A quantitative interspecies comparison of the respiratory mucociliary clearance mechanism.

Collectively coordinated ciliary activity propels the airway mucus, which lines the luminal surface of the vertebrate respiratory system, in cranial direction. Our contemporary understanding on how the quantitative characteristics of the metachronal wave field determines the resulting mucociliary transport is still limited, partly due to the sparse availability of quantitative observational data. We employed high-speed video reflection microscopy to image and quantitatively characterize the metachronal wave field as well as the mucociliary transport in excised bovine, porcine, ovine, lapine, turkey and ostrich samples. Image processing techniques were used to determine the ciliary beating frequency (CBF), the velocity and wavelength of the metachronal wave and the mucociliary transport velocity. The transport direction was found to strongly correlate with the mean wave propagation direction in all six species. The CBF yielded similar values (10-15 Hz) for all six species. Birds were found to exhibit higher transport speeds (130-260 [Formula: see text]m/s) than mammals (20-80 [Formula: see text]m/s). While the average transport direction significantly deviates from the tracheal long axis in mammals, no significant deviation was found in birds. The metachronal waves were found to propagate at about 4-8 times the speed of mucociliary transport in mammals, whereas in birds they propagate at about the transport speed. The mucociliary transport in birds is fast and roughly follows the TLA, whereas the transport is slower and proceeds along a left-handed spiral in mammals. The longer wavelengths and the lower ratio between the metachronal wave speed and the mucociliary transport speed provide evidence that the mucociliary clearance mechanism operates differently in birds than in mammals.

Modeling nanoparticle-alveolar epithelial cell interactions under breathing conditions using captive bubble surfactometry.

Many advances have been made in recent years in cell culture models of the epithelial barrier of the lung from simple monolayers to complex 3-D systems employing different cell types. However, the vast majority of these models still present a static air-liquid interface which is unrealistic given the dynamic nature of breathing. We present here a method where epithelial lung cells are integrated into a system, the captive bubble surfactometer, which allows the cyclical compression and expansion of the surfactant film at the air-liquid interface, thus modeling the dynamics of breathing. We found that cellular uptake of deposited gold nanoparticles was significantly increased under the dynamic (breathing) conditions of compression and expansion as compared to static conditions. The method could be very useful for studying nanoparticle-alveolar lung cell interactions under breathing conditions for applications in nanomedicine and toxicology.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NP>chitosan DNA NP=DNA unloaded chitosan NP>control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Surfactant Protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model.

BACKGROUND: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. METHODS: SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. RESULTS: SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. CONCLUSION: These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

Effects of flame made zinc oxide particles in human lung cells - a comparison of aerosol and suspension exposures.

BACKGROUND: Predominantly, studies of nanoparticle (NPs) toxicology in vitro are based upon the exposure of submerged cell cultures to particle suspensions. Such an approach however, does not reflect particle inhalation. As a more realistic simulation of such a scenario, efforts were made towards direct delivery of aerosols to air-liquid-interface cultivated cell cultures by the use of aerosol exposure systems.This study aims to provide a direct comparison of the effects of zinc oxide (ZnO) NPs when delivered as either an aerosol, or in suspension to a triple cell co-culture model of the epithelial airway barrier. To ensure dose-equivalence, ZnO-deposition was determined in each exposure scenario by atomic absorption spectroscopy. Biological endpoints being investigated after 4 or 24h incubation include cytotoxicity, total reduced glutathione, induction of antioxidative genes such as heme-oxygenase 1 (HO-1) as well as the release of the (pro)-inflammatory cytokine TNFα. RESULTS: Off-gases released as by-product of flame ZnO synthesis caused a significant decrease of total reduced GSH and induced further the release of the cytokine TNFα, demonstrating the influence of the gas phase on aerosol toxicology. No direct effects could be attributed to ZnO particles. By performing suspension exposure to avoid the factor "flame-gases", particle specific effects become apparent. Other parameters such as LDH and HO-1 were not influenced by gaseous compounds: Following aerosol exposure, LDH levels appeared elevated at both timepoints and the HO-1 transcript correlated positively with deposited ZnO-dose. Under submerged conditions, the HO-1 induction scheme deviated for 4 and 24h and increased extracellular LDH was found following 24h exposure. CONCLUSION: In the current study, aerosol and suspension-exposure has been compared by exposing cell cultures to equivalent amounts of ZnO. Both exposure strategies differ fundamentally in their dose-response pattern. Additional differences can be found for the factor time: In the aerosol scenario, parameters tend to their maximum already after 4h of exposure, whereas under submerged conditions, effects appear most pronounced mainly after 24h. Aerosol exposure provides information about the synergistic interplay of gaseous and particulate phase of an aerosol in the context of inhalation toxicology. Exposure to suspensions represents a valuable complementary method and allows investigations on particle-associated toxicity by excluding all gas-derived effects.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro.

