The pathogenic effects of inhaled solid material depend primarily on achieving a sufficient lung burden 15. The lung burden is determined by the rates of deposition and clearance. Logically, for any dust or fibre, a steady-state dose level will be achieved when the rates come into balance. This is only true when the solid material does not interfere with the clearance mechanisms. In respect to the burden the chemical and physical properties of the material itself are important insofar as they influence deposition and clearance rates. Spherical solid material can be inhaled when its aerodynamic diameter is less than 10 micron. The smaller the particulates the deeper they can travel into the lung, particles smaller than 2.5 micron will even reach the alveoli. Ultrafine particles (nanoparticles with an aerodynamic diameter of less than 100 nm) are deposited mainly in the alveolar region. Fibres are defined as solid materials with a length to diameter ratio of at least 3:1. Their penetration into the lung depends on the aerodynamic properties. Fibres with a small diameter will penetrate deeper into the lungs, while very long fibres (>>20 micron) are predominantly stuck in the higher airways 161718192021.

The mucociliary escalator dominates the clearance from the upper airways; clearance from the deep lung (alveoli) is predominantly by macrophage phagocytosis. The mucociliary escalator is an efficient transport system pushing the mucus, which covers the airways, together with the trapped solid materials towards the mouth. The phagocytosis of particles and fibres results in activation of macrophages and induces the release of chemokines, cytokines, reactive oxygen species, and other mediators; this can result in sustained inflammation and eventually fibrotic changes. The phagocytosis efficiency can be affected by the (physical-chemical) characteristics of the solid material (see below); moreover, fibres too long to be phagocytized (fibres longer than the diameter of the alveolar macrophage) will only be cleared very slowly.

Laboratory exposure studies have shown that if the inhaled concentrations are low, such that the deposition rate of the inhaled particles is less than the mechanical alveolar macrophage-mediated clearance rate in the lung, then the retention half time is about 70 days (steady-state lung burden during continuous exposure). If the deposition rate of the inhaled particles exceeds this clearance rate, the retention half time is significantly increased, reflecting an impaired or prolonged alveolar macrophage-mediated clearance function with continued accumulation of lung burden (overload). Inhaled fibres, which are persistent in the alveoli, can interact with the pulmonary epithelial cells or even penetrate the alveolar wall and enter the lung tissue. These fibres are often described as being in the "interstitial" where they may lie between or within the cells making up the alveolar walls. Bio-persistent solid materials, certainly those particles containing mutagenic substances or asbestos fibres or silica, which remain for years in the lungs, increase the risk of developing cancer.

It has been reported recently that nanotubes show a sign of toxicity 22, confirmed in two independent publications by Warheit et al 23 and Lam et al 24, which demonstrated the pulmonary effects of single walled cabon nanotubes in vivo after intratracheal instillation, in both rats and mice. Both groups reported granuloma formation, and some interstitial inflammation. The research group of Warheit et al 23 concluded that these findings (multifocal granulomas) may not have physiological relevance, and may be related to the instillation of a bolus of agglomerated nanotubes. But for the authors of 24 their results indicate that if carbon nanotubes reach the lungs, they are much more toxic than carbon black and can be more toxic than quartz. These studies have to be read with some caution because a study by the National Institute for Occupational Safety and Health (NIOSH) showed that none or only a small fraction of the nanotubes present in the air can be inhaled 25.

Clearance from the lung depends not only on the total mass of particles inhaled but also on the particle size and, by implication, on particle surface, as shown in the following studies. A sub-chronic 3 months inhalation exposure of rats to ultrafine (~20 nm) and fine (~200 nm) titanium dioxide (TiO2) particles demonstrated that the ultrafine particles cleared significantly slower, showed more translocation to interstitial sites and to regional lymph nodes when compared to the fine TiO2 particles 26. By comparing carbon black particles of similar size and composition but with significant specific surface area difference (300 versus 37 m²/g), it was found that the biological effects (inflammation, genotoxicity, and histology) were dependent on specific surface area and not particle mass. Similar findings were reported in earlier studies on tumorigenic effects of inhaled particles. It was shown that tumour incidence correlated better with specific surface area than with particle mass 2728.

