Prof. Ülo Langel, University of Tartu
Sandrine Sagan, UPMC-Université Paris, France
Advances in biochemistry and molecular biology over the years, together with the human genome sequencing project, have increased our understanding of the genetics of numerous diseases and has led to the identification of many disease-causing genes. In order to treat these diseases by inhibiting, modulating or introducing new genes to the affected cells, appropriate drugs need to be transported into their point of action. Due to their physicochemical properties, many drugs are unable to enter cells and require delivery vectors which are efficient, safe, and resistant to degradation. Generally, there are two types of vehicles, viral and non-viral. The most studied non-viral delivery vectors are liposome-, polymer- and micelle- based systems, and more recently, cell-penetrating peptides (CPPs) have gained interest as well. CPPs are short amino acid sequences, consisting of up to 30 amino acids, capable of delivering bioactive cargos inside cells in an efficient and non-toxic fashion.
As of today, more than one hundred CPPs are available with varying physicochemical properties and internalization mechanisms. In order to sift out the most effective delivery vectors, their exact uptake mechanisms, kinetics and cargo delivery properties need to be determined.
Several cellular internalization pathways have been proposed for these molecules and the majority of studies conclude that CPPs utilize several uptake routes simultaneously with some favored more than others. The reports vary as internalization depends on the type and concentration of the CPP, the nature of the cargo, the specific cell membrane composition of the studied cell line, the intracellular target and other experimental conditions.
In the present thesis, uptake mechanisms of several common CPPs are characterized by studying their cytosolic uptake kinetics using fluorescent and bioluminescent cargos. In addition, transfection mechanisms of non-covalent CPP-oligonucleotide nanocomplexes are investigated by assessing the effects of hydrophobic CPP modifications and complex formation on their efficacy.
Kinetic uptake studies coupled with endocytic pathway studies enable to determine the involvement and extent of each uptake route in CPP internalization in a more transparent fashion compared to single-endpoint methods. We therefore assessed the cytoplasmic uptake kinetics and mechanisms of several common CPPs using a quenched fluorescence assay and a semi-biological bioluminescence assay. The chosen peptides displayed very different and concentration dependent uptake kinetic profiles that were strongly affected by endocytosis inhibitors. Both of the studies support the simultaneous involvement of several endocytotic pathways in their cellular uptake.
Physicochemical studies of CPP-cargo complexes can reveal the uptake mechanisms of these dynamic non-covalent conjugates that can be used in the further development of more potent delivery vectors. We assessed the role of CPP hydrophobicity in the delivery of splice-correcting antisense oligonucleotides (SCOs). In addition, we characterized the interactions between CPPs and siRNA to shed light on their transfection mechanism.
To study the hydrophobic effects on transportan 10 mediated splice-correcting antisense oligonucleotide delivery, we conjugated different fatty acids to the peptide and measured how these modifications affect the hydrodynamic size and transfection efficacy of CPP-oligonucleotide complexes. We determined the optimal hydrophobic CPP modification and nanocomplex size for maximal splice correction efficiency.
In order to elucidate the role of complex formation in CPP-mediated siRNA transfections, we utilized isothermal calorimetry in conjunction with dynamic light scattering studies. The results revealed that although the complexes are with the same size at acidic and physiological pH, the amount of CPPs in the nanocomplexes varies, indicating that the dynamic equilibrium between the peptides and the cargo is pH-dependent.
The results presented in this thesis exemplify the advantage of kinetic assays as well as the importance of studying the physicochemical properties of non-covalent CPP-oligonucleotide nanocomplexes in understanding CPP and CPP-cargo uptake mechanisms. This thesis brings out crucial aspects that have to be accounted for when designing novel delivery vectors for biotechnological and clinical applications.