BACKGROUND: Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (-NH2) and carboxyl (-COOH) surface modifications. RESULTS: Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. CONCLUSIONS: The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies.

Early failure of a highly cross-linked polyethylene inlay after total hip arthroplasty probably due to insufficient irradiation.

Highly cross-linked polyethylene (XLPE) is a major advance in total hip arthroplasty (THA), as it suffers from less wear and thus is associated with lower revision rates than standard ultra-high molecular weight polyethylene. Early failures are reported rarely, and associated with specific design or manufacturing issues. We report a case requiring early revision due to adverse reaction to polyethylene particles. Investigations identified insufficient irradiation as the most probable cause of failure. Here are reported the features of a clinical case with determination of the material properties of the retrieved XLPE liner and establishment of the appropriate calibration curves as reference. Periprosthetic joint infection could be ruled out with appropriate sampling as cause for the inflammatory periarticular tumour. Histology identified a massive macrophagic reaction to micrometric polyethylene particles. No component malposition was present, nor any third-body wear. The trans-vinylene index (TVI) indicated insufficient and potentially detrimental irradiation of the polyethylene, while gel content, crystallinity, melting temperature and oxidation index remained within expected ranges. Histologically proven failure of a XLPE THA liner was identifiable despite correct implantation of the components. The cause of failure most probably was an inadequate irradiation, as indicated by determination of the TVI. This case underscores the importance of histologic workup even in aseptic revisions, and of detailed analysis of retrievals. The calibration curves provided are essential for analysis of other retrievals.

Investigating the interaction of cellulose nanofibers derived from cotton with a sophisticated 3D human lung cell coculture.

Cellulose nanofibers are an attractive component of a broad range of nanomaterials. Their intriguing mechanical properties and low cost, as well as the renewable nature of cellulose make them an appealing alternative to carbon nanotubes (CNTs), which may pose a considerable health risk when inhaled. Little is known, however, concerning the potential toxicity of aerosolized cellulose nanofibers. Using a 3D in vitro triple cell coculture model of the human epithelial airway barrier, it was observed that cellulose nanofibers isolated from cotton (CCN) elicited a significantly (p < 0.05) lower cytotoxicity and (pro-)inflammatory response than multiwalled CNTs (MWCNTs) and crocidolite asbestos fibers (CAFs). Electron tomography analysis also revealed that the intracellular localization of CCNs is different from that of both MWCNTs and CAFs, indicating fundamental differences between each different nanofibre type in their interaction with the human lung cell coculture. Thus, the data shown in the present study highlights that not only the length and stiffness determine the potential detrimental (biological) effects of any nanofiber, but that the material used can significantly affect nanofiber-cell interactions.

Nanotoxicology: a perspective and discussion of whether or not in vitro testing is a valid alternative.

