Disulfide-Cleavage-Triggered Chemosensors and Their Biological

Review pubs.acs.org/CR

Disulfide-Cleavage-Triggered Chemosensors and Their Biological Applications Min Hee Lee,† Zhigang Yang,† Choon Woo Lim,‡ Yun Hak Lee,† Sun Dongbang,† Chulhun Kang,*,‡ and Jong Seung Kim*,† †

Department of Chemistry, Korea University, Seoul 136-701, Korea East−West Medical Science, Kyung Hee University, Yongin 446-701, Korea cleavage reactions mediated by the abundance of cellular free thiols, including glutathione (GSH), which is the most abundant thiol-containing small molecule. Another major biological disulfide compound is glutathione disulfide (GSSG), which is produced upon the oxidation of GSH.10 In fact, GSH can be oxidized to GSSG, which can be reduced back to GSH in the presence of the NADPH-dependent enzyme glutathione reductase (Figure 1B) to maintain the cellular redox homeostasis essential for cell growth and function.11−13 In the human body, intracellular GSH concentration is in the millimolar range (1−10 mM),14 but it is at micromolar (20−40 μM) levels15−17 in common fluids outside cells, such as plasma and other body fluids, with the exception of the fluid lining the lower part of the respiratory tract. GSH can also be translocated CONTENTS to the cell surface to play roles at the interface between the 1. Introduction 5071 extracellular space and the membrane.18−20 Interestingly, 2. Chromogenic and Fluorogenic Sensors 5072 intracellular GSH concentration is much higher in cancer 2.1. Thiol Detection 5072 cells than that in the corresponding normal cells; this feature 2.2. Metal Ion Detection 5078 may prove to be important in the development of anticancer 3. Advances in Drug Delivery Systems 5080 drug delivery systems (DDS).21−25 3.1. Disulfide-Based Prodrugs 5080 A disulfide bond can be formed via the oxidation of two 3.2. Disulfide-Based Nanocapsule Carriers 5089 thiols, as shown in Figure 1. It is relatively stable in mildly 3.3. Disulfide-Based Hydrogel Carriers 5096 oxidizing (e.g., oxygen or bloodstream) and physiological pH 3.4. Disulfide-Based Inorganic Carriers 5100 conditions26,27 but readily susceptible to the disulfide−thiol 4. Application of Disulfide Cleavage in Other Fields 5102 exchange reaction, the reductive disulfide cleavage reaction to 4.1. Fabrication of Nanoporous Materials 5102 generate two thiols (or dithiol), or the photocleavage reaction 4.2. Fabrication of Nanotubes 5102 via a radical mechanism. A more detailed description of the 4.3. Functionalization of Inorganic Surfaces 5104 thiol chemistry (high reactivity with metal ions) that comprises 4.4. Synthesis of Redox-Sensitive Macrocyles 5104 the basis of the technologies discussed here shall be introduced. 5. Conclusions 5104 The development of disulfide-based fluorochromogenic Author Information 5106 sensors has recently emerged as a research area of significant Corresponding Author 5106 importance, and these sensors have attracted a tremendous Notes 5106 amount of attention due to their high sensitivity and rapid Biographies 5106 response to thiols.28 A number of studies in this related field Acknowledgments 5107 have increased considerably in the past few years. Since 1959, References 5107 when 5,5′-dithiobis(2-nitrobenzoic acid), also known as Ellman’s reagent, was first used to quantify thiol content, various types of chemosensors that exhibit fluorescence 1. INTRODUCTION enhancement or ratiometric changes upon the cleavage of disulfide groups have been reported, which can be used to The disulfide bond (−S−S−) is an extremely valuable quantitatively detect and monitor changes in thiols in real time. functional group in a variety of chemical and biological agents Our research group has also introduced some disulfide-based that display potent reactivity or biological activities (e.g., fluorescent probes.29−33 antitumor activities).1−7 It has already been found in proteins, Disulfide-based functionalization for biological applications oxidized glutathione, and even in numerous natural products has been widely used in the development of chemosensors, including some drugs (e.g., mitomycin disulfides, leinamycin, 8,9 etc). Disulfide bonds existing in proteins are relatively oxidative in the extracellular space; however, such disulfide Received: August 25, 2012 bonds can be rarely found inside cells because of the disulfide Published: April 12, 2013 ‡

© 2013 American Chemical Society

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dx.doi.org/10.1021/cr300358b | Chem. Rev. 2013, 113, 5071−5109

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Disulfide-based units are increasingly being used in the functionalization and fabrication of nanomaterials (e.g., quantum dots, gold nanoparticles, carbon nanotubes, silica materials, and other inorganic materials) due to the reversible redox property that allows them to switch between thiol and disulfide forms. The disulfide group acts as a linker to attach different functional moieties to the surface of the nanoparticle or carbon nanotube via a covalent or noncovalent interaction, and the obtained supramolecules can be used to investigate cellular processes at the molecular level by means of microinjection into cells. Disulfide-containing compounds have also been utilized to synthesize macromolecules with cavities or hollow materials, because of the corrosion of the disulfide bond in the presence of thiols, which can afford various shapes of nanomaterials. However, to the best of our knowledge, disulfide cleavagebased chemical and biological applications have not been reviewed yet. In the present review, we mainly summarize the recent progress in the use of disulfide cleavage-based compounds for chemosensing, in the development of prodrugs, hydrogels, and nanocarriers, and in material fabrication, and we discuss the applications of such compounds in bioimaging, drug delivery, and incorporation into organic or inorganic nanomaterials.

prodrugs, polymer hydrogels, nanomaterial carriers, and other agents, because disulfides are biodegradable in the presence of

Figure 1. (A) Reversible changes between the thiol and disulfide forms; (B) glutathione (GSH) and glutathione disulfide (GSSG).

