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

iological identity of the particles is greatly influenced (in some
cases likely completely determined) by the proteins, and not the
materials. Figure 1 makes the issue clear by showing the uptake
of silica with (and without) serum proteins. The relative
amounts are enormous. It is important to note that uptake is
dependant even on the type of serum used, and these
differences have been studied and linked to different coronas.
Clearly, the bare material surface is the wrong parameter.
Similar observations are being made for many nanomaterials
and situations. It is not possible to explain in great detail, but
using new experimental methods it is also now possible to
‘read’ the corona around particles in organelles inside the cell.
Evidently we need to shift considerably towards modelling of
the particle and its adhering proteins, and the interaction of this
object with biological membranes and barriers in the current
program.
Uptake of nanoparticles into cells
Small molecules typically distribute across living organisms such
that molecules ‘dissolve and distribute’ in organs (very crudely
speaking) according to near-to-equilibrium physiochemical
principles in which quasi equilibrium rate constants dominate.
Whilst this is a great over simplification, it carries with it the
heart of the matter. For example, a small molecule dye will
essentially ‘dissolve’ (diffuse) across a biological membrane.
When the source is removed, if there are no highly specific and
high affinity interactions in the environment (for example, inside
a cell) to retain the molecules, there will be a rapid flow out of
the cell (across the cellular membrane again) according to
chemical potential considerations. This is all nicely illustrated in a
very simple in vitro cell model in Figure 2A where uptake and
export of a molecular dye are tracked by fluorescence flow
cytometry. 5
Figure 1. Comparison of endocytosis of 50nm and 100nm Si0 2
nanoparticles at 100ug/ml in the presence (complete MEM) and
absence (Serum free Media) of serum proteins. Note the very
significant particle uptake in the absence of serum, compared to
the much lower uptake in the presence of serum. It has been
shown that serum reduces the non-specific interactions between
that nanoparticles and the cell surface. Other differences in details
of the uptake cannot be discussed here. Data from P1.
On the contrary, nanomaterials are too large to ‘dissolve’ across
membranes in a passive manner, and no such processes have
NanoSafetyCluster - Compendium 2012
(so far) been observed in all (our and other) experimental work
across many particles types down to sizes of 5nm. On the
contrary, nanoparticle uptake across the biological membrane is
rapid, and cellular energy dependent (see Figure 1B where we
show effects of cell energy depletion on nanoparticles uptake),
driven by active biological processes that are currently being
uncovered by various EU (including those of the current
Partners) and National programs around EU and US. Sufficient
preliminary information now exists 5, 6 for us to identify a broad
range of active biological processes (receptor mediated and
other) that are responsible for this uptake of nanoparticles.
Here it is sufficient to say that particles use a combination of
endogenous entry portals (receptors etc) and membrane
adhesion (followed by membrane turnover) together producing
internalization using the cells own energy.
Trafficking and clearance of nanoparticles at cellular level
Here again, radically new paradigms emerge, for unlike
chemicals (which may have wide and distributed access to the
intra-cellular space by similar dissolution processes)
nanoparticles have limited and managed access using
endogenous cellular pathways used to transport proteins and
other biomolecules. In some cases these processes lead to
nanoparticles being localized at very high concentrations in
particular organelles (for example lysosome is typical, as shown
in Figure 2C, and later on). Transport occurs only along
prescribed pathways, for which appropriate particle surface
signals are available - for example, in Figure 2D we show that
nanoparticles of a very similar substance to the dye in Figure 2A
(but in nanoparticulate form) are not cleared upon removal of
the extracellular nanoparticles source, but instead are trapped
(as far as we can tell ‘permanently’) inside lysosomes. This may
be visualized in a sequence of confocal fluorescence and EM
images from silica nanoparticles (see Figure 3) in which we see
events of uptake, and internalization, and final localization into
lysosomes. This is a very general paradigm we have seen in
many particles, cell types (and higher levels) that must be
accommodated in any model.
Figure 2. A. Uptake of green fluorescent dye (molecular) by A549
cells – no effect of energy depletion. B. Effect of cellular energy
depletion on uptake of 50nm nanoparticles of similar composition
to the dye. C. Confocal image showing the localisation of those
50nm nanoparticles in the lysosomes of A549 cells. D. Lack of
export of those 50nm nanoparticles from A549 following removal
of the particle source (I1), compared to rapid release of molecular
dye (YG). All data from Partner 1.5
Compendium of Projects in the European NanoSafety Cluster 199