Translocation Pathway of the Intratracheally Instilled Ultrafine Particles from the Lung into the Blood Circulation in the Mouse

Abstract

Recently, it has been demonstrated that ultrafine particles (UFPs) are able to translocate from the lung into the systemic circulation. Precise mechanisms of the anatomical translocation (crossing the air–blood barrier) of inhaled UFPs at the alveolar wall are not fully understood. In this study, we examined the translocation pathway of the intratracheally instilled ultrafine carbon black (UFCB) from the lung into the blood circulation in mouse. Electron microscopy demonstrated accumulation of intratracheally instilled UFCB in the large-sized gaps developing between the cytoplasmic processes of the alveolar epithelial cells, possibly as a result of shrinkage of cytoplasm, by receiving stimulus/signals generated and released following UFCB attachment on the alveolar epithelial cells. Occasional penetration of the accumulated UFCB into the alveolar basement membrane, exposing to the air space, was observed at the gap. These results suggest that inhaled UFPs may, in part, pass the air-blood barrier through the large-sized gap formed between the alveolar epithelial cells.

Introduction

Suspended particle matters, a component of air pollution, are ubiquitous in ambient urban and indoor air from multiple sources. Epidemiological studies indicate that peaks of ambient particulate air pollution are associated with an increase in pulmonary and cardiovascular morbidity and mortality (Samet et al., 2000). In fact, more people seem to die from cardiovascular than from pulmonary diseases during episodes of urban air pollution (Pope et al., 1999). In addition, the short-term effects of exposure to these particles have been recently described and include an increased susceptibility to ischemia (Pekkanen et al., 2002) and the occurrence of myocardial infarction (Peters et al., 2001).

Several mechanisms responsible for the cardiovascular disorders to inhaled particles have been hypothesized. Particles may cause inflammatory reactions in the lungs with a resultant systemic release of cytokines, which may influence cardiovascular endpoints (Seaton et al., 1995). Indeed, ambient particles are thought to enhance pulmonary inflammation associated with systemic hypercoagulability, generation of oxidants, activation of complement, increase in blood viscosity, and elevation of the plasma concentrations of fibrinogen and C-reactive protein (Donaldson et al., 2001, 2003). An alternative hypothesis is that the ultrafine particles (UFPs: particles <100 nm) translocate from the lungs into the circulation and thus influence cardiovascular endpoints more directly.

UFPs are mainly emitted from combustion engines (e.g., diesel-powered engines) and other high-temperature processes in the form of fractal-like aggregates composed of solid nanoparticles (Xiong and Friedlander et al., 2001). Nanotechnology, the new technology using nanometer-scale structures, which includes UFPs, has recently undergone increased development. Many nanotechnologies are still in the precompetitive stage, nanoscale materials (UFPs) are increasingly being used in optoelectronic, electronic, magnetic, medical imaging, drug delivery, cosmetic, catalytic, and materials applications. There is, however, a great concern as to the potential toxicity of the nanoscale materials that could enter the blood circulation (Hoet et al., 2004; Oberdörster et al., 2005; US Environmental Protection Agency, 2005).

Recent investigations provided evidence that the UFPs can be translocated from the lungs to the extrapulmonary organs (i.e., liver, heart, spleen, brain) via the blood circulation (Nemmar et al., 2001; Takenaka et al., 2001; Kreyling et al., 2002; Oberdörster et al., 2002). Indeed, Nemmar et al. (2001, 2002a) have reported that intratracheally instilled UFPs in the form of ultrafine ^99mTechnetium-labelled carbon particles (100 nm) or albumin nanocolloid particles (diameter < 80 nm) are quickly translocated from the lung into the systemic circulation in hamsters and humans; radioactivity was detected in blood at 1 minute and 5 minutes, respectively. In addition, morphological study showed instilled 30 nm gold particles in capillaries within 30 minutes’ postexposure (Berry et al., 1977) and inhaled ultrafine titanium dioxide particles at alveolar wall and in capillaries at 1 hour after the end of exposure (Geiser et al., 2005). UFPs in the blood circulation can directly or indirectly influence hemostasis or cardiovascular integrity and thus induce adverse cardiovascular endpoints more directly (Nemmar et al., 2002b, 2003a, 2003b). The exact pathway for this translocation, however, remains unclear.

