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Cell-penetrating homochiral cyclic peptides as nuclear-targeting molecular transporters.

The intracellular delivery of biologically active cargos by employing linear cell-penetrating peptides (CPPs) has been previously reported. Conjugation to linear cationic CPPs, such as TAT (trans-acting activator of transcription; a peptide derived from the HIV-1 transactivator protein), 3] penetratin, antennapedia, or oligoarginine, efficiently enhances the cellular uptake through different mechanisms. The cellular uptake and internalization of many CPPs along with the conjugated cargo occurs predominantly by an endocytic pathway that involves macropinocytosis, a caveolae pathway, clathrin-mediated endocytosis, or lipid-raft dependent endocytosis. Endosomal uptake represents a major challenge in targeted intracellular drug delivery since some compounds are trapped in endosomes and cannot reach the biological targets in the cytoplasm or nucleus. Thus, strategies that promote endosomal escape or avoid endosomal routes are required for improving bioavailability. Moreover, the nuclear delivery of cell-impermeable and water-insoluble molecules remains a major challenge. The nucleus is a desirable target because the genetic information of the cell and transcription machinery resides there. To date, most approaches for nuclear delivery of compounds have taken advantage of covalent conjugation, which requires release of the cargo from the conjugate and/or endosomal escape. There is therefore a need to develop alternative stable peptide carriers that avoid endosomal pathways and/or covalent conjugation. Compared to linear peptides that are susceptible to hydrolysis by endogenous peptidases, cyclic peptides are enzymatically more stable. The cell-penetrating properties and application of homochiral l-cyclic peptides in drug delivery remain unexplored. Previous studies on linear CPPs by our research group and others indicated that an optimal balance of positive charge and hydrophobicity is required for interactions with the cell membrane and deep penetration into the lipid bilayer. 4,8–10] Herein, we report the design and evaluation of amphipathic homochiral l-cyclic peptides for potential applications as CPPs and/or as molecular transporters of bioactive compounds. Eleven cyclic peptides, namely [WR]4, [FK]4, [AK]4, [EL]4, [RFEF]2, [EK]4, [ER]4, [FR]4, [RFE]3, [WR]3, and [WR]5 (Scheme 1), which contain l-amino acids, were synthesized by employing 9-fluorenylmethyloxycarbonyl (Fmoc) based peptide chemistry. The selection of the cyclic peptides was based on the presence of hydrophobic residues (e.g., W, F, L) and charged residues (e.g., K, R, E). We hypothesized that an optimal amphipathic cyclic peptide that contains appropriate residues, and that undergoes intermolecular and intramolecular interactions can act as a CPP and/or entrap and deliver a bioactive compound intracellularly. To examine the potential application of the cyclic peptides as molecular transporters, a model experiment was performed with lamivudine (( )-2’,3’-dideoxy-3’-thiacytidine, 3TC). 3TC is a nucleoside reverse transcriptase inhibitor that blocks HIV-1 and hepatitis B virus replication. The efficient cellular uptake of 3TC is critical for effective antiviral activity. To monitor the molecular transport ability of the cyclic peptides, a carboxyfluorescein derivative of 3TC (F-3TC) was synthesized. The cellular uptake of the fluorescently labeled 3TC (F3TC) was examined in the leukemia CCRF-CEM cell line in the presence or absence of cyclic peptides. After 1 h incubation at 37 8C, the cells were treated with trypsin to remove the cell-surface-bound drug. The cellular uptake of F3TC was monitored by fluorescence-activated cell sorting (FACS; Figure 1a) and fluorescence microscopy (Figure 1b). The cyclic peptides did not exhibit any cytotoxicity by using an MTT assay at an experimental concentration of 50 mm in four different cell lines, namely CCRF-CEM, HT-29, MDAMD-468, and SK-OV-3, thus showing consistent results (Figure S12). FACS and fluorescence microscopy showed significantly higher fluorescence signals in the cells treated with F-3TCloaded [WR]4 and [WR]5 compared to those treated with other F-3TC-loaded cyclic peptides and with F-3TC alone, thus suggesting that the uptake of F-3TC is facilitated by [WR]n (n = 4, 5) and is dependent on nature of amino acids. F3TC-loaded [WR]5 exhibited a cellular uptake that was approximately five times higher than that of F-3TC alone (Figure 1a). Phosphopeptides are valuable probes for studying phosphoprotein–protein interactions because these peptides mimic the interactions between the negatively charged phosphate group of phosphoproteins and positively charged amino acids in the binding pockets of a number of proteins. Studying negatively charged phosphopeptides in cellular systems is challenging because these peptides do not readily [*] Dr. D. Mandal, A. Nasrolahi Shirazi, Prof. K. Parang Department of Biomedical and Pharmaceutical Sciences University of Rhode Island 41 Lower College Road, Kingston, RI 02881 (USA) E-mail: kparang@uri.edu