Comparing the health effects of chronically inhaled TiO2 particles with distinctly different sizes, it is remarkable that the low exposure (10 mg/m³) study 29 resulted in a greater lung tumour incidence than the high exposure (250 mg/m³) study 30. The inhaled particles in both studies consisted of aggregated primary particles, with an aerodynamic diameter that was probably not very different. The primary particle size of the low dose study was 20 nm, while it was approximately 300 nm in the latter study.

In summary, most nano-sized spherical solid materials will easily enter the lungs and reach the alveoli. These particles can be cleared from the lungs, as long as the clearance mechanisms are not affected by the particles themselves or any other cause. Nano-sized particles are more likely to hamper the clearance resulting in a higher burden, possibly amplifying any possible chronic effects caused by these particles. It is also important to note that specific particle surface area is probably a better indication for maximum tolerated exposure level than total mass. Inhaled nano-fibres (diameter smaller than 100 nm) also can enter the alveoli and their clearing would, in addition, depend on the length of the specific fibre. Recent publications on the pulmonary effects of carbon nanotubes confirm the intuitive fear that nano-sized fibre can induce a rather general non-specific pulmonary response.

Reports on the surface properties of nanoparticles, both physical and chemical, stress that nanoparticles differ from bulk materials. Their properties depend heavily on the particle size. Therefore, nanoparticles are not merely small crystals but an intermediate state of matter placed between bulk and molecular material. Independently of the particle size, two parameters play dominant role. The charges carried by the particle in contact with the cell membranes and the chemical reactivity of the particle 31.

The impact of inhaled particles on other organs has only recently been recognised. Most research has concentrated on the possible consequences of particle related malfunction of the cardio-vascular system, such as arrhythmia, coagulation 46 etc. However, recent data support the concept that the autonomic nervous system may also be a target for the adverse effects of inhaled particulates 474811. Two complementary hypotheses explain the cardiovascular malfunctions after inhalation of ultra-fine particles. The first hypothesis explains the observed effects by the occurrence of strong (and persistent) pulmonary inflammatory reactions in the lungs, leading to the release of mediators (see above), which may influence the heart, coagulation, or other cardiovascular endpoints. The second hypothesis is that the particles translocate from the lungs into the systemic circulation and thus, directly or indirectly, influence haemostasis or cardiovascular integrity.

In the evaluation of the health effects of inhaled nanoparticles the translocation to the systemic circulation is an important issue. Conhaim and co-workers 49 found that the lung epithelial barrier was best fitted by a three-pore-sized model, including a small number (2%) of large-sized pores (400-nm pore radius), an intermediate number (30%) of medium-sized pores (40-nm pore radius), and a very large number (68%) of small-sized pores (1.3-nm pore radius). The exact anatomical location of this structure, however, remains to be established (see the review by Hermans and Bernard 50). Until recently, the possible passage of xenobiotic particles has not been attracting much attention, although, the concept is now gaining acceptance in pharmacology for the administration of macromolecular drugs by inhalation 51. Nemmar et al 11 studied the particle-translocation of inhaled ultrafine technetium (^99mTc) labelled carbon particles to the blood. These particles, which are very similar to the ultrafine fraction of actual pollutant particles, diffused rapidly – within 5 minutes – into the systemic circulation (Fig 4). The authors concluded that phagocytosis by macrophages and/or endocytosis by epithelial and endothelial cells are responsible for particle-translocation to the blood but other roots must also exist.

The literature on the translocation of very small particles from the lungs into the blood circulation is limited and often conflicting. A recent study has reported deposition and clearance over 2 h of an ultrafine (60 nm) ^99mTc labelled aerosol in human volunteers. No significant radioactivity was found in the liver (1–2 % of the inhaled radioactivity) but, unfortunately, no radioactivity measurements with blood were reported 52. In agreement with findings of Nemmar et al 11, Kawakami et al. 53 have reported the presence of radioactivity in blood immediately after inhalation of 99mTc-technegas in human volunteers. It is also known 54 that aerosolised insulin gives a rapid therapeutic effect although the pathways for this translocation are still unclear. In addition to human studies, in experimental animal studies, we 11 and others 55165657 have reported extra-pulmonary translocation of ultrafine particles after intra-tracheal instillation or inhalation. However, the amount of ultrafine particles that translocate into blood and extra-pulmonary organs differed among these studies. It has also been shown that, following intranasal delivery, polystyrene microparticles (1.1 micron) can translocate to tissues in the systemic compartment 58. A recent study 59 has provided, for the first time, morphological data showing that inhaled polystyrene particles are transported into the pulmonary capillary space, presumably by trans-cytosis. Another alley of translocation from the lungs towards other organs has been undertaken by Oberdörster et al 19. In inhalation experiments with rats, using 13C-labelled particles, they found that nano-sized particles (25 nm) were present in several organs 24 hours after exposure. The most extraordinary finding was the discovery of particles in the central nervous system (CNS). The authors examined this phenomenon further and found that particles, after being taken up by the nerve cells, can be transported via nerves (in this experiment via the olfactory nerves) at a speed of 2.5 mm per hour 56.

Passage of solid material from the pulmonary epithelium to the circulation seems to be restricted to nanoparticles. The issue of particle translocation still need to be clarified: both the trans-epithelial transport in the alveoli and the transport via nerve cells. Thus, the role of factors governing particle translocation such as the way of exposure, dose, size, surface chemistry and time course should be investigated. For instance, it would be also very important to know how and to what extent lung inflammation modulates the extra-pulmonary translocation of particles.

Long non-phagocytizable fibres (in humans longer than 20 micron) will not be effectively cleared from the respiratory tract. The main determinants of fibre bio-persistence are species specific physiological clearance and fibre specific bio-durability (physical-chemical processes). In the alveoli the rate at which fibres are physically cleared depends on the ability of alveolar macrophages to phagocytose them. Macrophages containing fibres longer than their own diameter may not be mobile and be unable to clear the fibres from the lung. The bio-durability of a fibre depends on dissolution and leaching as well as mechanical breaking and splitting. Long fibres in the lung can disintegrate, leading to shorter fibres that can be removed by the macrophages. Bio-persistent types of asbestos, where breakage occurs longitudinally, result in more fibres of the same length but smaller diameter. Amorphous fibres break perpendicular to their long axis 6061, resulting in fibres that can be engulfed by the macrophages.

It is self-evident that the slower the fibres are cleared (high bio-persistence), the higher is the tissue burden and the longer the fibres reside in a tissue the higher is the probability of an adverse response. A milestone was set by Stanton et al 6263 who undertook a series of experiments with 17 samples of carefully sized fibrous glass. They found that for mesothelioma induction in rats, the peak activity was in the fibres greater than 8 micron in length and less than 1.3 micron in diameter. These findings are known as the "Stanton hypothesis". However these results do not strictly indicate that all fibres longer than the lower threshold are equally active or that shorter fibres are not, although fibres less than 5 micron in length did not appear to contribute to lung cancer risk in exposed rats 64. Risk appears to increase with length, with fibres more than 40 micron in length imposing the highest risk. For the recent review see Schins 65.

The bio-durability of fibres with a diameter < 100 nm will probably not differ from larger inhalable fibres. Therefore, great caution must be taken in case of the contact with nano-fibres, Bio-durability tests must be performed before releasing any products containing them. Carbon nanotubes, which are of high technical interest, are one of the materials which need to be tested in depth concerning bio-persistence and cancer risk. The first toxicological studies indicated that carbon nanotubes can be a risk for human health 222324, while exposure assessment did indicate that these materials are probably not inhaled 25.

