Embed Size (px)
Citation preview
Lisa DeLouise
Nanomaterials EH&S –A Perspective
Panel 6 “Transformations in the organisms and in the environment: what do we measure and how do we develop testing strategies to measure impacts of
particles that may be transformed over time in the environment”
Lisa DeLouise, PhDUniversity of Rochester Medical Center
Departments of Dermatology and Biomedical Engineering
NNI Workshop on Nanomaterials and the Environment and InstrumentationOctober 6-7, 2009
Lisa DeLouise
Exposure?l Research should be guided by materials that are likely to be
economically important
l Obviously has driven EPA top 7 l single-walled carbon nanotubes, l multi-walled carbon nanotubes, l fullerenes, l cerium oxide, l silver, l titanium dioxide, l zero-valent iron.
State of the Science
Lisa DeLouise
The Problem is Much Bigger
l Evident technologies – pioneering QD consumer products
Clint Ballinger, CEO of Evident Technologies holds a selection of LED lights in this 2007 file photo.
(Photo by J.S. Carras)
2008 We'd like to introduce you to one of Evident's newest products - dotstrandTM Energy Efficient LED Lights - the world's first consumer product to utilize quantum dot technology.
Lisa DeLouise
Nanomaterials in Research
l From published literature it is clear the NP research increasing CNT 2x QD
l How to quantify exposure in academics and industrial R&D?
0
200
400
600
800
1000
1200
1400
1600
1800
2003 2004 2005 2006 2007 2008 1/1/09 to09/27/09
nanoparticle carbon nanotubequantum dot metal nanoparticle (x10)
Pubmed
Image from Evident Technologies WebsiteImage from Evident Technologies Website
CdSe
2-10 nm3
Can they penetrate through my gloves?
Lisa DeLouisehttp://www.siumed.edu/~dking2/intro/IN008b.htm
Histology - Healthy Human Epidermis
~100 mm
Under what conditions may NM penetrate?What cell types do they associate with?
Can we quantify it?
Lisa DeLouise
Skin Conditions Vary
l Different skin types (light/dark)l Follicular densityl Different barrier defectsl Mechanical cutsl Chemicalsl Environmental (UV, microbes,
allergens)l Disease
Lademann 2006
Lisa DeLouise
Hypotheses:
1. Size, shape, charge and surface energy are key NM properties that determine epithelial penetration upon first exposure
2. Composition, dissolution properties, and translocation determine toxicity secondarily
Are current methods & models sufficient to prove this? – No
Lisa DeLouise
State of the ScienceSkin Barrier Status and NM Detection Techniques
l Histology / Immunofluorescencel Slicing may introduce artifacts, slowl Background autofluorescence
l Transepidermal water loss (TEWL)l Only accepted methodl Measures inside-out barrier only
l Franz/Ussing Diffusion Studiesl TEM
l Limited tissue analysis, slow, expensivel Skin structures are also dark and smalll Need amplification strategy
l Flow cytometry (FACS)l Quantitative with good statistics l Destructive
l NIR microscopyl Major innovation and future http://www.odakecza.com/bilder/reviscisondarm.jpg
Lisa DeLouise
TEWL - UVB Induced Barrier Damage
* *** *** * *
*** *** *** ***
• UVB induced barrier impairment is quantifiable with TEWL quantified?• Can this be correlated to QD penetration levels?
*** *** *** ***
Lisa DeLouise
DAPI/QD Fluorescence
Brightfield/QD Fluorescence
180mJ/cm2 UVBDHLA QD 620
360mJ/cm2 UVBDHLA QD 620
Dose = (Exposure)x(Penetration)??
Lisa DeLouise
The Trouble Is...
Difficult to detect isolated particles above autofluorescence background
Lisa DeLouise
And on TEM……..
Colloidal Ag method used to amplify QD
Lisa DeLouise
Important Pointsl TEWL on UV-induced barrier (in vivo mouse) correlated with QD
penetration
l Need to resolve TEM/Histology results
l Whole tissue imaging preferred
l Quantification of NM penetration via visible fluorescence above tissue background is problematic
Lisa DeLouise
Fluorescence-activated cell sorting (FACS)
Fluorescence intensity used to quantify NM uptake
But….uptake or associated?
1
1.5
2
2.5
QD only Nair QD Tape strip QD
Fold change QD Median over Control
*
*
*p<0.0001
• Measure 20000 cells
• Quantify % of fluorescent cells or the relative fluorescence magnitude relative to control
QD applied to ex vivo skin 24 hr
Lisa DeLouise
State of the Science - Models
l Are tissue models appropriate (in vitro, ex vivo, in vivo)?l Are exposure methods relevant?
In vitro cell culture ubiquitously used to: Screen NM cytotoxictyUptake mechanisms
Lisa DeLouise
State of the Science – ModelsProblem #1 - Dose l Cytotoxicity studies done at acute NP exposure levels far greater than
is anticipated to occur.l Skin Cell QD toxicity >20 nMl 107 QD/cell
Zhang et al. Toxicology and Applied Pharmacology 228 (2008) 200–211
Is this realistic?
