Prof. Kenneth DawsonCentre for BioNano InteractionsWebsite: 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 6FP7SFI BionanoInteractHEA PRTLI4

EPA

HSE

EICEIN 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 IssuesCHEMICALS PARTITION NANOPARTICLES TAKEN UPCHEMICALS PARTITION NANOPARTICLES TAKEN UP

Risk, without HazardThe Cost of Doubt

IANH Round Robin(global problem, bottom up)

IDEAS AND PARADIGMs

The processes involved?

Nanoparticles utilise existing biological pathways (in new ways?)

chemicalsNanoscale objects

Energy Dependencepolystyrene 40nmDistinct localisation Clear energy dependence

Silica Easily EntersFinal Destination; LysosomesA549 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).

50 and 100nm Endo

1110.0181159420.0181159420.1161623540.1161623540.03888246040.0388824604

3.9045893724.07615341862.2191283290.12347225040.12347225040.2051704350.2051704350.30365269420.3036526942

5.05434782614.88604780433.1719128330.27255914920.27255914920.1306588730.1306588730.16925049490.1692504949

7.16425120776.62423568655.3075060530.25147468160.25147468160.5327231390.5327231390.28909281490.2890928149

9.09057971017.56976097839.0556900731.32723479271.32723479270.5861994990.5861994990.69690539890.6969053989

9.35748792279.591439688710.878934621.58379480111.58379480110.4311214670.4311214670.45781129780.4578112978

14.462560386511.281267370812.979418890.53867848310.53867848310.7684347930.7684347932.17958774872.1795877487

19.473429951712.9427459725.492736080.53252653080.53252653080.2524866670.2524866670.62485171780.6248517178

20.514492753618.768204558124.696125910.15117859450.15117859450.7910955780.7910955780.67478146650.6747814665

44.871980676322.962201222923.813559320.28895542750.28895542750.9463004390.9463004390.33685631420.3368563142

49.429951690823.669260700420.251815980.84963832490.84963832491.0133593451.0133593450.60948969310.6094896931

48.3043478261

49.5398550725

100nm December 1

100nm November 20

50nm November 11

Time (min)

Fold Increase Fluorescence Intensity

50 and 100nm exo

110.05238793330.05238793330.01381373370.0138137337

0.9805105050.96089372320.00671138950.00671138950.0516711830.051671183

0.91333394770.93200546220.01726716710.01726716710.01120736880.0112073688

0.9187246590.9317700240.01837487210.01837487210.00651591210.0065159121

0.86702911910.90881480440.01251055120.01251055120.04697972620.0469797262

0.86887209730.90186937890.01876582690.01876582690.00651591210.0065159121

0.79800958350.8560295710.02006900930.02006900930.03668458510.0366845851

0.75843162550.81254414470.01192411910.01192411910.0048217750.004821775

0.47765388870.5845222960.00084706860.00084706860.00931775430.0093177543

0.40476409880.48959363380.00045611380.00045611380.00762361720.0076236172

0.39591780320.46936949660.0150517570.0150517570.01029514110.0102951411

50nm Exocytosis

100nm Exo

Time (min)

Fold Decrease Fluorescence

Exocytosis 17hr Accumulation

Endo NaN3 4C

11000.00326608210.003266082110.00589255650.0058925565

2.66512702081.65819861430.01665381650.01665381650.35273687010.35273687011.64861111110.00481125220.0048112522

2.94226327941.90300230950.07838597110.07838597110.1763684350.1763684352.05972222220.13877773330.1387777333

3.89376443422.44033872210.24892317340.24892317340.28088306320.28088306322.24444444440.02678791880.0267879188

5.43648960742.58506543490.14058071420.14058071420.97982463910.97982463912.58055555560.1669962020.166996202

6.86605080833.58275596610.14697369590.14697369590.93736557140.93736557142.706250.04419417380.0441941738

8.87297921483.99769053120.04406185690.04406185690.07838597110.07838597113.49861111110.02441443870.0244144387

12.54965357974.71593533490.04406185690.04406185690.28741522750.28741522753.70.02946278250.0294627825

13.61200923795.11316397230.16330410650.16330410650.16330410650.16330410653.70138888890.03849001790.0384900179

15.94226327944.76905311780.61728952260.61728952260.27108481680.27108481683.53333333330.22391714740.2239171474

32.6882217090.30701172020.30701172021418

Control

4DegC

NaN3

Time (min)

Fold Increase Fluorescence Intensity

Endocytosis at 4C and with NaN3

NaN3 Exo

110010.05238793330.0523879333

1.00402943241.00402943240.03067887080.03067887080.03067887080.03067887080.9805105050.00671138950.0067113895

0.91617028730.91108969870.05215408030.05215408030.02949891420.02949891420.91333394770.01726716710.0172671671

0.91266643310.89269446390.023363140.023363140.00920366120.00920366120.9187246590.01837487210.0183748721

1.00455501050.86483882270.00731573070.00731573070.04011852330.04011852330.86702911910.01251055120.0125105512

0.9692536790.87902943240.02147520950.02147520950.02194719220.02194719220.86887209730.01876582690.0187658269

0.87885423970.74281709880.05144610640.05144610640.00117995660.00117995660.79800958350.02006900930.0200690093

0.84644358790.70366152770.00117995660.00117995660.02949891420.02949891420.75843162550.01192411910.0119241191

0.69542747020.57752277510.02949891420.02949891420.03893856670.0389385667

50nm Control 18hr Accumulation

50nm NaN3 18 Hr. Accumulation

December 17, 50nm Exo

Time (minutes)

Fold Decrease in Fluorescence

Control and NaN3 Exocytosis, 50nm

50 and 100nm Serum Free Endo

110.0089696420.0089696420.00891311070.008913110711

47.627906976739.81512605046.23293248186.23293248182.2858185532.2858185532.2191283293.904589372

