SYNTHESIS AND EVALUATION OF AMPHIPHILIC PEPTIDES AS NANOSTRUCTURES AND DRUG DELIVERY TOOLS

Author

Naser Ali Sayeh, University of Rhode IslandFollow

Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Keykavous Parang

Abstract

Intracellular delivery of cell-impermeable compounds in a variety cells using delivery systems have been extensively studied in recent years. Obtaining desirable cellular uptake levels often requires the administration of high quantities of drugs to achieve the expected intracellular biological effect. Thus, improving the translocation process across the plasma membrane will significantly reduce the quantity of required administered drug and consequently minimize the side effects in most of the cases. Efficient delivery of these molecules to the cells and tissues is a difficult challenge. Compounds with low cellular permeability are commonly considered to be of limited therapeutic value. Over the past few decades, several biomedical carriers, such as polymers, nanospheres, nanocapsules, liposomes, micelles, peptides and dendrimers have been widely used to deliver therapeutic and diagnostic agents to the cells. Biomaterials generated from nano-scale compounds have shown some promising data for delivery of many compounds in a number of diseases, such as viral infections, cancer, and genetic disorders. Although much progress has been achieved in this field, many challenges still remain, such as toxicity and limited stability.

Liposomes suffer from poor stability in the bloodstream and leakage during storage. They tend to aggregate and fuse with or leak entrapped drugs, especially highly hydrophilic small molecules. For solid lipid nanoparticles (SLNs), drug expulsion after polymorphic transition during storage, inadequate loading capacity, and relatively high water content of the dispersions have been observed.

Poly lactic-co-glycolic acid (PLGA) degrades in the body producing its original monomers of lactic acid and glycolic acid, which are the by-products of various metabolic pathways. However, this acidic microenvironment that occurs during degradation could negatively affect the stability of the loaded compound. Dendrimers can carry drugs as complexes or as conjugates although one limitation lies in the effort of controlling the rate of drug release. The encapsulated or complexed drugs tend to be released rapidly (before reaching the target site) and in the dendrimer–drug conjugates, it is the chemical linkage that controls the drug release. Thus, future studies in this field are urgently required to create more efficient and stable biomaterials.

Peptides are considered as efficient vectors for achieving optimal cellular uptake. The potential use of peptides as drug delivery vectors received much attention by the discovery of several cell-penetrating peptides (CPPs). The first CPPs, discovered in 1988, were sequences from HIV-1 encoded TAT protein, TAT (48–60), and penetrated very efficiently through cell membranes of cultured mammalian cells. CPPs are a class of diverse peptides, typically with 8–52 amino acids, and unlike most peptides, they can cross the cellular membrane with more efficiency. CPPs have also shown to undergo self-assembly and generate nanostructures. The generation of self-assembled peptides and nanostructures occur through various types of interactions between functional groups of amino acid residues, such as electrostatic, hydrophobic, and hydrogen bonding. Appropriate design and functionalization of peptides are critical for generating nanostructures.

Chemically CPPs are classified into two major groups: linear and cyclic peptides. It has been previously reported that linear peptides containing hydrophilic and hydrophobic amino acids could act as membrane protein stabilizers. These compounds are short hydrophilic or amphiphilic peptides that have positively charged amino acids, such as arginine, lysine or histidine, which can interact with the negative charge phospholipids layer on the cell membrane and translocate the cargo into the cells.

Conjugation to cationic linear CPPs, such as TAT, penetratin, or oligoarginine efficiently improves the cellular uptake of large hydrophilic molecules, but the cellular uptake is predominantly via an unproductive endosomal pathway. Therefore, the biological effect is very limited, as the compounds are trapped in these compartments and cannot reach their biological targets in the cytoplasm or the nucleus. Mechanisms that promote endosomal escape or avoid endosomal route are required for improving bioavailability. Highly cationic CPPs preferentially interact with particular cell types, have limited plasma half-life, show toxicity, do not cross multicellular barriers such as vasculature epithelia or the blood-brain barrier, and efficient cargo delivery requires 9-15 arginine residues. Highly cationic CPPs are, therefore not ideal small molecule drug delivery vehicles. Linear CPPs are susceptible to hydrolysis by endogenous peptidases. Conjugation to cationic CPPs, such as TAT, penetratin, or oligoarginine efficiently improves the cellular uptake of large hydrophilic molecules, but the cellular uptake occurs predominantly via an unproductive endosomal pathway. Therefore, the biological effect is very limited, as the compounds are trapped in these compartments and cannot reach their biological targets in the cytoplasm or the nucleus.

