Chunying [email protected]
National Center for Nanoscience and Technology, China CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety
Understanding the Interactions of Nanoscale Materials with Biological Systems
by Integrated Techniques
Various types of nanoparticles used in biomedical research
Cai, W.; Gao, T.; Hong, H.; Sun, J. Nanotechnology, Science and Applications 2009, 2, 1.A. H. Faraji, P. Wipf / Bioorg. Med. Chem. 17 (2009) 2950–2962
How to design & screen the safe How to design & screen the safe nanomaterials for the need of biomedical and nanomaterials for the need of biomedical and other industrial applicationother industrial application
www.minervaclassics.com
Promising advances in
nanomedicine
industrial application
Toxic effects of nanoparticle exposure
The Janus Faces of Nanoparticles
Donaldson & Seaton, J Nanosci Nanotech 7(2007)4607-4611
Cross blood-brain barrier –impair healthPulmonary toxicityprivacy concernslimited understanding……
Entry and target tissues for uptake of engineered nanoparticles
Outline
Understanding Interactions of Nanoscale Materials with Biological Systems
The ability of NPs for biological barriers
Pulmonary responses after Long-term retention of nanoparticles
The Role of Nanoparticles During cell Mitosis
Specific responses by different types of cells
Key factors influence the nano-bio interactions.
Nose-to-Brain transfer? Blood Brain Barrier?
Possibility and Ability of inhaled Nanoparticle Entering into Brain directly?
Is the olfactory neuronal pathway efficient for translocatinginhaled UFPs to the central nervous system ?
How do physico-chemical characteristics of NPs influence uptake and translocation?
Are there any toxicological consequences?
Ti retention in Brain and Lung tissuesTi retention in Brain and Lung tissuesNasal Instillation vs. Oral administration
Control 25 nm 80 nm 155 nm0
200
400
600
800
Intranasal (30d) Oral (28d)
Ti c
onte
nt in
Bra
in (n
g/g)
Exposure Groups
BrainBrain
Control 25 nm 80 nm 155 nm0
300
600
900
Ti c
onte
nt in
Lun
g (n
g/g)
Exposure Groups
Intranasal (30d) Oral (28 d)LungLung
TiO2 Accumulation: Nasal Instillation: Brain > Lung
Oral Administration: Brain < Lung
Wang JX, Chen CY, et al, Toxicology, 2008
Fig. Titanium content in the olfactory bulb (A), cerebral cortex (B), hippocampus (C) and cerebellum (D) of mice (n=6) intranasally instilled 25 nm, 80 nm and 155 nm TiO2 particles for 2, 10, 20 and 30 d.
25 nm 80 nm 155 nm0
80
160
240
320
400
480
Control
Ti c
onte
nt in
olfa
ctor
y bu
lb (n
g/g)
Exposure Groups
2 d 10 d 20 d 30 d
A
25 nm 80 nm 155 nm0
80
160
240
320
400
480
Control
Ti c
once
nt in
cer
ebra
l cor
tex
(ng/
g)
Exposure Groups
2 d 10 d 20 d 30 d
B
25 nm 80 nm 155 nm0
80
160
240
320
400
480
Control
Ti c
onte
nt in
cer
ebel
lum
(ng/
g)
Exposure Groups
2 d 10 d 20 d 30 d
D
25 nm 80 nm 155 nm0
80
160
240
320
400
480
Control
Ti c
onte
nt in
hip
poca
mpu
s (n
g/g)
Exposure Groups
2 d 10 d 20 d 30 d
olfactory bulbolfactory bulb cerebral cortexcerebral cortex
hippocampushippocampus cerebellumcerebellum
Cortex
OlfactoryHippocampus
Cerebellum
Ti distribution in different brain areas
Advantages:Simultaneous multi-element determinationthe information in tiny areas and thin slices. improve the sensitivity and space resolutionNon-destructive
Beamsize: 20X20 μm2
3X5 μm2
MicrobeamMicrobeam SRSR--XRF mapping techniquesXRF mapping techniques
μSRXRF mapping facilityInstitute of High Energy Physics
Synchrotron radiation
Accumulation of nano-TiO2 in mice olfactory bulb and brain following intranasal administration by SR-XRF mapping.
