Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells.

Polyethyleneimine (PEI) has been used extensively for transient gene expression (TGE) in mammalian cell cultures. However, the relationship between DNA/PEI complex preparation and their biological activity has not been fully established. Here, a systematic study of DNA/PEI complexes, their physicochemical properties during formation and their transfection efficiency was performed on a virus-like particle (VLP) production platform. The same chemically defined cell culture medium for DNA/PEI complex formation was used as an alternative to simple ionic solutions to minimize changes in complex properties during transfection. Upon formation, an initial concentration of 1E + 10 DNA/PEI complexes/mL underwent partial aggregation with an average size of 300 nm. The participation of NaCl ions in the evolution of complexes was analyzed by X-ray spectroscopy, stressing the relevance of complexing media composition in TGE strategies. After 15 min incubation, 250 complexes plus aggregates per cell were estimated at the time of transfection. Such heterogeneous preparations cannot be easily characterized; subsequently, nanoparticle tracking analysis (NTA) and cryo-electron microscopy were combined to achieve a complete picture of the preparation. Finally, the contribution of each DNA/PEI complex subpopulation was tested by drug inhibition endocytosis. Interestingly, all complexes delivered DNA efficiently and high size aggregates, which enter through macropinocytosis, when inhibited presented a major contribution to transfection efficiency. There is a need to understand the physicochemical factors that participate in DNA delivery protocols. Hence, this study provides new insights into the characterization of DNA/PEI complexes that will assist in more productive and reproducible TGE strategies.

Quantitative colocalization analysis of DNA delivery by PEI-mediated cationic polymers in mammalian cells.

Although cationic polymers are widely used for DNA delivery, the relationship between the properties of the formed complexes and their biological activity is not fully understood. Here, we propose a novel procedure consisting of superresolved images coupled with quantitative colocalization to analyse DNA release in living cells. This work compares the different workflows available in a quantitative colocalization study of DNA delivery using polyethylenimine as transfection reagent. A nimble workflow with deconvolution in three-dimensional images was developed. Among the different colocalization coefficients, Manders' colocalization coefficient was the best to track the complexes. Results showed that DNA/polyethylenimine complexes were tightly interacting at the time of transfection and their disassembly was observed between 2 and 10 h after their uptake. Heterogenicity was found in the intracellular fate of each complex. At 24 h, some complexes were still present underneath the nuclear envelope. Overall, this study opens the door for particle tracking assessment with three-dimensional imaging at intracellular level. LAY DESCRIPTION: DNA delivery technologies in living cells are of high relevance in the biotechnology field. The transient expression of a gene of interest enables the production of a wide range of new therapeutic candidates for clinical purposes. However, the introduction of an exogenous DNA construct into a cell culture requires the use of certain vehicles that protect the DNA from host cell DNases and deliver it into the cell nucleus. From the different systems available, polyethylenimine (PEI) has been extensively used in transient gene expression strategies for the last three decades. However, the intracellular fate of the formed DNA/PEI complexes and the DNA release from the complexes is still poorly understood. In this work, we propose the application of combined superresolved images through mathematical deconvolution to colocalization studies of DNA/PEI complexes evolution in living mammalian cell cultures. Both specimens were covalently labelled with Cy3 and Cy5 dye, respectively, and the kinetics of its disassembly process within the cells was tracked over the time. Because of the specific features of the formed-complexes, a comparative study of the different colocalization coefficients was performed towards optimizing the analysis of these particles with confocal microscopy. Besides, the 3D imaging of the process allowed the direct visualization of a partial DNA/PEI complexes disassembly and the location of those complexes underneath the nuclear envelope during the cell production phase (24 h after the uptake).

[Clinico-radiological correlation of pulmonary complications of pediatric HIV infection]. / Correlación clínico-radiológica de las complicaciones pulmonares de la infección VIH en Pediatría.

Fifty-five percent of children with HIV infection, aged two months to ten years, were admitted at our hospitals because of respiratory conditions. Pulmonary complications found at admission in these children were lymphoid interstitial pneumonitis, Pneumocystis carinii pneumonia, fungal over-infection, tuberculosis, and bacterial complications. Also, non-specific infectious bronchial conditions, probably of viral origin. The most representative chest-X rays of these pulmonary conditions were analyzed; together with data from clinical records a clinico-radiological diagnosis was obtained.