Despite the many proposed advantages related to nanotechnology, there are increasing concerns as to the potential adverse human health and environmental effects that the production of, and subsequent exposure to nanoparticles (NPs) might pose. In regard to human health, these concerns are founded upon the plethora of knowledge gained from research relating to the effects observed following exposure to environmental air pollution. It is known that increased exposure to environmental air pollution can cause reduced respiratory health, as well as exacerbate pre-existing conditions such as cardiovascular disease and chronic obstructive pulmonary disease. Such disease states have also been associated with exposure to the NP component contained within environmental air pollution, raising concerns as to the effects of NP exposure. It is not only exposure to accidentally produced NPs however, which should be approached with caution. Over the past decades, NPs have been specifically engineered for a wide range of consumer, industrial and technological applications. Due to the inevitable exposure of NPs to humans, owing to their use in such applications, it is therefore imperative that an understanding of how NPs interact with the human body is gained. In vivo research poses a beneficial model for gaining immediate and direct knowledge of human exposure to such xenobiotics. This research outlook however, has numerous limitations. Increased research using in vitro models has therefore been performed, as these models provide an inexpensive and high-throughput alternative to in vivo research strategies. Despite such advantages, there are also various restrictions in regard to in vitro research. Therefore, the aim of this review, in addition to providing a short perspective upon the field of nanotoxicology, is to discuss (1) the advantages and disadvantages of in vitro research and (2) how in vitro research may provide essential information pertaining to the human health risks posed by NP exposure.

Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles.

This study addresses the cellular uptake and intracellular trafficking of 15-nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air-liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG-coated than citrate-stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150-1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin- and clathrin-mediated endocytosis by methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG-coated than citrate-stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MbetaCD. With prolonged exposure times, both NPs are preferentially localized in larger-sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG-coated NPs in the cytosol.

Fluorescent-magnetic hybrid nanoparticles induce a dose-dependent increase in proinflammatory response in lung cells in vitro correlated with intracellular localization.

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.

Active uptake of dendritic cell-derived exovesicles by epithelial cells induces the release of inflammatory mediators through a TNF-alpha-mediated pathway.

Dendritic cells (DCs) can release hundreds of membrane vesicles, called exovesicles, which are able to activate resting DCs and distribute antigen. Here, we examined the role of mature DC-derived exovesicles in innate and adaptive immunity, in particular their capacity to activate epithelial cells. Our analysis of exovesicle contents showed that exovesicles contain major histocompatibility complex-II, CD40, and CD83 molecules in addition to tumor necrosis factor (TNF) receptors, TNFRI and TNFRII, and are important carriers of TNF-alpha. These exovesicles are rapidly internalized by epithelial cells, inducing the release of cytokines and chemokines, but do not transfer an alloantigen-presenting capacity to epithelial cells. Part of this activation appears to involve the TNF-alpha-mediated pathway, highlighting the key role of DC-derived exovesicles, not only in adaptive immunity, but also in innate immunity by triggering innate immune responses and activating neighboring epithelial cells to release cytokines and chemokines, thereby amplifying the magnitude of the innate immune response.

Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

The adsorption of biomolecules to multi-walled carbon nanotubes is influenced by both pulmonary surfactant lipids and surface chemistry.

BACKGROUND: During production and processing of multi-walled carbon nanotubes (MWCNTs), they may be inhaled and may enter the pulmonary circulation. It is essential that interactions with involved body fluids like the pulmonary surfactant, the blood and others are investigated, particularly as these interactions could lead to coating of the tubes and may affect their chemical and physical characteristics. The aim of this study was to characterize the possible coatings of different functionalized MWCNTs in a cell free environment. RESULTS: To simulate the first contact in the lung, the tubes were coated with pulmonary surfactant and subsequently bound lipids were characterized. The further coating in the blood circulation was simulated by incubating the tubes in blood plasma. MWCNTs were amino (NH2)- and carboxyl (-COOH)-modified, in order to investigate the influence on the bound lipid and protein patterns. It was shown that surfactant lipids bind unspecifically to different functionalized MWCNTs, in contrast to the blood plasma proteins which showed characteristic binding patterns. Patterns of bound surfactant lipids were altered after a subsequent incubation in blood plasma. In addition, it was found that bound plasma protein patterns were altered when MWCNTs were previously coated with pulmonary surfactant. CONCLUSIONS: A pulmonary surfactant coating and the functionalization of MWCNTs have both the potential to alter the MWCNTs blood plasma protein coating and to determine their properties and behaviour in biological systems.