A B C80 nm
155 nmA B C
Low High
TiO2 nanoparticles could be transfered via the secondary and tertiary olfactory pathways to reach most parts of brain.
Wang JX et al，High Energy ad Nucl Physics, 2005Wang JX et al, JRNC, 2007, Wang et al, 2008
Olfactory Nerve Olfactory Nerve Translocation pathwaysTranslocation pathways
A control group; B 25nm group;
C 80nm group; D fine group
Histopathological examinationHippocampus
enlarged and elongated pyramidal cell soma the stratum pyramidale was irregularNissl body decreased or disappeared.
GFAP-positive glial cells in the hippocampus of murine brainActivation of Astrocytes
Wang JX, Chen CY, et al, Tox Lett, 2008
Immunochemical examination
×50
×200
A
×200
B
×200
C
control 80 nm 155 nm0
40
80
120
160 **
Cou
nts
of a
stro
cyte
s pe
r 1m
m2
Exposure Groups
D
Concentrations of Cu in discrete murine brain regions after intranasally instilling copper nanoparticles 21 d.
0
2
4
6
8
10
12
14
16
18
20#
* #*# *#
*#
40 mg/kg10 mg/kg
Olfactorybulb Hippocampus Cerebralcortex Cerebellum Striatum
Control 1 mg/kg
*
#
Cu accumulation in various brain regions Cu accumulation in various brain regions
Monoamine neurotransmitters Monoamine neurotransmitters changes in the brainchanges in the brain
Olfactory bulb Cortex Hippocampus Striatum Cerebellum0
50
100
150
200
250
300
350
400
450
****
*
unit:
μg/
mg
wet
tiss
ue
Control L-Dose M-Dose H-Dose
DA DOPAC HVA
0
30
60
90
120
150
180
210
**
**
*
*
*
unit:
μg/
mg
wet
tiss
ue
Control L-Dose M-Dose H-Dose
dopamine
norepinephrine (NE)
Chen et al, Nanotoxicology, 2011
Cu nanoparticle
Question: What are differences of species?The significance for humans still needs to be established. Area: Rodents, the olfactory mucosa comprises 50% of total nasal mucosal surface
Human, 5% of the total nasal mucosal surface
Rat brain, Dorsal viewHuman brain, ventral view
olfactory bulbolfactory bulb
cerebral cortexcerebral cortex
cerebrumcerebrum
Extrapolation from Rodents to Human
Can nasal administration be a new way for pharmaceutical treating neural diseases ?
Outline
Understanding Interactions of Nanoscale Materialwith Biological Systems
The ability of NPs for biological barriersPulmonary responses after Long-term
retentionThe Role of Nanoparticles During cell MitosisSpecific responses by different types of cells
Key factors influence the nano-bio interactions
Pulmonary responses after Long-term retention of inhaled Toner particles
The Concentrations of PM2.5 and PM10 in collecting particles from different indoor environments.
