Translocation Pathway of the Intratracheally Instilled C60 Fullerene from the Lung into the Blood Circulation in the Mouse: Possible Association of Diffusion and Caveolae-mediated Pinocytosis

Abstract

Ultrafine particles are ubiquitous in ambient urban and indoor air from multiple sources and may contribute to adverse respiratory and cardiovascular diseases. Recently, it has been demonstrated that ultrafine particles (UFPs) are translocated from the lung into the systemic circulation. The exact pathway, however, for the translocation in the lung remains unclear. In this study, we examined the translocation pathway of intratracheally instilled C60 fullerene particles from the lung into the blood circulation in the mouse. Using light microscopy, aggregated particles of fullerene were observed in the capillary lumen in the lung and the pulmonary lymph nodes immediately after instillation. Electron microscopic analysis demonstrated an increased number of pinocytotic vesicles (caveolae) of various sizes in the type 1 alveolar epithelial cells (AEC) and endothelial cells; occasional caveolae containing some particulate substances were observed. In addition, particles of various sizes were observed throughout the structure of the air-blood barrier (ABB). These findings suggest that fullerene particles may pass the ABB by both diffusion and caveolae-mediated pinocytosis, resulting in immediate translocation into the systemic circulation.

Introduction

There has been great interest in the engineering application of nanoscale materials, and recent studies have been conducted to investigate their potential toxicity and safety. Nanotechnology has vast potential in uses such as fuel cells, optical and electronic materials, superconductors, personal care products, and drug delivery devices in the treatment of debilitating diseases such as human immunodeficiency syndrome (Akagi et al. 2007). On the other hand, epidemiologic studies have shown consistent associations between exposure to ultrafine particles (UFPs) and acute increases in morbidity and mortality rates (Iwai et al. 2005; Nemmar et al. 2003). In addition, short-term effects—such as susceptibility to ischemia (Mills et al. 2007; Pekkanen et al. 2002; Pope et al. 2006) and thrombosis (Mills et al. 2007; Pekkanen et al. 2002) and the occurrence of myocardial infarction (Peters et al. 2001)—of exposure to particles have recently been described.

Ultrafine particles, that is, particles with a diameter less than 100 nm, have been shown to be translocated from the lungs into the blood circulation through the air-blood barrier (ABB) (Geiser et al. 2005; Kreyling et al. 2006). Ultrafine particles translocate from the lungs into the circulation and thus directly influence cardiovascular end points. However, the exact translocation pathway is not fully understood. There are two hypotheses about the translocation pathway of nanoparticles at the ABB. Recent investigations have suggested that UFPs as well as serum molecules are translocated through the ABB via passive uptake (diffusion) or caveolae-mediated pinocytosis (Geiser et al. 2005; Oberdörster et al. 2005). A number of studies with different particle types have addressed the different translocation mechanisms (Oberdörster et al. 2005; Shimada et al. 2006). These studies indicated that particle size and surface chemistry (coating), and possibly charge, govern translocation across epithelial and endothelial cell layers. In this study, we focused on the translocation mechanism of one of the carbon-based nanoparticles, fullerene (C60). Fullerene has attracted a great deal of interest because of its unique chemical and physical properties (Baker et al. 2008). Some reports suggested its potential toxicity (Oberdörster 2004; Sayes et al. 2005; Sayes et al. 2007), with the release of fullerene into the environment resulting in possible ecological implications (Oberdörster 2004; Sayes et al. 2005).

There has not been sufficient morphological evidence of translocation of UFPs in the lung. In addition, there are few reports on the electron microscopic study of the translocation of instilled UFPs at the ABB (Geiser et al. 2005; Inoue et al. 2009; Shimada et al. 2006). The rapidity of the translocation process of inhaled UFPs at the ABB may be responsible for the difficulty of morphological detection of translocation. The purpose of this study was to demonstrate the precise translocation pathway of intratracheally instilled fullerene at the ABB by light and electron microscopy.

Materials and Methods

Morphology of C60 Fullerene Particles

Morphological examination of C60 fullerene particles was performed with a transmission electron microscope (Model 100CX Japan Electron Optical Laboratory, Tokyo, Japan) and a scanning electron microscope (Model X-650, Hitachi, Tokyo, Japan). Particles suspended in phosphate-buffered saline (PBS) were placed onto grids supported by collodion film. Samples were dried, stained with 1% osmium tetroxide, and examined by transmission electron microscopy. Samples were also examined by scanning electron microscope.

