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Live-cell Vibrational Spectroscopy for Nanotoxicity and Nanomedicine

S. K. Sundaram, B. J. Riley, T. J. Weber, C. A. Sacksteder, B. J. Harrer

Pacific Northwest National LaboratoryRichland, WA 99352, USA

J. M. PetermanSimplex Scientific

Middleton, WI 53562, USA

Keynote at NanotechInsight2009, Barcelona, SpainMarch 28 – April 2, 2009

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• Introduction– Nanotoxicity– Need rapid screening– Biomarker discovery

• ATR - FTIR spectroscopy– Transmission vs. ATR– Challenges and solutions

• Particokinetics - Silica settling• Silica settling on C-10 cell and interacting• Connection to high level cellular processes• Summary• Acknowledgements

Outline

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• The adverse health effects of particulate matter (PM) are well documented– Pulmonary– Cardiovascular

• Nanoparticle toxicity studies encompass complex processes – Partico/chemico/biokinetics– Composition-dependent

- Intentional functionalization- Unintentional functionalization and contaminants

• Impossible to test every variable - needs a rapid screening tool

Health Effects

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Biomarkers of particulate matter toxicity may not adequately predict nanoparticle toxicity (Warheit et al., Toxicol. Sci. 77:117-125, 2004).

What method can be used to rapidly detect biologically active nanoparticles?

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Need for Rapid and Cost Effective Global Profiling Technologies: Mode of Action is Unclear

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Comparison of Modalities

– IR Spectroscopy Modality - Can track IR-observable changes in viable cells in

real-time without the need for a specific probe- Expected to provide a semi-quantitative if not

quantitative way to know when to look for emerging biomarkers based on real-time monitoring of IR spectra

- Expected to provide new supporting bonding data by correlating IR spectral data to fluorescence imaging data of known cell responses to model toxicants

–Fluorescence Imaging Modality- Real-time sensitivity- Dependent on availability of probes for pre-determined

pathways/responses

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Why Combine IR Spectroscopy and Fluorescence Microscopy Capabilities?

–Difficult to interpret IR spectra when the origin of the IR-observable signal is unknown

–Fluorescence capability can exploit a battery of established fluorescence-based assays to improve our understanding of IR observable signals- e.g., changes in pH, membrane fluidity, organelle

status, etc…

–Fluorescence imaging modality will train the IR spectroscopy modality

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Electromagnetic Radiation

Wavelength

Ultraviolet Visible Near Infrared Infrared

200 nm 400 nm 700 nm 2500 nm 10000 nm

4000 cm-114400 cm-125000 cm-1

50000 cm-1Frequency

High Energy Low Energy

1000 cm-1

• Infrared region: – Low energy radiation– Causes net change in the dipole moment– Changes amplitude of rotation, stretching, and bending of

molecules– Non-symmetric bonds (H2O, CH, CH2, NH, NH3)

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Monitoring Cellular Response By FTIR Spectroscopy

• Cells make a good model system.- Respond to a wide range of materials- Provide complex response- Rapid screening- Cost effective- Animal welfare

• Emphasis placed on detecting biological activity of nanomaterial– No bias in the interpretation of biological activity– Detect biologically active nanoparticles without knowledge of

cellular targets a priori– Do not need a contrast agent or specific reagent to detect

biological activity– Near real-time (minutes)

• Prioritize materials for further investigation

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Transmission vs. ATR Modes of FTIR Spectroscopy

cell cellcell

Transmission Attenuated Total Reflectance (ATR)

cell cellcell

IR Detector IR Detector

IR Source

IR Source

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ATR-FTIR Spectroscopy

Material n (RT)ZnSe 2.40ZnS 2.20AMTIR 2.50Ge 4.0Water 1.330Silica 1.460

( ) 212212210

sin2 nndP

−=

θπλ

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Light Source

Light Detector Sample holder

ZnSe crystal

ZnSe crystal

ATR-FTIR Spectroscopy (continued)

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Unheated plate

Heated plate

ATR-FTIR Spectroscopy(continued)

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A Dedicated FTIR Facility

Thermo Nicolet 4700 FTIR bench

Thermo Continuµm IR microscope

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Problems in ATR Measurements: How to Improve?

ZnSe Crystal

Cell Membrane

1 µm3-10 nm

Cytosol

Functionalization layer (~ 5 Å)

IR SourceIR Detector

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Particokinetics - In Vitro

• Shape, size, and density affect settling rate.

