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. © 2013 Elsevier B.V.

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition / Pozzi, Daniela; C., Marchini; F., Cardarelli; F., Salomone; Coppola, Stefano; M., Montani; M., Elexpuru Zabaleta; M. A., Digman; E., Gratton; Colapicchioni, Valentina; Caracciolo, Giulio. - In: BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES. - ISSN 0005-2736. - STAMPA. - 1838:3(2014), pp. 957-967. [10.1016/j.bbamem.2013.11.014]

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition

POZZI, DANIELA;COPPOLA, STEFANO;COLAPICCHIONI, VALENTINA;CARACCIOLO, Giulio
2014

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. © 2013 Elsevier B.V.
2014
gene delivery; transfection barrier; lipid nanoparticle; lipoplex
01 Pubblicazione su rivista::01a Articolo in rivista
Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition / Pozzi, Daniela; C., Marchini; F., Cardarelli; F., Salomone; Coppola, Stefano; M., Montani; M., Elexpuru Zabaleta; M. A., Digman; E., Gratton; Colapicchioni, Valentina; Caracciolo, Giulio. - In: BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES. - ISSN 0005-2736. - STAMPA. - 1838:3(2014), pp. 957-967. [10.1016/j.bbamem.2013.11.014]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/543675
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