Elsevier

Advanced Drug Delivery Reviews

Volume 55, Issue 2, 10 February 2003, Pages 199-215
Advanced Drug Delivery Reviews

Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities

https://doi.org/10.1016/S0169-409X(02)00179-5Get rights and content

Abstract

The first disulfide linkage-employing drug conjugate that exploits the reversible nature of this unique covalent bond was recently approved for human use. Increasing numbers of drug formulations that incorporate disulfide bonds have been reported, particularly in the next generation macromolecular pharmaceuticals. These are designed to exploit differences in the reduction potential at different locations within and upon cells. The recent characterization of a novel redox enzyme in endosomes and lysosomes adds more excitement to this approach. This review focuses on understanding where and how the disulfide bond in the bioconjugate is reduced upon contact with biological milieu, which affects delivery design and the interpretation of the delivery strategies.

Introduction

Attachment of cellular and subcellular targeting moiety, delivery-enhancing molecules, or functional entities to drugs or their delivery systems has become an essential and important approach in the field of modern drug delivery. Its need is more acute for the strategies required for macromolecular delivery, as unique problems arise as the molecular weight of the drugs increases, including changes in cytotoxicity, pharmacokinetics, dynamics and metabolism. For bioconjugates, the nature of the linker between the pharmacologic agent and the delivery-augmenting moiety dictates the degree of successful delivery and its outcome. In this review, we have attempted to focus only on covalent linkages, rather than the less stable yet sometimes advantageous non-covalent linkages such as high affinity ligand–receptor interactions and electrostatic complexation.

Among various covalent linkages, the primary focus of this review is the readily reversible yet relatively stable linkage of disulfide bonds. As there exist several reviews on this subject as well as comparisons among various covalent linkages utilized for drug delivery (for reviews on bioconjugate strategies see Refs. [1], [2]), the attention here is directed to the mechanism of disulfide bond reduction after the drug delivery system contacts the biological milieu. The key subjects of this review are the questions of where and how the disulfide bond in the bioconjugate is reduced, which impacts the design strategy as well as interpretation of the experimental results. After reviewing the mechanistic aspects of this subject, we will briefly discuss the existing examples of disulfide linkage-employing drug delivery with the mechanism in mind.

A disulfide bond (–S–S–) is a covalent linkage which arises as a result of the oxidation of two sulfhydryl (SH) groups of cysteines or other SH-containing material. In bacterial and eukaryotic cells, they are often found in secretory proteins and exoplasmic domains of membrane proteins, which face a harsh extracellular environment. In eukaryotic cells, cysteines are correctly bridged in the endoplasmic reticulum (ER) via the disulfide bond, which functions primarily to fortify the protein tertiary structure. Two distinct characteristics that render this bond attractive in designing drug delivery systems are its reversibility and its relative stability in plasma. Covalently bonded disulfides can be formed spontaneously by autoxidation of sulfhydryls, primarily via oxidation upon exposure to air, which can reversibly be cleaved in the presence of reducing agents such as dithiothreitol (DTT) and β-mercaptoethanol. The presence of a high redox potential difference between the oxidizing extracellular space and the reducing intracellular space makes the disulfide bond intriguing as a potential delivery tool. Thus, the covalent linkage is dependent on the locale of the construct relative to the cellular compartments; a controlled cleavage and release of reduced components can occur upon cell entry. Indeed, a number of bacterial toxins, such as diphtheria and cholera toxins and plant toxins such as ricin, consist of two protein subunits linked via a disulfide bond. They take advantage of the reversible breakage of the disulfide bond during the process of translocation across cellular membranes into the cytosol of host cells [3]. Studies on toxin conjugates using disulfide bonds were published as early as the late 1970s [4], [5], [6], and ever since the disulfide-based bioconjugation approach has been a popular conjugation method applied in a variety of cellular drug delivery systems. Successful applications of thiol-based conjugation have obtained targeted delivery and enhanced cytosolic delivery, improved pharmacokinetics and increased stability. Moreover, such conjugates are being used more frequently in polypeptide or protein-based systems and in plasmid gene and oligonucleotide formulations, some of which are discussed later in this review. These macromolecular agents are membrane-impermeant, due to their large molecular size or polyanionic nature, and are typically endocytosed by cells. However, despite the substantial biological evidence for reductive cleavage of disulfide bonds occurring in the endocytosed substrates, the subcellular locations of these putative reduction sites and the mechanisms by which endocytosed macromolecules are reduced remain poorly understood [7]. This review focuses primarily on recent discoveries regarding the disulfide reduction in the endocytic pathway, and on the related disulfide conjugation-based delivery strategies for macromolecules. In particular, we concentrate on the redox enzymes and small redox buffers that reduce disulfides at cell surfaces, in various endocytic compartments, and in the cytosol.

Section snippets

Reducing cytosolic space and oxidizing ER space

From the perspective of cellular drug delivery, access to the cytosolic space of eukaryotic cells is restricted primarily to hydrophobic small drugs, which have relatively high membrane partition coefficients and permeability constants, i.e. they diffuse passively across the lipid membrane. Macromolecular drugs have low diffusivity, and the plasma membrane is the primary barrier to entering the cytosolic space. Their uptake via permeation through membrane is very low, and instead they are

Generating sulfhydryls through chemical linkers

To conjugate two molecules via a disulfide bond, thiol groups need to be introduced into both structures, unless endogenous thiols such as cysteines are already present. Different approaches are used to incorporate a thiol or cysteine moiety in a drug. For oligopeptides and oligonucleotides, it is easy to introduce a cysteine or to derivatize with a thiol group during chemical synthesis. It is more difficult for small drugs, however, since they rarely possess a thiol moiety for conjugation, and

Disulfide linkage-employing drug delivery systems

In addition to the advantage of site-specific conjugation, the reversible nature of the disulfide bond is exploited in a number of ways for drug delivery. As described in the previous sections, disulfides can be reduced by different mechanisms in different environments. Depending on the locale of a drug conjugate at which the reduction and cleavage of disulfides is anticipated, the delivery strategies are divided into three groups. Some examples are briefly discussed below.

Antibody-S-S-toxin

While many of the investigations reviewed thus far have been limited to demonstrating their feasibility in a cell culture model, in vivo studies of disulfide bond-based bioconjugates have centered around those of antibody-S-S-toxin, immunotoxins that target diseased cells [123]. These delivery schemes are based on the efficient internalization of antibodies before release of cytoxic components. Although the first-generation immunotoxins, murine monoclonal antibodies chemically coupled to

Conclusion

It is exciting to see the increasing number of drug formulations that have incorporated disulfide bonds in various distinct ways. Many of these include the next generation macromolecular pharmaceuticals that are still under in vitro optimization. Yet there are examples that have successfully reached the market or are in clinical trials. One of the most popular approaches has been to exploit the cellular reducing potential in the endocytic pathway to trigger the cleavage of disulfide bonds. Its

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