Gene therapy is emerging as a revolutionary alternative to conventional therapeutic approaches. However, its clinical application is still hampered by the lack of safe and effective gene delivery techniques. Among the plethora of diverse approaches used to ferry nucleic acids into target cells, non-viral vectors represent promising and safer alternatives to viruses and physical techniques. Both cationic lipids and polymers spontaneously wrap and shrink the genetic material in complexes named lipoplexes and polyplexes, respectively, thereby protecting it and shielding its negative charges. The development of non-viral vectors commenced more than two decades ago. Since then, some major classes of interesting molecules have been identified and modified to optimize their properties. However, the way towards the final goal of gene delivery, i.e. protein expression or gene silencing, is filled with obstacles and current non-viral carriers still have concerns about their overall efficiency. We strongly believe that the future of non-viral gene delivery relies on the development of multifunctional vectors specifically tailored with diverse functionalities that act more like viruses. Although these vectors are still a long way from clinical practice they are the ideal platform to effectively shuttle the genetic material to target cells in a safe and controlled way. In this review, after briefly introducing the basis of gene delivery and therapeutic applications we discuss the main polymeric and lipidic vectors utilized for gene delivery, focusing on the strategies adopted to overcome the major weaknesses inherent to their still limited activity, on the way towards ideal multifunctional vectors.

Cationic polymers are amongst the most utilized non-viral vectors for gene transfer owing to their ability to condense and protect the genetic material within polyplexes and to ferry it into cells. Quite a number of parameters, both related to the features of the vectors themselves (e.g. degree of branching, molecular weight, polydispersity) and to polyplexes (e.g. nitrogen to phosphate ratio (N/P), dose of complexes delivered, complexation buffer, etc.), are known to affect transfection behaviour. Consequently, some substantial discrepancy found in raw materials and in-home protocols across laboratories account for some disagreement and conflicting data about their performance. Hereinafter we provide a thorough chemical-physical and in vitro biochemical characterization, comparison, and optimization of the most widely used, commercially sourced polymers used in transfection, namely linear polyethylenimines (lPEIs), branched PEIs (bPEIs), linear poly-L-lysines (lPLLs), and polyamidoamine dendrimers (dPAMAMs). By means of a stepwise approach, we pinpointed the most effective molecular weight and complexation conditions specific to each of them and correlated the physicochemical features of polyplexes with their transfection effectiveness. Besides, taking separately into account the effects on transfection of the plasmid dose delivered to cells, the cell seeding density and the volume of the culture medium, we highlited a range of optimal conditions roughly specific to each studied polymer. Finally, we coped with the effect of the variation of these three parameters at once on the transfection effectiveness of lPEI and bPEI and pinpointed an array of settings specifically optimized to attain truly superior performances