The Cellular and Molecular Toxicity of Low Solubility Nanoparticles

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1 The Cellular and Molecular Toxicity of Low Solubility Nanoparticles Prof Vicki Stone Centre for Health and Environment Napier University, Edinburgh Safety of nanomaterials Interdisciplinary Research Centre

2 PM 10 Carbon based particles Traffic and industrial Many ultrafine Transition metals *** *** Sulphates/Nitrates Traffic/photochemical Mainly ultrafine 500nm Wind blown dust Mainly coarse Biological components Spores Pollen Mainly fine and course

3 Lung Defence Mechanisms mucus cilia Airway Alveolus Antioxidants Particles phagocytosed and cleared Gas exchange Particles trapped and cleared on mucociliary escalator Macrophage Blood vessel Particles

4 The Ultrafine Particle Hypothesis Particles Blood vessel Release of mediators by macrophages and epithelial cells INFLAMMATION Impaired movement Particles cross the epithelial barrier Seaton al The Lancet 345:

5 Ultrafine particle toxicology Neutrophils in bronchoalveolar lavage fluid X nm TiO nmtio 2 Ferin et al 1992 Am.J.Respir.Cell.Mol.Biol. 6: Time from start of exposure (weeks)

6 Accidental versus engineered nanoparticles Nanoparticles Source Exposure Toxicology Accidental Fossil fuel combustion E.g. Traffic, cooking. Low exposure to everyone Lots e.g. diesel, CB, welding fume etc Purposeful Bulk use of nanoparticles in industry e.g. carbon black Nanotechnology High exposure to workers followed by low exposure to everyone Virtually none Adapted from Ken Donaldson

7 Model nanoparticles 14 nm 254 m 2 /g 535nm CB 100nm 202nm 260 nm 8 m 2 /g 64nm 100nm Carbon black 500nm Polystyrene beads Low solubility, low toxicity materials.

8 Factors involved in NP toxicity 1. SIZE 10μm 1 μm 0.1μm Cilia 0.25μm diameter. 1.0 μm 0.1μm Bronchial epithelium

9 Factors involved in NP toxicity 2. PARTICLE NUMBER There are thousands more ultrafine particles / mg of dust than larger, respirable particles. 3. SURFACE AREA Particle surface area / mg is much greater for ultrafine particles than for larger, respirable particles. Fine carbon black 7.9 m 2 /g Ultrafine carbon black m 2 /g

10 Particle surface area and surface reactivity Duffin et al. (2002) Ann.Occ.Hyg.46; Neutrophils in BAL (x10 6 ) 10 5 Quartz 0 Relatively low toxicity particles Surface Area instilled (cm 2 ) Low toxicity low solubility particles straight line relationship between SA and neutrophils. Highly pathogenic particles highly reactive surface (eg quartz). do not sit on same line as low toxicity particles.

11 Nanoparticles, ROS and oxidative stress.

12 Reactive oxygen species production by carbon particles Supercoiled band intensity x nm CB 14 nm CB 6 4 * * Particle dose / 290 μl plasmid Stone et al Toxicol In Vitro 12:

13 Reactive oxygen species produced by nanoparticles DCF Fluorescence Intensity nm CB *** *** ** 260 nm CB Particle dose (mg/ml) DCF Fluorescence Intensity *** 64 nm 202 nm 535 nm Polystyrene particle diameter Wilson et al TAP 184: Brown et al TAP 175:

14 Effect of carbon particles on intracellular antioxidant content of A549 cells GSH (% of control) μg/mm 2 * Time (hours) 14 nm CB 260 nm CB Control Stone et al TIV 12:

15 Effects of Nanoparticles and PM 10 on Intracellular Signalling Pathways

16 Effect of particles on Ca 2+ signalling in human MonoMac 6 cells [Ca 2+ ] c Control ** 14 nm CB 260 nm CB Stone et al Eur Resp J 15: sec

17 The response to thapsigargin in human MM6 cells in the presence of CB and verapamil 260 nm CB 14 nm CB Ca 2+ Ca 2+ THAPSIGARGIN *** Ca 2+ VERAPAMIL Control Particles Particles + verapamil [Ca 2+ ] c nm after treatment with thapsigargin Stone et al Eur Resp J 15: sec

18 The effect of polystyrene nanoparticles on calcium signalling in macrophages Polystyrene bead diameter 535 nm 202 nm 64 nm Control [Ca 2+ ]c nm after treatment with thapsigargin 1500 sec Stone et al Inhal Tox 12 (suppl 3): ***

19 Calcium Signalling Inhibitors Calcium channel Ca 2+ Endoplasmic reticulum [mm] [nm] BAPTACa 2+ Ca 2+ Ca chellator W-7 Calmodulin Signalling Verapamil Ca 2+ channel blocker

20 The role of Ca 2+ in the induction of TNFα expression by carbon nanoparticles TNFα pg/ml *** Rat alveolar macrophages 4 hour treatments 0 Control 14nm CB 260nm CB 14nm CB Verap Verap 14nm CB BAPTA BAPTA Brown et al AJP 286; L344-L353

21 The role of Ca 2+ in the induction of TNFα mrna expression by CB nanoparticles GAPDH TNFα TNFα mrna (% GAPDH) Control 14nm CB 5 0 *** Control 14nm CB 260nm CB 14 CB Verap 260nm14nm CB CB Verap Verap 14 CB BAPTA BAPTA ** Verap 14nm CB BAPTA LPS BAPTA Brown et al AJP 286; L344-L353

