Prof. Kenneth Dawson

Centre for BioNano Interactions

Website: www.cbni.eu

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’

Acknowledgements

http://www.cbni.euCentre for BioNano Interactions

IRCSET

EU; FP 6FP7

SFI BionanoInteract

HEA PRTLI4

EPA

HSE

EI

CEIN NSF

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 NANOPARTICLES TAKEN UPCHEMICALS PARTITION ………………………NANOPARTICLES TAKEN UP

Risk, without HazardThe 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?)

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37C 4C NaN3

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FITC

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37C 4C NaN3

chemicals

Nanoscale objects

Energy Dependencepolystyrene 40nm

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Distinct localisation

Clear energy dependence

Silica Easily EntersFinal Destination; Lysosomes

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100nm December 1

100nm November 20

50nm November 11

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).

Different OrganellesDifferent situations

A549 cells exposed to 50 nm fluorescently-labelled silica nanoparticles for 24 hours.

nanoparticles

Zooming in

50 nm silica nanoparticles in multi-lameller vesicles inside A549 cell after 24 hours of exposure.

mitochondrion

Cellular Bio-Accumulation Deep Significance

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Exocytosis time, minutes

FIT

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plain polyscience YG dye negative invitrogenNanoparticle export kinetics

Typical small molecule

IDEAS AND PARADIGMS?

The Nano Objects Defined

New Paradigm; protein corona?

Size & surface charge matter!

Lipoproteins 100nm NH3 50nm NH3 100nm plain 50nm plain 100nm COOH 50nm COOH

Apolipoprotein A-I X X X X X X

Apolipoprotein A-II X X

Apolipoprotein A-IV X X X X X

Apolipoprotein B-100 X X X X

Apolipoprotein C-I X X X X

Apolipoprotein C-III X X X

Apolipoprotein D X X X

Apolipoprotein E X X X X

Apolipoprotein F X

Apolipoprotein L1 X X

Beta-2-glycoprotein 1 (apolipoprotein H) X X X X X

Impact on surface properties

Sample Medium ZetasizerZ-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

Adsorption of biomolecules changes surface charge which affects stability and aggregation behaviour.

Break-out session 1 – nanoparticle dispersion in media

proteins boundbiological fluids

1 = pregnancy zone protein

2 = unknown

3 = Apolipoprotein AIV

4 = Apolipoprotein E

5 = Apolipoprotein AI

6 = Apolipoprotein AII

7= …

8= …

About 45 identified so far

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2HSA

More hydrophobic More hydrophobic

Note Distinction Targetting to Nucleous!

DISCIPLINE, STANDARDS

Rational BioNanoScience

Nanoparticle synthesis Protein Corona

Functional impacts / toxicity

Disease / therapy

In vivo effects

Intracellular Visualization

Dispersion and characterisation

Many Serious (not widely kown) Problems

Sds-gel (4% stacking gel and 10% resolving gel)

particles

Free dye (<6 kda)

Much of the literature in this area questionable!

Impurities, cleaning, disperson, stability, etc etc

Standards we can Trust

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Commercial samples leak - free dye!

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

Sigma Ludox CL 420883Partner 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) 34.30 18.44 52.34 17.99

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.

Comet Assay – fully reproducible across multiple sites

Barnes, et al., Nano Letters, 2008, 8, 3069.

A proteinA 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

2m20-41_C25SSNFLNSYVSGFHPSDIEVDLLKG

The Nature of Dialysis-Related Amyloidosis

• aggregation of 2-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 joints-restricted 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 over-production can lead to the onset of AD.

Fibrillation of proteins in thepresence of nanoparticles

Fibrillation of proteins in thepresence of new nanoparticles

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Nanoparticles Can also quench Fibrillation!!