Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
Biosafety of yNanoparticles:
Evaluation of Nanomaterials By a Suite of Cellular Assays
Mellisa Theodore1, Nathan Boggs1, Bakhtiyor Rasulev PhD2,Jerzy Leszczynski PhD2 and Joany Jackman PhD1
Science & Technology Business Area
Jerzy Leszczynski PhD , and Joany Jackman PhD
1- JHU APL2- Jackson State University
Biosafety of Nanoparticles yOur Approach
Is there a need for concern? Nanoparticles are being used in many industries A variety of shapes and surface chemistry exist at this time- little is known about
the effect of nanoparticles on the host Carbon nanotubes have shown asbestos like health risks Carbon nanotubes have shown asbestos-like health risks Colloidal silver ingestion causes argyria Pharmaceutical, biological research, medical applications
“Wait and see” methodology not acceptable E d f t b th i t t id ti Exposure and fate both very important considerations
What do we want to know? If biomedically-relevant nano-materials can cause damage to cells, cell pathways or
genomic DNA If DNA damage correlates to organ specific effects or cytotoxicity How are we investigating the concerns? Building on a suite of in vitro cellular assays for hazard assessment of nanoparticles
i bi k i i hi h i h l i i bi k f h d
Milton S. Eisenhower Research Center (MERC)
Prescreening biomarkers in vitro which might translate to in vivo biomarkers of hazard assessment
Predicatively modeling cellular effects of nanoparticles Science and Technology Business Area (S&T BA)
2
Need for Concern?Industrial Problem:Industrial Problem: Nanoparticles May Not Be Eliminated by HEPA Filters
High Efficiency Particulate Air Filter99 9% efficient at 0 3 micron
How HEPA filters work:99.9% efficient at 0.3 micron
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 3
NOTE: Efficiency drops in this size range and …CRITICAL ISSUE
Need for Concern? Nanoparticles: Protection? Fate? Elimination?
Nanoparticle size and respiratory disposition(Maynard and Kuempel, 2005)
…this is size where greatest deposition ...and cannot be eliminated by
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 4
g poccurs in lungs filtration in the kidneys
Need for Concern?Medical and Industrial Problem: Nanostructures Have Been Linked ToMedical and Industrial Problem: Nanostructures Have Been Linked To Cancer
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 5
Research Question: What is relationship of chemistry, nanoparticle size and shape to risk?
Need for Concern?Hazards of non toxic dust: Effects may not be acute
Pulmonary alveolar proteinosis is a rare disease in which a type of protein builds up in the air sacs (alveoli) of the lungs, making b hi diffi lbreathing difficult
In some cases, the cause of pulmonary alveolar proteinosis is unknown
In other cases it is associated with infection or an immune problem
It also can occur with cancers of the blood system, and afterthe blood system, and after exposure to high levels of dust
This rare disorder generally affects people 30 - 50 years old and is seen in men more often than in
Milton S. Eisenhower Research Center (MERC)
seen in men more often than in women
Science and Technology Business Area (S&T BA) 6
How are we investigating?Suite of Assays and Cells
Assays- Cellular ScreenAssays Cellular Screen Acute measures of toxicity
MTT Assay- cellular metabolic activity Live/Dead Assay- cellular membrane integrity
Long-term measures DNA Ladders; Flow Cytometry (Annexin-V)- apoptosis/ necrosis Flow Cytometry- Cell cycle analysis/ growth kinetics Cytokine Detection Assay- multiplex immunoassay for human cytokines
Cells Lines HL60- promyloblast- peripheral blood THP1- monocytes- peripheral blood HepG2- hepatocyes- liver A549- alveolar epithelial-lung MCF7- breast adenocarcinoma- mammary gland
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 7
Nanoparticle Variations Nanoparticles are significantly different in all aspectsNanoparticles are significantly different in all aspects Our studies will consider the following distinctions in particles: Composition Size Size Shape (aspect ratio) Surface charge
