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Prof. Kenneth Dawson Centre for BioNano Interactions

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. IRCSET EU; FP 6 FP7. CEIN NSF. SFI

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Prof. Kenneth Dawson Centre for BioNano Interactions

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  1. 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

  2. Acknowledgements IRCSET EU; FP 6 FP7 CEIN NSF SFI BionanoInteract HEA PRTLI4 EPA HSE EI http://www.cbni.eu Centre for BioNano Interactions

  3. 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

  4. The Durable Issues CHEMICALS PARTITION ………………………NANOPARTICLES TAKEN UP CHEMICALS PARTITION NANOPARTICLES TAKEN UP

  5. 1st reports of Nanotoxicity Risk, without HazardThe Cost of Doubt

  6. IANH Round Robin(global problem, bottom up)

  7. IDEAS AND PARADIGMs The processes involved?

  8. Nanoparticles utilise existing biological pathways (in new ways?)

  9. 1 1 5 5 2 6 2 6 4 4 3 3 [5] K15= [1] chemicals Nanoscale objects

  10. Energy Dependencepolystyrene 40nm Distinct localisation Clear energy dependence

  11. Silica Easily EntersFinal Destination; Lysosomes 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).

  12. Different OrganellesDifferent situations nanoparticles A549 cells exposed to 50 nm fluorescently-labelled silica nanoparticles for 24 hours.

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

  14. Cellular Bio-Accumulation Deep Significance Nanoparticle export kinetics Typical small molecule

  15. IDEAS AND PARADIGMS? The Nano Objects Defined

  16. New Paradigm; protein corona?

  17. Size & surface charge matter!

  18. Impact on surface properties Adsorption of biomolecules changes surface charge which affects stability and aggregation behaviour. Break-out session 1 – nanoparticle dispersion in media

  19. 1 2 HSA 3 4 5 6 More hydrophobic More hydrophobic proteins bound biological 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

  20. Note Distinction Targetting to Nucleous!

  21. DISCIPLINE, STANDARDS

  22. Protein Corona Nanoparticle synthesis Dispersion and characterisation Disease / therapy Functional impacts / toxicity Rational BioNanoScience IntracellularVisualization In vivo effects

  23. particles Free dye (<6 kda) Sds-gel (4% stacking gel and 10% resolving gel) Many Serious (not widely kown) Problems Commercial samples leak - free dye! Much of the literature in this area questionable! Impurities, cleaning, disperson, stability, etc etc Standards we can Trust .

  24. 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

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

  26. A protein A chain of amino acid residues Hydrophobic Hydrophilic Charged

  27. A folded protein The structure is given by the sequence of amino acids

  28. Surface plasmon resonance of nanoparticles in biological fluids

  29. b2m20-41_C25S SNFLNSYVSGFHPSDIEVDLLKG

  30. 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 joints-restricted movement / pain / cavities (cysts)

  31. 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.

  32. Fibrillation of proteins in the presence of nanoparticles

  33. Fibrillation of proteins in the presence of new nanoparticles Ab1-40 (2.5mM) Nanoparticles Can also quench Fibrillation!! Thioflavin T fluorescence

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