Designing peptide-based scaffolds as drug delivery vehicles

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Abstract

Methods for delivering drugs into cells remain an important part of the process of designing drugs. One promising approach is the concept of loligomers, synthetic peptides composed of a branched polylysine core harboring identical arms. Loligomers are typically synthesized with eight arms, each carrying peptide signals guiding their import and localization into cells. The most important advantage of loligomers is the multivalent presentation of targeting signals resulting from a tentacular arrangement. Multivalency increases the efficiency of import and intracellular routing signals as compared to similar linear peptides. Secondly, it reduces and delays the impact of peptide degradation in terms of cellular processing and compartmentalization. The vectorial delivery of nucleus-directed loligomers into cells has recently been confirmed by microscopy and flow cytometry studies. Practical uses of loligomers as intracellular vehicles include the import of plasmid DNA into cells, the conjugation of chemical groups, such as photosensitizers for use in photodynamic therapy, and the incorporation of cytotoxic T-lymphocyte (CTL) epitopes with a view to creating synthetic vaccines. Branched peptides such as loligomers represent simple and versatile molecular vehicles with potential applications in a wide variety of drug design approaches.

Introduction

One of the most promising areas of research for the potential treatment of diseases such as cancer and various genetic disorders rests upon the specific delivery of DNA or other macromolecules into certain cell types. For this to be realized, vectors that enable the efficient internalization of macromolecules into cells must first be developed. The routing of macromolecules into specific organelles also represents a desirable feature that needs to be encoded by delivery vectors.

In the case of gene delivery, there are two basic strategies for the import of DNA into cells [1], each with its own set of advantages and drawbacks. The first approach is to incorporate the gene of interest into intact viral particles, which typically are engineered to be deficient in replication. This approach takes advantage of natural properties of viruses in directing nucleic acids into the nucleus of cells and promoting gene expression. This strategy is showing some promise particularly in the area of gene therapy. Viruses however have their own limitations, in terms of delivery agents. The production of infective viral particles, before the viral gene delivery vehicle can be assembled, is a costly and complex process. Issues such as their antigenicity, size (tissue penetration), limitations to the size of genes that can be delivered, and even their ability to selectively deliver DNA to all targeted cells, are well-known parameters that remain to be tackled. Moreover, viruses are only useful as DNA delivery vectors and cannot target other molecules, such as drugs, to cells.

A second family of gene delivery strategies encompasses all non-viral approaches where DNA is either physically delivered to cells [2] or non-covalently associated with molecular aggregates such as liposomes [3] or polymers such as poly-l-lysine [4], acting as cell import agents. For example, DNA is typically compacted by cationic agents such as polylysine resulting in the masking of negative charges of the phosphodiester backbone, thereby protecting the DNA from nucleases. Moreover, compaction of DNA by polycations in conjunction with ligands, such as antibodies, proteins, peptides, drugs or carbohydrates, results in the internalization of the ensuing complexes by cells [1], [5]. Non-viral approaches typically lack the efficiency of viruses to enter cells and exploit cellular mechanisms to express copies of specific genes or antisense messages, an important feature in gene therapy strategies. Practical solutions to the delivery of macromolecules as well as small drugs may lie in the development of synthetic virus mimics able to utilize cellular functions while being much smaller than viruses. Of all the non-viral delivery approaches that have been investigated to date, peptide-based vehicles offer the broadest range of options in terms of guiding the delivery of macromolecules to and into cells. A large number of molecular assemblies incorporating one or more such signals at present can be constructed and act as vehicles to modulate the localization, processing and duration of a macromolecule inside cells. One class of multivalent synthetic peptide scaffolds, termed loligomers, is reviewed here.

Section snippets

Loligomer construction

Synthetic peptide scaffolds incorporating drugs and cell localization signals are one type of technology that offers novel solutions to the challenge of improving the efficiency of macromolecule delivery into cells [6]. In particular, the structure of multiple antigenic peptides (MAPs) is based on a branched polylysine scaffold, first developed by Tam [7], [8], [9], that can be rapidly assembled on a solid phase support and provides a practical starting point for designing delivery vehicles.

Loligomers and photodynamic therapy

Photodynamic therapy (PDT) is a targeted treatment modality where small, photoactive compounds, called photosensitizers, accumulate into cells and are selectively activated by light, leading to the production of toxic species and cell death [17], [18]. Focusing the action of photosensitizers to a unique intracellular target may enhance their cytotoxicity. The routing of the porphyrin-based photosensitizer chlorin e6, to the nucleus of cells for example has recently been shown to significantly

Loligomers as gene delivery agents

The uptake by loligomers of large molecular entities such as plasmids was demonstrated using vectors harboring reporter genes suggesting that such constructs can act as non-viral, non-lipophilic transfection agents [11], [12]. In particular, nucleus-directed loligomers readily associate with plasmids to form complexes that can be imported into cells. The chemical coupling of nucleic acids to peptides [21] was not necessary since the negatively charged backbone of DNA interacts non-covalently

Loligomers and vaccine design

The development of non-viral, peptide-based constructs able to elicit protective in vivo cytotoxic T-lymphocyte (CTL) responses represents a major challenge in designing effective future vaccines. We have recently incorporated CTL epitopes into loligomer constructs (CTL-loligomers) leading to their import, processing and presentation as complexes with major histocompatability complex (MHC) class I molecules on the surface of antigen-presenting cells (K. Kawamura, R.-C. Su, P. Ohashi, J.

General considerations

The discussion up to this point has been limited to the potential uses of loligomers. However, there are some important general considerations when evaluating the applicability of these branched peptides. The challenge in synthesizing loligomers and the need to better characterize such constructs were discussed in Section 2. Additionally, the technical parameters associated with solid phase peptide synthesis place limitations on the size and nature of signals and peptides that can be

Conclusions

An important goal in drug design today is the development of moieties that enable small compounds, and macromolecules, to be targeted to cells. Once within the target cell, it might also be beneficial to promote the localization of the molecule to a specific organelle, such as the nucleus. Loligomers have been shown to carry out these functions efficiently with a minimal design strategy and few side-effects in their actions. Moreover, their structure and chemical assembly enables them to carry

Acknowledgements

Financial support for this work has been provided by the Canadian Breast Cancer Research Initiative and the National Cancer Institute of Canada with funds from the Canadian Cancer Society.

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