Copolymers composed of low-molecular-weight polyethylenimine (PEI) and amphiphilic Pluronics are safe and efficient non-viral vectors for pDNA transfection. A variety of Pluronic properties provides a base for tailoring transfection efficacy in combination with the unique biological activity of this polymer group. In this study, we describe the preparation of new copolymers based on hydrophilic Pluronic F68 and PEI (F68PEI). F68PEI polyplexes obtained by doping with free F68 (1:2 and 1:5 ) allowed for fine-tuning of physicochemical properties and transfection activity, demonstrating improved in vitro transfection of the human bone osteosarcoma epithelial (U2OS) and oral squamous cell carcinoma (SCC-9) cells when compared to the parent formulation, F68PEI. Although all tested systems condensed pDNA at varying polymer/DNA charge ratios (N/P, 5/1-100/1), the addition of free F68 (1:5 ) resulted in the formation of smaller polyplexes (<200 nm). Analysis of polyplex properties by transmission electron microscopy and dynamic light scattering revealed varied polyplex morphology. Transfection potential was also found to be cell-dependent and significantly higher in SCC-9 cells compared to the control bPEI25k cells, as especially evident at higher N/P ratios (>25). The observed selectivity towards transfection of SSC-9 cells might represent a base for further optimization of a cell-specific transfection vehicle.