Timing, hosts and locations of (grouped) events of NanoImpactNet

distinguishes between the interaction of particulate and soluble Zn
with the membrane surface.
Figure 4: Shows Oscillatoria princeps incubated with
SiO2. Cells separated by septa (arrowed). Right
filament partially covered by SiO2, left filament
completely covered.
A simple geometric model (see Figure 5) has been developed to
describe the adsorption of particles on to a supported membrane.
The model is directly transposed from the experimental data of the
packing of silica nanoparticles on to a supported membrane. The
model has one adjustable parameter which is the maximum
distance between the particle surface and the DOPC layer where
there is an interaction between the particle and the DOPC.
Figure 5.
(a) Model (top view) of
silica nanoparticles
binding to DOPC is
presented in this picture.
(b) Triangle ABC, where
AB=AC=BC=2R and A,B and
C are the centres of three
nanoparticles abutting
each other in (a).
(c) Vertical section
through (b) showing
nanoparticle touching the
DOPC surface at point A, h
is 'interfacial layer
thickness'.
(d) Model (horizontal
sectional view) of
nanoparticles bound to
DOPC showing radius, r, of
'interfacial contact area'.
WP3: Interactions with in vitro models. These studies are directed
to nanoparticle interactions at both the cellular level and the tissue
level. The test systems will be established on in vitro models. The
cellular level will include test systems ranging from tissues and
cultured cells to DNA. The tissue level includes nerve axons from
the squid consisting of a single axon and glia, and ascidian embryos
(rapidly developing chordate embryos to 12 hrs). The principle is to
understand how the nanoparticles affect the structure and
function of these systems using both real time assays and electron
microscopy. The in vitro work is led by Anton Dohrn and is spread
NanoSafetyCluster - Compendium 2012
between Anton Dohrn and Leeds (WP 3). Anton Dohrn has
extensive facilities in electron microscopy and biophysical and
molecular biological techniques and considerable world expertise
in electrophysiology.
Some of the most exciting recent work carried out by WP3 has
been on the effect of ZnO particles on membrane proteins. NPs
provided by WP2 were tested directly on HEK cells that
heterologously express the hERG K + channel. This gave us the
opportunity of assessing the impact of the NPs on defined
membrane proteins directly. The range of concentrations used was
0.1-10 mg ml -1 for both SiO 2 (dialyzed and non -dialyzed) and ZnO.
Cells were held at -70 mV under voltage clamp and hERG K +
channels were activated by patterns of voltage steps which
produced outward ionic currents which were subject to biophysical
analysis (Figure 6). The channel activity was stable for at least an
hour without run- down although experiments were normally
carried out in the first 20 minutes. Examination of the hERG current
kinetics (activation / inactivation and peak currents revealed no
effect of SiO 2 up to 10 mg mL -1 but a notable selective effect of ZnO
on channel kinetics (Figure 6). To establish if this effect was due to
release of Zn 2+ ions from the NPs, we carried out experiments
where increasing concentrations of ZnCl 2 were added and the peak
currents measured. As can be seen in Figure 7, increasing the
concentration of Zn 2+ begins to block the channel only in the mM
range. The effect of the NPs in Figure 6 is the opposite to this, i.e.
they increase the current. Therefore the NP effect cannot be due
to residual Zn 2+ .
Figure 6. ZnO removes the fast inactivation of hERG and is so doing
increases the amplitude of the current at positive voltages. The
graph shows the extracted values for current voltage relations of
the steady state K + current under different voltage steps.
Figure 7. Dose response of hERG peak currents in various
concentrations of Zn 2+ . The EC 50 of Zn 2+ on hERG is estimated
to be of the order of 1 mM. The results represent the mean
and standard deviation of results from at least five
experiments.
Compendium of Projects in the European NanoSafety Cluster 7