The rapidity of the translocation process of UFPs makes it unlikely that phagocytosis by macrophages and/or endocytosis by epithelial and endothelial cells are responsible for the passage (Nemmar et al., 2002a). Translocation of macromolecules including protein at the air–blood barrier may occur by means of noncoated or flask-shaped invaginations termed caveolae: the phenomenon may be an active rather than a passive event (Gumbleton, 2001). Although several hypotheses as to the mechanism for the inhaled particle translocation have been addressed (Takenaka et al., 2001; Oberdörster, 2004; Geiser et al., 2005), there has not been sufficient morphological evidence to support these hypotheses. In addition, there are few reports on the electron microscopic study of the translocation of the inhaled UFPs at the air-blood barrier; Geiser et al. (2005) recently demonstrated no membrane-bound ultrafine titanium dioxide particles in the cytoplasm of epithelial and endothelial cells and fibroblasts at the alveolar wall.

The purpose of this study was, first, to demonstrate the precise translocation pathway of intratracheally instilled UFPs at the air-blood barrier by light and electron microscopy, and second, to elucidate the mechanisms of particle transfer from lung to the pulmonary lymph node.

Materials and Methods

Animals

Ten-week-old female ICR mice weighing 29 to 34 g were obtained from CLEA JAPAN Inc. (Tokyo, Japan). Animals were kept at around 25°C and pelleted food and water were available ad libitum throughout the experiment. All animal experiments were performed according to the National Institute for Environmental Studies guidelines for animal welfare.

Particle Suspension

Ultrafine carbon black particles (UFCB) (Printex 90, 14 nm diameter) (Degussa, Frankfurt, Germany), was obtained as a kind gift from Degussa Japan (Osaka, Japan). Particles-PBS (phosphate-buffered saline, 0.1 M, pH 7.4) suspensions were prepared (see below) at the concentration of 20 mg/ml and sterilized by autoclave.

Intratracheal Instillation of the UFCB Particle Suspension

The preparations were sonicated for 1 minute and 30 seconds and always vortexed immediately (<1 minute) prior to intratracheal instillation. Mice were anesthetized by an intraperitoneal injection of xylazine (Celactal; Bayer, Leverkussen, Germany), 3 mg/kg body weight and ketamine hydrochloride (Ketalal; Sankyo Pharmaceutical, Tokyo, Japan), 75 mg/kg body weight. A volume of 0.05 ml of PBS-particle suspension containing 1 mg particles was instilled intratracheally via a plastic, blunt cannula, 1.20 mm gauge diameter, followed by 0.15 ml of air. 0.05 ml of PBS was instilled into the lungs of control groups of mice.

Histopathology

At 0, 5, 10, 30 minutes, 1, 2, 6, 12, and 24 hours following single exposure, the animals were sacrificed under diethylether anesthesia (n = 10 at 5, 10, 30 minutes, n = 5 at 0 minute, 1, 2, 6, 12 hours, n = 12 at 24 hours) (control groups: n = 1 at each point), followed by immediate dissection. On dissection, trachea was immediately exposed and 0.5 ml of 10% formalin was instilled gently with the use of syringe via the tracheal cannula at low pressure; formalin injection was carried out within 15 seconds after death. Thoracic cavity was opened by cutting the thoracic wall. The lungs were then excised with hearts. Lungs, pulmonary lymph nodes, livers, and spleens were collected and fixed in 10% neutral-buffered formalin for 24 hours. Lungs and pulmonary lymph nodes of 2 mice sacrificed 24 hours after instillation were immediately frozen with liquid nitrogen and stored at −80°C for immunohistochemical examination. Transverse sections (right cranial and accessory lobes) and longitudinal sections (right caudal and middle lobes and left lobe) were obtained from the formalin-fixed tissues of lungs. These sections and pulmonary lymph nodes were routinely processed, embedded in paraffin for histopathological and immunohistochemical examinations. Sections of approximately 3 μm in thickness were cut and stained with hematoxylin. Selected sections were cut and used for immunohistochemistry.

Antibodies used in this study were rat monoclonal antibodies specific for mouse determinants for ye2/36hlk (react with mouse H-2 1-A antigen: mouse MHC class II, B lymphocytes, monocytes and activated T cells) (1: 10 in PBS) (SEROTEC, Oxford, UK), Cl;A3-1 (react with 160 kD membrane protein of the F4/80 antigen: mouse monocytes/macrophages) (1:20 in PBS) (BMA Biochemicals Ag, Augst, Switzerland), MIDC-8 (react with 50 kD or 66 kD cytoplasmic antigen: mouse dendritic/interdigitating cells) (1:100 in PBS) (SEROTEC, Oxford, UK), NLDC-145 (react with 205 kD integral membrane glycoprotein: CD205/DEC-205, mouse dendritic cells) (1:25 in PBS) (SEROTEC, Oxford, UK) and a rabbit polyclonal antibody specific for human determinant for factor VIII (1:100 in PBS) (DAKO, Glostrup, Denmark). As secondary antibodies, the biotin-labelled goat anti-rat IgG (1:400 in PBS) (VECTOR Laboratories, Federal, Argentina), goat anti-rabbit IgG (1:400 in PBS) (DAKO, Glostrup, Denmark) and peroxidase-labelled donkey anti-rat IgG (1:500 in PBS) (CHEMICON International Inc., Temecula, CA) were used.

For factor VIII, ye2/36hlk and F4/80 immunohistochemistry, paraffin-embedded sections were immunostained by using avidin-biotin comprex (ABC) method, in which labelled Streptavidin biotin (LSAB) kit (DAKO, Glostrup, Denmark) was included. After deparaffinization of the sections, the sections were treated with 0.05% proteinase K. The sections were incubated with 3% H₂O₂ to quench endogenous peroxidase activity and then with 10% normal goat serum for 5 minutes with microwave (MW) treatment at 200 w (microwave oven: M1-77, Higashiya Iryo Kikai Co. LTD., Tokyo, Japan) to inhibit nonspecific reactions. Thereafter the sections were reacted with primary antibody for 20 minutes with MW treatment. The peroxidase-conjugated goat anti-rabbit IgG diluted at 1:400 (DAKO, Glostrup, Denmark) was reacted to sections for 7minutes with MW treatment as a secondary antibody (Kimoto et al., 2001). The positive reactions resulted in brown staining with a DAB-H₂O₂ solution [0.02% (w/v) 3, 3′-diaminobenzidine tetrahydrochloride (DAB) and 0.01% (v/v) H₂O₂ in 0.05 M Tris-HCl buffer, pH 7.6] for 30 minutes, and the sections were counterstained with hematoxylin.

For MIDC-8 and NLDC-145 immunohistochemistry, the cryosections 5 μm thick were placed on slides, air-dried and were fixed in pure acetone for 10 minutes, and then in formol calcium solution (Ezaki et al., 1995) for 2 minutes after rehydration in PBS. After washing in PBS and incubation with a blocking solution (Block Ace, Dainippon Seiyaku, Tokyo, Japan) for 10 minutes, sections were incubated with the first monoclonal antibody over night at 4°C. Thereafter, each step was followed by 3 times washing with PBS for 2 minutes. Bound monoclonal antibody was detected with an peroxidase-labelled second antibody for 1 hour at room temperature, and sections were fixed further with 1% glutaraldehyde in distilled water for 7 minutes (Matsuno et al., 1996). Labelled cells were colored brown with DAB substrate and 0.01% H₂O₂. Sections were counterstained with hematoxylin and mounted in Aquatex (DAIDO SANGYO Co. Ltd, Japan). As negative control, PBS was substituted for primary antibodies.

Electron Microscopy

Electron microscopy was performed on the lung tissues fixed by formalin inflation via the trachea from mice sacrificed 3 minutes and 8 minutes after instillation. The lungs were cut into 1–2 mm³ cubes. The small blocks were rinsed in 0.1 M PBS, pH 7.4, postfixed for 1 hour in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, dehydrated in alcohol, and embedded in epoxy resin. Semithin (1μm thick) sections were stained with 1% toluidine blue in distilled water to select and locate interesting areas for electron microscopic examination. Ultrathin sections stained with 3% uranyl acetate in distilled water and lead citrate (Reynolds’ lead citrate: 1.33 g lead nitrate and 1.76 g sodium citrate in 30 ml of distilled water) were examined under JEM-100CX electron microscope (Japan Electron Optical Laboratory, Tokyo, Japan).