Crohn's disease is characterised by transmural inflammation of the gastrointestinal tract. It is of unknown aetiology, but it is suggested that a combination of genetic predisposition and environmental factors play a role. Particles (0.1–1.0 micron) are associated with the disease and indicated as potent adjuvants in model antigen-mediated immune responses. In a double-blind randomised study, it has been shown that a particle low diet (low in calcium and exogenous microparticles) alleviates the symptoms of Crohn's disease 76. Although there is a clear association between particle exposure and uptake and Crohn's disease, little is known of the exact role of the phagocytosing cells in the intestinal epithelium. It has been suggested that the disruption of the epithelial barrier function by apoptosis of enterocytes is a possible trigger mechanism for mucosal inflammation. The patho-physiological role of M cells is unclear; e.g., it has been found that in Crohn's disease M cells are lost from the epithelium. Other studies found that material uptake (endocytose) capacity of M cells is induced under various immunological conditions, e.g. a greater uptake of particles (0.1 micron, 1 micron and 10 micron diameter) has been demonstrated in the inflamed colonic mucosa of rats compared to non-ulcerated tissue 7778 and inflamed oesophagus 79.

Diseases other than of gut origin also have marked effects on the ability of GIT to translocate particles. The absorption of 2-micron polystyrene particles from the PP of rats with experimentally induced diabetes is increased up to 100-fold (10% of the administered dose) compared to normal rats 80. However, the diabetic rat displayed a 30% decrease in the systemic distribution of the particles. One possible explanation for this discrepancy is the increased density of the basal lamina underlying the GI mucosa of diabetic rats that may impede particle translocation into deeper villous regions. This uncoupling between enhanced intestinal absorption and reduced systemic dissemination has also been observed in dexamethasone treated rats 81.

From the literature cited above it is clear that engineered nanoparticles can be taken up via the intestinal tract. In general the intestinal uptake of particles is better understood and studied in more detail than pulmonary and skin uptake. Because of this advantage it is maybe possible to predict the behaviour of some particles in the intestines but precaution should be taken. For those nanoparticles designed to stabilise food or to deliver drug via intestinal uptake other, more demanding, rules exist and should be followed before marketing these compounds.

Glass fibres and Rockwool fibres are widely distributed man-made mineral fibres because of their multiple applications, mainly as insulation materials, which have become important for replacing asbestos fibres. In contact with the skin, these fibres can induce dermatitis through the mechanical irritation. Why these fibres are such strong irritant has not been examined in detail. In occlusion irritant patch tests in humans it was found that Rockwool fibres with a diameter of 4.20 ± 1.96 micron were more irritating than those with a mean diameter of 3.20 ± 1.50 micron. The fact that "small" fibres can cause strong skin irritation has been known for a long time, e.g. itching powder. It is also commonly accepted that some types of man made fibres can easily induce non-allergic dermatitis. Although this is common knowledge, it is not clear what makes these fibres irritants. In search for reports on skin irritation caused by fibres with a diameter of < 100 nm no information could be found, indicating that more research is needed.

Epidemiological studies have reported a close association between particulate air pollution and cardiovascular adverse effects such as myocardial infarction 95. The latter results from rupture of an atherosclerotic plaque in the coronary artery, followed by rapid thrombus growth caused by exposure of highly reactive subendothelial structures to circulating blood, thus leading to additional or complete obstruction of the blood vessel. Nemmar et al 96 studied the possible effects of particles on haemostasis, focusing on thrombus formation as a relevant endpoint. Polystyrene particles of 60 nm diameter (surface modifications: neutral, negative or positive charged) have a direct effect on haemostasis by the intravenous injection. Positively charged amine-particles led to a marked increase in prothrombotic tendency, resulting from platelet activation. A similar effect could be obtained after the intratracheal administration of these positively charged polystyrene particles, which also caused lung inflammation 97. It is important to indicate that the pulmonary instillation of larger (400 nm) positive particles caused a definite pulmonary inflammation (of similar intensity to 60 nm particles), but they did not lead to a peripheral thrombosis within the first hour of exposure. This lack of effect of the larger particles on thrombosis, despite their marked effect on pulmonary inflammation, suggests that pulmonary inflammation by itself was insufficient to influence peripheral thrombosis. Consequently, the effect found with the smaller, ultrafine particles is most probably due, at least in part, to their systemic translocation from the lung into the blood.

