Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities
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
References (134)
- et al.
Receptor-mediated and enzyme-dependent targeting of cytotoxic anticancer drugs
Pharmacol. Ther.
(1999) Targeted drug conjugates: principles and progress
Adv. Drug Deliv. Rev.
(2001)- et al.
Penetration of protein toxins into cells
Curr. Opin. Cell Biol.
(2000) - et al.
Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate
J. Biol. Chem.
(1977) - et al.
Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes
J. Biol. Chem.
(1990) - et al.
Thioredoxin and thioredoxin reductase
Methods Enzymol.
(1995) Thioredoxin and glutaredoxin systems
J. Biol. Chem.
(1989)Glutathione deficiency produced by inhibition of its synthesis, and its reversal: applications in research and therapy
Pharmacol. Ther.
(1991)Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum
Semin. Cell Dev. Biol.
(1999)- et al.
Protein disulfide-isomerase in rat exocrine pancreatic cells is exported from the endoplasmic reticulum despite possessing the retention signal
J. Biol. Chem.
(1990)
Redox control of exofacial protein thiols/disulfides by protein disulfide isomerase
J. Biol. Chem.
Protein disulfide isomerase mediates integrin-dependent adhesion
FEBS Lett.
Secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes
J. Biol. Chem.
One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell
Cell
Cell-mediated reduction and incomplete membrane translocation of diphtheria toxin mutants with internal disulfides in the A fragment
J. Biol. Chem.
Cell penetration of diphtheria toxin. Reduction of the interchain disulfide bridge is the rate-limiting step of translocation in the cytosol
J. Biol. Chem.
Cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in Chinese hamster ovary cells
J. Biol. Chem.
A site-directed mutagenesis analysis of tNOX functional domains
Biochim. Biophys. Acta
Thioredoxin expression and localization in human cell lines: detection of full-length and truncated species
Exp. Cell Res.
Antigen unfolding and disulfide reduction in antigen presenting cells
Semin. Immunol.
Protein disulfide isomerase. A multifunctional protein resident in the lumen of the endoplasmic reticulum
J. Biol. Chem.
Disulfide spacer between methotrexate and poly(d-lysine). A probe for exploring the reductive process in endocytosis
J. Biol. Chem.
Measurement of phagosome-lysosome fusion and phagosomal pH
Methods Enzymol.
Mechanisms and catalysts of disulfide bond formation in proteins
Trends Biotechnol.
Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action
J. Biol. Chem.
Molecular and biochemical characterization of a novel gamma-interferon-inducible protein
J. Biol. Chem.
Receptor proximity, not intermolecular orientation, is critical for triggering T-cell activation
J. Biol. Chem.
A diverse set of oligomeric class II MHC-peptide complexes for probing T-cell receptor interactions
Chem. Biol.
Pseudomonas exotoxin A mutants. Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner
J. Biol. Chem.
Tumor targeting using anti-her2 immunoliposomes
J. Controlled Release
Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase
Blood
Improvements in protein PEGylation: pegylated interferons for treatment of hepatitis C
J. Controlled Release
Application of membrane-active peptides for non-viral gene delivery
Adv. Drug Deliv. Rev.
Melittin enables efficient vesicular escape and enhanced nuclear access of non-viral gene delivery vectors
J. Biol. Chem.
The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems
J. Biol. Chem.
PEG grafted polylysine with fusogenic peptide for gene delivery: high transfection efficiency with low cytotoxicity
J. Controlled Release
Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes
Dev. Biol.
Preparation of protein conjugates via intermolecular disulfide bond formation
Biochemistry (Moscow)
Chimeric toxins: toxic, disulfide-linked conjugate of concanavalin A with fragment A from diphtheria toxin
Proc. Natl. Acad. Sci. USA
Oxidized redox state of glutathione in the endoplasmic reticulum
Science
High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function
Proc. Natl. Acad. Sci. USA
Renal and hepatic toxicity of trichloroethylene and its glutathione-derived metabolites in rats and mice: sex-, species-, and tissue-dependent differences
J. Pharmacol. Exp. Ther.
Physiological functions of thioredoxin and thioredoxin reductase
Eur. J. Biochem.
Dissecting the mechanism of protein disulfide isomerase: catalysis of disulfide bond formation in a model peptide
Biochemistry (Moscow)
Glutathione
Annu. Rev. Biochem.
Thiol/disulfide exchange equilibria and disulfide bond stability
Exp. Neurol.
Presence of closely spaced protein thiols on the surface of mammalian cells
Protein Sci.
Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells
J. Cell Biol.
Cell surface protein disulfide-isomerase is involved in the shedding of human thyrotropin receptor ectodomain
Biochemistry (Moscow)
Membrane-bound protein disulfide isomerase (PDI) is involved in regulation of surface expression of thiols and drug sensitivity of B-CLL cells
Exp. Hematol.
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