Date of Award


Degree Type


Degree Name

Doctor of Philosophy in Pharmaceutical Sciences


Biomedical and Pharmaceutical Sciences

First Advisor

Keykavous Parang


Over the past two decades, drug delivery systems have become a subject of major interest in the pharmaceutical industry for the treatment of different diseases, such as cancer, viral infections, and genetic disorders. A wide range of compounds show high binding affinity and great potential in in vitro non-cellular assays through direct interacting with molecular targets. However, many compounds show low potency in cell-based assays due to their limited delivery across the cell membrane. The physicochemical properties of several compounds, such as size, poor water solubility, hydrophobic nature, and negative charge, limit their cellular uptake significantly. Thus, there is an urgent need in design and synthesis of novel molecular transporters for efficient delivery of effective compounds to cellular targets.

The recent growth of nanotechnology products will expand the current resources of therapeutics for the pharmaceutical industry in the next few years. The use of nanotechnology in drug delivery is widely expected to change the future of the pharmaceutical and biotechnology industries. The application of nanotechnology in drug delivery has led to the discovery of nano Drug Delivery Systems (nano-DDS).

Nanocarriers exhibit unique properties and take advantage of specific physiochemical behavior of nanoparticles. Properties such as, magnetism, conductivity, melting and boiling points, and chemical and biological reactivity become different at nano scale due to the quantum mechanical behavior of extremely small structures at molecular dimensions. Furthermore, nanoparticles behave differently since they take advantage of extraordinary high surface area to mass ratio, leading to increased surface interactions and distinct biological performance. Nanoparticles have the potential to be manipulated through changing their size, electronic, and hydrophobic nature. Employing nano-sized carriers offer several advantages, including enhanced intracellular delivery of poorly water-soluble drugs, targeted delivery, transporting relatively large biologically important molecules across the cell membrane, delivery of a combination of drugs to overcome drug resistance, and transporting drugs through challenging epithelial and endothelial barriers.

Thus, design and development of nano-sized pharmaceutical carriers has become attractive for chemists, biologists, and physicists. Functionalized Nano-DDS are designed to deliver and release cargo drugs intracellularly more efficiently than the currently available systems, leading to the enhanced drug tissue bioavailability and eventually therapeutics efficacy.

Among all nano-DDS, Cell-Penetrating Peptide (CPP)-mediated intracellular DDS has been widely used for the enhanced delivery of water insoluble drugs, negatively charged molecules (e.g., DNA, siRNA, phosphopeptides), and proteins. However, the application of (CPP) mediated intracellular DDS in in vivo models has been challenging due to their inherent toxicity to normal cells and organs. Several studies have been performed to facilitate the intracellular delivery of a wide range of low-molecular weight and macromolecular drugs using carriers through the cell cytoplasm bypassing the endocytic pathway to avoid lysosomal degradation. In this process, although the drug is delivered into cytoplasm protected, it still needs to be transported to a certain organelles, such as mitochondria or nuclei, where therapeutic function occurs. Several CPPs promote the delivery of drugs through receptor-mediated endocytosis. However, this mechanism requires high affinity between the drug-carrier complex and the target in the cell membrane for endocytosis. This process will be followed by energy-dependant formation of endosomes. However, this method suffers from entrapment and release challenges. After delivery of the molecular cargo into cells through endocytic pathway, and its entrapment in endosome, the cargo molecules or drug may end up in lysosome and degraded by the lysosomal enzymes. Thus, a limited amount of the drug can reach the cytoplasm because of inadequate endosomal escape and lysosomal degradation. As a result, although numerous CPPs exhibit promising results in in vitro assays, they fail in vivo because of the poor bioavailability.

Furthermore, the nuclear delivery of cargo drugs with known CPPs has been unsuccessful. The nucleus of a cell is an important target for drug delivery systems, due to presence of the genetic information and transcription machinery. An efficient cellular uptake of the drug is highly desired in nucleus where it can interact with nucleic acids. The nuclear targeting delivery is challenging. The designed drug-CPP complex must meet several requirements for nuclear delivery including efficient cell internalization by receptor-mediated endocytosis (RME), escaping endosomal/lysosomal pathways, acquiring a nuclear localization signal to communicate with nuclear pore complexes, and eventually, being sufficiently small to enter the nuclear membrane. Not many CPPs have been found efficient for the nuclear delivery applications.

