Amphipathic, alpha-helical antimicrobial peptides

Gene-encoded antimicrobial peptides are an important component of host defense in animals ranging from insects to mammals. They do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. They are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. A particularly abundant and widespread class of antimicrobial peptides are those with amphipathic, alpha-helical domains. Due to their relatively small size and synthetic accessibility, these peptides have been extensively studied and have generated a substantial amount of structure-activity relationship (SAR) data. In this review, alpha-helical antimicrobial peptides are considered from the point of view of six interrelated structural and physicochemical parameters that modulate their activity and specificity: sequence, size, structuring, charge, amphipathicity, and hydrophobicity. It begins by providing an overview of how these vary in peptides from different natural sources. It then analyzes how they relate to the currently accepted model for the mode of action of alpha-helical peptides, and discusses what the numerous SAR studies that have been carried out on these compounds and their analogues can tell us. A comparative analysis of the many alpha-helical, antimicrobial peptide sequences that are now available then provides further information on how these parameters are distributed and interrelated. Finally, the systematic variation of parameters in short model peptides is used to throw light on their role in antimicrobial potency and specificity. The review concludes with some considerations on the potentials and limitations for the development of alpha-helical, antimicrobial peptides as antiinfective agents.     

Cell-penetrating and cell-targeting peptides in drug delivery

During the last decade, the potential of peptides for drug delivery into cells has been highlighted by the discovery of several cell-penetrating peptides (CPPs). CPPs are very efficient in delivering various molecules into cells. However, except in some specific cases, their lack of cell specificity remains the major drawback for their clinical development. At the same time, various peptides with specific binding activity for a given cell line (cell-targeting peptides) have also been reported in the literature. One of the goals of the next years will be to optimize the tissue and cell delivery of therapeutic molecules by means of peptides which combine both targeting and internalization advantages. In this review, we describe the main strategies that are currently in use or likely to be employed in the near future to associate both targeting and delivery properties.     

Amphipathic alpha helical antimicrobial peptides.

Antimicrobial peptides (AMPs) that assume an amphipathic alpha helical structure are widespread in nature. Their activity depends on several parameters including the sequence, size, degree of structure formation, cationicity, hydrophobicity and amphipathicity. The analysis of numerous natural AMPs provided representative values for these parameters and led to a sequence template with which to generate potent artificial lead AMPs. Sequences were then varied in a rational manner, using both natural and nonproteinogenic amino acids, to probe the individual roles of each parameter in modulating biological activity. A high cationicity combined with a stabilized amphipathic alpha helical structure conferred enhanced cidal activity towards all the cell types considered, and was a requirement for Gram-positive bacteria and fungi. An elevated helicity also correlated with increased hemolytic activity. The structural requirements for activity against several Gram-negative bacteria were instead considerably less stringent, so that it persisted in peptides in which formation of a helical structure and/or amphipathicity were impeded. Either a reduced charge or a reduced hydrophobicity resulted in generally inactive peptides. These observations, combined with the kinetics of bacterial membrane permeabilization and time-killing are discussed in terms of currently accepted models of action for this type of peptide. The simple guidelines obtained in this study allowed the design of highly active shortened AMPs and may be generally useful in the development of this type of peptides as anti-infective agents.     

Cloaking cytolytic peptides for liposome-based detection and potential drug delivery

Potent cytolytic peptides with specific tethering and cloaking sites have been synthesised and used to release payload from liposomes in a quantitative manner. A functionally located cloaking site has been modified specifically by simple conjugation without adversely affecting the cytolytic properties of the peptide. The cytolytic activity of modified peptides was then efficiently (>98%) cloaked and uncloaked by ligand-protein or hapten-antibody interactions. The principle of a dual response peptide has been demonstrated using an avidin-cloaked pH-sensitive peptide. Biospecific cloaking/uncloaking provided a new sensitive (approximately 12 pmol) homogeneous diagnostic and also appears potentially suited to bioresponsively targeted release of antimicrobial, anticancer and other drugs now delivered using liposomes.     

Cloaking cytolytic peptides for liposome-based detection and potential drug delivery

Potent cytolytic peptides with specific tethering and cloaking sites have been synthesised and used to release payload from liposomes in a quantitative manner. A functionally located cloaking site has been modified specifically by simple conjugation without adversely affecting the cytolytic properties of the peptide. The cytolytic activity of modified peptides was then efficiently (>98%) cloaked and uncloaked by ligand-protein or hapten-antibody interactions. The principle of a dual response peptide has been demonstrated using an avidin-cloaked pH-sensitive peptide. Biospecific cloaking/uncloaking provided a new sensitive (∼12 pmol) homogeneous diagnostic and also appears potentially suited to bioresponsively targeted release of antimicrobial, anticancer and other drugs now delivered using liposomes.     

