We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Mechanistic Understanding of Gene Delivery Mediated by Highly Efficient Multicomponent Envelope-Type Nanoparticle Systems / D. Pozzi, C. Marchini, F. Cardarelli, A. Rossetta, V. Colapicchioni, A. Amici, M. Montani, S. Motta, P. Brocca, L. Cantù, G. Caracciolo. - In: MOLECULAR PHARMACEUTICS. - ISSN 1543-8384. - 10:12(2013 Nov), pp. 4654-4665.

Mechanistic Understanding of Gene Delivery Mediated by Highly Efficient Multicomponent Envelope-Type Nanoparticle Systems

S. Motta;P. Brocca;L. Cantù;
2013-11

Abstract

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.
nanoparticles ; gene delivery ; transfection ; endosomal escape
Settore FIS/07 - Fisica Applicata(Beni Culturali, Ambientali, Biol.e Medicin)
MOLECULAR PHARMACEUTICS
http://pubs.acs.org/doi/abs/10.1021/mp400470p
Article (author)
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/235831
Citazioni
  • ???jsp.display-item.citation.pmc??? 15
  • Scopus 45
  • ???jsp.display-item.citation.isi??? 47
social impact