20.324.1Continue collecting particles in a conference room (48h)
36.952.7Continue collecting particles in a photocopy room (48h)
54.033.3Collecting particles in an office with intermittent printing
18.319.6The Background of particles in an office (No Printing)
Concentration of PM10（μg/m3)
Concentration of PM2.5（μg/m3)
The Name of Experiment
Characterization of toner particles
Original toner
After printing
Long – term Retention of Inhaled Toner Particles in the Lung Tissues
Saline control
day 9
Saline control
days 28 and 84
Toner exposure
day 9 and 28
Toner exposure
day 56 and 84
Bai R, Zhang L, Chen C, Tox lett，2010
Long – term Retention of Inhaled Toner Particles in the Lung Tissues induced the pulmonary inflammatory
9 d 28 d 56 d 84 d 0
2000
4000
6000
8000
10000 **
*
IL-1
β (p
g/m
g pr
ot)
Time (days)
Control Saline Toner
*
9 d 56 d 84 d 0
2000
4000
6000
8000
10000
12000 *
IL-6
(pg/
mg
prot
)
Time (days)
Control Saline Toner
*
9 d 28 d 56 d 84 d 0
5
10
15
20
****
Tota
l Cel
l Num
bers
(105 )
Time (days)
Control Saline Toner ***#
9 d 28 d 56 d 84 d 0.0
0.1
0.2
0.3
0.4
Tota
l Pro
tein
Con
tent
(g/L
)
Time (days)
Saline Toner *
Bai R, Zhang L, Chen C, Tox lett，2010
Outline
Understanding Interactions of Nanoscale Materials with Biological Systems
The ability of NPs for biological barriers
Pulmonary responses after Long-term retention
The Role of Nanoparticles During cell MitosisSpecific responses by different types of cells
Key factors influence the nano-bio interactions.
The Role of Nanoparticles During the Mitotic Phase
Scholey, J. M. et al ,Nature, 2003
InterphaseMitosis
prophasemetaphaseanaphasetelephase
Cell division
NanoparticlesCarboxyl-modified (COOH-PS) amino-modified (NH2-PS) polystyrene particles various sizes (50, 100, 500 nm in diameter) fluorescence conjugation
The dynamics and Intracellular Trafficking of PS Particles in live cells
Liu, Li, Zhao, Chen, Biomaterials, 2011
Time-lapse observation of 100 nm COOH-PS nanoparticles in mitotic NIH 3T3 cells
Spatial distribution of COOHof COOH--PS nanoparticles PS nanoparticles at different phase of mitosis in GFPin GFP--actin NIH 3T3 cellsactin NIH 3T3 cells
No effect on the reorganization of the chromosome and actin cytoskeleton
Liu, Li, Zhao, Chen, Biomaterials, 2011
Localization of NH2-PS nanoparticles in GFP-actin NIH 3T3 cells
Localization of NH2-PS nanoparticles in GFP-histone HeLa cells
Spatial distribution of NH2of NH2--PS nanoparticles PS nanoparticles at different phase of mitosis in GFPin GFP--actin NIH 3T3 cellsactin NIH 3T3 cells
Hela cells, tubulin tracker red, PS NPs Orange，Chromosome Green
Effect on the Organization of Mitotic Spindle and whole cell cycle
Localization of COOH-PS Nanoparticles and tubulin in fixed HeLa cells
Liu, Li, Zhao, Chen, Biomaterials, 2011
Localization of NH2-PS Nanoparticles and tubulin in live HeLa cells
Effect on the Organization of Mitotic Spindle and whole cell cycle
Liu, Li, Zhao, Chen, Biomaterials, 2011
Interphase
Mitosis
Intercellular localization of PS NanoparticlesIntercellular localization of PS Nanoparticles
LysosomeLysosome
Liu, Li, Zhao, Chen, Biomaterials, 2011
24 48 720
20
40
60
80
100
120
cell
viab
ility
(% to
the
cont
rol)
incubation time (h)24 48 72
0
20
40
60
80
100
120
cell
viab
ility
(% to
the
cont
rol)