74.651162790764.58263305326.53464535156.534645351513.482261211313.48226121133.1719128335.0543478261

707.3121916843375.990196078446.53042371246.530423712134.5978908862134.59789088625.3075060537.1642512077

969.3530655391576.648459383852.843151165752.843151165786.95462177786.9546217779.0556900739.0905797101

1085.9281183932784.9663865546122.0947675118122.094767511810.878934629.3574879227

783.3361522199833.453781512638.153867377338.153867377313.583580687313.583580687312.9794188914.4625603865

1071.3044397463838.228991596636.246323502436.246323502449.316241432449.316241432425.4927360819.4734299517

24.6961259120.5144927536

23.8135593244.8719806763

20.2518159849.4299516908

48.3043478261

49.5398550725

50m serum free

100nm serum free

50nm with serum

100nm With Serum

Time (minutes)

Fold increase in FLuorescence

Endocytosis in Serum Free Media

50nm HI vs NHI

110.04543531250.04543531250.00982092750.0098209275

8.55366098297.75347222220.18156402810.18156402810.05401510130.0540151013

30.784353059230.10069444440.646821850.646821850.86424162150.8642416215

148.6459378134140.560185185215.909547959515.90954795958.03633987898.0363398789

Heat Inactivated

Non Heat Inactivated

Concentration (ug/ml)

Fold Increase Fluorescence Intensity

50nm HI vs. NHI 24 Hour Accumulation

100nm HI and NHI

110.04543531250.04543531250.00982092750.0098209275

8.29488465415.27893518520.26383522830.26383522830.4891070050.489107005

31.007689735924.1406251.1659044121.1659044120.5278748540.527874854

68.458709461763.58854166672.41064395092.41064395090.75866665060.7586666506

Heat Inactivated

Non Heat Inactivated

Concentration (ug/ml)

Fold INcrease FLuorescence Intensity

100nm SiO2 23 Hour Accumulation

Sheet1

Sheet2

Sheet3

Different OrganellesDifferent situationsA549 cells exposed to 50 nm fluorescently-labelled silica nanoparticles for 24 hours.nanoparticles

Zooming in50 nm silica nanoparticles in multi-lameller vesicles inside A549 cell after 24 hours of exposure.mitochondrion

Cellular Bio-Accumulation Deep SignificanceNanoparticle export kineticsTypical small molecule

IDEAS AND PARADIGMS?

The Nano Objects Defined

New Paradigm; protein corona?

Size & surface charge matter!

Lipoproteins100nm NH350nm NH3100nm plain50nm plain100nm COOH50nm COOHApolipoprotein A-IXXXXXXApolipoprotein A-IIXXApolipoprotein A-IVXXXXXApolipoprotein B-100XXXXApolipoprotein C-IXXXXApolipoprotein C-IIIXXXApolipoprotein DXXXApolipoprotein EXXXXApolipoprotein FXApolipoprotein L1XXBeta-2-glycoprotein 1 (apolipoprotein H)XXXXX

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

SampleMediumZetasizerZ-Ave [nm]PDITEM Size [nm]Zeta Pot. [mV]pH20nmH2O58.090.33428.54 -41.47.5240nmH2O73.310.2336.16 -35.87.45100nmH2O119.80.004117.3-42.37.54200nmH2O202.50.011177.5 -54.27.5320nmcMEM42.350.545--18.97.2640nmcMEM76.180.251--19.67.21100nmcMEM1310.258--18.77.26200nmcMEM221.70.146--20.57.25

proteins boundbiological fluids1 = pregnancy zone protein

2 = unknown

3 = Apolipoprotein AIV

4 = Apolipoprotein E

5 = Apolipoprotein AI

6 = Apolipoprotein AII

7=

8=

About 45 identified so far

Note Distinction Targetting to Nucleous!

DISCIPLINE, STANDARDS

Rational BioNanoScience

In vivo effectsIntracellular Visualization

Many Serious (not widely kown) Problems Much of the literature in this area questionable!Impurities, cleaning, disperson, stability, etc etcStandards we can Trust .Commercial samples leak - free dye!

SUMMARY

It matters (people, economy, science)Nanoscale objects are processed by organisms-new world, here to stayNeed to conserve effort, not compete (on platforms),get the right people involvedNeed paradigms, and to get at key ideas quicklyTime is short, data generation (without strategy) expensive (all ways)We have to do it right, and to agreeScience policing it own literatureProtocols for Round RobinsSome basic things eg. nanomaterial standards

Round-robin approaches 3T3-fibroblasts incubated for 24 h with 40 g/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 sitesBarnes, et al., Nano Letters, 2008, 8, 3069.

Sigma Ludox CL 420883Partner XPartner YTail%DNATail%DNA6h6hMeanSDMeanSD40 g/ml silica NP6.245.127.447.50Dialysate8.468.866.428.56Negative control (DMEM)7.265.996.127.31Positive control (H202)34.3018.4452.3417.99

A proteinA chain of amino acid residuesHydrophobic Hydrophilic Charged

A folded proteinThe structure is given by the sequence of amino acids

Surface plasmon resonance of nanoparticles in biological fluids

b2m20-41_C25SSNFLNSYVSGFHPSDIEVDLLKG

The Nature of Dialysis-Related Amyloidosisaggregation of b2-microglobulin

monomer freely circulates at constant level

renal failure: normal kidney functions inhibited - 2m 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 Alzheimers

Previous research suggests Amyloid Beta protein (A) plays central role in pathogenesis of disease.A 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 nanoparticlesAb1-40 (2.5mM)Thioflavin T fluorescenceNanoparticles Can also quench Fibrillation!!