Experimental Animals

Twenty-four female ICR mice (ten or eleven weeks of age) weighing 29 to 34 g were obtained from CLEA JAPAN Inc. (Tokyo, Japan). Female mice were chosen to compare the results from previous studies from our laboratory (Inoue et al. 2009; Shimada et al. 2006). Animals were kept at 25°C, and pelleted food and water were available ad libitum throughout the experiment. All animal experiments were performed according to the Tottori University guidelines for animal welfare.

Particle Suspension

Particles of C60 fullerene (0.68 nm in diameter) (Material Technologies Research Ltd., Cleveland, OH, USA) were used in this study. Suspensions of particles in PBS were prepared at the concentration of 12.5 mg/mL or 20.0 mg/mL and sterilized by autoclave at 120°C. The instillation dose of particles was determined in accordance with previous studies in our laboratory so that changes induced by different kinds of nanoparticles could be compared (Inoue et al. 2009; Shimada et al. 2006).

Intratracheal Instillation

The preparations were sonicated for one minute and thirty seconds and always vortexed immediately prior to intratracheal instillation. All mice were anesthetized by an intraperitoneal injection of xylazine (Celactal; Bayer, Leverkusen, Germany), 3 mg/kg body weight, and ketamine hydrochloride (Ketalal; Sankyo Pharmaceuticals, Tokyo, Japan), 75 mg/kg body weight, or nembutal, 5 mg/100 g body weight.

The mice were divided into six groups (n = 3) according to the sacrificed time after instillation: zero minutes, five minutes, one hour, six hours, twenty-four hours, or seven days. Mice were instilled with a volume of 0.05 mL of particle-PBS suspension containing 625 μg (for animals that were sacrificed after zero or five minutes after instillation) or 1000 μg (for animals that were sacrificed after one hour, six hours, twenty-four hours, or seven days after instillation) of particles intratracheally via a cannula, followed by 0.15 mL of air. Six mice were instilled with a volume of 0.05 mL of PBS (control groups: n = 1 at each point). All intratracheal instillation procedures took three seconds.

The mice from all groups were sacrificed following a single exposure.

Histopathology

At zero, five minutes, one hour, six hours, twenty-four hours, and seven days following the single exposure, the animals were sacrificed under diethylether anesthesia. All lobes of the lungs and pulmonary lymph nodes were then collected and fixed in 10% neutral buffered formalin. Fixed tissues of lungs and pulmonary lymph nodes were routinely processed and embedded in paraffin for histopathological examination. Sections of approximately 3 μm in thickness were cut and stained with hematoxylin and eosin. The histological examination was performed by two pathologists.

Electron Microscopy

Electron microscopy was performed on glutaraldehyde-fixed lungs from mice sacrificed five minutes after instillation. Cubes of 1 to 2 mm³ were made from each lobe. The small blocks were rinsed in 0.1 M phosphate buffer (pH = 7.4), postfixed for one hour in 1% osmium tetroxide, dehydrated in alcohol, and embedded in epoxy resin. Semithin (1 μm thick) sections were stained with 1% toluidine blue to select and locate interesting areas for electron microscopic examination. Ultrathin sections stained with uranyl acetate and lead citrate were examined under a TEM-100CX electron microscope (Japan Electron Optical Laboratory, Tokyo, Japan).

Results

Morphology of the C60 Fullerene Particles

Transmission electron microscopy demonstrated fullerene particles with aggregated state in various sizes (Figure 1). Scanning electron microscopy showed clusters of particles of varying shapes and sizes (Figure 2).

Histopathology

Aggregates of the instilled fullerene particles were occasionally found in the alveolar lumen of the lungs of the exposed animals (Figure 3). There were no signs of inflammation except for the increase in activated hypertrophic macrophages attached to the surface of the alveolar lumen. Aggregated particles adherent to the endothelial cells and red blood cells were occasionally observed in the alveolar capillaries (Figure 4).

Fullerene particles, which were confined to the cytoplasm of lymphocyte-like mononuclear cells, were demonstrated in the pulmonary lymph nodes immediately after instillation (Figure 5). No histopathological findings such as tissue damage, hemorrhage, or inflammation related to particle instillation were observed in all the examined tissues throughout the time of observation. No difference in the grading of pathological changes between lobes of the lung was observed. No time-related changes including pathology and spatial distribution of the particle were observed in the lung and pulmonary lymph node throughout the time of observation.

