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

Sammanfattning: High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and 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. In the third paper, a CPP is C-terminally 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 transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.