title: Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: interplay between nanostructure and composition.
abstract: Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.
authors.affiliation: Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Pozzi
authors.affiliation: Department of Bioscience and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Marchini
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Cardarelli
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy; NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Salomone
authors.affiliation: Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy; Department of Anatomy, Histology, Forensic Medicine and Orthopedics, "Sapienza" University of Rome, Via A. Borelli, 50, 00161 Rome, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Coppola
authors.affiliation: Department of Bioscience and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Montani
authors.affiliation: Department of Bioscience and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy.
authors.forename: M Elexpuru
authors.initials: ME
authors.lastname: Zabaleta
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, 3120 Natural Sciences 2, Irvine, CA 92697-2715, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Digman
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, 3120 Natural Sciences 2, Irvine, CA 92697-2715, USA.
authors.forename: E
authors.initials: E
authors.lastname: Gratton
authors.affiliation: Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Colapicchioni
authors.affiliation: Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy. Electronic address: giulio.caracciolo@uniroma1.it.
authors.forename: G
authors.initials: G
authors.lastname: Caracciolo
mesh_terms: D000818:Animals; D016466:CHO Cells; D002412:Cations / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000008:administration & dosage / Q000737:chemistry*; D011992:Endosomes / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D053761:Nanocomposites / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D010873:Pinocytosis; D011479:Protamines / Q000378:metabolism
chemical_list: D002412:Cations; D008055:Lipids; D008081:Liposomes; D011479:Protamines; D004247:DNA
keywords: Gene delivery; Lipid nanoparticle; Lipoplex; Transfection barrier