Mechanisms that promote endosomal escape or avoid endosomal route are required for improving bioavailability. Highly cationic CPPs preferentially interact with particular cell types, have limited plasma half-life, show toxicity, do not cross multicellular barriers such as vasculature epithelia or the blood-brain barrier, and efficient cargo delivery requires 9-15 arginine residues. Highly cationic linear CPPs are, therefore, have not become optimized as small molecule drug delivery vehicles.

On the other hand, cyclic peptides containing hydrophilic and hydrophobic amino acids have shown greater potential as drug delivery tools due to their enhanced chemical and enzymatic stability. Parang's laboratory has reported that Amphiphilic Cyclic Peptides (ACPs) containing positively charged arginine and hydrophobic tryptophan residues as potential candidates for drug delivery. Cyclic peptides have several benefits compared to linear peptides, such as rigidness of structure and stability against proteolytic enzymes. 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. Cyclosporine, gramicidin S, polymoxin B, and daptomycin are well-known examples of cyclic peptide drugs. Parang's laboratory designed amphiphilic cyclic CPPs containing alternative tryptophan and arginine residues as the positively charged and hydrophobic residues, respectively. The peptides were efficient in improving the cellular delivery of anticancer and antiviral drugs.

The cellular uptake mechanism of CPPs into cells is still a matter of some debate. The cellular entry of CPP can be influenced by the type of CPP, the cell line, the nature of the cargo, and the conditions of incubation. As described above, linear CPPs pass through the plasma membrane mostly via an energy-independent or endocytosis pathway.

Moreover, the cellular delivery of CPP-conjugated molecules also occurs through endosomal pathway and a strong enzymatic degradation and an inadequate cytoplasmic release of intact molecules from the conjugates are expected, thus leading to an inefficient transfer into the cytoplasm. The best strategy to overcome this issue is to designing CPP that by pass the endosomal uptake or by increasing the escape rate from the endosome to improve the intracellular delivery of CPP-attached molecules.

Parang laboratory has reported the cellular uptake of a number of cyclic peptides independent of endocytotic pathway. The extraordinary ability of cyclic peptides containing tryptophan and arginine, [WR]₄ and [WR]₅ to spontaneously translocate across bilayers independent of an energy source is distinctly different from the behavior of the well-known, highly cationic CPPs, such as TAT and Arg9, which do not translocate across phospholipid bilayers, and enter cells mostly by active endocytosis.

Alternatively, researchers have found that an effective cellular delivery vector can be improved developed by conjugating a CPP with a fatty acid chain. Amphiphilic peptides have also become a subject of major interest as potent antibacterial agents. Antimicrobial peptides (AMPs) are produced naturally by bacteria and are considered as the first line of host defense protecting against microbial infection. Various types of AMPs have been discovered, such as defensins, cecropins, magainins and cathelicidins, with significant different structures and bioactivity profiles.

These peptides are reported to be effectors and regulators of the innate immune system by increasing production and release of chemokines, and enhance wound healing and angiogenesis. They are able to suppress biofilm formation and induce the dissolution of existing biofilms. Thus, design of new AMPs with higher activity are urgently needed.

Although a number of cyclic peptides were discovered and reported as efficient cellular delivery agents and/or antimicrobial agents, a more systematic investigation is required to identify design rules for optimal entrapment, drug loading, and stability.

The balance of many small forces determines the overall morphology, size, and functionality of the structures. A deeper understanding of these factors is required for guiding future research, and for customizing cyclic peptides for drug loading and cellular delivery applications.