Results

Histopathology

The majority of the instilled UFCB were found in alveolar lumen. Numbers of particle-laden alveolar macrophages, which were F4/80 positive, were observed throughout the time course of observation (Figure 1). In addition, particle-laden cells, positive frequently with NLDC-145 and occasionally with MIDC-8, were observed in the alveolar lumina and interstitium (Figure 2). Occasional monocyte-like cells containing particles were encountered in the capillary lumen (Figure 3). Hemorrhage or inflammation were not observed throughout the time course of observation.

UFCB, present in the cytoplasm of the mononuclear cells, was demonstrated in the pulmonary lymph nodes at 24 hours after instillation. No visible particles were detected during the observation time except for 24 hours after instillation. The particle-laden cells were shown in the medullary and/or paracortical areas (T cell area) of the lymph nodes (Figure 4). The particle-laden cells were negative for F4/80, ye2/36hlk monoclonal, MIDC-8 and NLDC-145 polyclonal antibodies. No UFCB were observed in the liver nor spleen at light microscopic level.

Electron Microscopy

Electron microscopy demonstrated numerous free UFCB in the alveolar lumen. The amount and distribution pattern of UFCB particles in the alveoli differed among specimens; aggregated instilled particles were in the lumen, phagolysosoms in alveolar macrophages, and on the surface of the alveolar epithelial cells. Alveolar epithelial cell type I, on the surface of which aggregates of UFCB were frequently demonstrated, showed frequent fragmentation resulting in large-sized gaps developing between the cytoplasmic processes of the alveolar epithelial cells; size of the gap was not consistent ranging from 0.03 to 3 μm. These findings were observed in both samples taken at 3 and 8 minutes after instillation. Alveolar basement membranes, exposing to the alveolar air space, were observed at the gap. Aggregates of UFCB were also observed at the large-sized gap formed between the edges of the sparsely elongated cytoplasm of alveolar epithelial cells type I (Figure 5). Occasional UFCB were observed in the matrix of the basement membrane at and around the site of the large-sized gap (Figure 6). UFCB were also shown scattering on the surface of the occasional red blood cells in the capillary lumen in these lesions (Figure 7).

Endothelial cells of the alveolar capillaries occasionally showed excessive vesiculation (Figure 8) and bleb formation at immediately postinstillation. Although very rare, particles were occasionally observed in the vesicles in the cytoplasm of the endothelial cells at the site of alveolar wall where aggregates of UFCB were present (Figure 9). UFCB particles, which appeared to be not membrane bound, were also observed occasionally in the thin cytoplasm of the endothelial cells. These findings were observed in both samples taken at 3 and 8 minutes after instillation and not observed in control lung tissue.

Red blood cells, with particles scattered on their surface membrane, were frequently observed in the capillary lumen of the alveolar wall (Figure 10).

Discussion

This study demonstrated the location of the intratracheally instillated UFCB in the lung tissue at light and electron microscopic level. In the alveoli, most instilled UFCB particles were found in the alveolar lumen and phagolysozomes in alveolar macrophages. These findings are consistent with those of other studies on exposure to the UFPs by intratracheal instillation (Lauweryns and Baert, 1974; Adamson and Bowden, 1978; Takenaka et al., 2001). Elimination of these exposed particles from the alveolar region may take place by 3 major routes: (a) elimination of particles through the tracheobronchial tree with the mucocilliary clearance system, followed by subsequent ingestion into gastrointestinal tract and excretion in feces; (b) transfer of particles into the pulmonary lymph nodes (Strom et al., 1989); and (c) translocation of particles into the blood circulation (Takenaka et al., 2001). Recent major opinion about the clearance mechanism of the inhaled fine particles is that larger size particles are phagocytosed by alveolar macrophages followed by elimination by tracheobronchial tree or where macrophages remain in the interstitium, and smaller size particles (UFPs) are dissolved rapidly in lipid and/or fluid lining the alveolar wall and enter the blood capillaries (Takenaka et al., 2001; Nemmar et al., 2002a; Oberdörster et al., 2005;). However, the literature on the translocation of UFPs from lung into the blood circulation is limited and still conflicting. Recently, in the inhalation experiment on nonsmoking healthy human volunteers, Nemmar et al. (2002a) demonstrated that UFPs (radiolabelled UFCB particles 5–10 nm in size) could translocate from the lung into the blood circulation; radioactivity was detected in blood as early as 1 minute after exposure, reached a maximum between 10 and 20 minutes. There are also other tracer studies supporting this hypothesis (Nemmar et al., 2001; Takenaka et al., 2001; Kreyling et al., 2002; Oberdörster et al., 2002). In the present study, particles were observed in the capillary lumen in alveolar walls immediately postinstillation with UFCB. Also, 30 nm gold particles (Berry et al., 1977) and ultrafine titanium particles 22 nm in diameter (Geiser et al., 2005) were also observed in the capillary lumen at 30 minutes and 1 hour after exposure, respectively. These morphological findings are in agreement with the tracer studies, which showed systemic distribution of the UFP exposed to the lung.