CPPs can be classified into two major classes, linear and cyclic CPPs. Most reported CPPs are linear peptides that are flexible in solution and contain up to 10 amino acids. However, cyclization of peptides has been employed as a strategy to generate constrained structures. The rigidity imposed by cyclization reduces the flexibility and causes the system to adopt a restricted numbers of molecular conformations. Peptide cyclization has become a unique approach to generate conformations not available to linear peptides. Cyclic and linear peptides containing an equal number of similar amino acids create different geometries leading to different affinities for similar targets. Furthermore, cyclic CPPs containing specific amino acids have shown to have a different cellular uptake mechanism compared to linear CPPs. While most of the linear CPPs undergo endocytosis pathways in cellular uptake, some of the cyclic CPPs have endocytosis-independent uptake and target nucleus. Thus, cyclic peptides can be designed to be used as nuclear delivery vehicles of anticancer compounds targeting DNA. Functionalizing cyclic peptides with tumor targeting moieties can be used as a strategy for selective cancer cell targeting and improving nuclear targeting of anticancer drugs.

Cyclic CPPs can be also covalently conjugated to active drug cargos to generate prodrugs. Prodrugs are chemically modified analogs of an active metabolite that can improve pharmacokinetics and pharmacodynamics (PK/PD) properties of the drug. Prodrug strategy could offer several advantageous, such as enhancing water solubility, drug delivery, and chemical stability, and reducing toxicity. However, chemical transformation to the active drug is required in the presence of different intracellular enzymes to convert prodrugs to their corresponding pharmacologically potent moieties in in vivo systems. Cyclic peptide-drug conjugates can be used as alternative prodrug approach for improving delivery of compounds with limited cellular uptake.

This dissertation focuses on a class of cyclic peptides as intracellular molecular transporters that can be used as prodrugs or peptide-capped gold nanoparticles. We hypothesized that the combination of alternate hydrophobic tryptophan and positively charged arginine or lysine residues in the sequence of the cell-penetrating cyclic peptide was critical for improving the cell-penetrating properties of the system. Furthermore, the presence of arginine and tryptophan facilitated the formation of gold nanoparticles. The molecular transporting efficiency of the peptide alone and peptide-capped gold nanoparticles was evaluated for a broad range of molecular cargos including anti-HIV drugs, anticancer agents, and negatively charged phosphopeptides.

The cyclic peptides containing alternate tryptophan and arginine residues and their corresponding peptide-capped gold nanoparticles enhanced the delivery of water insoluble drugs and negatively charged biologically important molecules. A broad range of parameters including concentration, toxicity, time, and different sequences of amino acids were explored to optimize the cellular uptake. Several characterization methods were used to determine the interaction between drugs and carrier through the formation of the complex. Different methods were also used to investigate the mechanism of the cellular uptake.

This work presents examples where prodrugs containing linear peptides are compared with their corresponding cyclic peptides in terms of biological properties and in delivery of different drugs. For example, cyclic peptides containing certain amino acids generated gold nanoparticles more efficiently compared to their corresponding linear peptides. Moreover, cyclic peptide capped gold nanoparticles exhibited higher molecular transporting potency when compared with the linear counterparts. This dissertation will be discussed in four manuscripts:

The objective of Manuscript І (published in Molecular Pharmaceutics 2013, 10(5), 2008-2020) was to evaluate a cyclic octapeptide containing arginine and tryptophan for their ability to transport negatively charged phosphopeptides. Phosphopeptides are important compounds for studying protein-protein interactions. Phosphopeptides suffer from their limited cellular uptake due to the presence of negatively charged phosphate group in their structure. The hypothesis of this manuscript is that cyclic peptide [WR]4 containing positively charged arginine can interact with negatively charged cell-impermeable phosphopeptides and improve their cellular uptake. Furthermore, the positively charged arginine and hydrophobic tryptophan can interact with negatively charged and hydrophobic residues in the cell membrane phospholipids and improve cell permeability of phosphopeptides. The cellular uptake of several biologically important phosphopeptides such as GpYLPQTV, NEpYTARQ, AEEEIY GEFEAKKKK, PEpYLGLD, pYVNVQNNH2, and GpYEEI was evaluated in the presence and absence of [WR]4. The cyclic peptide enhanced the cellular uptake of all phosphopeptides significantly in human leukemia cells (CCRF-CEM) after 2 h incubation. Confocal microscopy studies in live cells confirmed that the mixture of F′-PEpYLGLD and [WR]4 was localized in cells and showed higher cellular uptake than the phosphopeptides alone. Several tools including Transmission Electron Microscopy (TEM) and Isothermal Calorimetry (ITC) were used to understand the interaction between the drug and carrier. TEM results showed that the phosphopeptide-drug complex (PEpYLGLD-[WR]4) formed specific nano-sized structures with the 125 × 60 nm dimensions. The ITC investigation proved an exothermic interaction driven by entropy. The result of this paper showed that the presence of the cyclic peptide enhanced the cellular uptake of phosphopeptides significantly. To this end, it was discovered that [WR]4 can be employed as a cellular delivery tool of negatively charged phosphopeptides through non-covalent interaction. However, the evaluation of the covalent conjugation between the drug and cyclic peptide remained unexplored.

Manuscript ІІ (published in Molecular Pharmaceutics 2013, 10(2), 488-499) embarked on taking advantage of both the cell-penetrating properties of the peptide and prodrug strategy. The hypothesis underlying this project is that cyclic CPP-drug conjugates can be used as potential prodrug to improve the cellular delivery of anticancer compounds. Doxorubicin (Dox), a potent anticancer drug, was covalently linked with the cyclic peptide containing alternate tryptophan and arginine ([W(RW)4]) to afford their corresponding prodrug cyclic [W(RW)4]−Dox. To study the effect of the cyclic nature of the peptide, linear peptide attached to Dox (linear (RW)4−Dox) was also synthesized for comparative studies. The biological activities of cyclic [W(RW)4]−Dox and linear (RW)4−Dox prodrugs including their cellular uptake and anticancer potency were compared in cell-based assays. Comparative antiproliferative assays between covalent (cyclic [W(RW)4]−Dox and linear (RW)4−Dox) and the corresponding noncovalent physical mixtures of the peptides and Dox were performed. Cyclic [W(RW)4]−Dox inhibited the CCRF-CEM (62−73%), ovarian adenocarcinoma (SK-OV-3) (51−74%), colorectal carcinoma (HCT-116) (50−67%), and breast carcinoma (MDA-MB-468) (60−79%) cells at a concentration of 1 μM after 72−120 h of incubation. Cyclic [W(RW)4]−Dox exhibited higher antiproliferative activity than linear (RW)4−Dox in all cancer cells after 72 h.. Furthermore, the cellular uptake of [W(RW)4]−Dox was observed to be higher than that of the linear prodrug after 24 h incubation in SK-OV-3 cells, suggesting that the cyclic nature offers major advantageous when compared with the corresponding linear ones. The result of this manuscript revealed that cyclic prodrug (cyclic [W(RW)4]−Dox) can be used as a potential prodrug candidate for enhancing the cellular retention of the drug in the treatment of ovarian cancer.

