eunjoo kim, ph. d
DESCRIPTION
Toxicity analysis of PbS QDs using nano -sized vesicles ( exosome ) se creted from HEK293 cells. Eunjoo Kim, Ph. D. Daegu Gyeongbuk Institute of Science and Technology (DGIST), Republic of Korea. Quantum dots. RUSNANO Corporation, http://en.rusnano.com/. Nanocrystals - PowerPoint PPT PresentationTRANSCRIPT
Toxicity analysis of PbS QDs using nano-sized vesicles (exosome)
secreted from HEK293 cells
Eunjoo Kim, Ph. D.Daegu Gyeongbuk Institute of Science and
Technology (DGIST), Republic of Korea
Quantum dots
RUSNANO Corporation, http://en.rusnano.com/
Nanocrystals 2-10 nm diameter, small enough
to exhibit quantum mechanical properties
Fluorophore Easily modified by polymers or
chemicals with such as –SH Semiconductors, agents for medi-
cal imaging
Exosomes
Donor cell Recipient cell
Early endosome
Fusion
Endosome
Roles of exosome contents• Cell-to-cell signaling• Membrane trafficking• Representing the disease status
and pathological condition of cel-lular origin Proteins
Released from most types of mammalian cells Different cells release different exosomes. Potential source of biomarkers for tissue condi-
tions The encapsulated molecules by exosomes can
be delivered over long distance. They play a key role in cell-cell communication
during disease progress such as cancer and neurodegeneration.
Isolated from circulating fluids: serum, urine, CSF and cell culture media
Properties of exosomes in biological signaling
provides new circulating biomarkers for in vivo and in vitro
makes possible to predict the cellular and molecular mechanism of toxicity efficiently
is an emerging field for high-throughput screening of disease biomarkers
is applied to human toxicology
Toxicology with exosomes
To provide nanotoxicological data for PbS QDs To propose a toxicologic pathology using exo-
some biomarkers
Aims of this study are..
2 Exposure of PbS-MPA to HEK293 cells
1 Preparation of MPA-coated PbS
4 Analysis of molecular markers: miRNA & proteins
3 Exosome isolation
5 Identification of Induced & de-pressed molecules
6 Functional analysis
7 Prediction of toxicologic pathology in vitro
Synthesis of PbS particles
50 nm
(a)
20 nm
(b)
10 nm
(c)
5 nm
(d)
JCPDS: 03-065-0692
20 30 40 50 60 70 80 90
Cou
nts
2(degree)
(111)
(200)
(220) (222)
(400)(331)
Akhtar, et al. J. Mater. Chem., 2010, 20, 2336-2344
0 1 2 3 40
10
20
30
40
50
Numb
er of
Parti
cles
Particle Size (nm)
Dave=2.15 nm (n=92)
PbS quantum dots with < 5 nm particle sizes were prepared by the colloidal chemistry method according to the theory of fast nucleation at high temperature and slow growth at low tempera-ture. Sodium sulfide was used as a sulfur precursor (odorless and less noxious). Oleic acid was used as a stabilizing agent to control the particle growth and it assisted in the formation of mono-dispersed PbS QDs.
Surface modification PbS by MPA
223 11 nm
3-mercaptopropionic acid (MPA)
TEM image DLS analysis
FT-IR analysis
500 1500 2500 3500 20
30
40
50
60
70
80
90
100
Frequency (cm-1)
%T
ran
smis
sio
n
500 1500 2500 3500 80
85
90
95
100
105
Frequency (cm-1)
% T
ran
smis
sio
n
PbS PbS-MPA
Cytotoxicity of PbS-MPA
0 1.5 15 1500
20
40
60
80
100
120
140
TCMK1THP1ALM12
Ce
ll p
rolif
era
tio
n (
%)
µg/ml
0 2 6 25 100 4000
20
40
60
80
100
120
Ce
ll p
rolif
era
tio
n (
%)
µg/ml
HEK293
Isolation and characterization of exosomes
109 84 nm
50 m
Bio-TEM
DLS
CD63
CD9
←53KD
←28KD
Exosome markers
System Bioscience Inc.http://www.systembio.-com/
Profiling of miRNA
System Bioscience Inc., http://www.systembio.-com/
• In SBI miRNA analysis kit, 380 primers found in exosomes were provided
Differentially expressed exosome miRNAs (DEGs)