As to the possible mechanism of the UFPs translocation at the air-blood barrier, there are 3 major hypotheses: (1) cell mediated active transportation: phagocytosis by macrophages and/or endocytosis by alveolar epithelial cells and endothelial cells; (2) passive transportation: diffusion; (3) active or passive transportation through the pore (caveolae) in the cytoplasm of endothelial cells or gaps between the alveolar epithelial cells (Heckel et al., 2004; Oberdörster et al., 2005).

Recently, morphological studies demonstrated the presence of inhaled polystyrene particles (240 nm in diameter) within the cytoplasm of type I and II alveolar epithelial cells and monocytes present in the capillary lumen at 30 minutes after inhalation treatment. These findings suggest that exposed particles, 240 nm in diameter, which is within a range of fine particles (100 nm–1000 nm), may be internalized by invagination of the cytoplasm of the alveolar epithelial cells, then transferred from the alveolar space to pulmonary capillary lumen by transcytosis (Kato et al., 2003). On the other hand, UFPs that were smaller than 100 nm have been shown to translocate the air-blood barrier as fast as 1 minute after inhalation (Nemmar et al., 2002a). The rapidity of the process makes it unlikely that phagocytosis by macrophages and/or endocytosis by alveolar epithelial cells and endothelial cells are involved in the process of particle translocation to the blood (Nemmar et al., 2002a). Increased number of pinocytic vesicles in endothelial cells at the site of particle aggregation were observed in this study. However, pinocytic vesicles containing particles were infrequently observed in endothelium and never observed in type I alveolar epithelial cells. Therefore, the role of endocytosis and/or caveolae-mediated translocation by alveolar epithelial cells and endothelial cells, if it takes place, may be of less importance than that of “gap-fenestration pathway,” which was observed in this study, in UFP translocation from lung alveolar space to the capillary lumen.

There are experimental data suggesting the existence of functional pores at the air-blood barrier (Conhaim et al., 1988). The air-blood barrier consists not only of tight intercellular junctions that allow passage of only water and electrolytes but also of a smaller number of large leaks that allow passage of particles up to nearly 400 nm in radius (Conhaim et al., 1988). Lung epithelial barrier was best fitted by a 3-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) (Conhaim et al., 1988). This concept is gaining acceptance in pharmacology for the administration of macromolecular drugs by inhalation (Crandall and Matthay, 2001).

In the present study, electron microscopy clearly showed large-sized gaps developing between the cytoplasmic processes of the alveolar epithelial cells; significant amounts of UFCB attaching to the alveolar basement membrane, exposed to the air space, were observed at the gap. No large-sized gaps were observed in any of control tissue examined. These findings indicate that instilled UFCB particles induce formation of larger-sized gaps between the cytoplasmic processes of alveolar epithelial cells possibly as a result of shrinkage of cytoplasm by receiving stimulus/signals generated and released following UFCB attachment. The findings, that UFCB were observed in the matrix of basement membrane at and around the site of the large-sized gap indicate that UFPs could rapidly and directly pass the alveolar-airway barrier through the gap and penetrate through the denuded basement membrane. Alveolar epithelial cells may have been damaged by physical or chemical stimulus generated by the attachment of UFCB on the basement membrane in terms of both their morphology and function. Histopathological results showed no signs of tissue injuries including hemorrhage or inflammation throughout the time course examined. Function of the endothelial cells such as active/passive transportation of molecules may, however, be altered without morphological evidence of cell damage. A possible translocation mechanism from basement membrane to capillary lumen is that UFPs can pass through the small fenestrae of endothelial cells and be taken into capillary lumen. This is in accordance with the hypothesis reported in the perfluoroisobutylene exposure study (Lehnert et al., 1993). The hypothesis is also in agreement with pharmacological macromolecular kinetics study of Crandall and Matthay (2001) and may be plausible, considering the rapidity of the translocation of UFPs (Nemmar et al., 2002a).

Incorporation of inhaled polystyrene particles (240 nm diameter) by monocytes within the capillary lumen was demonstrated similarly to that observed in the alveolar lumen, where alveolar macrophages recognize and take up larger size particles (Kato et al., 2003). In the present study on UFPs (UFCBs, 14 nm in size), occasional monocyte-like cells containing particles were encountered in the capillary lumen in the lung at the light microscopic level.

Red blood cell membranes contain membrane proteins, such as band 3 and glycophorin, which normally extends through the entire bilayer of its membrane (Bruce et al., 1994). Band 3 protein, anion exchanger of the red blood cell membrane, is responsible for the exchange of chloride and bicarbonate across the plasma membrane, a process necessary for respiration. Band 3 protein may be present on the red blood cell membrane in a scattering pattern. In addition, binding of some natural or chemical materials including mannan, sugar moiety extracted by cell wall of Candida albicans or stilbene compounds (DIDS, SITS) to band 3 protein has been reported (Watanabe, 2003). Red blood cells, whose surfaces carry a negative charge, adhere to each other when they are treated with cationic reagents (Nishiguchi et al., 1998). Red blood cells pretreated with antibody against band 3 protein do not show adhesion following the treatment with the cationic reagents. Adhesion between red blood cells induced by cationic reagents was due to changes in the charge on the membrane surface that involves polysaccharide chains and membrane surface proteins including band 3 protein (Nishiguchi et al., 1998).

In the present study, electron microscopy demonstrated a novel finding of the instilled UFCBs: the particles were frequently shown to be attached on the surface of the red blood cell membrane in a scattering pattern, which may be similar to the pattern of band 3 protein. Geiser et al. (2005) also demonstrated electron microscopic findings suggesting the uptake of the inhaled UFPs by red blood cells. UFPs, which penetrated into the basement membrane, may have passed through the capillary endothelial cells possibly by way of the small fenestrae and adhered to the surface of red blood cells. If UFCBs used in this study carry a cationic charge, the particle may easily bind to band 3 protein and may induce adhesion of red blood cells, leading to thrombus formation, which may be one of the factors responsible for the heart failure. Dendritic cells comprise a system of potent antigen presenting cell that occupy discrete portions of nonlymphoid and lymphoid organs that are interconnected by defined pathways of cellular traffic. One of the main functions of dendritic cells are thought to be the acquisition of antigens in peripheral tissues and their transport to draining lymph node for presentation as processed peptides to T cells (Matsuno et al., 1996). Strong antigen presenting cell function may reflect either high levels of MHC-peptide complexes (“signal 1”) and/or an abundance of T cell adhesion and costimulatory molecules (“signal 2”) and both signal 1 and 2 are abundant on T cell area dendritic cells (Inaba et al., 1997).

It would be plausible that UFPs taken up by these phagocytic cells and translocate to the pulmonary lymph node may have potential in modulation of the immune system. Detailed in vivo and in vitro studies including estimate of Th1/Th2 balance in T cell population are required in addition to the epidemiological study in judgment of the effects on the immune system of interaction with UFPs.

In conclusion, the results of our study indicated that inhaled UFPs could translocate into the blood circulation possibly through the “gap-fenestration pathway” induced at the air–blood barrier, and adhere to the surface of the red blood cell. Key steps may be involved in the UFPs translocation process (Figure 11): (1) formation of the large-sized gap between the type I alveolar epithelial cells; (2) passive transfer into the denuded basement membrane; and (3) translocation into the capillary lumen with the use of endothelial fenestration or transcytosis. These events may be followed by the systemic circulation following adherence of the UFPs to the cell membrane of the red blood cells.

The translocation process addressed in this study may not be applicable to all kinds of UFPs. Mechanisms of particle translocation may differ among each kind of UFP, dependent upon factors such as the route of exposure, dose, size, surface chemistry, and time course after exposure.