Electron Microscopy

Instilled fullerene particles were frequently observed in the alveolar lumen and in the cytoplasm of alveolar epithelial cell (AEC) type 1, basement membrane, cytoplasm of endothelial cells, and intravascular lumina. Electron microscopy demonstrated numerous free fullerene particles in the alveolar lumen and capillary lumen (Figure 6). Ruffling formed on the surface of AEC type 1, and an increased number of pinocytotic vesicles (caveolae) of various sizes in the AEC type 1 and endothelial cells were frequently observed in the lungs of mice exposed to both fullerene particles (Figure 7) and PBS. Instilled particles, which were aggregated in various sizes, were also demonstrated throughout the structure of ABB (Figure 8). Particulate substances were also observed in the vesicles (caveolae) of the AEC type 1 and endothelial cells at the site of the alveolar wall on which diffusion of fullerene particles were shown (Figure 9). Occasional invagination of AEC type 1, which suggests the process of small vesicle formation, was observed (Figure 9). There were occasional red blood cells (RBCs), which had particles internalized or attaching in a scattering pattern on the surface of the membrane in the capillary lumen of the alveolar wall (Figure 10). These findings were observed in both samples taken at zero and five minutes after instillation and not observed in lung tissues from control animals.

Discussion

The general opinion about the clearance mechanism of inhaled fine particles is that larger particles are phagocytized by alveolar macrophages followed by elimination via the tracheobronchial tree or that they remain in the interstitium, and that smaller particles (UFPs) are dissolved rapidly in the lung and enter the blood capillaries (Nemmar et al. 2001; Nemmar et al. 2002). Phagocytosis is one of the special functions of macrophages. However, it is believed that very small particles are not readily detected by alveolar macrophages. A previous study demonstrated that carbon black particles (14 nm in diameter) were frequently observed in phagolysozomes in alveolar macrophages (Shimada et al. 2006). In contrast, in this instillation study, no alveolar macrophages that had phagolysozomes containing fullerene particles were observed. It is yet to be elucidated whether UFPs are recognized and engulfed (phagocytized) by alveolar macrophages.

In their recent inhalation study using healthy nonsmoking human volunteers, Nemmar et al. (2002) demonstrated that UFPs (radiolabeled ultrafine carbon particles 5–10 nm in size) can translocate from the lung into the blood circulation; radioactivity was detected in blood after only one minute and reached a maximum between ten and twenty minutes. There are also other studies using tracers supporting this hypothesis (Nemmar et al. 2001; Semmler et al. 2004; Takenaka et al. 2001). In the present study, fullerene particles were observed in the alveolar lumen and in the capillary lumen; the findings were demonstrated at both the light and electron microscopic level. Light microscopic examination of lung tissues taken immediately after exposure showed occasional lymphocytic cells containing particles in the pulmonary lymph node. These findings suggest instilled particles may rapidly translocate through the ABB and enter the systemic blood circulation, resulting in spread to other organs and tissues in the body. Thus, the present morphological study supports the tracer studies, which, using quantitative methods including chemical analysis of radiolabeled elements, suggested the translocation of UFPs to the blood circulation (Nemmar et al. 2001; Nemmar et al. 2002; Semmler et al. 2004).

The literature on the translocation of UFPs from the lungs into the blood circulation is limited and still conflicting. As to the possible mechanism of UFP translocation at the ABB, there are two major hypotheses: (1) passive transportation: diffusion, and (2) cell-mediated active transportation: endocytosis by alveolar epithelial cells and endothelial cells. The diffusion pathway, by which gaseous substances are known to go through the ABB, may be suggested as one of the UFP translocation pathways. It is believed that rapidity of the gas diffusion process may be responsible for the difficulty in morphological detection of any particulate substances translocating the ABB by diffusion. Inhaled TiO₂ particles, however, were shown to pass through the ABB via passive uptake (diffusion) (Geiser et al. 2005). Also, diffusion of PEGylated cyanoacrylate nanoparticles in the rat brain was demonstrated after intravenous injection (Calvo et al. 2001). In this study, instilled fullerene particles, which were aggregated in various sizes, were also demonstrated throughout the structure of the ABB as early as five minutes after instillation, suggesting that the diffusion process may be involved as a translocation pathway. Small molecules with no electrical charge can directly pass the lipid bilayer by diffusion (Bruce et al. 2008). If the fullerene particles are uncharged, particles may also pass the ABB by diffusion.

There are other theoretical routes for UFP translocation. According to the experimental data, the ABB consists not only of tight junctions, but also of functional pores that allow passage of particles up to nearly 400 nm in radius (Conhaim et al. 1988). Transportation of ultrafine carbon particles through the gap formed in and between the elongated cytoplasm of alveolar epithelial cells has been suggested (Shimada et al. 2006). Transport via such structures, as suggested for lung-blood substance exchange, may be one of the potential mechanisms for UFP translocation. Caveolae are morphologically evident as “smooth-coated” or “noncoated” omega- or flask-shaped invaginations of the plasma membrane with a diameter of 50 to 100 nm that are connected to the plasmalemma by a neck-like structure (Gumbleton et al. 2003). Caveolae-mediated pinocytosis is one of the endocytosis systems that is known to mediate the extensive transcellular shuttling of serum proteins from the bloodstream into tissues across the endothelial cell layer (Conner and Schmid 2003).

