Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well studied CPP, PepFectl4, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.