• Settling time impacts timing/magnitude of response

• Media “dose” is different than dose to the cell

Time

Res

pons

e

Screen Compare Prioritize

In vitro

J. G. Teeguardian, P. M. Hinderliter, G. Orr, B. D. Thrall, and J. G. Pounds, “Particokinetics In Vitro: Dosimetry Considerations for In Vitro Nanoparticle Toxicity Assessments,” Toxicol. Sci., 95, pp 300-312 (2007).

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Particokinetics - Silica Particles

1 µm silicasettling experiment

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Particokinetics - Silica Particles(continued)

0

20

40

60

80

100

120

0 50 100 150 200Time (minutes)

Exp

erim

enta

l (pe

ak a

rea

unde

r 110

7 cm

-1)

0.E+00

1.E+07

2.E+07

3.E+07

4.E+07

5.E+07

6.E+07

Sim

ulat

ion

(num

ber o

f set

tled

parti

cles

)

1µm silica (measured)1µm silica (calculated)

1 µm silica settling model

J. G. Teeguardian, P. M. Hinderliter, G. Orr, B. D. Thrall, and J. G. Pounds, “Particokinetics In Vitro: Dosimetry Considerations for In Vitro Nanoparticle Toxicity Assessments,” Toxicol. Sci., 95, pp 300-312 (2007).

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Nano Silica Particles SettlingHigh dose of 10 nm silica after 2 hrs of settling

Low dose of 10 nm silica after 2 hrs of settling

Primary silica peak~ 1107 cm-1

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43

1

2

(1) Asymmetric CH3 stretch(2) Asymmetric CH2 stretch(3) Symmetric CH3 stretch(4) Symmetric CH2 stretch

Scans show a negative absorbance ofcells following exposure to silica particlesas compared to untreated cells.

Zoom in!

~2 hrs after exposure to high dose of 10nm silica

~22 hrs after exposure to low dose of 10nm silica

The changing of the CH2 andCH3 stretching peaks revealthat the membrane isbecoming soluble. This wasalso reported by Bureau, et al.

B. Bureau, X. H. Zhang, F. Smektala, J.-L. Adam, J. Troles, H.-l. Ma, C. Boussard-Ple`del, J. Lucas, P. Lucas, D. Le Coq, M. R. Riley, J. H. Simmons, “Recent advances in chalcogenide glasses,” Journal of Non-Crystalline Solids, 345&346, pp 276-283 (2004).

Nano Silica Particles Settling

21A. M. Melin, A. Allery, A. Perromat, C. Bébéar, G. Déléris, B. Barbeyrac. Fourier transform infrared spectroscopy as a new tool for characterization of mollicutes. Journal of Microbiological Methods. 2004, 56, 73-82.A. Pacifico, L. A. Chiriboga, P. Lasch, M. Diem. Infrared spectroscopy of cultured cells: II. Spectra of exponentially growing, serum-deprived and confluent cells. Vibrational Spectroscopy. 2003, 32, 107-122.

1400 cm-1-COO- symmetric stretch (Pacifico et al, 2003)

1455 cm-1CH2/CH3 deformation modes (Pacifico et al, 2003)

1546 cm-1N-H bending vibration (amide II) (Melin et al, 2004)

1625, 1640, 1657 cm-1β-sheets, α-helices (Melin, 2001)

A. M. Melin, G. Perromat, G. Déléris. 2001. Effect of radical attach on bacteria: an application of FT-IR spectroscopy. Applied Spectroscopy 55:23-28.

Zoom in!

~2 hrs after exposure to high dose of 10nm silica

~22 hrs after exposure to low dose of 10nm silica

Nano Silica Particles Settling

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Can We Detect Induced Biological Activity In Live Cells By FTIR?

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Ratio Spectra To Identify Differences

No difference

numerator

denominator

Difference Spectra

TargetWavenumber

24

-20

-10

0

10

20

30

40

165016521654165616581660

Wavenumbers (cm-1)

LPS

:Con

trol r

atio

1652:C=O stretch vibration

2 Hr6 Hr8 Hr24 Hr

Temporal Changes in RAW 264.7 Cells Treated With LPS

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Macrophage Activation

• Wavenumber 1652 frequently modulated by endotoxin treatment– C=O

• Endotoxin induces an inflammatory response in macrophages– reactive oxygen and nitrogen species (oxidative/

nitrative stresses) - Cox-2- iNOS

• Oxygen free radical damage to proteins results in protein carbonyl formation– C=O

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Hypothesis

Inflammatory processes can be detected in live cells by FTIR.