thiols or in reducing milieus. Biodegradable organic and inorganic delivery materials have drawn attention in the fields of chemistry and biology for decades because of their compatibility to various situations like chemosensing and drug delivery and have been incorporated into an extensive variety of drug delivery strategies.34,35 Compared to other drug delivery methods,36−41 disulfide-based strategies have demonstrated higher therapeutic performance due to the advantages of the disulfide bond over others in terms of biocompatibility, stability in the bloodstream, and cleavage by disulfide reductase or metabolic thiols. Disulfide conjugation of a drug molecule to a carrier allows it to be conveyed to its site of action with high efficiency; therefore, the drug can accumulate to higher concentrations in the target tissue. However, a large number of drugs and drug carriers do not contain disulfide functional groups and should be modified to attach a disulfide linkage between the drug and carrier. Drugs and suitable carriers possess primary amines or carboxylic acid groups that connect to units with disulfide groups. Most of the thiol linkers used in disulfide conjugations are based on thiols (or disulfides) with a short alkyl spacer terminating in either a primary amine or a carboxylic acid. Furthermore, disulfide-functionalized nanoparticle and organic polymer hydrogels could be utilized as delivery carriers that have also been rapidly developed in the past several years.42−44 Disulfide-based encapsulating drug carriers seem to be more resistant to reduction, as these capsular structures are held together by a series of disulfide bonds rather than by a single linkage. Moreover, even if a disulfide bond is cleaved in an encapsulating carrier, fragments or payload would be not instantly released. In most cases, capsulated fragments are assembled together in this way rather than via a single disulfide linkage. This allows the free thiol formed after reduction to remain in a position to break other disulfide linkages through intramolecular reactions. In the field of biomedicine, disulfide-based hydrogels are important polymer materials as drug delivery carriers or tissue regeneration scaffolds, due to their high water content, permeability, and molecular diffusivity. Biodegradation is considered a critical requirement for most hydrogel applications. Hydrogels are generally expected to degrade during or after tissue formation for many tissue regeneration applications. The ideal hydrogel should degrade completely once the new tissue is formed. A number of disulfide bonds incorporated into the hydrogel can control the biodegradation time because of the slow cleavage of the disulfide bond.

2. CHROMOGENIC AND FLUOROGENIC SENSORS 2.1. Thiol Detection

Since 1959, 5,5′-dithiobis(2-nitrobenzoic acid), also called Ellman’s reagent (1, Figure 2), has been used as the most

Figure 2. Ellman’s reagent (1).

popular reagent for quantification of free or covalently modified thiol groups on proteins.45 This reagent reacts rapidly and completely with thiols in solution or with accessible protein thiols to release an equivalent amount of a highly chromogenic product, 5-thio-2-nitrobenzoate (TNB) (ε = 14 150 M−1 cm−1 at 412 nm). To improve a drawback of Ellman’s reagentthe fact that it can be easily hydrolyzed under basic environments, a common condition for thiol detectionPei et al. introduced 5-(2aminoethyl)dithio-2-nitrobenzoate (2, Figure 3).46 Indeed,

Figure 3. Thiol-assisted disulfide bond cleavage reaction of 2.

even under basic aqueous solutions 2 shows the necessary stability for minimizing non-thiol-dependent reactions; hence, it can be used for thiol detection and enzyme kinetic assays in the whole range of pH without significant interference due to hydrolysis. The reaction of 2 with thiols follows similar kinetics to that of Ellman’s reagent. 5072


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Figure 4. Proposed disulfide bond cleavage reaction of 3 with thiols.

Figure 5. FRET-based thiol sensors 4−7.

were shown to emit fluorescence, presumably caused by the disulfide cleavage reaction, allowing quantification of thiols using flow cytometry. Moreover, the fluorescence intensity in the cells dramatically decreased upon pretreatment with Nethylmaleimide (NEM), a thiol-blocking reagent, confirming that sensor 3 is responsive to intracellular GSH levels. The excitation energy of one fluorophore (donor) can be transferred through a nonradioactive quantum process to a

For the imaging of intracellular thiols, a number of disulfidebased fluorescent probes have been investigated. In 2008, Chmielewski et al. constructed a disulfide-based rhodamine 3 (Figure 4) that reacts with thiols to produce intense emission in aqueous solution and in live cells.47 The disulfide bond is cleaved by the nucleophilic thiolate of the thiol, which induces the breakdown of the neighboring carbamate bonds to generate the fluorescent rhodamine. Upon incubation with 3, HeLa cells 5073


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Figure 6. Proposed disassembly mechanism of 8 by GSH.

neighboring fluorophore (acceptor) that is within 100 Å of the donor, if there is good overlap between the donor emission and acceptor absorption spectra. This energy transfer process is called fluorescence resonance energy transfer (FRET). The FRET off−on sensing system provides advantages over conventional fluorescence detection methods based on single fluorophores in that it has greater sensitivity and less background interference. In this regard, Daniel et al. described FRET-based probes 4 and 5 (Figure 5), composed of fluorescein (an energy donor) and rhodamine (an energy acceptor), which are connected via a disulfide linkage.48 When the probe is excited at a wavelength selective for donor excitation, rhodamine emits fluorescence via FRET; however, upon disulfide reduction, the acceptor is distant from the donor and FRET does not occur. Therefore, thiols can be quantified by excitation at 520 nm, which excites the fluorescein moiety alone. Probes 4 and 5 were also able to penetrate into cell membranes and react with thiols in the cytoplasm. Using this system, the authors demonstrated that an Escherichia coli strain that lacks GSH synthesis has decreased thiol levels and that zebrafish embryos maintain unusually high thiol levels in the chorion. Karuso et al. developed a FRET-based probe (6) consisting of two fluorescein moieties linked by a disulfide linker for the determination of glutathione reductase (GR) activity.49 Owing to its small Stokes’ shift, a fluorescein moiety can act as an acceptor for emission from the other moiety in the probe, and thereby the fluorescence of the probe is quenched. Cleavage of the disulfide bond by thiols allows the two fluorescein molecules to diffuse apart, resulting in a decrease in FRETmediated quenching and strong enhancement of fluorescence intensity at 520 nm. Probe 6 was applied to the GR activity assay and showed a sensitivity enhancement of 2 orders of

magnitude in comparison with the conventional colorimetric GR activity assay, which is sufficiently sensitive to detect GSH or glutathione transferase activity in living cells. Yu et al. prepared a fluorescent probe (7) using a porphyrin− coumarin scaffold for detecting thiols based on FRET, as depicted in Figure 5.50 In a 1:1 (v/v) solution of phosphatebuffered saline (PBS), pH 7.4, and ethanol, upon excitation of the coumarin moiety at 350 nm, 7 displayed the characteristic emission of porphyrin at 658 nm with little fluorescence at 459 nm, at which coumarin emission is expected. This observation was attributed to FRET between the coumarin and porphyrin moieties. However, upon the addition of thiol to the solution, significant fluorescence enhancement was observed at 459 nm, with a concomitant decrease in emission at 658 nm, firmly suggesting that the FRET is turned off by the presumed disulfide cleavage reaction by the thiol. Furthermore, the authors demonstrated that 7 is suitable for ratiometric fluorescence imaging of thiols in living HeLa cells. Qu et al. described a FRET-based molecular beacon 8, illustrated in Figure 6.51 Compound 8 is comprised of two parts, A and B; part A (the stem) is covalently linked to a fluorophore (FAM, 6-carboxyfluorescein)/quencher (TAMRA, tetramethylrhodamine) pair at the 5′- and 3′-termini of the hairpin structure to form a quenched format, and part B (the loop) consists of ssDNA sequences and includes a disulfide bond. Upon the addition of GSH, the fluorophore/quencher is completely disassembled via cleavage of the disulfide bond, leading to fluorescence enhancement at 516 nm upon excitation at 495 nm. Otherwise, the intensity would be quenched by FRET. According to its linear response to the GSH concentration, the detection limit was evaluated to be 1.0 × 10−9 M. The presence of thiols in a human erythroleukemia cell line were successfully demonstrated using 8. 5074