Initial Studies: Initial Studies: Gold nanoparticles characterized by NIST 2nm,10nm, 30nm, 60nm
R lt h f H G2 d A549 ll li Results shown for HepG2 and A549 cell lines Follow up Experiments: Comparison of Carbon nanotubes and Fullerenes
I ll i b i
Milton S. Eisenhower Research Center (MERC)
Immune cell responses to incubations ROS Evaluations
Science and Technology Business Area (S&T BA) 8
Results: MTT ASSAY 24H-72H
Assay results shown up to 72H of incubation with nanoparticles- little death observed compared to positive control (cell death induced by H2O2)
0 7
0.8
0.910nm 100nM
10nm 10nM
( y )
0 35
0.4
0.45
0.5
0.6
0.7
ce a
t 570 10nm 1nM
30nm 100nM
30nm 10nM 0.25
0.3
0.35
ance
0.3
0.4
0.5
bsor
banc
30nm 10nM
30nm 1nM
60nm 100nM
60nm 10nM0.15
0.2
0.25
Abs
orb
0
0.1
0.2A 60nm 10nM
60nm 1nM
Positive Control
N ti C t l 0
0.05
0.1
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 9
0 Negative Control 024H 72H
Results: LIVE / DEAD ASSAY 24H-72HAssay results shown up to 72H of incubation with nanoparticles- little death observed compared to positive control (cell death induced by H2O2)
90%
100%
24 H Live Dead‐ 10, 30, 60nm Gold Nanoparticle Incubations
y
90%
100%
72H Live Dead‐ 10, 30, 60nm Gold Nanoparticle Incubations
40%
50%
60%
70%
80%
40%
50%
60%
70%
80%
90%
Fluo
rescen
ce0%
10%
20%
30%
0%
10%
20%
30%
40%
% F
24H 72H
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 10
24H 72H
Results:CELL CYCLE EFFECTS
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 11
Normal Cell Cycle G2 phase Block G1 Phase Block*
The Inflammatory Response
Fever
Vascular leakage
Milton S. Eisenhower Research Center (MERC)
gIncreased blood flow
Results: 17-Plex Cytokine Assay
Cytokine Concentration Gold Nanoparticle ChallengeCytokine Concentration, Gold Nanoparticle Challenge (72hr)
NC
8 0010.0012.0014.00
pg/m
L)
PC10-10010-1010 1
0 002.004.006.008.00
ntra
tion
(p 10-130-10030-1030-10.00
IL-1
bet
a
IL-2
IL-4
IL-5
IL-6
IL-7
IL-8
IL-1
0
IL-1
2(p
70)
IL-1
3
IL-1
7
G-C
SF
GM
-CS
F
IFN
-g
amm
a
MC
P-1
(MC
AF
)
MIP
-1b
eta
TNF
-al
pha
Con
ce
Cytokine
30 160-10060-1060-1
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 13
Cytokines appear to have been induced by Nanoparticles…
Results: 17-Plex Cytokine Assay
Cytokine Concentration, LPS Challange Compared to Gold Nanoparticles
•Comparison to LPS induced
10
15
20
25
entra
tion
(pg/
mL)
NCPC10nm - 100nM10nm- 10nM10nm- 1nM30nm- 100nM30nm- 10nM30nm- 1nM
•Comparison to LPS induced cells shows higher induction in positive controls
0
5
IL-2 IL-12 G-CSF GM-CSF
48H Cytokines
Conc
e
60nm- 100nM60nm- 10nM60nm- 1nMLPS Control
•Cytokine induction in immune cells is much higher than in hepatocytes
Positive Control (10ng/mL LPS from K. pneumoniae)
35404550
g/m
L)
1 hour3 hour6 hour
05
101520253035
Con
cent
ratio
n (p 9 hour
12 hour18 hour
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 14
0
IL-2
IL-4
IL-6
IL-8
IL-1
0
GM
-CSF
IFN
-g
TNF-
a
IL-1
b
IL-5
IL-7
IL-1
2(p
70)
IL-1
3
IL-1
7
G-C
SFM
CP-
1(M
CAF
)M
IP-1
b
Results: Prescreening Biomarkers in vitro and Predicative Modeling Investigation of cytokine responses using modeling is necessary
for prescreening of potential biomarkersfor prescreening of potential biomarkers When placed into model Analyzing cytokine assay data- noticed that response values
for 2nm and 30nm particles were less then for 10nm and forfor 2nm and 30nm particles were less then for 10nm and for 60nm
This was noticed for almost all cytokine assays Selected IL-7 assays have the same distribution of values y
(expected that the response value should increase with decreasing or increasing nanoparticle size)
Wanted to find any rule for experimental data distribution and then find parameters that change noticeably with theand then find parameters that change noticeably with the cluster size increasing
By analyzing the data for IL-7 assay -found some interesting distribution of dataM d li Eff t C d t d b
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 15
