Proteomic and Lipidomic Analysis of Nanoparticle Corona upon Contact with Lung Surfactant Reveals Differences in Protein, but Not Lipid Composition.

Pulmonary surfactant (PS) constitutes the first line of host defense in the deep lung. Because of its high content of phospholipids and surfactant specific proteins, the interaction of inhaled nanoparticles (NPs) with the pulmonary surfactant layer is likely to form a corona that is different to the one formed in plasma. Here we present a detailed lipidomic and proteomic analysis of NP corona formation using native porcine surfactant as a model. We analyzed the adsorbed biomolecules in the corona of three NP with different surface properties (PEG-, PLGA-, and Lipid-NP) after incubation with native porcine surfactant. Using label-free shotgun analysis for protein and LC-MS for lipid analysis, we quantitatively determined the corona composition. Our results show a conserved lipid composition in the coronas of all investigated NPs regardless of their surface properties, with only hydrophilic PEG-NPs adsorbing fewer lipids in total. In contrast, the analyzed NP displayed a marked difference in the protein corona, consisting of up to 417 different proteins. Among the proteins showing significant differences between the NP coronas, there was a striking prevalence of molecules with a notoriously high lipid and surface binding, such as, e.g., SP-A, SP-D, DMBT1. Our data indicate that the selective adsorption of proteins mediates the relatively similar lipid pattern in the coronas of different NPs. On the basis of our lipidomic and proteomic analysis, we provide a detailed set of quantitative data on the composition of the surfactant corona formed upon NP inhalation, which is unique and markedly different to the plasma corona.

Atomic force microscopy and analytical ultracentrifugation for probing nanomaterial protein interactions.

Upon contact with the human body, nanomaterials are known to interact with the physiological surroundings, especially with proteins. In this context, we explored analytical methods to provide biologically relevant information, in particular for manufactured nanomaterials as produced by the chemical industry. For this purpose, we selected two batches of SiO(2) nanoparticles as well as four batches of CeO(2) nanoparticles, each of comparably high chemical purity and similar physicochemical properties. Adsorption of serum proteins and bovine serum albumin (BSA) was quantified by SDS-PAGE in combination with densitometry and further investigated by atomic force microscopy (AFM) and analytical ultracentrifugation (AUC). The protein adsorption to SiO(2) nanoparticles was below the limit of detection, regardless of adjusting pH or osmolality to physiological conditions. In contrast, the four CeO(2) nanomaterials could be classified in two groups according to half-maximal protein adsorption. Measuring the work of adhesion and indention by AFM for the BSA-binding CeO(2) nanomaterials revealed the same classification, pointing to alterations in shape of the adsorbed protein. The same trend was also reflected in the agglomeration behavior/dispersibility of the four CeO(2) nanomaterials as revealed by AUC. We conclude that even small differences in physicochemical particle properties may nevertheless lead to differences in protein adsorption, possibly implicating a different disposition and other biological responses in the human body. Advanced analytical methods such as AFM and AUC may provide valuable additional information in this context.

Influence of vitamin E TPGS poly(ethylene glycol) chain length on apical efflux transporters in Caco-2 cell monolayers.

D-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS 1000) is a widely used form of vitamin E. TPGS 1000 is comprised of a hydrophilic polar (water-soluble) head and a lipophilic (water-insoluble) alkyl tail. TPGS 1000 has been used as a solubilizer, an emulsifier and as a vehicle for lipid-based drug delivery formulations. Most recently, TPGS 1000 has been recognized as an effective oral absorption enhancer. An enhancing effect is consistent with a surfactant-induced inhibition of P-glycoprotein (P-gp), and perhaps other drug transporter proteins; however, the exact inhibition mechanism(s) remain unclear. Therefore, in an attempt to generate additional knowledge, we have synthesized and tested various TPGS analogs containing different PEG chain length (TPGS 200/238/400/600/1000/2000/3400/3500/4000/6000). These results demonstrate a relationship between TPGS PEG chain length and influence on rhodamine 123 (RHO) transport in Caco-2 monolayers, a relationship which may be illustrated using a Weibull distribution.

Large porous particle impingement on lung epithelial cell monolayers--toward improved particle characterization in the lung.

