| Author |
Pozzi, D. Salomone, F. Zabaleta, M. Elexpuru Digman, M. A. Gratton, E. Coppola, S. Montani, M. Colapicchioni, V. Marchini, C. Caracciolo, G. Cardarelli, F. |
| Description |
Author Affiliation: Pozzi D ( Department of Molecular Medicine, 'Sapienza' University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy.); Marchini C ( Department of Bioscience and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy.); Cardarelli F ( Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.); Salomone F ( Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy); Coppola S ( Department of Molecular Medicine, 'Sapienza' University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy); Montani M ( Department of Bioscience and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy.); Zabaleta ME ( Department of Bioscience and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy.); Digman MA ( Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, 3120 Natural Sciences 2, Irvine, CA 92697-2715, USA.); Gratton E ( Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, 3120 Natural Sciences 2, Irvine, CA 92697-2715, USA.); Colapicchioni V ( Department of Molecular Medicine, 'Sapienza' University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy.); Caracciolo G ( Department of Molecular Medicine, 'Sapienza' University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy. Electronic address: giulio.caracciolo@uniroma1.it.) |
| 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 3ß-[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. |