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A ratiometric fluorescent probe can eliminate most ambiguities by self-calibration of two emission bands. In this regard, Zhang et al. reported the colorimetric and ratiometric fluorescent probe 9 for thiols and their bioimaging applications as revealed in Figure 7.52 For probe 9, 4-aminonaphthalimide

penetrate the tissue. Two-photon microscopy (TPM) is certainly one of the techniques that allow this. Since TPM employs two near-infrared photons as the excitation source, it has many advantages over single-photon technology, such as increased penetration depth and prolonged observation time, thereby allowing efficient tissue imaging. Figure 9 illustrates an example of the detection of thiols deep inside live tissues using 11, which has been studied by Cho et

Figure 7. ICT-based thiol probe 9.

was chosen as a fluorophore because of its marked intramolecular charge transfer (ICT) function and desirable photophysical properties such as large Stokes’ shift, high quantum yield, long emission wavelength, and stability to pH variation. Upon the addition of thiols, 9 displays red-shifted emission (485 nm → 533 nm) and a color change (350 nm → 435 nm) from colorless to yellow. Furthermore, this probe allowed the determination of GSH by a ratiometric fluorescence method with a detection limit of 28 μM; more importantly, distinct ratiometrical fluorescence changes of 9 were observed in HeLa cells. In a continuation of ratiometrical fluorescence sensors, Murthy et al. presented a new probe, 3-hetaryl-7-thiolcoumarin (10), which can reversibly and ratiometrically measure the intracellular thiol−disulfide equilibrium (Figure 8).53 The coumarin moiety in probe 10 has a thiol group directly conjugated to its extended aromatic π system. Its spectroscopic properties are thus very sensitive to the redox state of the thiol. In its reduced state, 10 shows thiolate-extended π conjugation with an absorption band at 448 nm, and it reversibly reacts with thiols to produce 10-SR, which has an absorption band at 380 nm. Thereby, the ratio of 10 to 10-SR can be estimated from the emission ratio (Fex448/Fex372) at 490 nm. The reduced thiolate form (10) has an emission ratio (Fex448/Fex372) of 5, whereas for the oxidized disulfide form (10-SR) it is only 0.5. This ratiometric fluorescence change associated with probe 10 has been applied to measure the dynamic redox status of the whole cell. To detect thiols deep inside live tissues, the excitation and emission beams used in the detection should be able to

Figure 9. Disulfide-based two-photon probes 11 and 12 for detecting thiols deep inside live tissues.

al. Probe 11 bears 2-methylamino-6-acetylnaphthalene and disulfide as the TP fluorophore and thiol reaction site, respectively.54 In MOPS buffer solution (pH 7.2), 11 undergoes a disulfide cleavage reaction in the presence of thiols, without any significant interference from other biologically relevant analytes and variations in pH. Upon the cleavage reaction, 11 exhibits a new emission maximum at 505 nm upon excitation at 780 nm, caused by two-photon excited fluorescence (TPEF). The increased TPEF of 11 by cellular thiols has also been observed both in HeLa cells and in rat hippocampal tissues at depths of 90−180 μm. Subsequently, Cho et al. synthesized a ratiometric TP probe (12) for mitochondrial thiols.55 This probe has 6-(benzo[d]thiazol-2′-yl)-2-(N,N-dimethylamino)naphthalene as the TP fluorophore, a disulfide group as the thiol reaction site, and triphenylphosphonium salt (TPP) as the mitochondrial targeting group. Probe 12 specifically stains the mitochondria of HeLa cells, with ratiometric emissive color changes from blue (Fblue, 425−475 nm) to yellow (Fyellow, 525−575 nm) in the presence of cellular thiols. Upon excitation at 740 nm, the image ratio (Fyellow/Fblue) of HeLa cells with 12 was 1.24. However, when the intracellular GSH production increased on treatment with α-lipoic acid, the image ratio increased to 2.73,

Figure 8. Proposed reaction mechanism for the detection of GSH/GSSH ratio using 10. 5075


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Figure 10. Chemical structures of 13 and 14 and schematic representation of hepatocyte-targeted imaging of 13 through ASGP-R-mediated endocytosis into hepatocytes and cleavage of the disulfide bond to induce fluorescence changes.

and when the cells were treated with N-ethylmaleimide (NEM), a well-known thiol-blocking agent, it decreased to 0.77. The visualization of mitochondrial thiol levels in live cells and living tissues at depths of 90−190 μm was also demonstrated. The liver is the most important organ for thiol metabolism. It provides GSH to the bloodstream and other organs, such as the kidneys, lungs, and intestines, and thus plays a critical role in maintaining the organismal redox balance.56 Moreover, the liver-dependent intertissue flow of GSH plays an important role in cancer metastasis to remote sites.57 Therefore, the development of fluorescent probes that allow real-time fluorescence imaging of reduced thiols both in vitro and in vivo in liver cells is crucial for the early diagnosis of metastatic cancer, as well as for the screening of drug candidates that might display antineoplastic activity. Kim et al. reported a hepatocyte-targeting fluorescent probe 13, which incorporates a thiol-specific cleavable disulfide bond, a masked naphthalimide fluorophore, and a single galactose moiety as the hepatocytetargeting unit (Figure 10).29 For the hepatocyte targeting ability

of 13, the authors exploited the fact that the asialoglycoprotein receptor (ASGP-R) is uniquely expressed on the plasma membrane of mammalian hepatocytes and selectively recognizes terminal galactose residues on desialylated glycoproteins, allowing the selective internalization of the latter species within the hepatocyte via receptor-mediated endocytosis.58,59 Thus, the galactose subunit in 13 serves to guide the probe to hepatocytes, while the disulfide-linked naphthalimide moiety provides fluorescence emission at 540 nm when exposed to cellular thiols as a result of disulfide cleavage. Confocal microscopic imaging experiments indicate that probe 13, but not the galactose-free control compound 14 shown in Figure 10, is preferentially taken up by HepG2 cells through galactosetargeted, ASGP-R-mediated endocytosis. The liver-selective targeting ability of 13 has also been confirmed in vivo in rats. Furthermore, the potential utility of probe 13 in pathogenic states and as a possible screening tool for agents that can manipulate oxidative stress has been demonstrated in HepG2 cells. 5076


