ABSTRACT
PURPOSE: Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. MATERIALS AND METHODS: The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy. RESULTS: Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. CONCLUSIONS: These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.
Subject(s)
Caveolae/metabolism , DNA/chemistry , Macromolecular Substances/chemistry , Polyamines/chemistry , Androstadienes/pharmacology , Animals , Biological Transport/drug effects , COS Cells , Caveolae/drug effects , Chlorocebus aethiops , Chlorpromazine/pharmacology , Cholera Toxin/metabolism , Cholera Toxin/pharmacokinetics , Clathrin-Coated Vesicles/drug effects , Clathrin-Coated Vesicles/metabolism , Endocytosis/drug effects , Enzyme Inhibitors/pharmacology , Flow Cytometry , Fluorescent Dyes/chemistry , Genistein/pharmacology , Luciferases/genetics , Luciferases/metabolism , Macromolecular Substances/metabolism , Macromolecular Substances/pharmacokinetics , Methacrylates/chemistry , Microscopy, Fluorescence , Nocodazole/pharmacology , Nylons/chemistry , Polyelectrolytes , Polyethyleneimine/chemistry , Transfection/methods , Transferrin/metabolism , Transferrin/pharmacokinetics , Wortmannin , beta-Cyclodextrins/pharmacologyABSTRACT
The lamellarity of liposomes influences to a great extent the encapsulation efficiency, the efflux rate of liposomally encapsulated material, and the fate of a drug after cellular uptake. 31P-NMR in combination with the use of chemical shift reagents has been described for the determination of lamellarity of liposomes and this study was performed to evaluate the applicability of 31P-NMR analysis as published in the past. To date, very few details about the required conditions throughout the measurements are known. In this study the influence of various parameters on the measurement, such as different buffers with changing ion concentrations, varying pH and different shift reagents at increasing concentrations was investigated. Results were discussed by using cryo-electronmicroscopy as a reference method. The data of this study show that 31P-NMR might not result in the correct determination of liposome lamellarity, depending on the experimental settings and the shape of the liposomes.
Subject(s)
Liposomes/chemistry , Indicators and Reagents/chemistry , Magnetic Resonance Spectroscopy , Manganese/chemistry , Microscopy, Electron/methods , Phosphorus IsotopesABSTRACT
An oligonucleotide of 20 bases, complementary to a region of the sodium/myo-inositol cotransporter (SMIT) mRNA, was used to investigate the uptake efficiency and activity of transferred antisense oligonucleotides with regard to substrate uptake. We compared the efficiency of oligonucleotide delivery after application of either free or liposome-encapsulated material. Delivery of liposome-encapsulated material (marker or oligonucleotides) into astrocytoma cells and primary astrocyte cultures was more effective with pH-sensitive liposomes [dioleoylphosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate (CHEMS)] than with non-pH-sensitive liposomes (soy lecithin) or free material in solution. Antisense activity was evaluated by determination of myo-inositol uptake and detection of SMIT transcripts by RT-PCR. Encapsulation of oligonucleotides in pH-sensitive liposomes increased the inhibition of inositol uptake at least 50-fold compared with application of free oligonucleotides in solution.