In this study, pinocytic vesicles were morphologically recognized as caveolae. Ruffling formed on the AEC and high numbers of caveolae in both AEC type 1 and endothelial cells from the lungs of mice exposed to fullerene particles and PBS were observed in the electron microscopic study. Histopathological results showed no tissue injuries, including hemorrhage or inflammation, throughout the study. Alveolar epithelial cells may have been stimulated physically or chemically by fullerene particles, PBS, or both in both morphology and function. Results from a recent study showed that the change in cellular morphology and in plasma membrane composition (plasma-lemma vesicular density) in endothelial cells was observed as a response to mechanical stimuli subsequent to an increase in extravascular water (Daffara et al. 2004). An increased number of caveolae was observed in both particle- and PBS-instilled groups, which suggests that suspension itself might up-regulate the expression of caveolae. Indeed, results obtained by Daffara et al. (2004) demonstrated that caveolae were expressed under edema to remove water.

It should be considered that the amount of translocation of ultrafine particles by using this physical transportation system from the lung into the blood circulation might increase under the pathological condition that causes edema in the lung. Endothelial cell function such as active/passive transportation of molecules may also have been altered, as shown by the increase in the number of caveolae in this study. In addition, invagination of AEC was occasionally observed; such transient vesicle formation or fusions between adjacent vesicles would provide for a series of discontinuous pathways allowing the transcytosis of fullerene particles at the ABB. Caveolae-mediated transcytosis by both AEC and endothelial cells may play a role in translocation of UFPs from the airspace to the blood circulation at the ABB. In the previous ultrafine carbon particle study, gap formation in alveolar epithelial cells was observed (Shimada et al. 2006). In contrast, findings suggesting diffusion and/or caveolae-like pinocytotic transportation were observed in this study; pore- or gap-like structures were not observed. Diffusion of fullerene particles was also reported in the study using a model cell membrane (Qiao et al. 2007). These different structural changes observed in the air-blood barrier may result from the interactions between the biological membrane and different exposed particles.

The caveolae-mediated translocation system has been implicated in the internalization of small molecules and ions through a receptor-mediated mechanism, or potocytosis (Gumbleton et al. 2003). Folate, non-enveloped DNA virus SV-40, and chorela toxin are known to be internalized in cultured cells using this mechanism (Gumbleton et al. 2003). Using intravenous administration of albumin-coated gold nanoparticles in rodents, Mehta et al. (2004) demonstrated receptor-mediated transcytosis (albumin-binding proteins) via caveolae in cultured endothelial cells. In this study, particulate substances were occasionally shown in a manner of attachment on the inner surface of the vesicles; these findings imply that fullerene particles may also be bound to some kind of receptor on the cell membrane.

In this study, electron microscopic analysis suggested that fullerene particles may be translocated at the ABB by two different mechanisms, diffusion and caveolae-mediated pinocytosis. The small vesicles (50–60 nm in diameter) carry little fluid-phase volume (Conner and Schmid 2003). In addition, Qiao et al. (2007) reported that the fullerene molecule can easily pass into the model cell membrane by Brownian diffusion, and the mean translocation time was on the order of a few milliseconds. The rapidity of the process makes it unlikely that phagocytosis by macrophages and endocytosis by alveolar epithelial cells and endothelial cells are mainly involved in the process of particle translocation to the blood (Nemmar et al. 2002); diffusion may play a major role in fullerene translocation at the ABB. It must also be considered that proteins are very likely to bind rapidly to the particles, which then may affect the further metabolic fate of the particles in terms of their adhesion, residence time on the epithelium or uptake, and even penetration through the epithelium (Geiser et al. 2005).

In the present study, occasional particles internalized in red blood cells were observed in the capillary lumen. Similar findings were also observed in an inhalation study of rats exposed to 0.02 μm TiO₂ particles (Geiser et al. 2005). It has yet to be determined whether fullerene particles internalized or attaching on the surface of membrane of red blood cells are carried to the other organs, including the liver, heart, kidney, and even in brain, by the circulating red blood cells.

In conclusion, the results of our study indicated that inhaled UFPs could translocate into the blood circulation by diffusion and caveolae-mediated pinocytosis at the ABB, resulting in immediate invasion (translocation) into the systemic circulation.