Further memorandum by the Medical Research
Council Human Nutrition Research Unit
INTRODUCTION
When cells fail to recognise surface molecules
or molecular structure of small particles, the fate of these particles
may be determined by their physical size and no longer by their
chemical composition. Nanoparticulate, nanosized and nanostructured
are then descriptors that relate to a dominant characteristic.
NATURAL EXPOSURE
The human gut has been exposed to non-biological
particles of varying sizes for millennia. For example, dietary
ferritin is a small nanoparticle ( )
of 13 nm diameter when whole and 2.5 nm as the smallest
core sub-unit, while dust and soil nanoparticles tend to be hundreds
of nm in diameter/length ( ). Four
uptake (absorption) mechanisms have been proposed in the gastrointestinal
tract (Figure 1):
1. Through "regular" epithelial cells
(gut-lining cells) via a route termed endocytosis ("engulfing"
the particle). Very small particles—tentatively generally
<20nm in diameter.
2. M cell uptake (transcytosis) at the surface
of intestinal lymphoid aggregates. This is the quintessential
pathway for gut particle uptake and is very well described, especially
for large nanoparticles (> 100 nm), although smaller particles
are also likely to be able to access this route. M cells have
a "surveillance" role in the gut and are specialised
in particle uptake.
3. Persorption. Volkhemer's concept of passage
through "gaps" at the villous tip following loss of
enterocyte(s) to the gut lumen. Small and large nanoparticles
potentially access this route but its quantitative validity is
unclear.
4. Putative paracellular (between cell) uptake.
Generally junctional complexes are unlikely to allow even the
smallest of nanoparticles to permeate but certain drugs and/or
dietary situations, and especially diseases, may alter this situation
allowing influx of very small nanoparticles. Theoretical pathway
as it stands.
Figure 1: Schematic representation of different
routes for particle uptake in the small intestine. The numbers
refer to those pathways described in the text. Uptake via (1)
regular epithelial cells (2) M cells of the lymphoid aggregate
(3) persorption and (4) the theoretical paracellular pathway.
Regardless of mechanisms, it is clear that ingested
particles across the nano-range (0-1000 nm) will be absorbed
to some extent into both the circulation and the gut tissue itself.
Percentage absorption will depend on many factors (eg size, surface
charge, host gut permeability, etc). But even if only 0.1 per
cent of a total 1013 ingested particles is absorbed, that corresponds
to 109 particles absorbed/day.
From the circulation, particles will be retained
by cells in the liver and other vascular organs. From the gut
tissue, cells can migrate systemically with their cargo (eg particles),
especially to mesenteric lymph nodes. The persistence or degradation
of particles at any site depends upon the physico-chemical characteristics
of the particles but even undegradable particles have some clearance
through cellular-shedding in the gut and lung.
MAN-MADE
PARTICLE EXPOSURE—CURRENTLY
Silicates, aluminosilicates, titanium dioxide
and carrageenan are among the typical man-made, or at least man-modified,
particles that the human gut is now exposed to, especially in
the Western world, on a daily basis. Exposure has been for decades-as
food additives mainly. Except at MRC-HNR there is little research
on the gut-associated effects of these although some appear to
accumulate in gut tissue. Nonetheless, studies to-date suggest
that, overall, these particles are safe and even if they can be
shown to have any adverse effects it will almost certainly be
in a small minority with a different genetic make-up. However
there is no evidence for this currently.
The above particles are almost all in the larger
nano-range (being > 100 nm diameter/length). There is,
in the UK, no evidence currently for the significant intake of
new/man-made small nano-sized particles, although, increasingly
at the global level, proposals for this are made in industry and
in research studies.
MAN-MADE
PARTICLE EXPOSURE—FUTURE
"Nanosizing" can have a variety of
commercial advantages for certain foods, supplements (especially),
medicines, food packaging and other materials that may be ingested.
However, in many cases, the "nanosized" foods will undergo
simple gastrointestinal digestion prior even to meeting any cells
(Figure 2). Examples include "nano-salt" (1) and probably
some "nano-micelles" (2). However, even with nano-micelles
that are absorbed whole, they will undergo fairly rapid cellular
degradation and are likely to be recognised for their molecular
structure rather than their nanosize. Indeed it should be noted
that yoghurt and milk are foods containing nano-micelles (40-300 nm)
of casein that occur in large abundance in the intestinal lumen
upon ingestion. For competitive commercial reasons, as well as
the potential to lose scientific/toxicological focus, it would
seem sensible that such foods are considered separately with regards
to further "nano-legislation".
Figure 2: 1, Some nanoformulated materials,
eg nanosalts, are likely to be digested in the gut before any
cellular exposure. 2, Micellar nanoformulations may partially
degrade in the gut or be absorbed whole, but are likely to be
rapidly broken down in cells. 3, In contrast, truly or transiently
persistent nanoparticles are likely to lose any surface adsorbed
material in the stomach, but may themselves remain intact, and
then, later in the gut, could (depending on size, surface charge
etc.) adsorb other soluble luminal molecules before cellular uptake.
Thus, in the case of micellar nanoparticles
it is highly likely that the constituent molecules would dictate
toxicity, rather than their aggregated nature to form a nano-micelle,
although this latter property could influence bio-distribution.
In the final scenario in Figure 2, novel nanoparticles
may be bio-persistent, either transiently as there is gradual
cellular breakdown, or truly persistent as they can only be cleared
with the sloughed cells, as noted above. If the latter process
is slower than the rate of uptake then particles may accumulate.