Particles induce apical plasma membrane enlargement in epithelial lung cell line depending on particle surface area dose.

BACKGROUND: Airborne particles entering the respiratory tract may interact with the apical plasma membrane (APM) of epithelial cells and enter them. Differences in the entering mechanisms of fine (between 0.1 microm and 2.5 microm) and ultrafine (

Diesel exhaust particles modulate the tight junction protein occludin in lung cells in vitro.

BACKGROUND: Using an in vitro triple cell co-culture model consisting of human epithelial cells (16HBE14o-), monocyte-derived macrophages and dendritic cells, it was recently demonstrated that macrophages and dendritic cells create a transepithelial network between the epithelial cells to capture antigens without disrupting the epithelial tightness. The expression of the different tight junction proteins in macrophages and dendritic cells, and the formation of tight junction-like structures with epithelial cells has been demonstrated. Immunofluorescent methods combined with laser scanning microscopy and quantitative real-time polymerase chain reaction were used to investigate if exposure to diesel exhaust particles (DEP) (0.5, 5, 50, 125 mug/ml), for 24 h, can modulate the expression of the tight junction mRNA/protein of occludin, in all three cell types. RESULTS: Only the highest dose of DEP (125 mug/ml) seemed to reduce the occludin mRNA in the cells of the defence system however not in epithelial cells, although the occludin arrangement in the latter cell type was disrupted. The transepithelial electrical resistance was reduced in epithelial cell mono-cultures but not in the triple cell co-cultures, following exposure to high DEP concentration. Cytotoxicity was not found, in either epithelial mono-cultures nor in triple cell co-cultures, after exposure to the different DEP concentrations. CONCLUSION: We concluded that high concentrations of DEP (125 mug/ml) can modulate the tight junction occludin mRNA in the cells of the defence system and that those cells play an important role maintaining the epithelial integrity following exposure to particulate antigens in lung cells.

Toxic effects of brake wear particles on epithelial lung cells in vitro.

BACKGROUND: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. RESULTS: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. CONCLUSION: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress.

Virosomes can enter cells by non-phagocytic mechanisms.

Phagocytosis of fine particles (1 microm) by macrophages is a ligand-receptor-mediated, actin-based process, whereas the entering of smaller particles (< or = 0.2 microm) in macrophages occurs also by other mechanisms. Virosomes with a diameter of 0.12-0.18 microm are widely used as carrier systems for drugs, vectors, and plasmids in cancer therapy or for vaccines. We investigated their interactions with airway cells, in particular penetration into monocyte-derived macrophages. The microscopic analysis of phagocytic cells incubated with virosomes and polystyrene particles showed that virosomes and particles penetrated cells even in the presence of cytochalasin D, a drug inhibiting actin-based phagocytosis. The charge of the virosomes and particles did not influence their penetration. Also, different inhibitors of endocytotic pathways did not prevent the particles and virosomes from penetrating into the cells. Additionally, to study the ability of virosomes to overcome the epithelial airway barrier, a triple cell co-culture model composed of epithelial cells, monocyte-derived macrophages and dendritic cells of the respiratory tract was used. We found virosomes and polystyrene particles in both populations of antigen-presenting cells, monocyte-derived macrophages, and dendritic cells, in the latter even if they were not directly exposed. In conclusion, virosomes are readily taken up by monocyte-derived macrophages, both by conventional phagocytosis and by actin-independent mechanisms. Further, they can penetrate the airway barrier and reach resident dendritic cells. Therefore, virosomes are promising vaccine candidates.