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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.

Although there is a modest body of literature on the absorption of inhaled pharmaceuticals by normal lungs and some limited information from diseased lungs, there is still a surprising lack of mechanistic knowledge about the details of the processes involved. Where are molecules absorbed, what mechanisms are involved, how well are different lung regions penetrated, what are the determinants of metabolism and dissolution, and how best can one retard the clearance of molecules deposited in the lung or induce intracellular uptake by lung cells? Some general principles are evident: (1) small hydrophobic molecules are absorbed very fast (within tens of seconds) usually with little metabolism; (2) small hydrophilic molecules are absorbed fast (within tens of minutes), again with minimal metabolism; (3) very low water solubility of the drug can retard absorption; (4) peptides are rapidly absorbed but are significantly metabolized unless chemically protected against peptidases; (5) larger proteins are more slowly absorbed with variable bioavailabilities; and 6) insulin seems to be best absorbed distally in the lungs while certain antibodies appear to be preferentially absorbed in the upper airways. For local lung disease applications, and some systemic applications as well, many small molecules are absorbed much too fast for convenient and effective therapies. For systemic delivery of peptides and proteins, absorption may sometimes be too fast. Bioavailabilities are often too low for cost-effective and reliable treatments. A better understanding of the determinants of pulmonary drug dissolution, absorption, metabolism, and how to target specific regions and/or cells in the lung will enable safer and more effective inhaled medicines in the future.

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Multiple particle tracking (MPT) methodology was used to dissect the impact of nanoparticle surface charge and size upon particle diffusion through freshly harvested porcine jejunum mucus. The mucus was characterised rheologically and by atomic force microscopy. To vary nanoparticle surface charge we used a series of self-assembly polyelectrolyte particles composed of varying ratios of the negatively charged polyacrylic acid polymer and the positively charged chitosan polymer. This series included a neutral or near-neutral particle to correspond to highly charged but near-neutral viral particles that appear to effectively permeate mucus. In order to negate the confounding issue of self-aggregation of such neutral synthetic particles a sonication step effectively reduced particle size (to less than 340 nm) for a sufficient period to conduct the tracking experiments. Across the polyelectrolyte particles a broad and meaningful relationship was observed between particle diffusion in mucus (×1000 difference between slowest and fastest particle types), particle size (104-373 nm) and particle surface charge (-29 mV to +19.5 mV), where the beneficial characteristic promoting diffusion was a neutral or near-neutral charge. The diffusion of the neutral polyelectrolyte particle (0.02887 cm S(-1)×10(-9)) compared favourably with that of a highly diffusive PEGylated-PLGA particle (0.03182 cm(2) S(-1)×10(-9)), despite the size of the latter (54 nm diameter) accommodating a reduced steric hindrance with the mucin network. Heterogeneity of particle diffusion within a given particle type revealed the most diffusive 10% sub-population for the neutral polyelectrolyte formulation (5.809 cm(2) S(-1)×10(-9)) to be faster than that of the most diffusive 10% sub-populations obtained either for the PEGylated-PLGA particle (4.061 cm(2) S(-1)×10(-9)) or for a capsid adenovirus particle (1.922 cm(2) S(-1)×10(-9)). While this study has used a simple self-assembly polyelectrolyte system it has substantiated the pursuance of other polymer synthesis approaches (such as living free-radical polymerisation) to deliver stable, size-controlled nanoparticles possessing a uniform high density charge distribution and yielding a net neutral surface potential. Such particles will provide an additional strategy to that of PEGylated systems where the interactions of mucosally delivered nanoparticles with the mucus barrier are to be minimised.

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.

The multidrug resistant (MDR) transporter P-glycoprotein (Pgp) is constitutively expressed in normal tissues, where its spatial distribution defines it as an important element reducing the systemic exposure and tissue access of potentially harmful xenobiotics. We sought to determine whether P-gp is functionally expressed within alveolar epithelium of lung, in particular within the predominant cell type of this barrier, the alveolar epithelial (AE) type I cell. By immunohistochemistry, MDR-1/ mdr-1 P-gp was localized to luminal membranes of AE type I epithelium within normal human and rat lung tissue. Using a primary rat cell culture model affording study of AE type II to AE type I differentiation, we observed increased expression (reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunoflow cytometry techniques) of mdr-1a and mdr-1b P-gp in the cultures as they adopted an AE type I phenotype; freshly isolated AE type II cells were negative for mdr-1/P-gp. The functionality of P-gp within the AE cultures was demonstrated by a flow cytometric accumulation-retention assay using rhodamine-123 as substrate, and also by the polarized transport of vinblastine across confluent AE type I monolayers (basal-to-apical permeability was 3-fold that of apical-to-basal permeability), which was found to be comparable with the P-gp transport barrier presented by Caco-2 cell monolayers. The implications of localizing P-gp within alveolar epithelium is of significance to studies of fundamental respiratory cell biology as well as to further clarifying the nature of the barrier to xenobiotic transfer from alveolar airspace to pulmonary interstitium and capillary blood.P-Glycoprotein (P-gp) is a member of the ATP-binding cassette superfamily of membrane transport proteins that mediates the vectorial movement across cell membranes of a wide range of physicochemically diverse solutes (Stouch and Gudmundsson, 2002). In humans, two P-gp-related genes have been cloned and subsequently termed MDR1 and MDR3 (for review, see Ambudkar et al., 1999). The MDR1/P-gp gene product is recognized in particular to actively efflux from a cell a diverse range of cytotoxic drugs, a characteristic that is an important facet in the multidrug resistant (MDR) cell phenotype. Current evidence suggests that the MDR3/P-gp gene product does not contribute to an MDR phenotype.In rodents, three P-gp-related genes have been identified and designated mdr-1a, mdr-1b, and mdr-