparticle Characterization

Cell Culture

Bare
Nanoparticle
Characterization

Sample
Preparation

Flocculation
Particle Tracking

Simulations
Modeling
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A Multi-laboratory Effort

CSTL MSEL

Modeling Microscopy

Cell Culture Specimen

Preparation

Analytical
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Initial Test Nanoparticles and Metrology

CSTL MSEL
Extracellular Metrology Intracellular Metrology
Cell Culture Ultrastructure
Particle TEM TEM (sectioned material)
Characterization
SEM SEM (sectioned material)
FIB (sectioned material)
Cryomicroscopy
Dimensional Metrology SEM SEM
Gold
TEM TEM
nanoshell
DLS
USAXS
Particle Imaging SEM
TEM
Fluorescence Fluorescence Fluorescence
AFM AFM AFM
Transport Modeling x x x

Quantum Particle Placement Optical Tweezers Optical Tweezers

Dot Particle Localization Fluorescence Fluorescence

Particle Removal Optical Tweezers

 MEL

A Multi-laboratory Effort

CSTL MSEL

Transmission Electron Microscopy Single Particle Analysis

Modeling
• NEW Aberration Corrected TEM
Microscopy
and Manipulation
• Potential New NIST competence
Scanning Electron Microscopy
• NEW Environmental SEM
•Potential New NIST competence
Optical Tweezers Nanometrology
• Single nanoparticle manipulation
in biological materials
•Potential New NIST competence
Atomic Force Microscopy of
Cell Culture Specimen
3-D reconstruction of
biological materials cells and particles
Preparation

Analytical
 MEL

A Multi-laboratory Effort

CSTL MSEL

Transmission Electron Microscopy Single Particle Analysis

Modeling
• NEW Aberration Corrected TEM
Microscopy
and Manipulation
• Potential New NIST competence
Scanning Electron Microscopy
• NEW Environmental SEM
•Potential New NIST competence
Optical Tweezers Nanometrology
C Fe

• Single nanoparticle manipulation

in biological materials
•Potential New NIST competence
K Si

Atomic Force Microscopy of

Cell Culture Specimen
3-D reconstruction of
biological materials cells and particles
Preparation
O Overlay

STEM EDS maps with 50 nm resolution at 5mm WD

Analytical
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A Multi-laboratory Effort

CSTL MSEL

Modeling Microscopy

Nanometrology
NOVA Nanolab - dynamic
sectioning of biological
material for
nanoparticle analysis Focused Ion Beam
Cell Culture
and metrology Specimen
• NOVA Nanolab
• Potential New NIST competence Biological Sample Preparation
Preparation
Biological sample • UMd
preparation
for electron microscopy
• Potential New NIST competence Analytical
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A Multi-laboratory Effort
Ultra-Small Angle X-ray Scattering (USAXS)
CSTL
USAXS compliments
MSEL
the microscopy
methods, providing
Modeling Microscopy
statistically relevant data on
populations of particles under in situ
conditions
USAXS SLIT-SMEARED GEOMETRY Nanometrology
sample APS
photodiode E
FLOW-CELL

detector ion
I chamber
Si (111) mirrors SCHEMATIC
Io
Si (111) collimating 33ID
Si (111)
analyzer crystals 2D slits undulator
monochromator
crystals

Cell Culture Specimen

Advantages of USAXS facility
Preparation
• 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)
Analytical
X-ray Scattering
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A Multi-laboratory Effort
Dynamic Light Scattering (DLS)
CSTL MSEL

Modeling Microscopy

Nanometrology

Cell Culture Specimen

Preparation

Dh = Hydrodynamic diameter of SCK

Analytical
Dynamic Light Scattering
 MEL

Outline

CSTL MSEL

• Measurement Challenge
• Technical Strategy
• Issues and Barriers
• Dissemination of Results
• Milestones
• Resources and Leverages
 MEL
Issues/Barriers

CSTL MSEL

• Dynamic Light Scattering (DLS) of particles

results in the hydrodynamic diameter which isMicroscopy Measurements
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 5 nm size Pt particles in their original
done environment (fuel cell)
– But will result in different values.

• Resolution of these measurement differences

is imperative
– Important part of this competence effort
 MEL

Issues/Barriers

CSTL MSEL Modeling

Numerous proposed mechanisms and controversy in the scientific
literature motivates intensive experimental and computational study of
these types of assembly processes.
1) Endocytosis and the Encapsulation of Nanoparticles by Clathrin
There is currently no systematic experimental or
2) Computational of Transport
computational approach and
to Configurational Properties of
predict how nanoparticles
might affect normal biological processes.
Nanoparticles
3) Influence of Particle-Substrate Interactions on the Rate of Diffusion
4) Matrix-Driven Translocation of Nanoparticles

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.
 MEL

Issues/Barriers

CSTL MSEL

Can we actually manipulate the nanoparticles

and place them where we want them?

cell
liposome

Manipulation of cells and liposome using optical tweezers

Fig. 6 Manipulation of cells and liposome using optical tweezers.
 MEL

Outline

CSTL MSEL

• Measurement Challenge
• Technical Strategy
• Issues and Barriers
• Dissemination of Results
• Milestones
• Resources and Leverages
 MEL

Technology Transfer and Dissemination

CSTL MSEL

• 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
 MEL

Measures of Success: Milestones

CSTL MSEL

2006__ • Intercomparison of metrology techniques

• Test particle uptake in cell cultures

2007__ Demonstration of dual beam sectioning

Initial nanoparticle transport model

2008__ • Demonstration of cryomicroscopy capabilities

• Demonstration of a robust measure of dispersion

2009__ • Demonstration of 3D reconstruction for biomedical applications

• 3D chemical and dimensional metrology of test nanoparticles
2010__
• Development of a 3D particle nanometrology infrastructure
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Summary

CSTL MSEL

This Competence at NIST will provide:

• Development of an innovative toolbox for metrology of
GOAL:
nanoparticles in biological systems
– Currently does
Develop a 3Dnotparticle
exist nanometrology
infrastructure
• A strong group by 2010
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
 MEL

Outline

CSTL MSEL

• Measurement Challenge
• Technical Strategy
• Issues and Barriers
• Dissemination of Results
• Milestones
• Resources and Leverages
 MEL

Resource Requirements

CSTL MSEL

Budget
Total
Competence
STRS
Year MEL CSTL MSEL IE Funding
Labor
Request
(Labor + IE)
($ in thousands)
2006 400K 300K 300K 1.0M 1.2M 2.2M
2007 400K 300K 300K 1.0M 600K 1.6M
2008 400K 300K 300K 1.0M 300K 1.3M
2009 400K 300K 300K 1.0M 200K 1.2M
2010 400K 300K 300K 1.0M 200K 1.2M
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Leverages

CSTL MSEL

• 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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CSTL MSEL

Thank you
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CSTL MSEL

Thank you