Transcript the biomolecular corona approach

A RATIONAL APPROACH TO
INTERACTION BETWEEN NANOSCALE OBJECTS AND
LIVING ORGANISMS?
‘Time is short-we need to move-and there is a lot to do’
Prof. Kenneth Dawson
Centre for BioNano Interactions
Website: www.cbni.eu
Acknowledgements
CEIN NSF
IRCSET
SFI
BionanoInteract
EU;
FP 6
FP7
HEA PRTLI4
EPA
HSE
EI
http://www.cbni.eu
Centre for BioNano Interactions
Why does it Matter?
effective and safe implementation nanoscale science improve human condition
– Innovations IT, energy storage, energy harvesting
telecoms, construction, textiles, etc; safe implementation
– New approaches to diagnose human disease
– New approaches to cure human disease
NEW FUNDAMENTAL LENGTH SCALES
Less than 100nm enter cell, less than 40nm enter nucleus, less than
35nm pass Blood Brain Barrier
The Durable Issues
CHEMICALS
PARTITION
CHEMICALS
PARTITION
NANOPARTICLES TAKEN UP
………………………NANOPARTICLES TAKEN UP
Risk, without Hazard
The Cost of Doubt
1st reports of Nanotoxicity
IANH Round Robin(global
problem, bottom up)
IDEAS AND PARADIGMs
The processes involved?
Nanoparticles utilise existing
biological pathways (in new ways?)
37C
NaN3
250
1
5
chemicals
200
FITC log
2
4C
6
150
100
4
50
3
0
[5]
K15=
[1]
0
50
100
150
200
250
Exposure time, minutes
37C
4C
NaN3
30
1
25
5
FITC log
2
20
6
15
Nanoscale
objects
10
4
3
5
0
0
50
100
150
Exposure time, minutes
200
250
300
Energy Dependence
polystyrene 40nm
37C
4C
NaN3
35
Cell Fluorescence Intensity
30
25
20
15
10
5
0
0
30
60
90
120
150
180
210
Exposure time, minutes
Distinct localisation
Clear energy dependence
240
Silica Easily Enters
Final Destination; Lysosomes
Fold Increase Fluorescence Intensity
60
50
100nm December 1
40
100nm November 20
30
50nm November 11
20
10
0
0
500
1000
1500
2000
2500
Time (min)
A549 cells exposed to 50 and 100nm
fluorescently-labelled silica nanoparticles.
Top: Uptake curves from flow cytometry.
Right: Confocal microscopy images of
localisation of 50nm particles (24 hours).
3000
3500
Different Organelles
Different situations
nanoparticles
A549 cells exposed to 50 nm fluorescently-labelled silica nanoparticles for 24 hours.
Zooming in
mitochondrion
50 nm silica nanoparticles in multi-lameller vesicles inside A549 cell after 24 hours
of exposure.
Cellular Bio-Accumulation
Deep Significance
Nanoparticle export kinetics
plain polyscience
YG dye
negative invitrogen
1,4
1,2
FITC log
1
0,8
0,6
0,4
Typical small molecule
0,2
0
0
200
400
600
800
Exocytosis time, minutes
1000
1200
1400
IDEAS AND PARADIGMS?
The Nano Objects Defined
New Paradigm; protein
corona?
Size & surface charge
matter!
Lipoproteins
Apolipoprotein A-I
100nm NH3
50nm NH3
100nm plain
50nm plain
100nm COOH
50nm COOH
X
X
X
X
X
X
Apolipoprotein A-II
X
Apolipoprotein A-IV
X
X
X
Apolipoprotein B-100
X
X
Apolipoprotein C-I
X
X
X
X
Apolipoprotein D
X
X
X
X
X
Apolipoprotein F
X
Apolipoprotein L1
X
Beta-2-glycoprotein 1
(apolipoprotein H)
X
X
X
X
X
Apolipoprotein C-III
Apolipoprotein E
X
X
X
X
X
X
X
X
X
X
X
Impact on surface properties
Adsorption of biomolecules changes surface charge which affects
stability and aggregation behaviour.
