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.