Examples could speculatively include, nano-silver, nano-clays
and nano-silica. Depending upon their size, surface charge etc.,
ingested particles may adsorb (to their surface) other soluble
molecules, including bacterial toxins, from the gut lumen, and
carry these across into cells (Figure 2:3). Probably the larger
nanoparticles are better at this.
Particle Toxicity: Factors and Why Nanoparticles?
A number of poorly predictable properties dictate
particle toxicity—eg crystalline structure, surface reactivity,
dissolution characteristics, adsorptive properties etc. So, for
example the  -quartz form of silicon
dioxide is a toxic particle while the amorphous form of silicon
dioxide is not. A second example, mediated by a similar process
to that of quartz, is that nano-particulate hydroxyapatite may
be toxic to cells while some other forms of nano-particulate calcium
phosphate are considered less so.
Particle shape can also affect particle toxicity.
Thus asbestos, erionite and some man-made nanotubes appear toxic
due to their high aspect-ratio or "needle-like" shapes.
Finally, size. This is often poorly understood.
The large majority of particles are fairly inert/non toxic unless
they have some specific property, as noted above. In the absence
of any "special property", particle toxicity can be
considered in two simple forms:
(1) Direct toxicity. Normally mediated through
"free radical" activity and, in this case, smaller particles
are considerably more active than the same mass of larger particles.
This appears to be a surface area phenomenon. However, just because
this can happen, we must ask does it happen? Many experiments
use such unrealistic particle doses that extrapolation to lower
doses, that represent real exposures, may be artefactual. The
result of drinking a bottle of whisky one evening tells us little
about the result of one drink per evening over a few months. Secondly,
most tissues, including the gut and circulation, are armed with
complex and replenishable antioxidant defences to combat such
acute (short-term) exposures. In doing so, however, there may
be downstream costs (long-term).
Accumulating evidence suggests that lung exposure
to nanoparticles is linked with an increased risk of chronic cardiovascular
disease. A second potential lesson from the lung is that certain
individuals (eg those with asthma) can experience an exacerbation
of disease upon acute exposure to an abnormally high environmental
dose of particles (eg at peaks of urban pollution). However it
is the view of these authors (but not the wider community) that
this latter phenomenon, as opposed to the chronic systemic effects,
could be more related to the large nanoparticle fraction () than it is to the small fraction (), leading onto the second potential mechanism
of general particle toxicity.
(2) Large nanoparticles (or aggregated small
ones) can make good cellular "adjuvants" such that an
immune response to a protein/allergen/antigen is enhanced or "polarised"
when exposure is in the presence of a particle. Contact between
the allergen/antigen and the particle (eg adsorption) appears
important.
What is Special about Nanoparticles?
Three things. First, as detailed above, in the
absence of a "special property" for particle toxicity,
all particles will be more directly toxic to cells as small nanoparticles
than as larger ones. The pros and cons of this observation are
noted above. Secondly, as a rough guide, particles <100 nm
diameter will be taken up by cells through a different pathway
to that of larger particles (Figure 3), meaning that they will
access different cellular compartments and have different cellular
effects. Again, "induction of free radicals" versus
"adjuvant activity" are the basic differing outcomes.
Thirdly, very small nanoparticles are especially mobile and motile
and may access all areas of the body including even the brain
and all areas of the cell including even the nucleus (being smaller
than nuclear pores). It is this latter property that probably
makes very small nanoparticles most worrisome to scientists and
hence the translation of this concern (but not the knowledge of
why) to the public.
Figure 3: Schematic representation of cellular
particle uptake for large particles via 1. active phagocytosis
or engulfing of large particles and 2. macropinocytosis which
is a different type of active particle capture. These events are
triggered by the size of the particle. 3. small particles are
taken up by constitutive pinocytosis and are processed by the
cell in a different fashion.
Finally, it should be noted that in the absence
of specific particle toxicity there is no logical reason to assume
that, in the gut, smaller nanoparticles will always have worse
adverse health effects than larger ones or that either will have
any adverse health effects at all. It will depend on many other
variables including host genotype, persistence, dose, and ability
to adsorb gut luminal molecules. And thus there is no logical
reason to use 100 nm as a cut-off for adverse effects, even
though, as discussed, this size discrimination may help determine
the type of cellular effect.
OTHER IMPORTANT
FACTORS
(1) Particles may aggregate so that their behaviour during
at least part of the exposure process is more typical of large
particles () even though their
single unit is as a small particle ().
This is especially true for small nanoparticles.
(2) Particles are rarely seen by cells in
their "native form". Most particles readily adsorb to
their surface molecules and ions from their environment. In the
gut, particle surfaces may be "cleared" in the acid
and enzyme-active area of the stomach but re-adsorb material further
down the G.I. tract. In this environment, bacterial proteins and
carbohydrates are especially common.
(3) Classical toxicity or toxicology studies
may be poor or even misleading at deciphering particle toxicity
following oral exposure. In particular, long-term (decades) effects
and host genotypes cannot be mimicked in animal studies. Instead
a "logic algorithm" and some targeted in vitro tests
may be more useful.
(4) Nanotechnology may actually serve to
make some materials less toxic. For example, MRC-HNR is developing
a transiently stable nano-formulation of supplemental iron which
should exhibit much less toxicity to the intestinal mucosa, and
therefore side-effects, than the current common therapeutic supplements,
namely ferrous sulphate and other ferrous salts.
ACKNOWLEDGEMENT
We thank Drs Laetitia Pele and Nuno Faria for
their invaluable contribution during the preparation of this document.
March 2009
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