Sample
Medium
Zetasizer
Z-Ave [nm]
PDI
TEM Size [nm]
Zeta Pot. [mV]
pH
20nm
H2O
58.09
0.334
28.54
-41.4
7.52
40nm
H2O
73.31
0.23
36.16
-35.8
7.45
100nm
H2O
119.8
0.004
117.3
-42.3
7.54
200nm
H2O
202.5
0.011
177.5
-54.2
7.53
20nm
cMEM
42.35
0.545
-
-18.9
7.26
40nm
cMEM
76.18
0.251
-
-19.6
7.21
100nm
cMEM
131
0.258
-
-18.7
7.26
200nm
cMEM
221.7
0.146
-
-20.5
7.25
Break-out session 1 – nanoparticle dispersion in media
proteins bound
biological fluids
1 = pregnancy zone protein
1
2
HSA
2 = unknown
3
3 = Apolipoprotein AIV
4
4 = Apolipoprotein E
5
6
5 = Apolipoprotein AI
More hydrophobic
More hydrophobic
6 = Apolipoprotein AII
7= …
8= …
About 45 identified so far
Note Distinction
Targetting to Nucleous!
DISCIPLINE, STANDARDS
Rational BioNanoScience
Nanoparticle synthesis
Dispersion and
characterisation
Intracellular
Visualization
Disease / therapy
In vivo effects
Protein Corona
Functional impacts /
toxicity
Many Serious (not widely kown)
Problems
Commercial samples leak - free dye!
particles
Free dye (<6 kda)
Sds-gel (4% stacking gel and 10%
resolving gel)
Much of the literature in this area questionable!
Impurities, cleaning, disperson, stability, etc etc
Standards we can Trust
SUMMARY
•It matters (people, economy, science)
•Nanoscale objects are ‘processed’ by organisms-new world,
here to stay
•Need to conserve effort, not compete (on platforms),get the right
people involved
•Need paradigms, and to get at key ideas quickly
•Time is short, data generation (without strategy) expensive (all ways)
•We have to do it right, and to agree
•Science policing it own literature
•Protocols for Round Robins
•Some basic things eg. nanomaterial standards
Round-robin approaches
Comet Assay – fully reproducible across multiple sites
3T3-fibroblasts incubated for 24 h with 40 μg/ml 30 nm silica nanoparticles (Glantreo),
b) incubated for 24 h with the dialysate, c) negative control, d) positive control.
Sigma Ludox CL 420883
Partner X
Partner Y
Tail%DNA
Tail%DNA
6h
6h
Mean
SD
Mean
SD
40 μg/ml silica NP
6.24
5.12
7.44
7.50
Dialysate
8.46
8.86
6.42
8.56
Negative control (DMEM) 7.26
5.99
6.12
7.31
Positive control (H202)
18.44
52.34
17.99
34.30
Barnes, et al., Nano Letters, 2008, 8, 3069.
A protein
A chain of amino acid residues
Hydrophobic Hydrophilic Charged
A folded protein
The structure is given by the sequence of amino acids
Surface plasmon resonance of
nanoparticles in biological fluids
b2m20-41_C25S
SNFLNSYVSGFHPSDIEVDLLKG
The Nature of
Dialysis-Related Amyloidosis
• aggregation of b2-microglobulin
• monomer freely circulates at
constant level
• renal failure: normal kidney
functions inhibited - β2m
concentration increase 60 fold.
• This increase in concentration can
lead to formation of amyloid
plaques (clusters).
• Accumulation in patients jointsrestricted movement / pain /
cavities (cysts)
The Nature of Alzheimer’s
•Previous research suggests
Amyloid Beta protein (Aβ)
plays central role in
pathogenesis of disease.
•Aβ is derived from a
precurser protein.
•Its production is a normal
process but an overproduction can lead to the
onset of AD.
Fibrillation of proteins in the
presence of nanoparticles
Normalized Fluorescence Intensity
Thioflavin T fluorescence
Fibrillation of proteins in the
presence of new nanoparticles
Ab1-40 (2.5mM)
1.0
0.8
Nanoparticles
Can also quench
Fibrillation!!
0.6
0.4
0.2
0.0
0
100
200
300
Time / minutes
400