Date of Award


Degree Type


Degree Name

Doctor of Philosophy in Pharmaceutical Sciences


Interdepartmental Program

First Advisor

Keykavous Parang


Cell membrane is a barrier to be overcome for efficient delivery of therapeutics into a target site in cytoplasm or nucleus. The hydrophobic phospholipids are major components of the cell membrane that obstruct the transportation of therapeutics. Thus, various delivery systems, such as liposomes, nanoparticles and viral vectors, have been developed to transfer small molecules, peptides, proteins, and oligonucleotides across the membrane.

Negatively charged phosphopeptides, oligonucleotides, and siRNAs have emerged as potential therapeutic agents. Phosphopeptides mimic phosphoproteins, which give on/off signal to many enzymes through interactions with protein kinases. For example, phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) is an optimal peptide ligand for binding to the Src tyrosine kinase SH2 domain. Oligonucleotides have been introduced as antisense drugs to inhibit the translation of mRNA that transfers the coding information from genes. Small interfering RNA (siRNA)-based therapy has been also spotlighted since the discovery of RNA interference (RNAi) phenomenon. However, the cellular delivery of phosphopeptides, oligonucleotides, and siRNAs is a major obstacle despite many advantages of these compounds. Phosphopeptides contain negatively charged phosphate group, and/or negatively charged amino acids, such as glutamic acid or aspartic acid, in their sequences. Oligonucleotides and siRNAs are polymers composed of nucleosides, which are connected through negatively charged phosphodiester groups. These negatively charged molecules are hard to enter cancer cells by diffusion because cancer cell membranes are composed of negatively charged lipids. In addition, when a naked siRNA is administered in vivo, it

does not show efficient cellular uptake in most mammalian cells and is quickly disappeared in the blood. Thus, developing carriers to improve the cellular uptake delivery of negatively charged cell-impermeable compounds has become a subject of major interest. Novel strategies are urgently needed to circumvent the problems associated with the delivery of these compounds.

Cell-penetrating peptides (CPPs) have become one of the emerging vehicles for delivery of cargo drugs. CPPs are short hydrophilic or amphiphilic peptides that have plenty of positively charged amino acids, such as lysine or arginine, which can penetrate cell membranes. CPP-drug conjugates have been reported to help the cellular uptake of some drugs. Alternatively, they have been used as non-covalent drug delivery systems.

CPPs have been investigated for improving the intracellular delivery of negatively-charged molecules. By physical interaction between positive charges in CPPs and negative charges in phosphopeptides, oligonucleotides, and siRNAs, the cell penetration could be improved. Among many CPPs, arginine-rich peptides have been the subject of major focus because it has been known that the guanidine group of arginine side chain shows better interaction with the negatively charged phospholipid in the cell membrane. Tryptophan is also a key amino acid found in CPPs that enhances the interaction of peptides with lipids in the cell membrane.

Parang’s laboratory has previously shown that monocyclic CPPs containing alternative arginine and tryptophan have potential applications for drug delivery. Cyclic peptides have several benefits compared to linear peptides, such as stability against proteolytic enzymes and rigidness of structure. The rigidity of the structure can enhance the binding affinity of ligands toward receptors by reducing the freedom of possible structural conformations. Cyclic peptides are also present in nature and have been developed as therapeutics. Cyclosporin, gramicin S, polymoxin B, and daptomycin are well-known examples of cyclic peptides. Parang’s laboratory designed amphiphilic cyclic CPPs containing alternative tryptophan and arginine residues as the hydrophobic and positively charged residues, respectively. The peptides were efficient in improving the cellular delivery of anticancer and antiviral drugs.

In this dissertation, we designed novel classes of amphiphilic cyclic peptides for improving the intracellular delivery of cell-impermeable phosphopeptides, and their antimicrobial activities were investigated. The hypothesis of this dissertation is that amphiphilic cyclic peptides, having positively charged arginines on one side of structures and hydrophobic tryptophan (or fatty acid) on the other side, can enhance intracellular drug delivery and/or act as antimicrobial agents having synergy with other antibiotics.