Pollutant particles such as diesel exhaust particles (DEP), may cause a marked pulmonary inflammation within an hour after their deposition in the lungs. Moreover, intratracheal instillation of DEP promotes femoral venous and arterial thrombosis in a dose-dependent manner, already starting at a dose of 5 μg per hamster (appr. 50 μg/kg). Subsequent experiments showed that prothrombotic effects persisted at 6 h and 24 h after instillation (50 μg/animal) and confirmed that peripheral thrombosis and pulmonary inflammation are not always associated 97. Solid inhaled particles are a risk for those who suffer from cardiovascular disease. Experimental data indicate that many inhaled particles can affect cardiovascular parameters, via pulmonary inflammation. Nano-sized particles, after passage in the circulation, can also play a direct role in e.g. thrombogenisis.

Epidemiologic studies have provided valuable information on the adverse health effects of particulate air pollution in the community, indicating that nanoparticles act as an important environmental risk factor for cardiopulmonary mortality. Particle-induced pulmonary and systemic inflammation, accelerated atherosclerosis, and altered cardiac autonomic function may be part of the patho-physiological pathways, linking particulate air pollution with cardiovascular mortality. Also, it has been shown that particles deposited in the alveoli lead to activation of cytokine production by alveolar macrophages and epithelial cells and to recruitment of inflammatory cells. An increase in plasma viscosity, fibrinogen and C-reactive protein has been observed in samples of randomly selected healthy adults in association with particulate air pollution 959899.

A number of reports on cellular uptake of micro- and nano- sized particles has been published. Reports on particle uptake by endothelial cells 100101, pulmonary epithelium 1027910359, intestinal epithelium 5179 alveolar macrophages 10410510610757, other macrophages 8910876109, nerve cells 110 and other cells111 are available. This is an expected phenomenon for phagocytic cells (macrophages) and cells that function as a barrier and/or transport for (large) compounds. Except for macrophages, the health effects of cellular uptake of nanoparticles have not been studied in depth.

One of the promising alleys of nanotechnology is organ- or cell- specific drug delivery mediated by nanoparticles 112113114. It is expected that transport of nanoparticles across the blood-brain barrier (BBB) is possible by either passive diffusion or by carrier-mediated endocytosis. Coating of particles with polysorbates (e.g. polysorbate-80) results in anchoring of apolipoprotein E (apo E) or other blood components. Surface modified particles seem to mimic LDL particles and can interact with the LDL receptor leading to uptake by endothelial cells. Hereafter, the drug (which was loaded in the particle) may be released in these cells and diffuse into the brain interior or the particles may be trans-cytosed.

Also, other processes such as tight junction modulation or P-glycoprotein (Pgp) inhibition also may occur 115. Oberdörster et al 2002 reported the translocation of inhaled nanoparticles via the olfactory nerves 56. Drug delivery systems crossing the BBB are certainly welcome, but this also implicates that unintended passage through the BBB is possible; therefore good safety evaluations are needed.

It has been shown that nanoparticles, that enter the liver, can induce oxidative stress locally. A single (one day; 20 and 100 mg/kg) and repeated (14 days) intravenous administration of poly-isobutyl cyanoacrylate (PIBCA, a biodegradable particle) or polystyrene (PS, not biodegradable) nanoparticles induced a depletion of reduced glutathione (GSH) and oxidised glutathione (GSSG) levels in the liver, as well as inhibition of superoxide dismutase (SOD) activity and a slight increase in catalase activity. The nanoparticles did not distribute in the hepatocytes, implicating that the oxidative species most probably were produced by activated hepatic macrophages, after nanoparticle phagocytosis.

Uptake of polymeric nanoparticles by Kupffer cells in the liver induces modifications in hepatocyte antioxidant systems, probably due to the production of radical oxygen species 108. We have discussed above that nano-sized particles in the lung can induce, via the pulmonary inflammatory response as well as via spontaneously surface related reactions, oxidative stress. Besides pulmonary studies, not many have studied particle-induced oxidative stress in tissues. However, the authors 108 reported that the depletion in glutathione was not sufficient enough to initiate significant hepatocytic damage (no lipid peroxidation). It needs to be stressed that long-term studies are needed to prove the safe use of these nanoparticles because chronic depletion of the anti-oxidant defence can lead to severe health problems.