Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease

BACKGROUND: Inhalative nanocarriers for local or systemic therapy are promising. Gold nanoparticles (AuNP) have been widely considered as candidate material. Knowledge about their interaction with the lungs is required, foremost their uptake by surface macrophages and epithelial cells.Diseased lungs are of specific interest, since these are the main recipients of inhalation therapy. We, therefore, used Scnn1b-transgenic (Tg) mice as a model of chronic obstructive pulmonary disease (COPD) and compared uptake and localization of inhaled AuNP in surface macrophages and lung tissue to wild-type (Wt) mice. METHODS: Scnn1b-Tg and Wt mice inhaled a 21-nm AuNP aerosol for 2 h. Immediately (0 h) or 24 h thereafter, bronchoalveolar lavage (BAL) macrophages and whole lungs were prepared for stereological analysis of AuNP by electron microscopy. RESULTS: AuNP were mainly found as singlets or small agglomerates of <= 100 nm diameter, at the epithelial surface and within lung-surface structures. Macrophages contained also large AuNP agglomerates (> 100 nm). At 0 h after aerosol inhalation, 69.2+/-4.9% AuNP were luminal, i.e. attached to the epithelial surface and 24.0+/-5.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 35.3+/-32.2% AuNP were on the epithelium and 58.3+/-41.4% in macrophages. The percentage of luminal AuNP decreased from 0 h to 24 h in both groups. At 24 h, 15.5+/-4.8% AuNP were luminal, 21.4+/-14.2% within epithelial cells and 63.0+/-18.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 9.5+/-5.0% AuNP were luminal, 2.2+/-1.6% within epithelial cells and 82.8+/-0.2% in macrophages. BAL-macrophage analysis revealed enhanced AuNP uptake in Wt animals at 0 h and in Scnn1b-Tg mice at 24 h, confirming less efficient macrophage uptake and delayed clearance of AuNP in Scnn1b-Tg mice. CONCLUSIONS: Inhaled AuNP rapidly bound to the alveolar epithelium in both Wt and Scnn1b-Tg mice. Scnn1b-Tg mice showed less efficient AuNP uptake by surface macrophages and concomitant higher particle internalization by alveolar type I epithelial cells compared to Wt mice. This likely promotes AuNP depth translocation in Scnn1b-Tg mice, including enhanced epithelial targeting. These results suggest AuNP nanocarrier delivery as successful strategy for therapeutic targeting of alveolar epithelial cells and macrophages in COPD.

Type IV delayed-type hypersensitivity of the respiratory tract due to budesonide use: report of two cases and a literature review

Respiratory type-IV hypersensitivity reactions due to corticosteroids is a rare phenomenon. We describe two such cases. The first is a 37- year-old atopic woman who developed labial angioedema and nasal itching after the use of budesonide nasal spray. A month later, after the first puffs of a formoterol/budesonide spray prescribed for asthma, she noticed symptoms of tongue and oropharyngeal itching and redness with subsequent dysphagia, labial and tongue angioedema, and facial oedema. The second is a 15-year-old non-atopic woman who reported pruritic eruptions around the nostrils after using a budesonide nasal spray. A year later she presented with nasal pruritus with intense congestion and labial and facial oedema after using the same spray. Both patients were evaluated with patch-tests using the commercial T.R.U.E. test, a budesonide solution, and corticosteroid creams. Test evaluation was performed at 48 and 96 hours. In both patients, patch tests were positive to budesonide (++) on the second day. The first patient also had a positive (+) reaction to tixocortol-21-pivalate. All the other patch tests were negative. Clinicians should be aware that hypersensitivity reactions may occur during the use of nasal or inhaled corticosteroids.

Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles

The scope of this work was to examine in vitro responses of lung cells to secondary organic aerosol (SOA) particles, under realistic ambient air and physiological conditions occurring when particles are inhaled by mammals, using a novel particle deposition chamber. The cell cultures included cell types that are representative for the inner surface of airways and alveoli and are the target cells for inhaled particles. The results demonstrate that an exposure to SOA at ambient-air concentrations of about 10(4) particles/cm(3) for 2 h leads to only moderate cellular responses. There is evidence for (i) cell type specific effects and for (ii) different effects of SOA originating from anthropogenic and biogenic precursors, i.e. 1,3,5-trimethylbenzene (TMB) and alpha-pinene, respectively. There was no indication for cytotoxic effects but for subtle changes in cellular functions that are essential for lung homeostasis. Decreased phagocytic activity was found in human macrophages exposed to SOA from alpha-pinene. Alveolar epithelial wound repair was affected by TMB-SOA exposure, mainly because of altered cell spreading and migration at the edge of the wound. In addition, cellular responses were found to correlate with particle number concentration, as interleukin-8 production was increased in pig explants exposed to TMB-SOA with high particle numbers.

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.

The influence of pulmonary surfactant on nanoparticulate drug delivery systems

The pulmonary route is very attractive for drug delivery by inhalation. In this regard, nanoparticulate drug delivery systems, designed as multifunctional engineered nanoparticles, are very promising since they combine several opportunities like a rather uniform distribution of drug dose among all ventilated alveoli allowing for uniform cellular drug internalization. However, although the field of nanomedicine offers multiple opportunities, it still is in its infancy and the research has to proceed in order to obtain a specific targeting of the drug combined with minimum side effects. If inhaled nanoparticulate drug delivery systems are deposited on the pulmonary surfactant, they come into contact with phospholipids and surfactant proteins. It is highly likely that the interaction of nanoparticulate drug delivery systems with surfactant phospholipids and proteins will be able to mediate/modulate the further fate of this specific drug delivery system. In the present comment, we discuss the potential interactions of nanoparticulate drug delivery systems with pulmonary surfactant as well as the potential consequences of this interaction.

