Powerpoint Presentation for Sigma Xi Student Research Showcase

1. Effect of the Nano-Bio Interface on the
Genotoxicity of Titanium Dioxide Nanoparticles
and Associated Cellular Response
R u Y. Pr asad
aj
Ph.DC andi dat e
Envi r onm al Sci ence and Engi neer i ng D
ent epar t ment
U C G l l i ngs’ G obal School of Publ i c H t h
N i l eal
GPSF U ver si t y R
ni esear ch Day
Febr uar y 26, 2012
1

2. Brief History of Engineered Nanoparticles
“There’s Plenty of Room at the Bottom” – R char d Feynm N
i an, obel Laur eat e
Physi ci st , 1959
1985 – Fi r st 2001 – Pr esi dent Devel opm of
ent
nanopar t i cl e C i nt on est abl i shes
l Nanopar t i cl es f or :
synt hesi zed (car bon N i onal
at
f ul l er enes) Nanot echnol ogy
I ni t i at i ve
• D ug D i ver y
r el
• C onsum Pr oduct s
er
• I ndust r i al Pr oduct s
“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
Par t i cl es w t h a si ze di m
i ensi on bet ween 1 and 100
nmt hat shownovel pr oper t i es t hat ar e not f ound i n
bul k sam es of t he sam m er i al
pl e at
3

4. Titanium Dioxide Nanoparticles
C yst al St r uct ur es: Anat ase, R i l e and
r ut
Br ooki t e
H gh R r act i ve I ndex, W t e Pi gm at i on
i ef hi ent
4

5. Use of Titanium Dioxide Nanoparticles in Consumer
Products
Sunscreens : ~10-100 µg Ti/mg product (1-10%)
www.consumerreports.org
Weir et al. Environ Sci Technol 2012
5

6. Association of Physicochemical Properties with Toxicity
D t o sm l si ze di m
ue al ensi ons, l ar ge sur f ace ar ea t o
vol um r at i o, novel pr oper t i es, and pot ent i al
e
exposur e, i t i s i m t ant t o det er m ne ef f ect s on
por i
hum heal t h
an
Sayes et al. Tox Sci 2006
Ther e i s a need t o det er m ne physi cochem cal
i i
char act er i st i cs of nanopar t i cl es t hat can l ead t o adver se
6
ef f ect s

7. Nano-Bio Interface
For m i on of Pr ot ei n
at I nt er act i on w t h C l s
i el
C ona
or
Nel et al. Nature Materials 2009
7

8. Impact of Nano-Bio Interface on Genotoxicity
Degussa P25 Ti O 2
nanopar t i cl es
el ul edi :
C l C t ur e M um
-f
Ser um r ee K M
G
KB DM KF
• C l C t ur e m um
el ul edi • PBS + 0.6% BSA + 0.01% • C l C t ur e m um
el ul edi
(KG ) + 0.1% BSA
M DSPC (sur f act ant ) (KG ) + 10% FBS
M
• 2 m n pr obe
i • 20 m n cup soni cat i on
i • 2 m n pr obe
i
soni cat i on of st ock of st ock sol ut i on soni cat i on of st ock
sol ut i on • C r i f uged and r e-
ent sol ut i on
suspended i n cel l
cul t ur e m um(KG )
edi M
Ef f ect s on H an Lung Epi t hel i al C l s (BEAS-
um el
Prasad et al., 2013 ACS 2B)
Nano
8

9. Agglomeration: Physicochemical Characteristic
Impacted by the Nano-Bio Interface

10. Study Hypothesis
The t r eat m m umt hat el i ci t s t he
ent edi
sm l est nanopar t i cl e aggl om at es w l l
al er i
be associ at ed w t h cel l ul ar i nt er act i on
i
and genot oxi ci t y

11. Identification of the Protein Adsorbed to the Surface of the
Nanoparticles
KB DM KF
2
Ti O i n K 2 2
B Ti O i n D Ti O i n K
M F
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. Highest Cellular Uptake in KF Medium Determined by Flow
Cytometry
Fl owC om r y H st ogr am
yt et i s
Prasad et al., 2013 ACS Nano 14

15. Evaluation of Genotoxicity (Damage to the Genome)
D A D age
N am C om
hr osom D age
al am
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
C r ol
ont D aged
am
Prasad et al., 2013 ACS Nano
16

17. Micronuclei Induction Only in KF Medium
Prasad et al., 2013 ACS Nano
17

18. Increased % of S-Phase Cells in KF Medium
C l C e
el ycl
Prasad et al., 2013 ACS Nano
18

19. Summary
M umt hat el i ci t ed
edi
sm l est aggl om at es (K
al er F)
w associ at ed w t h hi ghest
as i
cel l ul ar upt ake, m cr onucl ei
i
f or m i on, and i ncr eased % of
at
cel l s i n S phase.
I n cont r ast , D A dam w
N age as
i nduced r egar dl ess of
aggl om at i on and cel l ul ar
er
upt ake
I nhal at i on exposur e t o
t i t ani umdi oxi de
nanopar t i cl es m not i nduce
ay
m cr onucl ei i n l ung
i
cel l s, whereas, serum proteins
in “blood” (f or
exam e, i ngest i on exposur e)
pl
Prasad et al., 2013 ACS Nano m act t o l ow
ay er 19

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?