Selection criteria: 1) Increment or decrement simultaneously in both of 5 and 50 g/ml 2) Cut-off for 2-fold changes in 5 or 50 g/ml 3) p<0.05
hsa
-miR
-54
5
hsa
-miR
-20
0a
hsa
-miR
-37
9
hsa
-miR
-27
b
hsa
-miR
-31
hsa
-miR
-45
0a
hsa
-miR
-18
1d
hsa
-miR
-51
2-3
p
hsa
-miR
-22
0c
hsa
-miR
-52
7
hsa
-miR
-19
9a
-5p
hsa
-miR
-52
5-5
p
hsa
-miR
-51
9a
hsa
-miR
-53
2-3
p
hsa
-miR
-54
8e
-15
-10
-5
0
5
10
550
Lo
g2
(ΔC
t)
g/ml
g/ml
Top functional networks
Diseases and disorders p-value# Mole-
cules
Cancer6.52E-04 - 4.38E-
0211
Organismal Injury and Abnormalities6.52E-04 - 2.51E-
028
Reproductive System Disease2.85E-03 - 4.95E-
024
Inflammatory Response2.18E-03 - 2.79E-
024
Associated Network Functions Score
Cancer, Organismal Injury and Ab-
normalities,
Reproductive System Disease
20
By Ingenuity Pathway Analysis (IPA)
2D gel electrophoresis of exosome proteins
Control
5 g/ml PbS-MPA 50 g/ml PbS-MPA
1
2 3
1) 1<2<3 or 1>2>3 2) 1 vs. 2 or 1 vs. 3, 2-fold cut off
Spot selection (29 spots)
40027102
4102
4106
40035004 5004
6201
4109
4001
72039305
A: Increased changes (19)
7007 81081103
1015204
2101
42068303
8603
8703
B: Decreased changes (10)
Protein spots identified by Maldi-TOF and PMF analysis
52049305
8603
8703
Spot No. Protein Expres-
sion Function
Re-sponse in can-
cer
9305
Leucine-rich repeat-con-taining
protein 23 isoform b (IR-R23b)
Increase Exosome protein Increase
5204 Keratin 5 (KRT5) Decrease
Breast cancer biomarker
(negative relation-ship)
Decrease
8603 Unnamed protein product Decrease -
8703 Albumin-like Decrease -
Confirmation of exosome biomarkers
0 5 50 (μM)
← p53
In Exosomes
← CD9
← KRT 5
← LRR23b
0 5 500
0.51
1.5
p53
QD-in (μg/ml)
p5
3/C
D9
0 5 500
0.51
1.52
LRR23b
QD-in (μg/ml)L
RR
C2
3/C
D9
0 5 500.5
1
1.5KRT 5
QD-in (μg/ml)
Cy
tok
era
tin
5/C
D9
Events in the origin of the exosomes, HEK293 cells: expression of protein biomarkers
In cells
← p53
← β-actin
← LRR23b
← KRT 5
0 5 50 (μM)
0 5 5001234
Cytokeratin 5
QD-in (μg/ml)
Cy
tok
era
tin
5/a
cti
n
0 5 500
0.51
1.52
p53
QD-in (μg/ml)
P5
3/a
cti
n
0 5 500
1
2
3
LRR23b
QD-in (μg/ml)L
RR
C2
3/a
cti
n
Comet assay for DNA damage
UV PBS PbS-MPA 5 μg/ml PbS-MPA 50 μg/ml
- 100 μm
Comet assay responses as indicators of carcinogen exposure.
Events in the origin of the exosomes, HEK293 cells: expression of cancer-related biomarkers (mRNA)
Control QD-in 5 μg/ml QD-in 50 μg/ml012345678
Human IL-8
Rel
ativ
e ra
tio
Control QD-in 5 μg/ml QD-in 50 μg/ml0
0.5
1
1.5
2
2.5
3
3.5
Human p53
Rel
ativ
e ra
tio
Control QD-in 5 μg/ml QD-in 50 μg/ml0
2
4
6
8
10
Human CXCL5
Rel
ativ
e ra
tio
*P<0.01,**P<0.001 vs Control
**
**
*
*
*
**
The exosome miRNA profiles for PbS-MPA to HEK293 cells showed 15 DEGs. These were primarily involved in cancer and organismal injury and abnor-malities.
LRR23b and KRT5 were identified as biomarkers of exosome proteins for PbS-MPA exposure. These proteins are also known as cancer biomarkers.
Comet assay clearly showed that DNA fragmentation was occurred, and supported the carcinogenic activity of PbS-MPA QDs.
The exosome derived biomarkers could represent the toxicological response of origin cells.
1. A toxicological response could be identified by genomic and proteomic analysis for secreted exosomes.
2. The eoxsome-based analysis could provide an effective tool for high-throughput screening (HTS) of biomarkers involved in possible toxicology.
3. The HTS of exosome biomarkers will be more efficient than that of the whole cells, because 1) they have less number of molecular contents, 2) are expected to secret critical biomarkers to represent the cellular state and communicate with other cells.
Conclusion
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