In Manuscript I (Submitted to Angewandte Chemie International Edition), we designed amphiphilic bicyclic peptides as cellular delivery agents. The objective of this manuscript was to design a novel class of bicyclic CPPs containing two monocyclic peptides of tryptophan and arginine amino acids. Two bicyclic peptides [W5G]-(triazole)-[KR5] and [W5E]-(-Ala)-[KR5] were synthesized by conjugation of two monocyclic peptides using click chemistry and amide synthesis, respectively. A corresponding linear peptide, W5G-(triazole)-KR5, and a monocyclic peptide with a linear component, [W5G]-(triazole)-KR5, were synthesized as controls. Among all peptides, [W5E]-(-Ala)-[KR5] improved the cellular delivery of fluorescein-labeled phosphopeptide, F-GpYEEI by 19.3-fold. Confocal microscopy showed that the corresponding fluorescein-labeled bicyclic peptide F-[KW4E]-(-Ala)-[KR5] was localized in the cytosol and nucleus in human ovarian adenocarcinoma (SK-OV-3) cells. Studying the cellular uptake of F-[KW4E]-(-Ala)-[KR5] in the presence of endoycytosis inhibitors indicated that the clathrin- and caveolin-dependent endocytosis were the main pathways for cellular uptake. [W5E]-(-Ala)-[KR5] enhanced the intracellular uptake of fluorescein-labeled phosphopeptide, F-GpYEEI by 4.5- and 3.0-fold compared to those of well-known cell-penetrating peptides (CPPs), TAT and CR7, respectively. The bicyclic peptide was able to improve antiproliferative activity of doxorubicin by 20%. Thus, this manuscript suggests that amphiphilic bicyclic peptides containing tryptophan and arginine can be utilized as a new class of cell-penetrating peptides and potential cellular delivery tools.

In Manuscript II (Submitted to Molecular Pharmaceutics), we investigated the role of fatty acylation and cyclization for intracellular transport of phophopepides in short-length polyarginine peptides Most of the reported arginine-rich CPPs to enhance intracellular drug delivery are linear peptides, and have more than seven arginines to retain cell penetrating properties. Herein, we synthesized penta and hexaarginine peptides (R5 and R6), and explored the effect of acylation and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5(6)-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. The molecular transporter property of fatty acylated cyclic peptides was compared with those of fatty acylated linear peptide and nonacylated cyclic peptide using flow cytometry. The combination of acylation and cyclization (dodecanoyl-[R5]) enhanced intracellular delivery of a fluorescence-labeled phosphopeptide (F′-GpYEEI) in human SK-OV-3 cancer cell line. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that of in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. A comparative FACS results showed that dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4-2.5 fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. In this manuscript, we found that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids.

In Manuscript III (to be submitted to Molecular Pharmaceutics), the antimicrobial activities of cyclic CPPs were investigated against multidrug resistant pathogens. Antimicrobial peptides and CPPs share similar structural features. Based on the intracellular delivery property of amphiphilic cyclic peptides in manuscript II, we synthesized several amphiphilic cyclic CPPs and their analogs, and investigated antibacterial activities against multidrug resistant pathogens. [R4W4] exhibited a potent antibacterial activity, exhibiting MIC value of 2.67 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA). Cyclic [R4W4] and the linear counterpart R4W4 exhibited MIC values of 42.8 and 21.7 μg/mL, respectively, against Pseudomonas aeruginosa. [R4W4] in combination with tetracycline enhanced the potency, by decreasing the MIC 4 fold (0.12 μg/mL), suggesting partial synergistic effect of the combination between [R4W4] and tetracycline against MRSA. Twenty-four hour time-kill studies evaluating [R4W4] in combination with tetracycline demonstrated bactericidal activity against MRSA and E. coli. [R4W4] showed cell-penetrating properties as expected, and exhibited more than 84% cell viability at 15 μM (20.5 μg/mL) concentration against three different human cell lines. This study suggests that amphiphilic cyclic CPPs, when used in combination with antimicrobials could provide additional benefit to defeat multi-drug resistant pathogens.

In summary, the studies in this dissertation provided insights and a deep understanding of applications of cyclic cell-penetrating peptides to enhance intracellular uptake of cargo drugs, and their antimicrobial activities as drug alone or combination with other antibiotics. Amphiphilic bicyclic peptides are the first reported bicyclic peptides as CPPs and molecular transporters. Acylated cyclic polyarginines showed that short polyarginines can be utilized as CPPs to have cell-penetrating properties by combining fatty acylation and cyclization. Moreover, this study provided a potential of amphiphilic cyclic CPPs as antimicrobial agents that their potency could be maximized by the combination with other antibiotics possibly through their drug delivery properties. Overall, these findings will be beneficial for the scientific community in academia and industry working in the area of designing molecular transporters of cell impermeable compounds, and cellular delivery.



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