Modeling Effort Conducted by: Interdisciplinary Center for Nanotoxicity ~ Jackson State University
Results: Prescreening Biomarkers in vitro and Predicative Modeling-g gIn silico approaches
SARs: Structure-
Physical Properties Toxicity
SARs: StructureActivity Relationships
QSARs: Quantitative Structure-Activity Relationships E i t l Bi ki tiRelationships Environmental
DistributionBiokinetic
Parameters
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 16
Modeling Effort Conducted by: Interdisciplinary Center for Nanotoxicity ~ Jackson State University
Results: Prescreening Biomarkers in vitro and Predicative Modeling
3-dimensional plot of data- difficult to see regularity
M d li Eff t C d t d b
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 17
Modeling Effort Conducted by: Interdisciplinary Center for Nanotoxicity ~ Jackson State University
Results: Prescreening Biomarkers in vitro and Predicative Modeling
When plotted on one axis, can see a sinusoidal curve, which is gradually damping
Looked for equation to fit the curve Among several thousands curve fitting functions
we found the following: we ou d e o ow g
Y(IL7)=7.88E-03·(X12·ATAN(X2))+3.64·(COS(X1)
ATAN(X2))+3.64
where Y(IL7) is response variable, IL-7 cytokine assay values, X1 – gold nanoparticle size in nm, X2 – concentration in nM
Need to check robustness of this equation with experimental data for other sizes of gold
inanoparticles
M d li Eff t C d t d b
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 18
Modeling Effort Conducted by: Interdisciplinary Center for Nanotoxicity ~ Jackson State University
TEM Images of Gold Nanoparticles10nm 30nm 60nm10nm 30nm 60nm
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 19
TEM Images of Gold Nanoparticles: Incubated with cells
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 20
Potential uptake of nanoparticles into cells
Conclusions: Well characterized gold nanoparticles have shown little/ no effect in acute
toxicity assays Further testing is scheduled for more cell lines and various types of
nanoparticlesD t f f ll i t ill b il d i t th d t b f Data from follow on experiments will be compiled into the database for continued modeling for prediction of relative risk of well characterized materials
Disruption of cellular pathways and biomarkers of exposure will be used toDisruption of cellular pathways and biomarkers of exposure will be used to develop predictive monitoring tools
Acknowledgements:Acknowledgements: APL: Melissa BetzNIST: Dr. Bryant Nelson, Dr. John ElliotJohns Hopkins University: Microscopy Facility
Milton S. Eisenhower Research Center (MERC)
Johns Hopkins University: Microscopy Facility
Science and Technology Business Area (S&T BA) 21
References:
Atsuya Takagi, Akihiko Hirose, Tetsuji Nishimura, Nobutaka Fukumori, Akio Ogata, Norio Ohashi, Satoshi Kitajima and Jun Kanno: “Induction of mesothelioma in p53+/− mouse by intraperitoneal application of multi-wall carbon nanotube”: J. Toxicol. Sci., Vol. 33: No. 1, 105-116. (2008)
HEPA Filtration Facts. D. F. Solutions. Minneapolis: 1-2 Maynard, A. and E. Kuempel (2005). "Airborne Nanostructured Particles and Occupational Health." Journal of
Nanoparticle Research 7(6): 587-614Nanoparticle Research 7(6): 587 614 Poland, C. A., R. Duffin, et al. (2008). "Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like
pathogenicity in a pilot study." Nat Nano 3(7): 423-428 Song, Y., X. Li, et al. (2009). "Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma."
European Respiratory Journal 34(3): 559-567 Wick, P., A. Malek, et al. (2009). "Barrier Capacity of Human Placenta for Nanosized Materials." Environ Health Perspect
( )118(3)
Image Sources http://www.atcc.org/Attachments/1761.jpg http://losangelespublicrelations.com/colloidal-silver-blue-man/0781 http://www.pharmacology2000.com/respiratory_anesthesiology/pulmonary_assessment/pulmonary_assessment1.htm https://tools.invitrogen.com/content/sfs/gallery/low/g001619.jpg
Milton S. Eisenhower Research Center (MERC) Science and Technology Business Area (S&T BA) 22