Thus, additional amphiphilic cyclic and linear peptides with variable electrostatic and hydrophobic residues were designed here to optimize drug encapsulation. The diversity in ring size, amino acid number, position and sequences, number of rings, net charge, and hydrophobicity of side chains in cyclic peptides will allow us to explore requirements for generating peptides with optimized drug encapsulation and to establish correlations between the structure of peptides with their drug entrapment properties. Thus, the general objective of this dissertation was to design and evaluate additional cyclic or amphiphilic peptides as nanostructures and to compare their efficiency in delivery of small molecules with the previously reported cyclic peptides containing tryptophan and arginine residues. This dissertation consists of three chapters.

Chapter 1. MANUSCRIPT І (published in Current Organic Chemistry 2014). The objective of this work was to design amphiphilic linear and cyclic peptides containing hydrophobic tryptophan W residues that were linked through a triazole ring to positively charged arginine (R) and lysine (K) residues. The peptides were synthesized through click chemistry between hydrophobic peptides containing alkyne and positively charged peptides containing azide groups. Their structures were investigated using solubility tests, circular dichorism (CD), and transmission electron microscopy (TEM), cytotoxicity assays. The conjugates showed minimal cytotoxicity in two cell lines. The secondary structures of both peptides were similar to a distorted α-helix as shown by CD spectroscopy. TEM imaging also showed that linear-linear (WG(triazole-KR-NH₂))₃ and cyclic-linear [WG(triazole-KR-NH₂)]₃ peptides formed nano-sized structures.

Chapter 2. MANUSCRIPT ІI (Submitted to Journal of Molecular Modeling). In this work, we investigated the structural and dynamical aspects of cyclic-linear peptide ([WG(triazole-KR-NH₂)]₃ and linear-linear peptide (WG(triazole-KR-NH₂))₃) formed nanostructures compared to a drug delivery system with [WR]₄. While [WR]₄ was found to be an efficient molecular transporter for small molecule drugs, such as lamivudine and dasatinib, cyclic-linear peptide ([WG(triazole-KR-NH₂)]₃ was inefficient. Molecular modeling was used to explain the differential behavior of these peptides. We showed how the morphology of these systems can affect the drug delivery efficiency. The result of this work provided insights about optimizing the amphiphilic cyclic-linear trizaolyl peptides with more efficient drug delivery capabilities.

Chapter 3. MANUSCRIPT ІII. The objective of this Chapter was to synthesize a different series of amphiphilic peptides for different objectives. First, the amphiphilic trizaolyl peptides in Chapter I were systematically modified by increasing the number of arginine and tryptophan sequence in cyclic and linear peptides. The rationale for the modification was to enhance the possibility of interaction with the cell membrane and therefore improving the cellular uptake process. Moreover, a new class of amphiphilic peptides consisting of tryptophan and glutamic acid were conjugated with a peptide containing arginine and lysine residues using Fmoc-based peptide chemistry. These peptides have an amide bond that generates more flexibility compared to a triazole ring. Finally, additional fatty acids with different length chains were conjugated with positively charged peptides and evaluated as antibacterial agents. Stearic acid (C16) and myristic acid (C14) were conjugated with peptides consisting of arginine azide and lysine amino acids to enhance the antibacterial activity.

In summary, the work in this dissertation provided insights about the synthesis and characterization of a new class of amphiphilic triazolyl peptides as drug delivery carriers and amphiphilic peptides as antibacterial agents. Molecular modeling was used to explain why triazolyl peptides were unable to enhance the delivery of small molecule drugs compared to the previously synthesized cyclic peptides [WR]₄ (Chapter 2) Modification of synthesized peptides in Chapter 1, by addition of more positively charged amino acids or reducing the rigidity by incorporating amide bonds instead of triazolyl groups can be used to improve the cell penetrating properties. Finally, we conjugated amphiphilic peptides with different fatty acids (Chapter 3) to investigate their application as antibacterial agents.

Recommended Citation

Sayeh, Naser Ali, "SYNTHESIS AND EVALUATION OF AMPHIPHILIC PEPTIDES AS NANOSTRUCTURES AND DRUG DELIVERY TOOLS" (2014). Open Access Dissertations. Paper 296.
https://digitalcommons.uri.edu/oa_diss/296