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Hydrogen sulfide (H2S), one of the reactive cellular thiols, also plays vital roles in regulating the intracellular redox status and other fundamental signaling processes involved in human health and disease. To precisely understand the cellular function of H2S, accurate and reliable measurement of H2S concentrations in biological samples is needed. Several H2Sspecific fluorescent probes have been developed on the basis of unique chemical reactions such as the reduction of an azido group60,61 and Michael addition−cyclization by H2S.62 Xian et al. presented a disulfide-based fluorescent probe 15 for the detection of H 2 S on the basis of the H 2 S-mediated benzodithiolone formation reaction (Figure 11).63 H2S can be

Figure 12. Paramagnetic thiol probes 16 and 17.

mM using 17, consistent with the value determined by Ellman’s reagent (3.71 ± 0.09 mM). Thus, both trityl-based thiol probes exhibited unique properties, enabling the measurement of thiols in biological systems. Figure 13 illustrates the quantum dot (QD)-based probe 18@QD, which detects GSH by manipulating its electron transfer pathway, developed by Santra et al.65 Probe 18@QD uses QDs (CdS:Mn/ZnS, core/shell) as the signaling unit, the electron acceptor carbon disulfide (CS2) as the linker, and dopamine as an electron donor. In the probe, the carbon atom of CS2 forms a chemical bond with the primary amine of dopamine, and the sulfur atoms directly form disulfide bonds with the ZnS surface of the QDs. The unmodified QDs exhibit bright yellow-orange emission at 592 nm (on state), whereas the probe 18@QD is nonfluorescent, which can be attributed to electron transfer from the electron-rich dopamine to the QDs (off state). However, cleavage of the disulfide bond in 18@QD by GSH allows the dopamine molecules to detach from the QDs, resulting in fluorescence recovery (on state). Mutus et al. described disulfide-linked gold nanoparticle (AuNP) clusters, 19@AuNP (Figure 14), which are able to react specifically with low molecular weight thiols (LMWT) but not with high molecular weight thiols (HMWT).66 Their strategy was to place a disulfide bond on the cluster in a steric environment accessible only to LMWT. For the synthesis of 19@AuNP, the surface of AuNP was first coated with dithiobis(succinimidylpropionate) (DSP), and then the resulting succinimidyl residues were reacted with the amino groups of GSSG, which is used as a disulfide-containing cross-linker. The monodispersiveness of 19@AuNP was confirmed by transmission electron microscopy. AuNP has a strong absorption band at 520 nm, whereas the AuNP cluster has a broad, red-shifted absorption band at 610 nm, with a deep blue color. However, when 19@AuNP is exposed to thiols, it changes to a monodisperse AuNP via cleavage of its disulfide bond, exhibiting a blue-shift from 610 to 520 nm as well as a color change from deep blue to red. Moreover, the rate of the disulfide cleavage reaction is inversely proportional to the size of the thiols. In the case of HMWT, such as protein thiols (bovine serum albumin and protein disulfide isomerase), the cleavage reaction rates of 19@AuNP were 170 000-fold slower than that of H2S.

Figure 11. H2S-assisted disulfide cleavage reaction of 15.

considered a nonsubstituted thiol that can undergo two nucleophilic reactions, whereas other thiols such as cysteine are monosubstituted thiols that can only undergo a single nucleophilic reaction. Accordingly, they designed probe 15 containing bis-electrophilic centers, a disulfide bond, and a ester group. They found that probe 15 (Φ = 0.003) reacted rapidly with H2S to generate fluorophore 15-A (Φ = 0.392) and benzodithiolone 15-B in good yields under mild conditions. In contrast, no reaction occurred with other biological thiols such as Cys or GSH. Furthermore, the efficiency of 15 in the measurement of H2S concentration has been demonstrated in aqueous buffers and plasma as well as in cells. An exogenous paramagnetic probe generating low-frequency electron paramagnetic resonance (EPR) signals has been indispensable in the measurement of various physiological parameters in vivo due to its high specificity, noninvasiveness, and good depth of magnetic field penetration in animal tissues. Zweier et al. reported for the first time the synthesis of trityl radical-conjugated disulfide biradicals 16 and 17 as paramagnetic thiol probes (Figure 12).64 The use of trityl radicals in the probes greatly facilitates thiol measurement based on the EPR signal, because the trityls have high stability in living tissues and produce a single narrow EPR line that enables high sensitivity and resolution for in vivo imaging. Both 16 and 17 exhibit a broad characteristic EPR signal at room temperature because of their intramolecular spin−spin interactions. However, the cleavage of the disulfide bond in the biradical in the presence of thiols facilitates the separation of the two radical moieties, resulting in an increase in the EPR signal intensity of the corresponding monoradicals. The moderately slow reaction between the biradicals and GSH (k2 = 0.3 M−1 s−1 for 16 and 0.2 M−1 s−1 for 17) allows in vivo measurement of GSH concentration without altering the redox balance in the biological system. The hepatic GSH concentration in rat was determined to be 3.49 ± 0.14 mM using 16 and 3.67 ± 0.24 5077


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Figure 13. QD-based probe 18@QD for GSH detection.

