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.

Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics

“…3E and 4E). The mechanism of action of LPD may that a core complex is formed with protamine and DNA, and this complex in conjunction with CLs then forms a LPD nanoparticle with a shell-core structure (9). Moreover, in our study, the size of the nanoparticles decreased following the addition of protamine to form the LPD complexes (Fig.…”

“…In addition, previous studies have demonstrated that CL-mediated gene transfer may be enhanced by the addition of natural polycations which act as DNA-condensing agents and form liposome-polycation-DNA complexes. Natural polycations, such as protamine sulfate (8,9), chitosan (10) and poly-L-lysine (11), have been shown to condense DNA and form nanoparticles which are mechanically stable, of uniform particle size and of controllable morphology. Protamine, which is non-toxic and is composed of natural cationic peptides, may provide unique membrane-translocating and nuclear-localizing activities owing to its abundant amino acid sequence (12).…”

Single-cell real-time imaging of transgene expression upon lipofection

“…The produced NLS-GFP is used as bright and photo-stable reporter of successful transfection. The "gold-standard" Lipofectamine M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT formulation (hereafter: LFN) is selected as a benchmark for lipid-based DNA transfection 9-11 and compared to the recently introduced alternative formulation made of the neutral lipid 1,2-Dioleoylsn-glycero-3-phosphoethanolamine (DOPE) and the cationic cholesterol derivative 3β-[N-(N′, N′-Dimethylaminoethane)-Carbamoyl]Cholesterol (DC-Chol) (hereafter: DC-Chol/DOPE) 12,13 . This latter has been classified as one of the most efficient vectors for transfection of DNA into cells 14,15 (a schematic representation of a typical experiment is reported in Fig.…”

Como estudar interações entre nanopartículas e sistemas biológicos

“…Faz-se necessário padronizar as concentrações dos inibidores utilizando marcadores específicos de cada via que depende do inibidor, do tipo celular e do tempo de exposição. Os marcadores mais utilizados são a transferrina para endocitose mediada por clatrina, 60 toxina da cólera para a endocitose mediada por caveolina 61 e dextrana para macropinocitose. 62 Os marcadores geralmente são disponíveis comercialmente já associados a um fluoróforo.…”