incubation time (h)24 48 72
0
20
40
60
80
100
120
cell
viab
ility
(% to
the
cont
rol)
incubation time (h)
24 48 720
20
40
60
80
100
120
cell
viab
ility
(% to
the
cont
rol)
incubation time (h)24 48 72
0
20
40
60
80
100
120
cell
viab
ility
(% to
the
cont
rol)
incubation time (h)24 48 72
0
20
40
60
80
100
120
cell
viab
ility
( %
to th
e co
ntro
l)
incubation time (h)24 48 72
0
20
40
60
80
100
120
cel
l via
bilit
y ( %
to th
e co
ntro
l)
incubation time (h)
24 48 720
20
40
60
80
100
120ce
ll vi
abili
ty (%
to th
e co
ntro
l)
incubation time (h)
50nm 100nm 500nm
NIH-3T3
Hela
TimeTime--Course and SizeCourse and Size--Dependent Dependent CytotoxocityCytotoxocity
Cytotoxicity: COOH-PS＜＜ NH2-PS
Liu, Li, Zhao, Chen, Biomaterials, 2011
Outline
Understanding Interactions of Nanoscale Materials with Biological Systems
The ability of NPs for biological barriers
Pulmonary responses after Long-term retention
The Role of Nanoparticles During cell Mitosis
Specific responses by different types of cells
Key factors influence the nano-bio interactions
SPR effects -molecular interaction
Drug and gene carriers
Application of Gold NPs in Biomedicine
SERS: Disease diagnosis and detection
Immunological Labeling
Labeling and tracking for Tumor
(NIR, X-ray CT imaging, SERS)
Thermotherapy agents and
temperature sensitive container
Optical extinction
Human pulmonary adeno-carcinoma cell (A549 cells)
Rat Bone marrow mesenchymal stem cells(MSC cells)
Normal human bronchial epithelial cell(16HBE cells)
Cell types
Wang, Chen, et al, Nano Letters, 2011
Fast Adsorption of serum proteins to Au NRs
400 500 600 700 800 900 1000
0.3
0.6
0.9
1.2
1.5
Abs
orba
nce
Wavelength (nm)
Au NRs in water FBS incubated Au NRs in PBS
5 min 30 min 1 h 3 h 6 h 9 h 12 h
b
(55.6±7.8 ) * (13.3 ±1.8) nmA thicker layer around nanorod
Serum protein adsorption can facilitate the internalization
Specific responses to cancer cells
Normal cells Stem cells
Cancercells
Wang, Chen, et al, Nano Letters, 2011, 11, 772–780
Changes in cell shape
Why cell-specific responses?
1. Internalized amounts of Au NRs?
2. Uptake pathways?
3. Intracellular trafficking?
Different Internalization of Au NRs
Caveolin, Clathrinand energy-dependent endocytosis
16HBE< A549 = MSC (cell volume)
Wang, Chen, et al, Nano Letters, 2011, 11, 772–780
Different Removal of Au NRs
Removal Cell viability
Wang, Chen, et al, Nano Letters, 2011
Intracellular localization
A549
MSC
Lysosme Mitochondria
A549: mitochondria, lysosomes/endosome16HBE: lysosomes/endosomeMSC: lysosomes/endosome
Wang, Chen, et al, Nano Letters, 2011, 11, 772–780
Different localization during exclusion in vitro
Wang, Chen, et al, Nano Letters, 2011, 11, 772–780
Increased lysosomal permeation by Au NRs in cancer cells
damage to the lysosomal membrane lead to further translocation of the Au NRs to other organelles
The integrity of lysosome
Wang, Chen, et al, Nano Letters, 2011, 11, 772–780
What is next for Au NRs?
For A549 cells,Decrease in mitochondrial membrane potentials.Increased intracellular ROS level
Wang, Chen, et al, Nano Letters, 2011, 11, 772–780
Cellular uptake and cytotoxicity of Au nanorods: The influence of surface chemistry and aspect ratio
The linear fitting of longitudinal plasmonicmaximum to aspect ratio calculated from data based on TEM images.
