Viral and Synthetic Vectors Have Divergent Approaches
Independent from origin or cargo, any shuttle for a nucleic acid must at least offer three functional elements, namely (i) binding of the cargo, (ii) initial cellular uptake, mostly through the endocytic pathway, and (iii) escape from the endosomal compartment and release into the cytosol to enable activity. Other functions such as reverse transcription or genome integration are optional and do not necessarily define the core delivery event.
Viruses naturally fulfill these requirements, but do so through complex protein structures. The fundamental building blocks of synthetic vectors, on the other hand, are rather simple polycations. The charge complementarity offered by these not only facilitates binding of the nucleic acid cargo, but also promotes cellular binding. For the disassembly of the materials inside the cytosol, the strength of the interaction can be tuned by making adjustments to the charge density of the carrier.
This main technology feature of the synthetic carriers, however, is surprisingly different from its natural role model. Viruses do use cationic structures for the condensation of the nucleic acid, but do not expose a lot of these charged elements on their surface. The reason for this is due to the fact that cells, serum proteins, and mucus are all polyanionic structures; their negative charge represents the ground state in biology.
As such, the introduction of polycationic structures into such systems leads to undirected interactions and the unwanted formation of aggregates. This limited the use of the first synthetic transfectants such as polyethylenimine (PEI, a polymer), or DOTAP (a cationic lipid), to in vitro applications, where the cells to be transfected had to be adherent. In such a 2D arrangement, the transfection complexes could bind to the cell surface without triggering aggregation.
Conversely, these early transfectants were not compatible with suspension cells, could not penetrate tissues and had to be used under serum-free conditions.
As well as aggregation, the use of cationic structures suffers from another unfortunate problem. When exposed to low pH conditions such as those found in the cellular endosome, many of the polycations built into the carrier molecule exhibit an increase in charge density as their pKa value falls. This means that the transfection complex becomes even more hydrophilic inside the endosomal compartment, hindering membrane transition and having the exact opposite effect to that required to enable intracellular delivery.