Background: The potential of gene therapy for treatment of neurological disorders can be explored using designed lipid-based nanoparticles such as liposomes, which have demonstrated ability to deliver nucleic acid to brain cells. We synthesized liposomes conjugated to cell-penetrating peptides (CPPs) (vascular endothelial-cadherin-derived peptide [pVec], pentapeptide QLPVM and HIV-1 trans-activating protein [TAT]) and transferrin (Tf) ligand, and examined the influence of surface modifications on the liposome delivery capacity and transfection efficiency of encapsulated plasmid DNA. The design of liposomes was based on targeting molecular recognition of transferrin receptor overexpressed on the blood–brain barrier (BBB) with enhanced internalization ability of CPPs. Methods: CPP-Tf-liposomes were characterized by particle size distribution, zeta potential, protection of encapsulated plasmid DNA, uptake mechanisms and transfection efficiencies. An in vitro triple co-culture BBB model selected the liposomal formulations that were able to cross the in vitro BBB and subsequently, transfect primary neuronal cells. The in vivo biodistribution and biocompatibility of selected formulations were also investigated in mice. Results: Liposomal formulations were able to protect the encapsulated plasmid DNA against enzymatic degradation and presented low hemolytic potential and low cytotoxicity at 100 nM phospholipid concentration. Cellular internalization of nanoparticles occurred via multiple endocytosis pathways. CPP-Tf-conjugated liposomes mediated robust transfection of brain endothelial (bEnd.3), primary glial and primary neuronal cells. Liposomes modified with Tf and TAT demonstrated superior ability to cross the barrier layer and subsequently, transfect neuronal cells compared to other formulations. Quantification of fluorescently labeled liposomes and in vivo imaging demonstrated that this system could efficiently overcome the BBB and penetrate the brain of mice (7.7% penetration of injected dose). Conclusion: In vitro screening platforms are important tools to enhance the success of brain-targeted gene delivery systems. The potential of TAT-Tf-liposomes as efficient brain-targeted gene carriers in vitro and in vivo was suggested to be related to the presence of selected moieties on the nanoparticle surface.