radical-dependent disulfide photocleavage. Subsequently, Ye et al. developed a Hg2+ sensing probe based on the pyrenemediated photolysis of the disulfide bond.68 They synthesized two thymine-rich oligonucleotides, 20 (5′-CTCTTATCTTGTT-/Pyr/-TCTCTTTTCTCTT-3′) and 21 (5′TTGTGTTATGTG-/FAM/-/SS/-/Dab/-TCTTGTTATGTG-3′), as shown in Figure 15. It is known that Hg2+ binds with thymine bases to form stable thymine−thymine mismatched complexes (T−Hg2+−T).69 Probe 20 contains two T-rich domains separated by a pyrene moiety, and probe 21 also has two T-rich domains separated by 6-carboxyfluorescein (FAM), disulfide, and Dabcyl (quencher of the FAM fluorophore) moieties. Initially, the fluorescence of FAM in 21 is quenched by the nearby Dabcyl moiety. In the presence of Hg2+ in a solution containing 20 and 21, nucleotides form a complex via the formation of T−Hg2+−T base pairs between the chains, which bring the disulfide linkage in 21 and the pyrene moiety in 20 close together. Then, the disulfide linkage in 21 is cleaved via pyrene-mediated photolysis upon light irradiation at 350 nm to release the cleavage products from the 21−Hg2+−20 complex, causing the fluorescence recovery of FAM and “turning on” the fluorescence for Hg2+ detection. This sensing mechanism based on pyrene−disulfide molecular engineering has numerous applications in biomedical and proteomics scenarios, with light-induced spatiotemporal control. Copper overload in biological systems plays an important role in several diseases, including Wilson’s disease.70 Impairment of copper transport through the hepatocytic membrane results in cytosolic copper accumulation and associated cellular injury.71 To efficiently lower the copper concentration in the overloaded liver, a compound that can act as a copper chelator should be targeted to the hepatocytes. In this context, Delangle et al. designed a bifunctional glycopeptide, 22 (Figure 16), that can target hepatocytes and lower intracellular copper.72 The bifunctional cyclodecapeptide scaffold has two independent faces. One face displays a cluster of carbohydrates to guide 22 to hepatocytes through ASGP-R-mediated endocytosis,58,59 whereas the second face is devoted to copper complexation with two cysteine residues. The cellular uptake of 22 and its ability to bind intracellular Cu+ were tested in hepatic cell lines. Indeed, 22 was selectively internalized into hepatocytes through ASGP-R-mediated endocytosis, and the disulfide bridge of 22 was cleaved by intracellular GSH to afford two reduced thiols, enabling 22 to chelate with Cu+ in the liver. Thus, 22 could be used as a prochelator that is activated in the reduced cellular environment to reduce copper overloading in the liver.

Figure 14. Synthesis of 19@AuNP and its color change in the presence of GSH.

2.2. Metal Ion Detection

Recently, Tan et al. demonstrated an interesting phenomenon in which pyrene induces efficient photocleavage of disulfide upon irradiation with 350-nm light.67 This is explained by the fact that pyrene can generate large amounts of radical cations upon irradiation at 350 nm, resulting in the enhancement of 5078


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Figure 15. Schematic illustration of Hg2+ detection via pyrene-mediated photolysis of disulfide bonds.

Figure 16. Schematic illustration of hepatocyte-targeted uptake and Cu+ chelation of 22.

Figure 17. Prodrug conjugates of 23a,b and 24a,b.

atoms of the disulfide bond in 9 provide high affinity toward Hg2+ ions. The fluorescence enhancement of 9 was proportional to the concentration of Hg2+ in the range of 0−150 μM, with a detection limit of 0.38 μM. In addition, it showed reversibility in the presence of EDTA.

Zhang et al. introduced a disulfide-linked naphthalimide dimer, 9, for detecting the Hg2+ ion.73 Compound 9 (Figure 7) shows a 7-fold enhancement in fluorescence intensity at 468 nm in the presence of Hg2+ ions in Tris-HCl solution (5 mM, pH 7.4) containing 50% (v/v) EtOH because the two sulfur 5079


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Figure 18. Cancer-targeted prodrug structures of 25−28.

3. ADVANCES IN DRUG DELIVERY SYSTEMS

In this regard, Johannes et al. reported two prodrug conjugates of SN38 (23a and 24a) linked to the nontoxic Bsubunit of the Shiga toxin (termed STxB) via a disulfide bond (Figure 17).75 STxB produced by intestinal pathogenic bacteria binds to the cellular toxin receptor glycolipid Gb3 and can be internalized into the cells. Interestingly, the Shiga toxin receptor, Gb3, is abundant in certain human cancers, including colorectal carcinoma. The authors of the study synthesized two versions of these conjugates, one with SN38 (a) and the other with biotin (b). For the biological evaluation of 23 and 24, the stabilities of the biotin versions (23b and 24b) were tested in different media. Compound 23b was found to be readily activated even in the absence of cells, thus precluding its use in vivo. In contrast, 24b was completely stable over extended periods of up to 48 h at 37 °C in all media including pure fetal calf serum. Enzyme-linked immunosorbent assay (ELISA) analysis of 24b demonstrated that disulfide cleavage was detectable in 6−24 h and was essentially completed in 48 h, in

3.1. Disulfide-Based Prodrugs

In recent times, the disulfide bond is being actively investigated as a component of targeted drug conjugates that can be concentrated in a targeted area where the drug’s activity is needed and release the drug upon selective cell internalization. In general, a drug is linked to a conjugate as an inactive prodrug via a cleavable linker and can be converted into the corresponding active parent drug by an activation process, such as enzymatic cleavage of the linker. The disulfide bond is suitable as a cleavable linker, since it can accommodate various types of chemicals, such as cytotoxic agents and tumor-targeting molecules, and can be cleaved by the abundant intracellular thiols. Moreover, it is stable during its journey to the target site, because thiols are scarce in blood circulation. In fact, GSH levels are 1000-fold higher in tumor cells than in the blood plasma.74 5080


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Figure 19. Proposed drug release mechanism of 29 and 30 with GSH.

Figure 20. Disulfide bond cleavage mechanism of 31a−c.

as the cytotoxic agent (Figure 18).76 This conjugate is internalized efficiently into tumor cells overexpressing cellsurface biotin receptors via receptor-mediated endocytosis (RME), followed by drug release via GSH-triggered disulfide cleavage. In order to monitor and validate the RME of 25, drug release, and drug binding to the target protein, three conjugates (26−28, shown in Figure 18) were synthesized. Biotin− fluorescein (26) was designed to characterize biotin-dependent RME. Biotin−linker−coumarin (27) was designed to confirm

Gb3-expressing HT-39 cells. On the basis of these observations, the targeting ability of 24a, as well as drug release, were tested, and the cytotoxicity of 24a was determined, with an IC50 value of 300 nM in Gb3-expressing HT-39 cells. In contrast, 24a showed no cytotoxicity in Gb3-negative CHO cells. It was therefore concluded that the prodrug 24a can be selectively internalized in Gb3-expressing cells and release active SN-38. Ojima et al. developed a prodrug conjugate (25) with biotin, a disulfide linker, and a second generation taxoid (SB-T-1214) 5081


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Figure 21. (A) Syntheses of 35 and 36. (B) Once in the extracellular milieu, the fluorescence emission and cytotoxicity of 35 are quenched (off). Upon target-specific internalization, enhanced fluorescence emission and cytotoxicity (on) occurs due to the cleavage of the disulfide bond. (C) Although 36 undergoes internalization, it remains biologically inactive (off) with quenched fluorescence, due to the absence of a cleaving mechanism.

