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

Thank you for your attention