Rational modifications of cell-penetrating peptides for drug delivery Applications in tumor targeting and oligonucleotide delivery

High molecular weight biomolecules are becoming increasingly important in the development of new therapeutics. However, the hydrophilic nature and size of such molecules creates a major limitation for their application – poor penetration through biological membranes. Over a decade ago, a new class of peptides, cell-penetrating peptides (CPPs), with membrane translocating ability, was discovered. They have shown remarkable capability to transport various hydrophilic macromolecules inside the cells in a relatively non-toxic fashion. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity when applied in vivo and restricted bioavailability resulting from endocytic uptake of CPPs and subsequent entrapment in endosomal compartments. This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs and evaluated for cargo delivery. In the third paper, a CPP is Cterminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be effectively transported inside the nearby cells. In paper IV, an amphipathic CPP, transportan 10 (TP10), has been N-terminally prolonged with a stearic acid tail and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide cargo delivery as commercial transfection agent LipofectamineTM 2000. Moreover, stearylTP10 displays superior characteristics, preserved efficacy in serum and insignificant toxicity to cells, to the cationic lipid. In conclusion, we believe that these results give new insights for improving drug delivery. More specifically, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.