Non-viral gene delivery strategies have attracted significant interest in the development of novel therapeutic approaches as well as for basic and applied research in vitro. Compared to popular viral vectors, the class of non-viral carriers, namely cationic lipids (CLs) and polymers (CPs) able to spontaneously interact with negatively charged nucleic acids (NAs) to give nanoparticles called complexes, is now witnessing a surge of interest within the scientific community because they are relatively safe, cost-effective, and they can be easily produced and functionalized even at large scale. However, their efficiency in achieving the delivery tasks is still too low to outperform their viral counterparts. The efficacy of non-viral vectors is a tradeoff between their ability to drive NAs into cells, thus allowing/inhibiting their expression, their inherent toxicity, and their ability to deliver genes to target cells. Extensive research effort has thus been put into developing novel ways to improve the efficiency of such a class of delivery systems. In this context, my Ph.D. aimed at developing innovative strategies to improve non-viral vector effectiveness. To this purpose, we dealt with the delivery issue from two different perspectives: on one hand, the modulation of the vector chemistry was disclosed as a way to develop multifunctional carriers with improved effectiveness; on the other hand, we sought to improve cell-(nano)particles' interactions through the mechanical modulation of the cell behavior in response to the delivery of non-viral vectors. The first part of this thesis was thus aimed at highlighting the importance of vector chemistry on the structure-function relationship of such kinds of materials, with a focus on lipid-based carriers. Furthermore, I dealt with the characterization of a novel class of lipid-based vectors to investigate the interconnection between their structure and ultimate gene transfer ability. This thesis next explored novel ways to improve the performances of polyethyleneimine (PEI), namely the gold standard polymer vector, both in linear (lPEI) and branched (bPEI) topography. First, a thorough investigation of all the experimental variables affecting the performances of PEI-based polyplexes was carried out to disclose the best working conditions of PEI-based carriers and improve the standardization of in vitro screening protocols. Next, I focused on the development of a vector-based approach to functionalize bPEI with targeting moieties to improve the vector's selectivity towards a specific cell type. We thus synthesized a series of bPEI conjugates incorporating targeting peptides to selectively deliver genes to vascular smooth muscle cells (vSMCs). Moreover, the targeting vectors were incorporated into a polyplex releasing matrix to enable their local and controlled release for cardiovascular-related approaches. Through the conjugation of an elastin-derived peptide sequence to the bPEI structure, we were able to improve the polymer's effectiveness on target vSMCs while leaving off-target cells unaffected, a fact that is especially relevant for the translation of non-viral gene delivery approaches in vivo. On the other side, a novel strategy based on the regulation of cell response to the delivery of nanoparticles was devised. Indeed, cells in vivo are constantly subjected to different environmental cues that govern some key cell functions. We thus investigated the application of an exogenous mechanical stimulus in terms of vibrational loading to cells undergoing transfection (i.e., the delivery of NAs utilizing non-viral vectors) using lPEI and bPEI-based polyplexes. Interestingly, mechanical stimuli applied to cells improved polyplex internalization by triggering the activation of clathrin-mediated endocytosis (CME), thus leading to greater transfection outcomes. This strategy outlined the importance of cell responses to exogenous cues on the ultimate internalization and expression of a gene of interest and set the stage for a novel way to deal with the non-viral delivery issue. Overall, the big picture drawn by this Ph.D. project highlighted the suitability of chemical-based approaches and cell-based approaches as promising ways to improve non-viral vector effectiveness. Further improvement in non-viral gene delivery research might be achieved by combining the strategies devised in this project. The development of multidisciplinary approaches taking into account both the delivery vector, the environment in which the delivery of genes takes place, and the cell response may thus pave the way to ever more effective strategies, and expedite the translation from the bench to the bedside of these materials.