Calculated aspect ratio: 1.2, 2.0, 3.0, 4.0
Aspect Ratio 1 2 3 4
Au NRs
CTAB‐1 CTAB‐2 CTAB‐3 CTAB‐4
PSS‐1 ‐ ‐ PSS‐4
PDDAC ‐1 ‐ ‐ PDDAC‐4
PSS(poly(sodium‐p‐styrenesulfate)
PDDAC(poly(diallyl dimethyl ammonium chloride)
CTAB(cetyltrimethyl ammonium bromide )
Collaboration with Prof. Xiaochun Wu
Au NRs of different coatings and shape
Au@PDDAC Au@PSS AuNRs
Coating and Shape dependent cellular uptake
Two photonluminescence(TPL) images of Au NRs with different surface coatings in MCF-7 cells.
Aspect ratio↑cellular uptake↓
PDDAC coated Au NRs are cells’ “favorite cookies”
Surface coating can affect the cytotoxicity of Au NRs
Surface coating dependent cytotoxicity
Qiu and Chen, Biomaterials, 2010, 31, 7606-7619
Shape cause no influence upon the cytotoxicity of Au NRs with toxic or non-toxic coatings.
Shape independent cytotoxicity
PEG-1 PEG-2 PEG-3 PEG-40
20
40
60
80
100
Cel
l Via
bilit
y (%
)
NCS-1 NCS-2 NCS-3 NCS-40
20
40
60
80
100
Cel
l Via
bilit
y (%
)
PEG: polyethylene glycolNCS: Newborn Calf Serum
Replacement by PEG
Replacement by serum protein
Qiu and Chen, Biomaterials, 2010, 31, 7606-7619
Selective Targeting of Gold Nanorods at the Mitochondria of Cancer Cells: Implications for Cancer Therapy
Full Assessment of Fate and Physiological Behavior of Nanomaterials in vivo
Caenorhabditis elegans (C. elegans) Important model system About 1000 somatic cells. A life cycle of about 3 days The body length: 1 mm.
Rat/mousecellZebra fishDrosophilaDaphnia
Worm Crawling
C. Elegan
Uptake & accumulationMetabolism Metabolism
DistributionElimination
ToxicityToxicityLethality Life spanBehavior
Comparison of toxicological effects of different types of QDs.
Larval development
life span
brood size
Qu, Tang, Chen, et al. Nano Lett, 2011
Body distribution of QDs in C. Elegans
BodyGut near tail
Tail
Head
BodyPharynx & Gut
60×objective
10×objective
Material：620 nm QDs (CdTe MPA)
Treatment: Adult feed with live OP50;
exposed 36h, wash and move to clean plate;
4day (96h) wash and move to clean plate;
4.5 day(108h) confocal image;
QDs cannot enter eggs and neonatal lava40×objective
QD distribution in intestinal GFP-labeled C. elegans.
Qu, Tang, Chen, et al. Nano Lett, 2011
Reproductive behavior and egg-laying difficulty after long time exposure.
Qu, Tang, Chen, et al. Nano Lett, 2011
In situ elemental analysis and degradation of QDs
Tail
Pharynx
50μm
Posteriordeirid
Anteriordeirid
Gonad
Intestine
100μm100μm
50μm
BF / QDs Selenium Zinc
0 μg/g 280 0 μg/g 50 0 μg/g 280 0 μg/g 50
50μm 50μm
C
E
G
D
F
H
e
g
f
h
a
b
c
ed
Se Zn
Se Zn
Se Zn
Se Zn
Se Zn
Se Zn
μ-XRF mappingOptical imageBF / QDs Selenium Zinc
μ-XRF mappingOptical imageExposure for 12h Exposure for 24h
12640 12660 12680 12700 Energy (eV)
Inte
nsity
(a.u
.) QDs materials
QDs in pharynx (a)
QDs in hindgut(b)
I Se K-edge Ⅰ Ⅱ
Qu, Tang, Chen, et al. Nano Lett, 2011
12640 12660 12680 12700
Inte
nsity
(a.u
.)