the release of fluorescent coumarin via disulfide cleavage. Biotin−linker−taxoid−fluorescein (28) was also prepared to validate the entire process of internalization by RME and drug release. As anticipated, 28 was internalized into L1210FR cells overexpressing the biotin receptors and released the fluorescent taxoid. More importantly, by confocal fluorescence microscopy, the authors found that the released fluorescent taxoid bound to microtubules, the target protein of the taxoid. Finally, they demonstrated that conjugate 25 exhibited selective internalization to L1210FR cells through RME and that taxoid release by GSH was efficient in 72 h of incubation. Moreover, in a cell viability test using the MTT assay, 25 showed high potency (IC50 = 8.8 nM) against L1210FR cells overexpressing biotin receptors, while the potencies against the biotin-receptornegative cell lines L1210 (IC50 = 522 nM) and WI38 (IC50 = 570 nM) were 59- and 65-fold lower, respectively. Recently, Kim et al. also presented a multicomponent synthetic strategy that allows direct fluorescence-based monitoring of targeted cellular uptake and release of a conjugated therapeutic agent.30 This work is based upon the

known fact that cyclic peptides containing an RGD (Arg-GlyAsp) sequence can be recognized and internalized by αvβ3 integrin, a well-known tumor-associated receptor. In this work, as depicted in Figure 19, a RGD peptide-appended naphthalimide camptothecin (CPT) prodrug (29) was first prepared. 29 is a multifunctional molecule composed of a disulfide bond as the cleavable linker, naphthalimide as the fluorescent reporter, a cyclic RGD peptide as the cancer targeting ligand, and CPT as the model active agent. Upon reaction with thiols in aqueous media at pH 7.4, disulfide cleavage occurs. This leads to the release of active CPT, as well as the generation of a new fluorescence emission at λem = 535 nm. To demonstrate the role of the RGD moiety in guiding 29 to the target cells, precursor 30 was also synthesized and tested. The authors of the study found that 29 was selectively internalized into tumor cells via αvβ3 integrin-mediated endocytosis. In the endoplasmic reticulum (ER), disulfide cleavage occurs upon exposure to cellular thiols, releasing active CPT with a concomitant fluorescence off−on signal response. 5082


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Figure 22. CPT release mechanism of 37 by a thiol-induced reduction.

The quenching mechanism by the intramolecular folate moiety is unclear; however, a fluorescent energy transfer is certainly ruled out, since the excitation spectrum of folic acid does not overlap with the emission spectrum of Dox. The authors of this study examined the cytotoxicity of Dox and the fluorescence change of 35 and its noncleavable analog (36) in both FRpositive (A549) and FR-negative (MCF7) cells. In A549 cells, the prodrug 35 was internalized via FR-mediated endocytosis followed by GSH-induced cleavage to release Dox-SH, the fluorescent and active drug species. The transfer of the cytotoxic Dox-SH to the nucleus is also monitored by its fluorescence. However, 36 showed insignificant fluorescence and no cytotoxicity, mainly due to its lack of a cleavable linker. In contrast, in MCF7 cells, both probes show negligible cytotoxicity, confirming that 35 is delivered through FRmediated internalization. Moreover, 35 was found to have excellent plasma stability (half-life of 47 h in mice), suggesting that this type of prodrug may be suitable for clinical trials. Low et al. synthesized a folate−peptide−camptothecin conjugate (37) with a disulfide linker (Figure 22).79 The clinical use of camptothecin (CPT) has been severely hindered by the ring-opening of its lactone, which results in an inactive carboxylate species. Its poor water solubility is another drawback in the development of CPT as a therapeutic agent. The approach to improve the chemical stability and water solubility of CPT in the study cited was to construct a folate conjugate linked to the −OH group of CPT via a hydrophilic peptide (Pte-γ-Glu-Asp-Arg-Asp-Asp-Cys) spacer containing a disulfide linker. The prodrug (37) exhibited enhanced water solubility and efficient release of CPT via the disulfide cleavage reaction. Moreover, 37 was demonstrated to possess high affinity for FR-expressing cells and to inhibit cell proliferation in

Wender et al. reported the development of three disulfidebased luciferin-transporter conjugates (31a−31c) for the realtime monitoring of cellular uptake and release.77 The conjugates contain luciferin, octaarginine as the transporter, and a disulfide linker, with a variable distance between the carbonyl group and the proximal disulfide linkage (Figure 20). The stabilities of 31a−31c in HEPES-buffered saline (pH 7.4) at 37 °C were estimated by measuring their decomposition products (luciferin, 32a−32c, and CO2) using high-pressure liquid chromatography (HPLC). The half-lives of the conjugates are 3, 11, and 33 h for 31a, 31b, and 31c, respectively. In contrast, the release of luciferin (34) and of the cyclic products formed by dithiothreitol (DTT)-mediated disulfide cleavage of the conjugates was completed in 1 min. When the conjugates 31b and 31c were incubated with prostate cancer cells transfected with a luciferase gene, strong luminescence was observed in 1 min, caused by luciferase activity utilizing luciferin from the disulfide cleavage of the conjugates. The cells treated with 31c showed lower initial luminescence and a slower rate of decay when compared to those treated with 31b; this can be attributed to the long-lived intermediate 33b. Therefore, the disulfide-based luciferin system allows the quantification of the uptake via various transporters in both transfected cells and transgenic animals. Perez et al. reported an activatable folate−doxorubicin conjugate prodrug (35), in which the folate moiety acts as both a targeting ligand toward cells expressing the folate receptor (FR) and a quencher for doxorubicin (Dox) fluorescence, as denoted in Figure 21.78 The fluorescence from Dox at 594 nm is quenched 5-fold when it is covalently linked to a folate unit using the dithiobis(succinimidyl propionate) (DSP) or disuccinimidyl suberate (DSS) linker. 5083


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Figure 23. FR-targeted prodrug conjugates 38−40.

