Jan Hoyer

Dimerization of a cell-penetrating peptide leads to enhanced cellular uptake and drug delivery

Summary Over the past 20 years, cell-penetrating peptides (CPPs) have gained tremendous interest due to their ability to deliver a variety of therapeutically active molecules that would otherwise be unable to cross the cellular membrane due to their size or hydrophilicity. Recently, we reported on the identification of a novel CPP, sC18, which is derived from the C-terminus of the 18 kDa cationic antimicrobial protein. Furthermore, we demonstrated successful application of sC18 for the delivery of functionalized cyclopentadienyl manganese tricarbonyl (cymantrene) complexes to tumor cell lines, inducing high cellular toxicity. In order to increase the potential of the organometallic complexes to kill tumor cells, we were looking for a way to enhance cellular uptake. Therefore, we designed a branched dimeric variant of sC18, (sC18)2, which was shown to have a dramatically improved capacity to internalize into various cell lines, even primary cells, using flow cytometry and fluorescence microscopy. Cell viability assays indicated increased cytotoxicity of the dimer presumably caused by membrane leakage; however, this effect turned out to be dependent on the specific cell type. Finally, we could show that conjugation of a functionalized cymantrene with (sC18)2 leads to significant reduction of its IC50 value in tumor cells compared to the respective sC18 conjugate, proving that dimerization is a useful method to increase the drug-delivery potential of a cell-penetrating peptide.

Peptide Vectors for the Nonviral Delivery of Nucleic Acids

Over the past two decades, gene therapy has garnered tremendous attention and is heralded by many as the ultimate cure to treat diseases such as cancer, viral infections, and inherited genetic disorders. However, the therapeutic applications of nucleic acids extend beyond the delivery of double-stranded DNA and subsequent expression of deficient gene products in diseased tissue. Other strategies include antisense oligonucleotides and most notably RNA interference (RNAi). Antisense strategies bear great potential for the treatment of diseases that are caused by misspliced mRNA, and RNAi is a universal and extraordinarily efficient tool to knock down the expression of virtually any gene by specific degradation of the desired target mRNA. However, because of the hurdles associated with effective delivery of nucleic acids across a cell membrane, the initial euphoria surrounding siRNA therapy soon subsided. The ability of oligonucleotides to cross the plasma membrane is hampered by their size and highly negative charge. Viral vectors have long been the gold standard to overcome this barrier, but they are associated with severe immunogenic effects and possible tumorigenesis. Cell-penetrating peptides (CPPs), cationic peptides that can translocate through the cell membrane independent of receptors and can transport cargo including proteins, small organic molecules, nanoparticles, and oligonucleotides, represent a promising class of nonviral delivery vectors. This Account focuses on peptide carrier systems for the cellular delivery of various types of therapeutic nucleic acids with a special emphasis on cell-penetrating peptides. We also emphasize the clinical relevance of this research through examples of promising in vivo studies. Although CPPs are often derived from naturally occurring protein transduction domains, they can also be artificially designed. Because CPPs typically include many positively charged amino acids, those electrostatic interactions facilitate the formation of complexes between the carriers and the oligonucleotides. One drawback of CPP-mediated delivery includes entrapment of the cargo in endosomes because uptake tends to be endocytic: coupling of fatty acids or endosome-disruptive peptides to the CPPs can overcome this problem. CPPs can also lack specificity for a single cell type, which can be addressed through the use of targeting moieties, such as peptide ligands that bind to specific receptors. Researchers have also applied these strategies to cationic carrier systems for nonviral oligonucleotide delivery, such as liposomes or polymers, but CPPs tend to be less cytotoxic than other delivery vehicles.

Fusion of a Short HA2-Derived Peptide Sequence to Cell-Penetrating Peptides Improves Cytosolic Uptake, but Enhances Cytotoxic Activity

Cell-penetrating peptides (CPP) have become a widely used tool for efficient cargo delivery into cells. However, one limiting fact is their uptake by endocytosis causing the enclosure of the CPP-cargo construct within endosomes. One often used method to enhance the outflow into the cytosol is the fusion of endosome-disruptive peptide or protein sequences to CPP. But, until now, no studies exist investigating the effects of the fusion peptide to the cellular distribution, structural arrangements and cytotoxic behaviour of the CPP. In this study, we attached a short modified sequence of hemagglutinin subunit HA2 to different CPP and analysed the biologic activity of the new designed peptides. Interestingly, we observed an increased cytosolic distribution but also highly toxic activities in the micromolar range against several cell lines. Structural analysis revealed that attachment of the fusion peptide had profound implications on the whole conformation of the peptide, which might be responsible for membrane interaction and endosome disruption.

Knockdown of a G protein-coupled receptor through efficient peptide-mediated siRNA delivery.

In recent years, therapeutic applications of siRNAs have come into the focus of pharmaceutical research owing to their potential to specifically regulate gene expression. However, oligonucleotides have to overcome a series of extracellular and intracellular barriers which is why delivery systems helping to overcome these barriers are desperately needed. A promising approach to transport nucleic acids beyond cellular membranes is the use of cell-penetrating peptides (CPPs), which are able to autonomously cross the plasma membrane. Recently, we synthesized branched derivatives of truncated human calcitonin (hCT) and identified them as efficient vehicles for non-covalent gene delivery. Here we describe two novel branched hCT-derivatives that are optimized for efficient intracellular delivery of siRNA by conjugation with either a fatty acid or an endosomolytic peptide sequence. As target we chose the human NPY Y₁ receptor (NPY1R), which belongs to the family of G protein-coupled receptors and thus constitutes a model for complex therapeutic targets related to various disorders. For instance, knockdown of Y₁ receptor expression offers a potential therapy for osteoporosis. We present a read-out system that allows for the quantitation of the induced knockdown of receptor expression on the protein as well as on the mRNA level. As a result of this study, we could show that the herein presented cell-penetrating peptides effectively transport siRNA into HEK-293 cells without inducing cytotoxicity and that the knockdown rates are comparable to those obtained by lipofection.

Controlled Release Technologies for RNAi Strategies in Regenerative Medicine

The ultimate goal in siRNA formulation for regenerative application centers around a gradual and sustained local release of intact siRNA which is adapted to the physiological requirements of the specific tissue. Although various issues in optimizing the siRNA delivery systems in terms of safety and efficiency still need to be addressed, progress towards the potential translation of RNAi-based therapies to clinical use has been made. An increasing number of promising siRNA-based gene therapies is currently in, or advancing towards clinical trials. Looking back to a period of only 17 years since the discovery of RNAi (Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC, Nature 391(6669):806–811. doi: 10.1038/35888, 1998) and the introduction of siRNAs as the underlying principle of RNAi (Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T, Nature 411(6836):494–498. doi: 10.1038/35078107, 2001), it is likely that regenerative applications will be explored in clinical studies soon as well.

Verbesserung der Bioverfügbarkeit von Organometallverbindungen

Metallocenes have been in the focus of intense research, due to their promising activity as anti-cancer therapeutics. However, these compounds suffer from only poor bioavailability that is caused by limited water solubility and restricted uptake in living cells. We describe one concept to circumvent these drawbacks using cell-penetrating peptides as tools to modulate physical properties and biological activity of organometallic complexes.