22 The effect of antioxidants on CB induced TNFα expression TNFα pg/ml ** Control 14nm CB 14nm CB Nacystelin Nacystelin TNFα mrna (% GAPDH) *** *** 0 Control14nm 14nm Nacystelin CB CB Nacystelin Brown et al AJP 286; L344-L353

23 Particle induced TNFα expression role of calcium and oxidants TNFα pg/ml ** Human monocyte derived macrophages 4 hour treatments Control 14 nm CB CB CB CB CB + verap + BAPTA + W-7 + Trolox Brown et al AJP 286; L344-L353

24 Activation of NFκB and promoter binding Degraded IκB NFκB P IκB P IκB P U DNA NFκB Phosphorylation NFκB mrna NFκB Promoter TRANSCRIPTION Nuclear membrane

25 Immunofluorescent staining of the p65 subunit of NFκB in human monocyte/macrophages 14 nm CB Control Brown et al AJP 286; L344-L353

26 Nuclear localisation of the p65 subunit of NFκB in human monocytes Nuclear p65 intensity (% control) 160 * Control CB CB CB CB CB CB + verap + W-7 + BAPTA + Nac + Trolox Brown et al AJP 286; L344-L353

27 Nanoparticles, calcium and oxidants Ultrafine, nanoparticles Macrophages Oxidative Calcium stress signalling Activation of NF-κB Production of pro-inflammatory proteins

28 J774 macrophages exposed to 14nm CB CONTROL Wilson et al., manuscript in prep 14 nm CB

29 Measurement of cytoskeletal stiffness using ferromagnetic particles Step twist Θ Twisting field B TW Magnetic particles within phagosomes 6 Hz Macrophage Fluxgate sensor Magnetic twisting device Aligned ferromagnetic microparticles ingested by macrophages, Detection by an array of magnetic fluxgate sensors (Förster device).

30 Impact of particles on the macrophage phagosome transport Relaxation b5 (drug/control) 1.6 * 1.4 * Relaxation b5 (drug/control) * $ $ $ 0 14nm CB 320 μg/million cells EC90 EC90 DEP UD 0 EC90 Verap BAPTA W7 Moeller et al., 2003 Manuscript submitted

31 Effects of nanoparticles and PM 10 on migration and phagocytosis

32 Chemotaxis Chamber Macrophages (J774.2) Filter (5 µm pore) Direction of migration Chemoattractant Actual Filter

33 Chemotaxis of J774 macrophages in response to epithelial cell conditioned medium Chemotaxis % of Control Particles & 200 Cells Particles Only * ZAS 250 nm TiO 2 Barlow et al Particle Fibre Toxicol 2: nm TiO nm CB 14 nm CB

34 Chemotactic response of J774 macrophages to epithelial cell conditioned medium % of Control ZAS > <5 Molecular Weight Fraction of Conditioned Medium kda Barlow et al Particle Fibre Toxicol 2:11.

35 Chemotactic response of J774 macrophages to serum treated with particles *** % of Negative Control *** 90 ZAS mg/ml 14nm CB 260nm CB 21nm TiO 2 250nm TiO 2 Barlow et al Toxicol Lett 155:

36 Nanoparticle uptake by macrophages

37 Phagocytosis Barlow et al Submitted.

38 Effect of TiO 2 and CB on phagocytosis % of cells that phagocytose >2 beads 8h exposure Renwick et al Toxicol Applied Pharmacol. 172:

39 Non-phagocytic cells Renwick et al Toxicol Applied Pharmacol. 172:

40 Cambridge multiwalled carbon nanotube

41 Nottingham Multiwalled carbon nanotubes

42 Control Carbon nanotubes Cambridge Commercial Nottingham

43 Effects of nanotubes on cell viability Apoptosis Necrosis % Total Cells % total cells 00 Control Control E PR UfCB LFA Triton TNF Nanotubes/fibres Nanotube Treatment ug/ml Nanotubes CB LFA Triton TNFα Necrotic Apoptotic 16μg/ml % Total Cells % total cells 00 Control Control E PR UfCB LFA Triton TNF Nanotubes/fibres Nanotube treatment 31.25ug/ml nanotubes Necrotic Apoptotic CB LFA Triton TNFα 31μg/ml

44 Effect of nanotubes and nanofibres on phagocytosis 90 Phagocytosis (% Total) ug/m l 31.25ug/m l 62.5ug/m l 0 Medium E21 PR19 Sample 9-01 Sample 3-01 Sample 1-03 Uf CB LFA Treatment All treatments induced a significant (p<0.05) inhibition

45 Summary Low toxicity, low solubility NP: Inflammation is related to surface area dose. Generate ROS leading to oxidative stress in epithelial and macrophage cells. Induce Ca 2+ influx in macrophages in vitro resulting in; activation of NFκB and AP1 up-regulation of pro-inflammatory cytokine expression Inhibition of phagosome transport

46 Summary Low toxicity, low solubility NP: Stimulate production of chemotaxins by epithelial cells. Rapidly taken up into macrophages. Inhibit subsequent phagocytosis of larger particles MWCNT: Morphology determines uptake by macrophages. Inhibit subsequent phagocytosis of E.Coli.

47 Napier University Dr David Brown Dr Martin Wilson Marieke Tuinman Dr Suzanne Patterson Janis Vamvakopolous Dr Peter Barlow Martin Clift University of Edinburgh Prof Ken Donaldson University of Cambridge Prof Alan Windle Dr Ian Kinloch UIF Dusseldorf Dr Rodger Duffin Dr Roel Schins Dr Paul Borm Dr Markus Denhart GSF Munich Dr Winfried Moeller Nottingham University Dan Walters THE COLT FOUNDATION