PURPOSE: The ability to optimize new formulations for pulmonary delivery has been limited by inadequate in vitro models used to mimic conditions particles encounter in the lungs. The aim is to develop a physiologically-relevant model of the pulmonary epithelial barrier that would allow for quantitative characterization of therapeutic aerosols in vitro. METHODS: Calu-3 human bronchial epithelial cells were cultured on permeable filter inserts under air-interfaced culture (AIC) and liquid-covered culture (LCC) conditions. Calu-3 cells grown under both conditions formed tight monolayers and appeared physiologically similar by SEM and immunocytochemical staining against cell junctional proteins and prosurfactant protein-C. RESULTS: Aerosolized large porous particles (LPP) deposited homogeneously and reproducibly on the cell surface and caused no apparent damage to cell monolayers by SEM and light microscopy. However, monolayers initially grown under LCC conditions showed a significant decrease in barrier properties within the first 90 min after impingement with microparticles, as determined by transepithelial electrical resistance (TEER) measurements and fluorescein-sodium transport. Conversely, AIC grown monolayers showed no significant change in barrier properties within the first 90 min following particle application. A dense mucus coating was found on AIC grown Calu-3 monolayers, but not on LCC grown monolayers, which may protect the cell surface during particle impinging. CONCLUSION: This in vitro model, based on AIC grown Calu-3 cells, should allow a more relevant and quantitative characterization of therapeutic aerosol particles intended for delivery to the tracheobronchial region of the lung or to the nasal passages. Such characterization is likely to be particularly important with therapeutic aerosol particles designed to provide sustained drug release in the lung.

16HBE14o- human bronchial epithelial cell layers express P-glycoprotein, lung resistance-related protein, and caveolin-1.

PURPOSE: To study the expression of P-glycoprotein (P-gp), lung resistance-related protein (LRP), and caveolin-1 (cav-1) in the human bronchial epithelial cell line 16HBE14o-. METHODS: The presence of P-gp, LRP, and cav-1 in 16HBE14o- cell layers was evaluated using immunocytochemical staining and visualization with confocal laser scanning microscopy (CLSM). Functionality of P-gp was determined by bidirectional transport of rhodamine-123 with and without a P-gp inhibitor, verapamil. Caveolae were visualized using transmission electron microscopy (TEM). Flux of fluorescein-Na was also studied as a paracellular transport marker. RESULTS: Immunocytochemical staining showed expression of P-gp localized at the apical membrane of 16HBE14o- cell layers. The flux of rhodamine 123 across cell layers exhibited a greater Papp value for the secretory (i.e., basolateral-to-apical) direction. This asymmetry disappeared in the presence of verapamil. CLSM provided evidence for the expression of LRP and cav-1. TEM further showed typically shaped caveolae at the apical and basolateral membranes. CONCLUSION: Cell layers of 16HBE14o- express drug transport systems that are also present in the human bronchus in vivo, indicating that the 16HBE14o- cell line may be a suitable candidate for an in vitro model for mechanistic studies of drug transport processes involved in the smaller airways.

Differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-C.

Human alveolar type II cells were isolated from lung tissue and cultured for several days. The morphology of cells was investigated at different time points postseeding and the synthesis of alveolar cell-type specific proteins was analyzed using different methods. The rationale of the study was to characterize a primary cell culture of human alveolar cells for the development of an in vitro model studying pulmonary drug delivery. In vitro test systems based on human cells are attracting increasing interest as important alternatives to animal-derived models because possible interspecies differences in alveolar cell biology and transport mechanisms cannot be excluded. In our study, both morphological characterization and marker protein synthesis of human alveolar cells in culture indicate the differentiation of isolated alveolar type II cells into epithelial monolayers consisting of alveolar type I-like and alveolar type II-like cells, which corresponds to the composition of the alveolar epithelium of the donor tissue. By using flow cytometry, immunofluorescence, immunoblotting and reverse transcriptase polymerase chain reaction (RT-PCR), we observed a shift in the synthesis of important marker proteins. Early cultures were characterized by low caveolin-1 and high Sp-C levels. In comparison, the protein biosynthesis of alveolar cells switched with time of culture to high caveolin-1 and low Sp-C levels. Based on the similarity between human alveolar epithelium and the development of our primary alveolar cell culture, we suggest that the culture may serve as a suitable model to study epithelial transport or cell biological processes in human alveolar cells.

Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies.

Air-interfaced culture (AIC) versus liquid-covered culture (LCC) conditions are known to have different effects on the differentiated phenotype of several cell types, including lung epithelial cells. We report the influence of culture conditions such as apical medium volume on the development of intercellular junctions in the human epithelial cell line 16HBE14o-. Immunofluorescence staining of the tight-junctional protein, ZO-1, has revealed its presence in cells grown in both AIC and LCC. However, only LCC-grown cells exhibit protein ZO-1 localized as a zonula-occludens-like regular belt connecting neighboring cells. The presence of typical tight junctions has been confirmed by electron microscopy. Immunostaining for occludin, claudin-1, connexin43, and E-cadherin has demonstrated intercellular junction structures only in the cells in LCC. These morphological findings have been paralleled by higher transepithelial electrical resistance values and similar fluxes of the hydrophilic permeability marker, fluorescein-Na, under LCC compared with AIC conditions. We conclude that the formation of functional 16HBE14o- cell layers requires the presence of an apical fluid volume, in contrast to other culture conditions for airway epithelial cells.