Lisa DeLouise
State of the Science – ModelsProblem #2 Validityl NM can interfere with standard assays used to quantify cytotoxicityl HEK line
SWCNT
QD-COOH 655
Lisa DeLouise
State of the Science – ModelsEx vivo Tissue Modelsl Pig, human skin – they are differentl Storage – frozen skin is deadl Application of materials – no standard vehicle
Lisa DeLouise
QD Ex vivo Pig Skin PenetrationFirst studies (2006) demonstrated high permeability levels and surface
chemistry dependent.
JP Ryman-Rasmussen, et al. Toxicol Sci 2006, 91, 159.
565nm PEG Invitrogen 565nm PEG-amine Invitrogen 565nm Carboxyl Invitrogen
Lisa DeLouise
QD Skin Penetration
More recent work (2008) ex vivo porcine skin suggests much lower levels of permeability
LW Zhang et al. Toxicol Appl Pharmacol 2008, 228:200211
621nm PMAO-PEG, 24 hours
• Is it NM shape?• Tissue processing?• Tissue Type?
Lisa DeLouise
FITC conjugated- NLS (PKKKRKV)
Confocal Images
Lisa DeLouise
l Standardized NM and viability assays
l Need to quantify tissue penetration at realistic exposure/dose levelsl short-term/high dose vs. long-term/low dose
l Need accurate models of barrier impaired skin (physical, chemical or diseased)
l Need methods to quantify tissue penetrationl Fluorescent techniques need to be calibrated- what is the detection limit?l How does quantum yield of NM effect detection?l Need novel NM amplification strategies.l Need NIR-based whole tissue imaging (no slicing, larger sample)l Can we take advantage of NIR two-photon imaging?
Science and Technology Barriers
Lisa DeLouise
l NIR Tissue Windowl Some NM exhibit strong absorbance in NIRl Two-photon excitation microscopy
State of the Science
R Weissleder Nat Biotechnol 2001, 19, 316.
Lisa DeLouise
State of the ScienceWild and Jones Environ. Sci. Technol. 2009, 43, 5290–5294 (Lancaster UK)
TPEM used to image TiO2, CeO2, and MWCNTs in living wheat rootsl Pump at 720 nml Image MWCNT ex 710 nm, em 300-390 nml Image TiO2 ex 720 nm, em 410-600 nml Wheat root ex 710 nm, em 500-530 nm
Discovered MWCNT pierces cell membrane and is a portal for transport of PAH
Lisa DeLouise
QD Limit of Detection
s
N =4prb
2Fout
WQrqd2 exp(-2ms 'z)F0
Fout = 8.89 ´10-4 mW
QD
Detector
Laser
F0 = 500mW
RA Wilson, J Zavislan Submitted to Optics Lett 2008
W = 2p 1- cos sin-1( 34 NA)( )( )
NA=numerical aperture
Lens Collection
4pW
Tissue Scattering
z=Tissue Depth
ms '= Scattering Coefficient
1e-2m s 'z
Beam Waist
rb2
rqd2
rb= radius of the laser beamrqd= radius of the QD being illuminated
Near-IR Wavelength
Q= Quantum Yield
Near-IR Wavelength
Lisa DeLouise
Preliminary Data: Reflectance
In Collaboration with the Zavislan Lab yellow arrow reference, red arrow areas of interest
Stratum CorneumStratum GranulosumStratum SpinosumStratum Basale
Lisa DeLouise
Acknowledgements
EHSC Pilot Project Funding
NIH / NIAID 5K25AI060884
DeLouise LabLisa BonannoLuke MortensenUt-Binh Thi GiangSupriya RavichandraSidarth Chelsea VirgileJ. Matt KauhleRenea FaulknorHong Zheng, PhD
Gunter Oberdorster, PhDAlison Elder, PhDJim Zavislin, PhD
Lisa DeLouise
EPAR833856
Development of an In Vitro Test and a Prototype Model to Predict Cellular Penetration of Nanoparticles
Grant July 1, 2008 through June 30, 2011
R833857“Effects of Surface Oxides on the Behavior of Carbon Nanotubes and their influence on the Mobility of Contaminants in Aquatic Environments”
Grant July 1, 2008 through June 30, 2011
R833858
Quantum Dot Weathering and its Effects on Microbial Communities
Grant July 1, 2008 through June 30, 2011
R833859
Analysis and Fate of Single-Walled Carbon Nanotubes and Their Manufacturing Byproducts in Estuarine Sediments and Benthic Organisms
Grant July 1, 2008 through June 30, 2011
R833860 Functionalized Metal Oxide Nanoparticles: Environmental Transformations and Ecotoxicity
Grant July 1, 2008 through June 30, 2011
R833861 Environmental Transport, Biodegradation, and Bioaccumulation of Quantum Dots and Oxide Nanoparticles
Grant July 1, 2008 through June 30, 2011
R833862 Bioavailability, Environmental Transformation, and Detoxification of Core/Shell Nanomaterials
