Gene delivery by cationic lipids: in and out of an endosome

Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in relation to eventual transfection efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes, facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery. To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems are required

Mechanism of Polyplex- and Lipoplex-Mediated Delivery of Nucleic Acids:Real-Time Visualization of Transient Membrane Destabilization without Endosomal Lysis

<p>Lipoplexes and polyplexes are widely applied as nonviral gene delivery carriers. Although their efficiencies of transfection are comparable, their mechanisms of delivery, specifically at the level of nudeic acid release from endosomes, are different. Thus, lipoplex-mediated release is proposed to rely on lipid mixing, as occurs between lipoplex and endosomal target membrane, the ensuing membrane destabilization leading to nucleic add delivery into the cytosol. By contrast, the mechanism by which polyplexes, particularly those displaying a high proton buffering capacity, release their nucleic acid cargo from the endosome, is thought to rely on a so-called "proton sponge effect", in essence an osmotically induced rupturing of the endosomal membrane. However, although a wealth of Indirect insight supports both these mechanisms, direct evidence Is still lacking. Therefore, to further clarify these mechanisms, we have investigated the interaction of lipo- and polyplexes with Hela cells by live cell imaging. As monitored over an incubation period of 2 h, our data reveal that in contrast to the involvement of numerous nanocarriers in case of lipoplex-mediated delivery, only a very limited number of polyplexes, that is, as few as one up to four/five nanocarriers per cell, with an average of one/two per cell, contribute to the release of nudeic acids from endosomes and their subsequent accumulation into the nucleus. Notably, in neither case complete rupture of endosomes nor release of intact polyplexes or lipoplexes into the cytosol was observed. Rather, at the time of endosomal escape both the polymer and its genetic payload are separately squirted into the cytoplasm, presumably via (a) local pore(s) within the endosomal membrane. Specifically, an almost Instantaneous and complete discharge of nucleic acids and carrier (remnants) from the endosomes is observed. In case of lipoplexes, the data suggest the formation of multiple transient pores over time within the same endosomal membrane, via which the cargo is more gradually transferred into the cytosol.</p>

Gene delivery by cationic lipid vectors: overcoming cellular barriers

Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the effciency of cationic-mediated gene delivery, although often suffcient for cell biological purposes, runs seriously short from a therapeutics point of view, as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement, there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including lipoplex–cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport into the nucleus. Current work indicates that a major obstacle involves the relative ineffcient destabilization of membrane-bounded compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices, entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most susceptible to lipoplex-induced destabilization, thereby allowing the most effcient release of DNA, a minimal requirement for optimizing non-viral vector-mediated transfection.