Energy (eV)
QDs materials
QDs in intestine(c)
QDs in rear of intestine (d)
QDs in hindgut(e)
Se K-edge Ⅰ Ⅱ
Understanding Interactions of Nanoscale Materials with Biological Systems
Nanotoxicology is a new and highlighted field, which opens a great opportunity and challenge to chemist, biologist, and toxicologist.
The rules are different for living matters when materials becomenanoscale. Some concepts of traditional toxicology need to be modified in nanotoxicology.
The ability of NPs for biological barriers
Key factors influence the nano-bio interactions.
All studies are a function of particle size, size distribution, shape, surface coating, pH, reactivity, vehicles, agglomeration / aggregation……
More issues will be taken into consideration ……
Models for Risk Evaluation of Nanoparticle Exposure
Bronchia InstillationBronchia Injection (without surgery)Nasal InstillationInhalation (ambient air)Blood vein injectionOral gavage
In vivo testing
The nematode The nematode C. elegansC. elegansabout 1000 somatic cells.about 1000 somatic cells.a life cycle of about 3 da life cycle of about 3 d1 mm1 mm
High-throughput screening
Drosophila
Primary and cell linesPrimary and cell lines
Daphnia magna
Zebra fish
Financial SupportFinancial Support
• National Nature and Science foundation of China (NSFC)
• Chinese Academy of Sciences (CAS)
• Ministry of Science and Technology of China (MOST)
Institute of High Energy Institute of High Energy PhysicsPhysics, CAS , CAS
Yuliang ZHAO, Prof.Yuliang ZHAO, Prof.Zhifang CHAI, Prof. Zhifang CHAI, Prof. Wei LI, PhDWei LI, PhDJiangxueJiangxue WANG, PhDWANG, PhDYuxiYuxi GAO, Dr. GAO, Dr. YufengYufeng LI, PhDLI, PhDBaiBai LI, TechnicianLI, Technician…………
National Center for National Center for Nanoscience and Technology Nanoscience and Technology (NCNST)(NCNST)
XiaochunXiaochun Wu, ProfWu, ProfGuangjunGuangjun NieNie, Prof, ProfZhiyongZhiyong Tang, ProfTang, ProfDong Han, Prof.Dong Han, Prof.Fang LAO, PhDFang LAO, PhDYing LIU, DrYing LIU, DrYang QIUYang QIU..……....
Collaborations & AcknowledgementsCollaborations & Acknowledgements
Institute of Biophysics, CASInstitute of Biophysics, CASTaotaoTaotao WEI, Dr.WEI, Dr...……....
Sept 4-7, Beijing, China
History2012, the 6th NT Conf., Beijing, China2010, the 5th NT Conf., UK 2008, the 4th NT Conf., Switzerland2007, the 3rd NT Conf., Italy2006, the 2nd NT Conf., USA2005, the 1st NT Conf., USA
Programme: The conference will be divided into sessions that focus on specific topics of all sciences for nano-bio interfaces. The paper presented at the conference will be published at a peer-reviewed SCI journal of nano-field.
Theme Covered: it includes but not limits to: Nanotoxicology, Nanobiotechnology, Nanomedicine, Bio-nanomaterials, Nanoecology, Nanochemistry, Nano standardization, etc.
Organizers: National Center for Nanoscience and Technology, China
Steering Committee Chair: Prof. Yuanfang LiuConference Chair: Prof. Yuliang ZhaoSecretary General: Prof. Chunying Chen
ContactNames: Drs. Rui CHEN & Motao ZHU Email: [email protected]: +86-10-82545526/Fax:+86-10-62656765National Center for Nanoscience and Technology, ChinaNo.11, Beiyitiao Zhongguancun 100190 Beijing
Supported by: MOST 973 program, National Natural Science Foundation of China, Chinese Academy of Sciences, Chinese Society of Toxicology
Thank you!Thank you!http://www.nanoctr.cnhttp://nanosafety.ihep.ac.cn
Prof. & Dr. Chunying [email protected]
