Arginine-rich cell-penetrating peptides are widely utilized as vectors for gene delivery. However, their transfection efficacy still needs to be optimized. To accomplish this, guanidinocarbonylpyrrole groups, which are tailor-made anion binding sites, were introduced into the side chains of tetralysine to obtain the peptide analogue 1. In contrast to the common strategy of adding a lipophilic tail to peptide vectors, this novel method most likely enhances transfection efficacy through more specific interactions between the binding motifs and DNA or the cell membrane. Tetrapeptide analogue 1 is thus the smallest peptidic transfection vector that has been reported to date. The transfection efficacy of 1, which on average has less than two positive charges under physiological conditions, is even better than that of polyethylenimine (PEI). Furthermore, 1 exhibits only negligible cytotoxicity, which makes it an interesting candidate for further development.

Functionalization of the tetracationic cyclic peptide (Ka)4 with a single guanidiniocarbonyl pyrrole (GCP) moiety, a weakly basic but highly efficient arginine analogue, completely alters the self-assembly properties of the peptide. In contrast to the nonfunctionalized peptide 2, which does not self-assemble, GCP-containing peptide 1 forms cationic nanofibers of micrometer length. These aggregates are efficient gene transfection vectors. DNA binds to their cationic surface and is efficiently delivered into cells.

Herein, a novel cationic peptide gemini amphiphile containing diacetylene motifs (DA P) is presented, which self-assembles into novel tadpole- and bola-shaped nanostructures at low concentrations and nanofibers at higher concentrations. Interestingly, the DA P assemblies can be polymerized into a fluorescent red phase but only during incubation with HeLa cells, most likely owing to the reorganization of the diacetylene chains of DA P upon interaction with the cell membrane. The red-fluorescent polymerized DA P assemblies can serve as a novel cell imaging probe. However, only vesicles, tadpole- and bola-shaped DA P assemblies can be translocated into HeLa cells, whereas the nanofiber-like DA P assemblies are trapped by the cell membranes and do not enter the cells. Hence, morphology-dependent cell imaging is observed.

The binding between peptides and DNA is often driven by entropic forces. We demonstrate herein a new approach to shift the thermodynamic profile of peptide/DNA binding from entropy to enthalpy driven. This eventually leads to higher compacted DNA aggregates which are important for gene transfection.