Immortalization of Human Alveolar Epithelial Cells to Investigate Nanoparticle Uptake

Abstract

Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro-surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 mm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 mm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-mm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cellassociated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.

The intensity of internalization and cytotoxicity of superparamagnetic iron oxide nanoparticles with different surface modifications in human tumor and diploid lung cells

“…Moreover, Ge et al [44] have reported recently that positively charged magnetite nanoparticles are more efficiently taken up into KB cells due to electrostatic attraction to the negatively charged cell membrane than negatively charged particles. In contrast, a preferential uptake of negatively charged particles into immortalized alveolar epithelial type 1 cells has been found [47]. Surface charge as an important parameter in biological activity of nanoparticles has been supported also by other authors [12,[48][49][50][51].…”

“…The mouse macrophages (RAW 264.7) were for example shown to internalized PEGmodified MNPs less efficiently than the human breast cancer cells [9]. Recent study has revealed that the human alveolar type 2 cells internalized nanoparticles less efficiently than the human alveolar type 1 cells [47]. Since A549 cells are representatives of the alveolar epithelial type 2-like cells [15] it is reasonable to suppose that this fact might underlie less efficient MNPs entrance into the human lung tumor cells compared with the human lung diploid cells.…”

The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles

“…Carboxylated 1 µm and 50 nm polystyrene particles were ingested to a higher degree by alveolar type I cells, 134 whereas Fazlollahi et al 135 showed preferential uptake of cationic polystyrene particles in MDCK cells. For quantum dots, some groups reported preferential uptake of anionic quantum dots, 136,137 and others that of positively charged quantum dots.…”

Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier

“…A new human ATI cell line (TT1), recently described by Tetley et al (van den Bogaard et al, 2009), was obtained through the immortalization of primary ATII and used as a model for inflammatory response studies and nanoparticle uptake. However, TT1 cells do not appear to develop tight intercellular junctions, and therefore still lack important barrier properties (van den Bogaard et al, 2009;Kemp et al, 2008). Other widely used lung cell lines are 16HBE14o-and Calu-3, but they are both of bronchial origin and therefore -although they build TEER of 300-600 Ω*cm 2 -are not suitable as a model of the alveolar epithelium (Ehrhardt et al, 2003;Forbes, 2003;Grainger et al, 2006).…”

“…Blank, F., Wehrli, M., Lehmann, A. et al (2011) viously reported immortalization approach with a thermo-sensitive mutant of the SV40 Large T-Antigen led to the development of an immortalized human lung cell line that is well suited for many applications, e.g., nanoparticle-uptake (Kemp et al, 2008) or inflammatory response (van den Bogaard et al, 2009). However, these cells also lack the capacity to form functional tight junctions, which are needed to build a formidable diffusion barrier also to smaller hydrophilic molecules.…”