Due to the lack of therapeutic treatments for genetic and acquired disorders, in the last decades the attention of researchers was focused on gene therapies. The emergent need for innovative treatments is due to the fact that existing therapies are outdated and nonspecific [1]. Gene therapies using non-viral vectors have shown promising behaviours, after viral vectors, in treating different types of melanoma[2]. The use of non-viral approaches has some advantages and disadvantages compared with viral therapies, in vitro testing on different types of cells proved that non-viral carriers exhibit low cytotoxicity with different degree of transfection efficiency depending on components used in vectors synthesis [3]. The most explored components are cationic compounds, especially polycationic compounds which showed a significant efficiency in delivering of nucleic acids (branched and linear polyethyleneimine) [4]. In this context, our studies were focused in obtaining non-viral vectors with improved transfection efficiency and low cytotoxicity by using combinatorial chemistry. Previously studies by our group in this field showed that the most suitable components to fulfil the desired properties are: squalene (a natural biocompatible lipid), benzene-1,3,5-tricarboxaldehyde (TA) (multifunctional core), branched polyethylenimine with low molecular weight (PEI) (positively charged polymer) and polyethylene glycol (PEG) (biocompatible polymer) which are connected together by reversible imine bonds [5,6]. The main aim of this study was to observe the influence of PEG from its composition on transfection efficiency. For this purpose, we used PEG of three molecular weights in different molar ratios. TEM and DLS studies showed that in aqueous media this type of systems adopts a core-shell structure forming hydrophobic/hydrophilic assemblies. The formation of the polyplexes between plasmid nucleic acid and non-viral vectors was proved by agarose gel electrophoresis assay, showing that, the obtained systems are able to bind the plasmid nucleic acid. The efficiency in transfection and cytotoxicity were tested in vitro on HeLa cell line and results showed that the type and content of PEG in obtained polyplexes possess a crucial role in delivering genetic material.