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Wavenumber 1652 cm-1 Temporal Profile Following LPS-Treatment

Time [Hr]

Fold

Incr

ease

J J

J

JJ J

J

0

5

10

0 2 4 6

Wavenumber 1652

iNOS

Cox-2

DAPI

Control LPS

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Indomethacin Effects on Peak 1652 cm-1

-37

17

165016521654-327

1217

165016521654

Fold

Cha

nge

LPSLPS +

Indomethacin

Wavenumbers (cm-1)

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Cox-2 Transfected Cells Treated With Arachidonic Acid

1652 cm-1

Time [Hr]

J JJ

J

J

0

2

4

6

8

0 2 4

Fold

Incr

ease + AA

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Carbonyl bond frequencies found in different functional groups

Group Frequency (cm-1) Functional Group Figure Reference

1610-1550 / 1420-1300 Carboxylate (carboxylic acid salt) 1

1680-1630 Amide 2

1690-1675/(1650-1600)a Quinone or conjugated ketone 3

1725-1700 Carboxylic acid 4

1725-1705 Ketone 5

1740-1725/(2800-2700)b Aldehyde 6

1750-1725 Ester 7

1735 Six-membered ring lactone 8

1760-1740 Alkyl carbonate 9

1815-1770 Acid (acyl) halide 10

1820-1775 Aryl carbonate 11

1850-1800/(1790-1740) Open-chain acid anhydride 12

1870-1820/(1800-1775) Five-membered ring anhydride 13

2100-1800 Transition metal carbonyls 14

a Lower frequency band is from the conjugated double bondb Higher frequency band characteristic of aldehydes, associated with the terminal aldehydic C-H stretch

REFERENCE: J. Coates. Interpretation of infrared spectra, a practical approach in Encyclopedia of Analytical Chemistry. R. A. Meyers (Ed.), pp. 10815-10837. Copyright © John Wiley & Sons Ltd, Chichester. 2000.

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MWCNT-Treated RAW 264.7 Cells

Exp 1

Exp 2

Exp 3

1083 O-P=O symmetric stretchphosphates

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Transformation and Analysis

Data

Information

Knowledge

Analysis

Model

Chemometrics• Discipline concerned with the

application of statistics and mathmatical methods of chemistry

• Complex, interrelated data sets

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Qualitative vs. Quantitative

• Mostly qualitative or semi-quantitative• Quantitative analysis

–Water absorption–Base-line shift–Temperature control–Atmosphere

• Chemometrics• Mechanisms• Integration for rapid screening

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Summary• Live cell FTIR spectroscopy is a promising tool to identify

biologically active nanomaterials– Intuitive temporal patterns of regulation– Changes are complex

- Complementary to genomic and proteomic approaches

• Functionalization monolayer chemistries support cell attachment and proliferation– Goal is to identify chemistry that is biologically inert or non-perturbing

to improve ATR spectroscopy in live cell experiments

• Particochembiokinetics – Can be studied in near real-time

• Development of pathway- or process-specific spectral profiles is needed to enable spectra interrogation– Cell cycle, Cell death, Target pathways, Toxicants with well defined

modes of action, etc.

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Acknowledgements

• Cheryl Cejka for support under TechnologyMaturation program

• Joel G. Pounds, Ellyn Murphy, EnvironmentalBiomarker Initiative (EBI)– Support of LDRD project

• Pacific Northwest National Laboratory (PNNL) is amultiprogram national laboratory operated byBattelle Memorial Institute for the United StatesDepartment of Energy under DE-AC06-76RLO1830.

Slide Number 1OutlineHealth EffectsSlide Number 4Need for Rapid and Cost Effective �Global Profiling Technologies: �Mode of Action is UnclearComparison of ModalitiesWhy Combine IR Spectroscopy �and Fluorescence Microscopy Capabilities?Slide Number 8Monitoring Cellular Response �By FTIR SpectroscopyTransmission vs. ATR Modes of �FTIR SpectroscopyATR-FTIR SpectroscopySlide Number 12ATR-FTIR Spectroscopy�(continued)A Dedicated FTIR FacilityProblems in ATR Measurements: �How to Improve?Slide Number 16Particokinetics - Silica ParticlesParticokinetics - Silica Particles�(continued)Nano Silica Particles SettlingNano Silica Particles SettlingNano Silica Particles SettlingCan We Detect Induced Biological Activity In Live Cells By FTIR?Ratio Spectra To Identify DifferencesSlide Number 24Macrophage ActivationHypothesisWavenumber 1652 cm-1 Temporal Profile Following LPS-TreatmentIndomethacin Effects on �Peak 1652 cm-1Cox-2 Transfected Cells Treated �With Arachidonic AcidCarbonyl bond frequencies found �in different functional groupsMWCNT-Treated RAW 264.7 CellsSlide Number 32Qualitative vs. QuantitativeSummaryAcknowledgements