In Manuscript III (published in Molecular Pharmaceutics, 2013, 10(2), 500-511), we investigated the application of cyclic peptides in generating gold nanoparticles. We hypothesize that cyclic peptides containing specific amino acids can be used as reducing agents and generate peptide-capped gold nanoparticles (CP-AuNPs) from gold ion (Au3+). Several cyclic peptides including [WR]4, [FK]4, [AK]4, [EL]4, [RFEF]2, [EK]4, and [FR]4 containing hydrophobic residues (W, F, L) and charged residues (K, R, E) where A = alanine, E = glutamic acid, F = phenylalanine, K = lysine, L = leucine, R = arginine, W= tryptophan were investigated for their ability to generate gold nanoparticles. Among all of them, only [WR]4 was discovered to be appropriate for the synthesis of CP-AuNPs. Flow cytometry studies showed that the cellular uptake of fluorescence-labeled anti-HIV drugs such as lamivudine, emtricitabine, and stavudine was significantly enhanced in human ovarian adenocarcinoma (SK-OV-3) cells in the presence of [WR]4-AuNPs. For instance, the cellular uptake of fluorescence labeled lamivudine-loaded [WR]4-AuNPs showed 12-fold higher cellular uptake compared to fluorescence labeled lamivudine alone in CCRF-CEM cells after 2 h incubation. The morphology and size of nanoparticles were characterized using TEM.. TEM results showed [WR]4-AuNPs form nano-sized structures with approximate size 5-50 nm. [WR]4-AuNPs (100 μM) did not show significant toxicity in CCRF-CEM, SK-OV-3, and normal human colon myofibroblast (CCD-18Co) cells after 24-72 h incubation. To this point, we understand that the cell-penetrating peptide-capped gold nanoparticles have potential to be used as prodrugs for delivery of antiviral and anticancer drugs through noncovalent conjugation. However, this strategy was required to be evaluated for non-cell penetrating peptides. Thus, we used a sequence of amino acids for designing a non-cell-penetrating peptide that generated peptide-capped gold nanoparticles with molecular transporting property.

In Manuscript ІV, (published in Molecular Pharmaceutics, DOI: 10.1021/mp400199e), a green method for the synthesis of non-cell penetrating peptide-capped gold nanoparticles was reported. We hypothesize that a non-cell penetrating peptide containing lysine and tryptophan namely linear (KW)5 and cyclic [KW]5 can be converted to a cell-penetrating molecular transporter upon generation of cyclic peptide-capped gold nanoparticles (linear (KW)5-AuNPs and cyclic [KW]5-AuNPs). A comparative flow cytometry results revealed that the cellular uptake of fluorescence-labeled anti-HIV drugs (emtricitabine (FTC) and lamivudine (3TC)) in CCRF-CEM cells, and a negatively charged cell-impermeable phosphopeptide (GpYEEI) in SK-OV-3 cells was significantly higher in the presence of cyclic [KW]5-AuNPs than that of linear (KW)5-AuNPs, parent cyclic [KW]5, and linear (KW)5 peptides. For example, the cellular uptake of F’-GpYEEI was enhanced 12.8-fold by cyclic [KW]5-AuNPs when compared with F’-GpYEEI alone. Microscopy results showed that fluorescence-labeled-3TC-loaded cyclic[KW]5-AuNPs were localized in nucleus in SK-OV-3 cells after 1 h incubation. However, linear (KW)5-AuNPs delivered the majority of fluorescence-labeled-3TC into cell cytoplasm. The results from this work suggest that non-cell penetrating peptides can be converted to efficient drug carriers by forming peptide-capped gold nanoparticles.

[Figure 1 can not be displayed here, refer to PDF]

In summary, the studies described in this dissertation provided insights about the application of a new generation of cyclic peptides for the delivery of a broad range of drugs and biomolecules. The cyclic peptide containing alternate tryptophan and arginine residues was found to be efficient molecular transporters and nuclear delivery agents (Figure 1). In addition, we have demonstrated that a non-cell penetrating cyclic peptide can be converted to an efficient intracellular drug transporter through generation and capping of the gold nanoparticles. A detailed investigation on the cyclic nature of the peptide compared to the linear one in terms of their chemical behavior and biological activity was provided. Overall, a concrete strategy has been established by developing non-toxic cyclic peptide molecular transporters that can be used in prodrug strategy or gold nanoparticle formation. The scientific knowledge was advanced in the area of application of cell-penetration peptides in prodrug strategy and designing nanocarriers.