1. Effect of the Nano-Bio Interface on the
Genotoxicity of Titanium Dioxide Nanoparticles
and Associated Cellular Response
Raju Y. Prasad
Ph.D Candidate
Environmental Science and Engineering Department
UNC Gillings’ Global School of Public Health
GPSF University Research Day
February 26, 2012
1

2. Brief History of Engineered Nanoparticles
“There’s Plenty of Room at the Bottom” – Richard Feynman, Nobel Laureate Physicist, 1959
1985 – First nanoparticle 2001 – President Clinton Development of
synthesized (carbon establishes National Nanoparticles for:
fullerenes) Nanotechnology Initiative
• Drug Delivery
• Consumer Products
• Industrial Products
“The 2013 Federal Budget provides $1.8 billion for
the National Nanotechnology Initiative (NNI)”
2

3. Definition of Engineered Nanoparticles
~1.6 m ~2 nm width
100 10-1 10-2 10-3 10-6 10-9
1-100 nm
meter decimeter centimeter millimeter micrometer nanometer
Particles with a size dimension between 1 and 100 nm
that show novel properties that are not found in bulk
samples of the same material
3

4. Titanium Dioxide Nanoparticles
Crystal Structures: Anatase, Rutile and Brookite
High Refractive Index, White Pigmentation
4

5. Use of Titanium Dioxide Nanoparticles in Consumer
Products
Concentration of nano Ti in consumer products
Sunscreens : ~10-100 µg Ti/mg product (1-10%)
Sunscreen w/o
Sunscreen w/ TiO2 nanoparticles
TiO2 nanoparticles Weir et al. Environ Sci Technol 2012
5

6. Association of Physicochemical Properties with Toxicity
Due to small size dimensions, large surface area to volume
ratio, novel properties, and potential exposure, it is
important to determine effects on human health
Sayes et al. Tox Sci 2006
There is a need to determine physicochemical characteristics of
nanoparticles that can lead to adverse effects
6

7. Nano-Bio Interface
Formation of Protein Corona Interaction with Cells
Nel et al. Nature Materials 2009
7

8. Impact of Nano-Bio Interface on Genotoxicity
Degussa P25 TiO2 nanoparticles
Cell Culture Medium:
Serum-free KGM
KB DM KF
• Cell Culture medium • PBS + 0.6% BSA + 0.01% • Cell Culture medium
(KGM) + 0.1% BSA DSPC (surfactant) (KGM) + 10% FBS
• 2 min probe sonication • 20 min cup sonication of • 2 min probe sonication
of stock solution stock solution of stock solution
• Centrifuged and re-
suspended in cell culture
medium (KGM)
Effects on Human Lung Epithelial Cells (BEAS-2B)
Prasad et al., 2013 ACS Nano
8

9. Agglomeration: Physicochemical Characteristic
Impacted by the Nano-Bio Interface
In liquid medium, TiO2 nanoparticles can agglomerate to form
larger particles. The agglomeration profile is affected by the
composition of the medium, therefore must be measured to
determine its role in genotoxicity
Two different media show different agglomeration profiles

10. Study Hypothesis
The treatment medium that elicits the smallest
nanoparticle agglomerates will be associated
with cellular interaction and genotoxicity

11. Identification of the Protein Adsorbed to the Surface of the
Nanoparticles
KB DM KF
TiO2 in KB TiO2 in DM TiO2 in KF
ladder
kDa
250
130
95
72
55
36
28
Tedja et al., 2012 ACS Nano, Lundqvist et al., 2008 PNAS 11

12. Size Measurements in Treatment Media
0h
KB
24 h DM
KF
Prasad et al., 2013 ACS Nano
12

13. No Significant Cytotoxicity in BEAS-2B cells
0 µg/ml 10 µg/ml 20 µg/ml 50 µg/ml 100 µg/ml MMS (100 µM)
Li ve:
C cei n-
al
AM
D ead:
Pr opi di um
I odi de
Prasad et al., 2013 ACS Nano 13

14. Methods: Cellular Uptake Determined by Flow Cytometry
Flow Cytometry Histograms
↑uptake in KF
blue
green
red
Prasad et al., 2013 ACS Nano 14

15. Methods for Evaluation of Genotoxicity
DNA Damage Chromosomal Damage
Binucleated cell
(BN)
treat 24 h 48 h
Add CytoSpin cells
cytochalasin B on glass slides
Micronucleated
Binucleated cell
(MN)
Fenech et al. Mutat Res-Fund Mol M 2000 , Kirsch-Volders et al. Mutat Res-Fund Mol M 2003, Singh et al. Mutat Res 1991 15

16. TiO2 Nanoparticles Induce DNA Damage in All Media
C r ol
ont D aged
am
Prasad et al., 2013 ACS Nano
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17. Chromosomal Damage Only in KF Medium
Prasad et al., 2013 ACS Nano
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18. Difference between DNA and Chromosomal Damage: Role of the
Cell Cycle
Cell Cycle
Increased % S phase cells in KF
Prasad et al., 2013 ACS Nano
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19. Summary
Medium that elicited smallest
agglomerates (KF) was associated
with highest cellular uptake,
micronuclei formation, and
increased % of cells in S phase.
In contrast, DNA damage was
induced regardless of
agglomeration and cellular
uptake
Inhalation exposure to titanium
dioxide nanoparticles may not
induce chromosomal damage in
lung cells, whereas, serum
proteins in “blood” (for example,
ingestion exposure) may act to
lower agglomeration and induce
micronuclei formation.
Prasad et al., 2013 ACS Nano
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20. Thank you
Dr. Rebecca Fry Dr. David DeMarini Dr. Ivan Rusyn Dr. Jim Swenberg Dr. Bill Kaufmann
Kathleen Wallace Dr. Carl Blackman Dr. Nana Nikolaishvili-Feinberg
Kaitlin Daniel Dr. Jorge Muniz Ortiz Dr. Stephanie Cohen
Alan Tennant Dr. Andrew Kligerman Mark Olorvida
Jim Campbell Dr. Robert Zucker Bentley Midkiff
Micaela Killius Dr. Steve Simmons Mervi Eeva
Sharon Leavitt Dr. Kevin Dreher Dr. Stephanie Smith-Roe
Ben Castellon Dr. Bellina Veronesi Lisa Smeester
Jenna Strickland Dr. Brian Chorley Dr. Kathryn Bailey
Danielle Ackerman Dr. Thomas Knudsen Dr. Bhavesh Ahir
Peggy Matthews Dr. Kirk Kitchin Dr. Julia Rager
Sarah Warren Dr. Will Boyes Alison Sanders
Nancy Hanley Dr. Bill Lefew Fry lab
Laura Degn Keith Tarpley
Kristen Sanders U.S. EPA NHEERL
Questions?