Figure 24. Proposed K-182 release mechanism of 41 by cellular reduction.

which was completed within 6 h and was confirmed by HPLC. Following positive in vitro and in vivo results and toxicological evaluation, 39 could be selected as a potential clinical candidate. Miyata et al. designed a FR-targeted prodrug, the thiolate histone deacetylase inhibitor (HDACI) 40, that shows potent HDACI activity under reducing conditions.82 Prodrug 40 displayed growth-inhibitory activity against FR-positive breast cancer cells, and the cellular uptake of 40 was demonstrated to be FR-mediated based on a competition experiment with free folic acid. The utilization of a disulfide bond to connect a thiolate HDACI with folic acid should be applicable to other anticancer drugs bearing a thiol group. Figure 24 illustrates a scheme by Nagaoka et al. for the development of a HDACI prodrug 41 to enhance the transfection efficiency of genes into human cells.83 It is known that cationic nanoparticles (NPs) bind electrostatically to negatively charged DNAs to form complexes. The complex can enter the cells via endocytosis and transfer its DNA portion into the nucleus. Upon the basis of the fact that HDACI increases transfection efficiency due to improved gene transfer in the cytoplasm, they attempted to combine NPs with HDACI to generate an efficient DNA carrier. An aliphatic chain

human KB cells (a human cervical cancer cell line that overexpresses the folate receptor) with an IC50 of 10 nM. Leamon et al. reported a novel folate conjugate of mitomycin C (38) that is produced by coupling folic acid−γ-cysteine to 7N-modified mitomycin C (MMC) via a disulfide bond (Figure 23).80 The unmodified MMC is very toxic to humans, and thus its use is limited to very infrequent dose schedules. In this aspect, the targeted disulfide-based prodrug 38 could be a better option for dosing humans with this drug class. In both in vivo and in vitro tests, 38 exhibited high affinity for FR and released active MMC via disulfide cleavage. The inhibition of DNA synthesis by the released MMC was measurable in FRpositive cells with an IC50 of ∼5 nM. However, no drug activity was measured in FR-negative cells and the IC50 value (>1000 nM) was also high. Simultaneously, Vlahov et al. also presented a water-soluble folate receptor (FR)-targeted conjugate 39 composed of folic acid, the potent cytotoxic molecule desacetylvinblastine (DAVB) monohydrazide, and a reducible disulfide bond, as shown in Figure 23.81 When 39 in PBS buffer (pH 7.4) was treated with 1 mM GSH at 37 °C, disulfide bond cleavage was observed with the concomitant release of the folic acid spacer, 5084


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Figure 25. Prodrug conjugates 42−44.

Figure 26. GSH-transporter-targeted prodrug conjugates 45 and 46.

transporters and release the active drug (dopamine or adamantamine) in the brain. Long et al. synthesized the disulfide-linked polyethylene glycol (PEG)−GSH conjugates 47 and 48 for GSH delivery (Figure 27).88 PEG is employed to protect the GSH moiety

exhibiting affinity for NPs was connected to the HDACI drug K-182 via a disulfide linker to release active HDACI under the reducing conditions of the cytosol. The NPs combined with 41 yielded a 2−4-fold improvement in gene expression efficiency compared to that of the original NPs. The enhancement of gene expression is due to the release of K-182 from 41 in the NPs. In the brain, the penetration of many antiviral drugs is limited at the blood−brain barrier (BBB).84 This is due to the presence of P-glycoproteins (P-gp) on the membrane of brain capillary endothelial cells. Specifically, P-gp transports a large variety of hydrophobic drugs out of the plasma membrane into the extracellular milieu before they reach the cytosol of brain capillary endothelial cells.85 Recently, Chmielewski et al. developed a set of dimeric prodrugs of abacavir, 42−44, that are designed to inhibit P-gp at the cell membrane and then release active abacavir, a HIV drug, in cellular reducing environments (Figure 25).86 The dimeric prodrugs interacted with P-gp at the membrane in a brain capillary model of BBB and efficiently inhibited its activity. The subsequent cleavage of the disulfide bond in the prodrugs by thiols facilitated the release of active abacavir, exhibiting anti-HIV activity in T-cellbased HIV assays. The release rate of abacavir was shown to decrease with the increasing number of methyl groups in the linkage. Vince et al. developed another set of prodrug conjugates, 45 and 46, that target GSH transporters at the BBB to deliver antiParkinson drugs (Figure 26).87 The prodrugs contain GSH as the carrier, adamantamine (45) or dopamine (46) as the active anti-Parkinson drug, and a disulfide bond as a cleavable linker. In the BBB cell model, prodrugs 45 and 46 were able to cross the BBB through their selective interactions with GSH

Figure 27. PEG−GSH conjugates 47 and 48.

from oxidation until it is delivered into the cells. GSH is oxidized to GSSG during oxidative stress in cells, thereby restricting the oxidation of other molecules. Therefore, it plays an antioxidant role to protect cells from oxidative stress. When administered to the SH-SY5Y human neuroblastoma cell line under oxidative stress induced by H2O2, 47 was not effective in protecting the cells; however, 48 was 100% effective. This is because the C−S bond of 48 can be cleaved to release GSH, but the corresponding bond in 47 is not cleavable. 5085


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Figure 28. PSMA-targeted prodrug conjugates 49−55. 5086


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Figure 29. Triazine-polymer-based prodrug conjugates 56 and 57.

while 57 contains both ester and disulfide linkages. For enhanced water solubility and biocompatibility, 2-kDa PEG chains are incorporated into the prodrugs. In the evaluation of their antitumor activities, 57 displayed higher mortality than 56. Intravenous delivery of 57 was performed by single, double, or triple dosing regimens with doses spaced 1 week apart. The doses varied from 50 to 200 mg of paclitaxel/kg. Tumor growth arrest and regression were observed over the 10-week treatment period without mortality in mice treated three times with 50 mg paclitaxel/kg. Receptor-mediated endocytosis (RME) is a major mechanism of selective uptake of impermeant molecules into cells, which would be highly valuable for DDS. In typical RME, an extracellular ligand designed to bind to its receptor on the cell membranes enters the cells as part of the receptor−ligand complex to form early endosomes. The inside of the particle is then acidified (pH ≈6) to allow the dissociation of the receptor molecule from the complex and subsequent recycling to the membrane. The particles containing the remaining ligands are further acidified to late endosomes and lysosomes (pH ≈5),

Low et al. reported the development of the prostate-specific membrane antigen (PSMA)-targeted disulfide-bridged chemotherapeutic agents 49−55 (Figure 28).89 PSMA is expressed on the plasma membranes of prostate cancer cells and other solid tumors but is largely absent from healthy tissues. PSMA thus constitutes an ideal candidate for tumor-specific targeting. As a PSMA-specific ligand, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) was used, which selectively binds to PSMA and enters human prostate cancer cells via PSMAmediated endocytosis. In addition, a variety of drugs associated with prostate cancer, such as CPT, verrucarin A (VrcA), didemnin B (Did B), tubulysin B (Tub), and DAVB, were tested in this study. The authors concluded that the conjugates 49−55 can be selectively internalized into prostate cancer cells through recognition by PSMA and deliver a variety of cytotoxic drugs upon thiol-induced disulfide cleavage. Simanek et al. reported the antitumor activities of triazine dendrimers (56 and 57) bearing paclitaxel, a well-known mitotic inhibitor, against mice with human prostate cancer (Figure 29).90 Paclitaxel is attached by an ester linkage in 56, 5087


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Figure 30. Early/recycling endosome-targeted conjugates 58−61.