Potential of cell penetrating peptides for cell drug delivery

The interest of the scientific community for cell penetrating peptides (CPP) has been growing exponentially for these last years, and the list of novel CPP is increasing. These peptides are powerful tools for the delivery of cargoes to their site of action. Indeed, several drugs that cannot translocate through the cell plasma membrane have been successfully delivered into cells when grafted to a CPP. Various cargoes have been linked to CPP, such as oligonucleotides, pharmacologically active drugs, contrast agents for imaging, or nanoparticles as platforms for multigrafting purposes… This review illustrates the fabulous potential of CPP and the diversity of their use, but their most interesting application appears their future clinical use for the treatment of various pathological conditions.     

The intracellular and nuclear-targeted delivery of an antiandrogen drug by carrier peptides

Cell permeable carrier peptides are currently of interest for their potential to improve the delivery of bioactive molecules into cells and to specific cellular compartments. We have investigated the activity of a derivative of the antiandrogen drug, bicalutamide, attached to the cell-permeable carrier peptide penetratin(R). We have used both disulfide (labile) and thioether (nonlabile) linkages to attach the bicalutamide derivative to the peptide in order to assess whether one type of chemistry has advantages over the other. In addition we have added a nuclear localization sequence (NLS) to the carrier peptide to investigate whether localization of the drug to the nucleus of the cell affects the activity of the drug. Biotin-labeled peptides were used to demonstrate that the carrier peptide is rapidly accumulated inside cultured cells, and that the incorporation of an NLS in the sequence results in its nuclear targeting. The bicalutamide derivative linked to carrier peptides via a disulfide-linkage exerted no greater antiproliferative effect in LNCaP cells, than the bicalutamide derivative alone. The bicalutamide derivative linked to the carrier peptide by a non-labile thioether linkage showed a similar activity profile. When the construct includes a nuclear targeting sequence, however, a markedly increased antiproliferative effect was observed. This study has thus shown that the activity of bicalutamide may be enhanced by the nonlabile attachment of a cell-permeable and nuclear-targeted peptide, which has implications for the development of novel antiandrogens for the treatment of prostate cancer.     

Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery

Topical or transdermal drug delivery is challenging because the skin acts as a natural and protective barrier. Therefore, several methods have been examined to increase the permeation of therapeutic molecules into and through the skin. One approach is to use the nanoparticulate delivery system. Starting with liposomes and other vesicular systems, several other types of nanosized drug carriers have been developed such as solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanoparticles and magnetic nanoparticles for dermatological applications. This review article discusses how different particulate systems can interact and penetrate into the skin barrier. In this review, the effectiveness of nanoparticles, as well as possible mode of actions of nanoparticles, is presented. In addition to nanoparticles, cell-penetrating peptide (CPP)-mediated drug delivery into the skin and the possible mechanism of CPP-derived delivery into the skin is discussed. Lastly, the effectiveness and possible mechanism of CPP-modified nanocarriers into the skin are addressed.     

Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery

Abstract

Topical or transdermal drug delivery is challenging because the skin acts as a natural and protective barrier. Therefore, several methods have been examined to increase the permeation of therapeutic molecules into and through the skin. One approach is to use the nanoparticulate delivery system. Starting with liposomes and other vesicular systems, several other types of nanosized drug carriers have been developed such as solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanoparticles and magnetic nanoparticles for dermatological applications. This review article discusses how different particulate systems can interact and penetrate into the skin barrier. In this review, the effectiveness of nanoparticles, as well as possible mode of actions of nanoparticles, is presented. In addition to nanoparticles, cell-penetrating peptide (CPP)-mediated drug delivery into the skin and the possible mechanism of CPP-derived delivery into the skin is discussed. Lastly, the effectiveness and possible mechanism of CPP-modified nanocarriers into the skin are addressed.     

Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer

Cell penetrating peptides (CPPs) are short amphipathic and cationic peptides that are rapidly internalized across cell membranes. They can be used to deliver molecular cargo, such as imaging agents (fluorescent dyes and quantum dots), drugs, liposomes, peptide/protein, oligonucleotide/DNA/RNA, nanoparticles and bacteriophage into cells. The utilized CPP, attached cargo, concentration and cell type, all significantly affect the mechanism of internalization. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now clear that CPP can mediate intracellular delivery via both endocytic and non-endocytic pathways. In addition, the orientation of the peptide and cargo and the type of linkage are likely important. In gene therapy, the designed cationic peptides must be able to 1) tightly condense DNA into small, compact particles; 2) target the condensate to specific cell surface receptors; 3) induce endosomal escape; and 4) target the DNA cargo to the nucleus for gene expression. The other studies have demonstrated that these small peptides can be conjugated to tumor homing peptides in order to achieve tumor-targeted delivery in vivo. On the other hand, one of the major aims in molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical agents of malignant transformation. For example, cell penetrating peptide aptamers might disrupt protein-protein interactions crucial for cancer cell growth or survival. In this review, we discuss potential functions of CPPs especially for drug and gene delivery in cancer and indicate their powerful promise for clinical efficacy.     

Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.     

Transdermal drug delivery

Transdermal drug delivery has made an important contribution to medical practice, but has yet to fully achieve its potential as an alternative to oral delivery and hypodermic injections. First-generation transdermal delivery systems have continued their steady increase in clinical use for delivery of small, lipophilic, low-dose drugs. Second-generation delivery systems using chemical enhancers, noncavitational ultrasound and iontophoresis have also resulted in clinical products; the ability of iontophoresis to control delivery rates in real time provides added functionality. Third-generation delivery systems target their effects to skin's barrier layer of stratum corneum using microneedles, thermal ablation, microdermabrasion, electroporation and cavitational ultrasound. Microneedles and thermal ablation are currently progressing through clinical trials for delivery of macromolecules and vaccines, such as insulin, parathyroid hormone and influenza vaccine. Using these novel second- and third-generation enhancement strategies, transdermal delivery is poised to significantly increase its impact on medicine.     

Lipophilic peptides for gene delivery

DNA transfections are widely performed in research laboratories and in vivo gene delivery holds the promise for curing many diseases. The synthetic carriers or vectors for DNA are typically cationic lipids. However, in biology, the recognition of nucleic acids by proteins involves both electrostatic and stacking contributions. As such we have prepared a series of new lipophilic peptide vectors that possess lysine and tryptophan amino acids for evaluation. These lipophilic peptides show minimal cytotoxicity and enhanced in vitro gene transfection activity.     

Seek & Destroy, use of targeting peptides for cancer detection and drug delivery

Accounting for 16 million new cases and 9 million deaths annually, cancer leaves a great number of patients helpless. It is a complex disease and still a major challenge for the scientific and medical communities. The efficacy of conventional chemotherapies is often poor and patients suffer from off-target effects. Each neoplasm exhibits molecular signatures – sometimes in a patient specific manner – that may completely differ from the organ of origin, may be expressed in markedly higher amounts and/or in different location compared to the normal tissue. Although adding layers of complexity in the understanding of cancer biology, this cancer-specific signature provides an opportunity to develop targeting agents for early detection, diagnosis, and therapeutics. Chimeric antibodies, recombinant proteins or synthetic polypeptides have emerged as excellent candidates for specific homing to peripheral and central nervous system cancers. Specifically, peptide ligands benefit from their small size, easy and affordable production, high specificity, and remarkable flexibility regarding their sequence and conjugation possibilities. Coupled to imaging agents, chemotherapies and/or nanocarriers they have shown to increase the on-site delivery, thus allowing better tumor mass contouring in imaging and increased efficacy of the chemotherapies associated with reduced adverse effects. Therefore, some of the peptides alone or in combination have been tested in clinical trials to treat patients. Peptides have been well-tolerated and shown absence of toxicity. This review aims to offer a view on tumor targeting peptides that are either derived from natural peptide ligands or identified using phage display screening. We also include examples of peptides targeting the high-grade malignant tumors of the central nervous system as an example of the complex therapeutic management due to the tumor’s location. Peptide vaccines are outside of the scope of this review.     

Sequence effect of self-assembling peptides on the complexation and in vitro delivery of the hydrophobic anticancer drug ellipticine

A special class of self-assembling peptides has been found to be capable of stabilizing the hydrophobic anticancer agent ellipticine in aqueous solution. Here we study the effect of peptide sequence on the complex formation and its anticancer activity in vitro. Three peptides, EAK16-II, EAK16-IV and EFK16-II, were selected to have either a different charge distribution (EAK16-II vs. EAK16-IV) or a varying hydrophobicity (EAK16-II vs. EFK16-II). Results on their complexation with ellipticine revealed that EAK16-II and EAK16-IV were able to stabilize protonated ellipticine or ellipticine microcrystals depending on the peptide concentration; EFK16-II could stabilize neutral ellipticine molecules and ellipticine microcrystals. These different molecular states of ellipticine were expected to affect ellipticine delivery. The anticancer activity of these complexes was tested against two cancer cell lines: A549 and MCF-7, and related to the cell viability. The viability results showed that the complexes with protonated ellipticine were effective in eradicating both cancer cells (viability <0.05), but their dilutions in water were not stable, leading to a fast decrease in their toxicity. In contrast, the complexes formulated with EFK16-II were relatively stable upon dilution, but their original toxicity was relatively low compared to that with protonated ellipticine. Overall, the charge distribution of the peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in hydrophobicity of the peptides significantly altered the state of stabilized ellipticine and increased the stability of the complexes. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.