title: Quantitative evaluation of DNA dissociation from liposome carriers and DNA escape from endosomes during lipid-mediated gene delivery.
abstract: Nonviral vectors are highly attractive for gene therapy from a clinical point of view, and cationic lipid nanoparticles in particular have generated considerable interest. However, despite considerable recent advances, problems associated with low transfection efficiencies remain to be resolved to fully meet the potential of these vectors. The trafficking of plasmid DNA (pDNA) from the extracellular space up to the nucleus is prevented by several barriers, including liposome/pDNA dissociation within the endosome and pDNA escape into the cytosol. The aim of this work was to develop and optimize a tool that could offer simultaneous quantitative information both on the intracellular dissociation of oligonucleotides from lipid nanoparticles, and on the DNA escape from endocytic compartments. The ability to follow in real time both of these processes simultaneously (in a quantitative manner) is expected to be of high value in the rationalization and conception of new lipid nanoparticle vectors for gene delivery for therapeutic purposes. To this effect, a combination of Förster resonance energy transfer (FRET) and colocalization microscopy was employed. We show that it is possible to distinguish between liposome/pDNA dissociation and depletion of DNA within endosomes, providing resolution for the detection of intermediate species between endocytic particles with intact lipoplexes and endosomes devoid of DNA because of DNA escape or degradation. We demonstrate that after endocytosis, exceptionally few endocytic particles are found to exhibit simultaneously DNA/lipid colocalization and low FRET (DNA/lipid dissociation). These results clearly point to an extremely short-lived state for free plasmid within endosomes, which either escapes at once to the cytosol or is degraded within the endocytic compartment (because of exposure of DNA). It is possible that this limitation greatly contributes to reduction in probability of successful gene delivery through cationic lipid particles.
authors.affiliation: 1 Institute of Biotechnology and Bioengineering, Centre for Chemical and Biological Engineering, Instituto Superior Técnico, Universidade de Lisboa , 1049-001 Lisbon, Portugal .
authors.forename: Salomé
authors.initials: S
authors.lastname: Magalhães
authors.forename: Sofia
authors.initials: S
authors.lastname: Duarte
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Monteiro
authors.forename: Fábio
authors.initials: F
authors.lastname: Fernandes
mesh_terms: D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D057054:Molecular Imaging / Q000379:methods*; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D014162:Transfection; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D005229:Fatty Acids, Monounsaturated; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D053835:Unilamellar Liposomes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane