ABSTRACT
This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe. A precise pump allowed their passage through the chamber under a range of shear stresses. The binding of polyethyleneimine (PEI)- and histidylated polylysine (His)-polyplexes was carried out over 30 min by time-lapse video microscopy. At 10 microg pDNA/ml in 10% serum, we found that 360+/-80 PEI- and 250+/-50 His-polyplexes were bound per 1000 cells at a shear stress of 0.3-1 dyn/cm(2). This number dropped to approximately 100 at 2 dyn/cm(2). These polyplexes exhibited differences in their interactions with the cell membrane. Concerning PEI-polyplexes, there was a shear threshold effect allowing a maximum binding at 0.06 dyn/cm(2) and a higher binding reduction (77%) at 5 microg/ml pDNA in 100% serum. The polyplex binding was augmented by 300% with PEI bearing tetraglucose moiety. This set-up is potentially helpful to screen a wide array of endothelial cells ligands prior in vivo experiments.
Subject(s)
DNA/administration & dosage , Endothelial Cells/metabolism , Gene Transfer Techniques , Polymers/administration & dosage , Cells, Cultured , DNA/metabolism , Humans , Particle Size , PlasmidsABSTRACT
BACKGROUND: Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. METHODS: After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. RESULTS: PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. CONCLUSIONS: We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes.
Subject(s)
Cell Polarity , Endothelium, Vascular/metabolism , Lung/blood supply , Polyethyleneimine/metabolism , Transfection/methods , Benzoxazoles , Cell Culture Techniques , Cell Line, Transformed , Cell Transformation, Neoplastic , Cells, Cultured , Endocytosis/drug effects , Endothelium, Vascular/cytology , Epithelial Cells/cytology , Flow Cytometry , Fluorescent Dyes , Gene Transfer Techniques , Histidine/chemistry , Humans , Luciferases/metabolism , Osmolar Concentration , Plasmids , Polyethyleneimine/chemistry , Polylysine/chemistry , Polylysine/metabolism , Quinolinium Compounds , Temperature , Trachea/cytologyABSTRACT
Knowledge of the entry mechanism and intracellular routing of polyplexes is of major importance for designing efficient gene delivery systems. We therefore investigated the internalization and trafficking of polyplexes in HepG2 cells. pDNA encoding the luciferase was complexed with histidylated polylysine (His-pLK), a polymer that requires acidic pH for pDNA endosomal release. Fluoresceinylated polyplexes (F-His-pLK or F-pDNA) were internalized by clathrin-dependent and -independent pathways. The latter most likely occurred by macropinocytosis since it was stimulated by phorbol myristate and blocked by dimethylamiloride. Intracellular routing of the plasmid was analyzed by confocal microscopy and flow cytometry. These data revealed that: (i) one part of the plasmid was present in vesicles that were not labeled with any known organelle-specific marker, (ii) the other part was in transferrin receptor-positive vesicles, and (iii) the plasmid was not transferred to late endosomes/lysosomes. Using luciferase activity as a readout for gene expression, we found that it was strongly reduced when macropinocytosis was stimulated, whereas macropinocytosis inhibitors had no effect. However, blocking clathrin-dependent internalization by chlorpromazine completely prevented gene expression. These findings demonstrate that: (i) macropinocytosis of polyplexes and (ii) plasmid recycling impair the transfection efficiency and (iii) clathrin-dependent endocytosis is the most productive route for transfection of HepG2 cells.