Grant July 1, 2008 through June 30, 2011
R833891Transformation and Fate of Manufactured Metal Oxide and Metal Nanoparticles in Aqueous Environments
Grant January 15, 2009 through January 14, 2012
R833892Platinum-Containing Nanomaterials: Sources, Speciation, and Toxicity in the Environment
Grant March 1, 2009 through February 29, 2012
R833893Bioavailability of Metallic Nanoparticles and Heavy Metals in Landfills
Grant April 1, 2009 through March 31, 2012
R834091Nanocavity sensor array for the isolation, detection and quantitation of engineered nanoparticles
Grant December 1, 2008 through November 30, 2011
R834092Influence of Water Quality on the Bioavailability and Food Chain Transport of Carbon Nanoparticles
Grant October 1, 2008 through September 30, 2011
R834093Interactions of Natural Organic Matter with C60 Fullerene and their Impact on C60 Transport, Bioavailability and Toxicity
Grant January 1, 2009 through December 31, 2011
R834094Environmental Behaviors of Solubilized Carbon Nanotubes in Aquatic Systems: Transformation, Sorption, and Toxicity Exposure
Grant September 1, 2008 through August 31, 2011
EPA Recent Awardshttp://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.rfa/rfa_id/461
Lisa DeLouise
State of the Science – Models
Nel’s “Hierarchical Oxidative Stress Paradigm”
If a link exists between an in vivo disease (eg. allergic airway inflammation) and a mechanistic
pathway at the cellular level (eg. oxidative stress) one can use a cell line (bronchial epithelial cells, macrophage) for NM high-throughput screening.
Nel and coworkers ACS Nano 2009 3(7) 1620.
ROS
Inflammation
CytotoxicityIn vitro cell culture ubiquitously used to:
• Screen NM cytotoxicty• Uptake mechanisms
How useful are these results?
Lisa DeLouise
UV exposed, 24hr after COOH, cryosection
40X•QD fluorescent evident in viable epidermis•What cell types do QD interact with?
Lisa DeLouise
• QD fluorescence associated with cell nucleus
• Are these aggregates?
UV exposed, 24hr after QD-COOHCryosection, DAPI
40X
Lisa DeLouise
State of the ScienceWhat is the appropriate dose metric - mass, surface
area, particle number, composition?
Pulmonary toxicity l Oberdörster et. al. (2005) - particle surface area l Wittmaack et. al. (2007) -particle number l Warheit et. al. (2009) - chemical reactivity
Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect. 2005;113:823–839.
Wittmaack K. 2007. In search of the most relevant parameter for quantifying lung inflammatory response to nanoparticle exposure: particle number, surface area, or what? Environ Health Perspect 115187–194
Warheit DB, Reed KL, Sayes CM. A role for nanoparticle surface reactivity in facilitating pulmonary toxicity and development of a base set of hazard assays as a component of nanoparticle risk management. Inhal Toxicol. 2009 Jul;21(S1):61-67.
Lisa DeLouise
Langmuir 2008, 24, 9194-9197
State of the Science –reactivity
Lisa DeLouise
Absorbance Spectrum (Company A)
0
0.05
0.1
0.15
0.2
400 500 600 700 800
Wavelength (nm)
Abs
orba
nce
(A.U
.)
QD520-TOL
QD620-TOL
Absorbance Spectrum (Company A)
0
0.05
0.1
0.15
0.2
400 500 600 700 800
Wavelength (nm)
Abs
orba
nce
(A.U
.)
QD520-TOL
QD620-TOL
Cysteine-QD620
Cysteamine-QD620
GSH-QD520
GSH-QD620
NAC-QD520
NAC-QD620
Tiopronin-QD520
Tiopronin-QD620
Absorbance Company A
Lisa DeLouise
Fluorescence Company A
0
50000
100000
150000
200000
250000
300000
420 470 520 570 620
Wavelength (nm)
Phot
olum
ines
cenc
e (A
.U.)
Cysteamine-QD520DHLA-QD520QD520-TOL
0
20000
40000
60000
80000
100000
120000
140000
160000
520 570 620 670 720
Wavelength (nm)
Phot
olum
ines
cenc
e (A
.U.)
Cysteamine-QD620DHLA-QD620QD620-TOL
Lisa DeLouise
Absorbance Spectrum (Company B)
0
0.05
0.1
0.15
0.2
400 500 600 700 800Wave length (nm)
Abs
orba
nce
(A.U
.)
QD620-TOLCysteamine-QD620DHLA-QD620GSH-QD620NAC-QD620Tiopronin-QD620MSA-QD620
Absorbance Company B
Lisa DeLouise
Fluorescence Company B
0
10000
20000
30000
40000
50000
60000
70000
80000
520 570 620 670 720
Wavelength (nm)
Ph
oto
lum
ine
sc
en
ce
(A
.U.)
CYS-QD620DHLA-QD620TP-QD620MSA-QD620NAC-QD620GSH-QD620QD620-TOL
Equal concentaions
