Comblike Polyethylenimine-Based Polyplexes: Balancing Toxicity, Cell Internalization, and Transfection Efficiency via Polymer Chain Topology.

Comblike polyethylenimines with varying degrees of polymerization of both the main and side chains as well as different grafting densities were evaluated as gene delivery vectors. They were able to condense linear and plasmid DNA into nanosized polyplex particles with dimensions and surface potentials in the 130-330 nm and -30 to +15 mV ranges, respectively, depending on the amine/phosphate (N/P) ratio. The polyplexes remained stable in aqueous and buffer solutions from several hours up to several days. The moderate colloidal stability was also manifested in a relatively broad size distribution (PDI typically above 0.2) and structural polymorphism observed by transmission electron microscopy. Both the neat polymers and polyplexes displayed low cytotoxicity in WISH cells as the relative cell viability was more than 60%. Experiments with lysosomal fluorescence staining revealed that the internalization pathways and, in turn, transfection efficiency of the polyplex nanoparticles depended on the polymer chain topology. The vector systems based on the polymers of denser structure can be considered to be promising systems for gene transfection in eukaryotic cells.

Cell proliferation in in vivo-like three-dimensional cell culture is regulated by sequestration of ERK1/2 to lipid rafts.

OBJECTIVES: Regulatory mechanisms of cell proliferation have been extensively studied as they represent major challenges when dealing with pathologies such as fibrosis, tumourigenesis or tissue regeneration. Numerous in vitro studies still exploit conventional, two-dimensional cell cultures where cells are forced to adhere to unnaturally stiff and flat surfaces of culture dishes. In the living organism, however, each cell is in contact with components of the extracellular matrix and/or neighbouring cells, thus creating a complex three-dimensional (3D) tissue structure. The current paper describes a native 3D culture of cells, based on the GD25ß1 fibroblast cell line, and its use for investigating cell proliferation in in vivo-like conditions. MATERIALS AND METHODS: Four-day post-confluent culture of GD25ß1 fibroblasts resulted in formation of a 3D system of cells embedded in naturally synthesized extracellular matrix. Morphological characterization of the culture was performed by histochemistry, immunohistochemistry and immunofluorescence. Viability/proliferation was assayed by MTT testing, FACS analysis and Western blotting for determination of expression levels and activation status of the relevant signalling molecules. RESULTS: GD25b1 fibroblasts, grown as 3D culture, gave rise to tissue-like structures characterized by low level of apoptosis, low senescence and development of 3D matrix adhesions, typical of living tissues. Transition to three-dimensionality led to a switch from exponential to linear culture growth, accompanied by accumulation of activated ERK1/2 into caveolin-containing raft domains. Disruption of raft domains as well as reverse transition from 3D back to monolayer culture led to release of phosphorylated ERK1/2 from rafts, activation of cyclin D1 expression and increase in proliferation levels. CONCLUSIONS: These results imply that under in vivo-like conditions, cells might achieve reduction of their proliferation level by sequestering activated ERK1/2 to lipid rafts.

Halothane-induced alterations in cellular structure and proliferation of A549 cells.

Genotoxicity, cytotoxicity or teratogenicity are among the well-known detrimental effects of the volatile anaesthetics. The aim of the present work was to study the structural changes, proliferative activity and the possibility of alveolar A549 cells to recover after in vitro exposure to halothane at 1.5 and 2.1mM concentrations. Our data indicated significant reduction of viability, suppression of mitotic activity more than 60%, and that these alterations were accompanied by disturbances of nuclear and nucleolar structures. The most prominent negative effect was the destruction of the lamellar bodies, the main storage organelles of pulmonary surfactant, substantial for the lung physiology. In conclusion, halothane applied at clinically relevant concentrations exerts genotoxic and cytotoxic effect on the alveolar cells in vitro, most likely as a consequence of stress-induced apoptosis, thus modulating the respiratory function.

Halothane does not directly interact with genome DNA of A549 cells.

Although the inhalation anaesthetics are commonly used in clinical practice, their toxic effects on the lung cells have not yet been well studied. Previous studies indicated strong genotoxic effect of some inhalation anaesthetics, applied at clinically relevant concentrations. The aim of the present study was to assess the extent of DNA damage, nuclear abnormalities and possibility of human A549 cells to recover after treatment with halothane at lower concentrations. The data obtained demonstrate that even lower halothane concentrations could induce DNA damage although the anaesthetic does not interact directly with DNA. We have found that irreversible impairment of the cell genome is initiated at a concentration as low as 1.5 mM. Part of the cell population displays some characteristics of stress-induced apoptosis, defining this concentration as threshold for cell survival. We suggest that the intracellular signalling pathway triggers the toxic effects of halothane.

Volatile anaesthetic halothane causes DNA damage in A549 lung cells.

The present study was performed to elucidate the extent of damage and the ability of lung epithelial cells to recover or to undergo apoptosis after in vitro treatment with the volatile anaesthetic halothane. The results obtained from the comet assay clearly show that halothane, applied at 3.0mM concentration, causes DNA and cell damage. Cells exhibited nuclear fragmentation and budding early after treatment and these events gradually increased during the next few days. The presence of a large number of mini-comets after single cell gel electrophoresis was found to represent apoptotic bodies with fragmented DNA. Our results demonstrate apoptosis-like changes after in vitro exposure of A549 cells to the volatile anaesthetic halothane. The majority of the affected cells did not recover and were directed to cell death.