Importance of size and surface area with respect to effect of particles on cytokine release in lung cells NANOMAT Conference 2009 Lillehammer, 18 may 2009 Magne Refsnes, Tonje Skuland, Marit Låg, Johan Øvrevik, Per Schwarze, Maurizio Gualtieri Norwegian Institute of Public Health

Nanoparticles (NPs)- different sizes, forms and chemical composition Spherical NPs Rods Fibres with modifications What characteristics are critical for posssible adverse health effects? Make NPs that minimize the health hazard, but keep the advantagous properties of NPs! Co-Ni

µm nm Ultrafine particles and Nanoparticles (NPs) Particulate matter, PM10 and PM2.5 Suh et al. (in press) - Nanoparticles: engineered particles- from nmeter - Small particles have a much larger surface area than larger particles related to mass -To what extent will the size and surface area determine the cellular responses? - Will particles of different sizes induce qualitative different responses? Definition of NPs

Importance of particle surface area for cytokine responses: effect of crystalline silica in epithelial lung cells Hetland et al. 2001

In vivo experiments- importance of surface area Mass instilled (  g) Mean PMN in lavage (millions) Dose expressed as mass instilled Courtesy K. Donaldsson, Edinburgh

Effect of surface area dose on IL-8 mRNA expression- effect of different particles Nanoparticles and fine particles examined in epithelial lung cells (A549) Linear relationship beween particle surface area and response- different slopes for the different particles A surface area dose treshold is suggested Monteiller et al.2009

Wonder –whether this is too simplified ! Nanoparticles have mostly been compared with larger particles How will the cellular responses be to different sizes of particles in the nanosize area? The deposition pattern of particles is very dependent on size- both for nano-sized particles and larger particles The particle uptake in cells will affect the targeting to the site of action- and this could be influenced by the particle size

In which parts of the airway system are the nanoparticles deposited? A complex deposition pattern in both the NP size area and at larger sizes The dependence of size vary in different parts of the respiratory system

Different uptake mechanisms depending on particle size How are the uptake patterns in the NP size area and for larger particles? Different mechanisms for different sizes! In macrophages versus epithelial cells? Is the uptake required for the responses, or are the responses initiated at the plasma membrane? Will the uptake be required for some responses, not for others? s IL-8 mRNA ↑ Nanoparticles Agglomerated Inhibitors Signals from the plasma membrane Particle uptake IL-8 signals

Model systems to further examine the importance of size Model systems: - BEAS-2B cells (bronchial epithelial human cells) - Primary rat alveolar epithelial cells (type 2) - Primary rat alveolar macrophages - THP-1 monocytes +/- differentiation End points: - Cytokines (IL-6, IL-8, MIP-2) - Cytotoxicity (PI/Hoechst-staining, LDH) Particles: - Polystyrene particles (50, 100, 200, 1000 nmeter) - Amorphous silica particles (30, 50, 100, 200 and 500 nmeter)

Effects of amorphous particles of different sizes on bronchial epithelial cells (BEAS-2B) Gualtieri -unpublished The 30, 50 and 100 nm more potent than 500 nmSiNPs The 200 nm SiNPs most potent The cytokine response is not proportional to particle surface area in the range from 30 to 200 nm

Effect of different sizes of silica particles in primary type 2 cells: MIP-2 At 25µg/ml: 50 and 200 nm> 30 and 100 nm> 500 nm

Polystyrene particles in primary type 2 cells: cytokine responses to different sizes in relation to increasing mass Polystyrene particles of 50, 85, 200 and 1000 nm The smallest particles most potent on a mass basis

Polystyrene particles in rat primary type 2 cells: IL-6 responses to different sizes in relation to surface area The 50, 85 and 200 nm silca particles induced similar IL-6 responses when related to surface area. The 1000 nm particles seemed most potent!

Further work! Determine mRNA expression profile of different sizes of NPs: Will an increased mRNA expression for cytokine genes (IL-6, MIP-2) be explained by the surface area? Will nano-sizes particles give an qualitatively different pattern of mRNA expression than larger particles?

Conclusions Overall, our data with silica are not in accordance to that the particle surface area is the only determinant in inducing cytokine responses, at least not in the size area from 30 to 200 nm. The relationship between size and cytokine response might vary between cell types and also type of particles. The explanation for a non-linear relationship between size and responses are unclear, but may indicate a differential handling of the particles, such as uptake and receptor interactions, of different particle sizes and types in different cells.

Effect of BSA on silica-induced IL-6 responses induced by different sizes of SiNPs BSA in the culturing medium is reducing the responses- means that the exposure conditions are very important Gualtieri- unpublished

Important end points to study the importance of particle size for health hazard: cytokine responses, ROS, recruitment of immune cells and cell death

Cell death induced by amorphous silica particles of different sizes Napierska et al NPs below 20 nmeter are cytotoxic at relative low concentrations- a qualitative or quantitative change compared to larger NPs?

Effect of particle surface area on depletion of gluthathion in epithelial lung cells Effect of nanoparticles and fine particles examined in A549 cells Linear relationship between particle surface area and response- different slopes for the different particles Monteiller et al. 2009

IL-6 from 4 independent experiments. Mean + SEM. Effect of different sizes of silica particles in primary type 2 cells: IL-6 -The silica particles induce IL-6 concentrations at much lower concentrations in these cells