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall

The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

Effectiveness of nitric oxide during spontaneous breathing in experimental lung injury

Inhaled nitric oxide (iNO) improves gas exchange in about 60% of patients with acute respiratory distress syndrome (ARDS). Recruitment of atelectatic lung areas may improve responsiveness and preservation of spontaneous breathing (SB) may cause recruitment. Accordingly, preservation of SB may improve effectiveness of iNO. To test this hypothesis, iNO was evaluated in experimental acute lung injury (ALI) during SB. In 24 pigs with ALI, effects of 10 ppm iNO were evaluated during controlled mechanical ventilation (CMV) and SB in random order. Preservation of SB was provided by 4 different modes: Unassisted SB was enabled by biphasic positive airway pressure (BIPAP), moderate inspiratory assist was provided by pressure support (PS) and volume-assured pressure support (VAPS), maximum assist was ensured by assist control (A/C). Statistical analysis did not reveal gas exchange improvements due to SB alone. Significant gas exchange improvements due to iNO were only achieved during unassisted SB with BIPAP (P <.05) but not during CMV or assisted SB. The authors conclude that effectiveness of iNO may be improved by unassisted SB during BIPAP but not by assisted SB. Thus combined iNO and unassisted SB is possibly most effective to improve gas exchange in severe hypoxemic ARDS.

Deposition, imaging, and clearance: what remains to be done?

Deposition and clearance studies are used during product development and in fundamental research. These studies mostly involve radionuclide imaging, but pharmacokinetic methods are also used to assess the amount of drug absorbed through the lungs, which is closely related to lung deposition. Radionuclide imaging may be two-dimensional (gamma scintigraphy or planar imaging), or three-dimensional (single photon emission computed tomography and positron emission tomography). In October 2009, a group of scientists met at the "Thousand Years of Pharmaceutical Aerosols" conference in Reykjavik, Iceland, to discuss future research in key areas of pulmonary drug delivery. This article reports the session on "Deposition, imaging and clearance." The objective was partly to review our current understanding, but more importantly to assess "what remains to be done?" A need to standardize methodology and provide a regulatory framework by which data from radionuclide imaging methods could be compared between centers and used in the drug approval process was recognized. There is also a requirement for novel radiolabeling methods that are more representative of production processes for dry powder inhalers and pressurized metered dose inhalers. A need was identified for studies to aid our understanding of the relationship between clinical effects and regional deposition patterns of inhaled drugs. A robust methodology to assess clearance from small conducting airways should be developed, as a potential biomarker for therapies in cystic fibrosis and other diseases. The mechanisms by which inhaled nanoparticles are removed from the lungs, and the factors on which their removal depends, require further investigation. Last, and by no means least, we need a better understanding of patient-related factors, including how to reduce the variability in pulmonary drug delivery, in order to improve the precision of deposition and clearance measurements.

Generation and characterization of stable, highly concentrated titanium dioxide nanoparticle aerosols for rodent inhalation studies

The intensive use of nano-sized titanium dioxide (TiO2) particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of TiO2 nanoparticles (NP) with biological systems ideally needs to be investigated using physico-chemically uniform and well-characterized NP. In this article, we describe the reproducible production of TiO2 NP aerosols using spark ignition technology. Because currently no data are available on inhaled NP in the 10–50 nm diameter range, the emphasis was to generate NP as small as 20 nm for inhalation studies in rodents. For anticipated in vivo dosimetry analyses, TiO2 NP were radiolabeled with 48V by proton irradiation of the titanium electrodes of the spark generator. The dissolution rate of the 48V label was about 1% within the first day. The highly concentrated, polydisperse TiO2 NP aerosol (3–6 × 106 cm−3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation, and number concentration. Extensive characterization of NP chemical composition, physical structure, morphology, and specific surface area was performed. The originally generated amorphous TiO2 NP were converted into crystalline anatase TiO2 NP by thermal annealing at 950 °C. Both crystalline and amorphous 20-nm TiO2 NP were chain agglomerated/aggregated, consisting of primary particles in the range of 5 nm. Disintegration of the deposited TiO2 NP in lung tissue was not detectable within 24 h.

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.

IgE, IgGa, IgGb and IgG(T) serum antibody levels in offspring of two sires affected with equine recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is a chronic lower airway disease of the horse caused by hypersensitivity reactions to inhaled stable dust, including mould spores such as Aspergillus fumigatus. The goals of this study were to investigate whether total serum IgE levels and allergen-specific IgE and IgG subclasses are influenced by genetic factors and/or RAO and whether quantitative trait loci (QTL) could be identified for these parameters. The offspring of two RAO-affected sires (S1: n=56 and S2: n=65) were grouped by stallion and disease status, and total serum IgE levels and specific IgE, IgGa, IgGb and IgG(T) levels against recombinant Aspergillus fumigatus 7 (rAspf7) were measured by ELISA. A panel of 315 microsatellite markers covering the 31 equine autosomes were used to genotype the stallions and their offspring. A whole-genome scan using half-sib regression interval mapping was performed for each of the IgG and IgE subclasses. There was no significant effect of disease status or sire on total IgE levels, but there was a significant effect of gender and age. rAspf7-specific IgGa levels were significantly higher in RAO-affected than in healthy horses. The offspring of S1 had significantly higher rAspf7-specific IgGa and IgE levels than those of S2. Five QTLs were significant chromosome-wide (P<0.01). QTLs for rAspf7-specific IgGa and IgE were identified on ECA 1, for rAspf7-specific IgGa and IgGb on ECA 24 and for rAspf7 IgGa on ECA 26. These results provide evidence for effects of disease status and genetics on allergen-specific IgGa and IgE.

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.