Cell-penetrating peptides, also known as protein transduction domain (PTD), have attracted interest as carriers for intracellular drug delivery. Originally, these short peptide sequences, typically with 5 to 30 amino acids, were derived from the trans-activating transcriptional activator of the HIV 1 [1, 2]. Cell-penetrating peptides have been used to transport numerous cargos across the cell membrane in vitro and in vivo  (e.g. drugs , nucleic acids , polymers , quantum dots , and proteins ). Although they have been studied for more than 25 years now , and have demonstrated considerable potential as carriers, some issues remain still unclear. For instance, cellular uptake efficiency can strongly depend on the different incubation conditions and the applied cell lines . The mechanism of uptake is intensively debated, although it is believed that the majority of PTDs enter the cell by some form of endocytosis, and many parameters can easily influence cellular internalization. The applied peptide concentration can alter the uptake from energy-dependent endocytosis at low concentration to direct translocation at elevated concentration [11, 12]. The work of Hirose et al. demonstrated that a particular combination of PTD and a small hydrophobic dye can lead to direct penetration, which highlighted the importance of the choice of payload when investigating or comparing different PTDs . The influences of the cargo and the combination of the peptide carrier could also explain some of the inconsistencies that exist in the PTD field, and it underlines that studies that aim to unveil the potential of particular peptide should first consider the cargo and the mode of attachment.
The aim of this study was to synthesize and characterize cell-penetrating peptide–drug conjugates, exploiting a novel crosslinking method and doxorubicin as a model drug. In addition, we compare two peptide carriers, which belong to different classes of PTDs, in order to study the influence of the nature of the peptide over the uptake efficiency and drug efficacy.
The two applied cell-penetrating peptides differ in their net charge at physiological pH and their secondary structure. First, a polyarginine, consisting of nine amino acids, eight arginines, and an N-terminal cysteine, was selected. Octaarginine is widely used in the context of arginine-rich PTDs, like HIV-1 Rev, HIV-1 transcriptional activator, HTLV-II Rex, and BMV Gag [2, 14]. Second, a proline-rich amphipathic cell-penetrating peptide [15, 16] containing three VELPPP units with a negative net charge was chosen. Various proline-rich sequences have been described in the literature and have shown the capacity to permeate cells and no cytotoxic effects, even at high concentrations [16-18]. Furthermore, the anionic cell-penetrating peptide bears a CGGW motif at its N-terminus, required for drug modification at the terminal cysteine.
As a model drug, we have chosen doxorubicin (DOX) for the attachment to the two PTDs, due to its intrinsic fluorescence, which enables the determination of cellular uptake, and intracellular distribution applying flow cytometry and fluorescence microscopy. Anthracycline drugs have a wide spectrum of activity against hematopoietic and solid tumors ; however, a reversible dose-dependent hematologic toxicity represents the acute dose-limiting toxicity, and their prolonged administration can induce cardiomyopathy and congestive heart failure . Another major drawback of doxorubicin is the onset of drug resistance; P-glycoprotein overexpressed by resistant cells is extruding the drug, causing low intracellular retention . Therefore, doxorubicin and its analogues have been modified in order to improve the drug's biological properties on the one hand and to reduce the severe side effects on the other hand. Doxorubicin modification can be performed either in a noncovalent fashion, encapsulated in liposomes, e.g. , or by alteration of the drugs structure with the addition of a crosslinker. Heterobifunctional crosslinkers are extensively applied in drug modifications, and their utilization has proven to be very important for the attachment of diverse carriers, such as monoclonal antibodies [23, 24], proteins , polymers , and peptides , to doxorubicin. Established conjugation technique is applied at the C-13 keto group by hydrazones, due to their fast hydrolysis in acidic environment existing in biological compartments like endosomes and lysosomes [28, 29]. However, the insufficient stability of the doxorubicin hydrazone conjugates has been reported even at physiological pH (7.4) , leading to the release of the free drug in the bloodstream. In order to overcome these difficulties, we have chosen a crosslinker capable of creating an oxime bond on doxorubicin's ketone, due to the higher hydrolytic stability of the oxime group . Additionally, thiol-containing carriers, like albumin proteins, have also been conjugated to anthracyclines utilizing functional groups that are highly specific for sulfhydryl groups, e.g. maleimides and pyridyl disulfides . The application of pyridyl disulfides is advantageous, because a disulfide bond between the linker and the cargo is formed, which can be reduced in the cytoplasm by glutathione to deliver the freight . Furthermore, pyridyl disulfides can serve as a protective group during synthesis to avoid undesired dimerization as well as an activating group for the thiol to facilitate disulfide formation . In contrast, maleimides react with a thiol via Michael addition; thus, a covalent bond is created that cannot be cleaved under physiological conditions. Therefore, we have selected a heterobifunctional crosslinker that contains a protected aminooxy group and pyridyl disulfide.