Figure 31. Proposed disulfide cleavage reaction (A) of 60 by GSH and schematic illustration (B) for the selective release of fluorophore 60-A from early/recycling endosomes by 58 into the cytosol and nucleus of mammalian cells.

endosomal system. For selective delivery into early/recycling endosomes, they synthesized four derivatives of the membrane anchor N-alkyl-3β-cholesterylamine, 58−61 (Figure 30). Glutamic acid residues in the linkers were also employed to localize the conjugates into endosomes. A pH-dependent membrane-lytic dodecapeptide (PC4) was added to confer membrane-disrupting ability. Compounds 60 and 61 contain a 5-carboxyfluorescein group linked to the linker moiety through disulfide and amide bonds, respectively. Since early/recycling endosomes contain oxidizing environments, the disulfide of 60 should be relatively stable in intact endosomes. However, disruption of the endosomal membranes by the PC4 moiety allows cytosolic GSH to encounter the conjugates. Thereby, the

where the ligands encounter degradation enzymes, including hydrolases.91 However, some viruses and other intracellular pathogens can escape from the endosomal transfer pathway to late endosomes/lysosomes by producing oligopeptides that disrupt endosomal membranes in a pH-dependent manner.92 In this regard, Peterson et al. reported several membranebound disulfide-linked conjugates designed to escape to early/ recycling endosomes from degradation in the late endosome system in living cells.93 They hypothesized that targeting to early/recycling endosomes, which are less acidic and less hydrolytically active than late endosomes/lysosomes, might endow a degradable chemical with a longer lifetime in cells, compared to delivery methods that penetrate deeper into the 5088


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disulfide bond of 60 is reductively cleaved, and the fluorophore 60-A is released into the cytoplasm and nucleus of the cell (Figure 31). However, replacement of the disulfide of 60 with the amide bond of 61 blocks the release of the fluorophore.

located inside the macrocyle, encapsulating the hydrophobic guest (Nile Red in the study). This cyclodextrin exhibited release of the guest molecules upon cleavage of the disulfide bond by the reducing reagent DTT. Further improvements in utilizing micelles that contain the S−S bond were investigated by Thayumanavan et al.95 They also used the idea of connecting the hydrophilic part to the hydrophobic part using a disulfide unit and forming a micellelike nanoassembly in aqueous media, as in micelles of 63 (Figure 33). They used the anticancer drug Dox as the hydrophobic guest, which was encapsulated by the hydrophobic part. Upon reduction, the 63@Dox micelle disassembled due to disulfide cleavage, resulting in the release of Dox. The rate of disassembly depended on the concentration of a reducing agent, GSH. This study not only demonstrated the release of the drug but also confirmed the cytotoxic effect on MCF-7 cells upon drug release by GSH in vitro. Lee et al. demonstrated the synthesis of shell cross-linked polymer micelles (SCMs) using a cross-linking agent containing disulfide bonds.96 Unlike other SCMs, which frequently use polyacrylate or polyacrylamide, micelles of 64 use PEG and poly(amino acid)s as their building blocks (Figure 34). The utilization of PEG has an advantage: it can prolong the in vivo circulation time of nanocarriers which are important in cancer therapy via the enhanced permeability and retention (EPR) effect. Micelles of 64 were shown to encapsulate the antifolate drug methotrexate (MTX) through self-assembly, and the release of MTX into the cellular cytoplasmic environment was evaluated in living cells. As the GSH level in A549 cells was increased, inhibition of cell proliferation was enhanced as well. Considering the fact that cancer cells have enhanced levels of GSH, such novel SCMs can potentially increase drug delivery efficiencies. Zhong et al. investigated a biodegradable micellar system with a shedable shell consisted of disulfide-linked dextran-βpoly(ε-caprolactone) diblock copolymer 65 (Figure 35).97 Dextran is a natural alternative to PEG and is often utilized in biomedical applications due to its aqueous solubility, biocompatibility, and nonfouling characteristics. Micelles of 65 showed high efficiency for Dox delivery into cells through endocytosis, with subsequent drug release upon encountering intracellular reducing reagents. The 65@Dox micelles react

3.2. Disulfide-Based Nanocapsule Carriers

Amphiphilic molecules can easily generate nanomicellar structures in which, in an aqueous environment, the surface consists of the hydrophilic moieties of the molecules whereas the interior is full of the hydrophobic ones, an ideal place to accommodate hydrophobic guests such as drugs. Whenever such contrasting moieties are linked by a disulfide bond, the molecules are susceptible to cleavage in reducing environments such as intracellular spaces, and the corresponding nanomicelles may act as carriers. Ravoo et al. introduced a derivative of cyclodextrin (62), a cyclic oligosaccharide. This macrocyclic compound has a disulfide bond connecting its hydrophobic substituents to the hydrophilic cyclodextrin and is capable of forming a nanoparticle vesicle (Figure 32).94 The hydrophobic moiety of 62 is

Figure 32. Cyclodextrin vesicles consist of bilayers of 62 (in which the hydrophobic tails are directed inward and the hydrophilic macrocyclic head groups are facing water) enclosing an aqueous interior.

Figure 33. (a) Amphiphilic unit with redox-sensitive disulfide functionality. (b) Schematic representation of drug release via GSH-triggered disassembly of amphiphilic polymer micelles. 5089


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Figure 34. Illustration of shell cross-linking in the 64@MTX micelle and facilitated drug release in response to cellular GSH.

Figure 35. Reduction-responsive biocompatible and biodegradable micelles (65) for the efficient triggered intracellular release of DOX.

The disassembly rate of Dox from 66@Dox nanomicelles depends on intracellular GSH levels; thereby, the drug delivery can differentially affect normal cells verses cancer cells, because cancer cells typically have 4-fold higher GSH concentrations than those seen in normal cells. Likewise, the 66@Dox nanomicelles exhibit specificity toward drug release in tumor environments, and demonstrate improved pharmacological efficacy in cell proliferation assays with MCF-7 cells. In disulfide-based nanocapsule carriers, small interfering RNAs and gene vectors may occupy the position of the

with DTT, resulting in the cleavage of their disulfide bonds. Cell experiments demonstrated the efficient delivery and release of Dox into the cytoplasm and nucleus of the cell with high efficacy. Shi et al. introduced a disulfide-based nanocapsule system utilizing PEG as the hydrophilic part. They synthesized a novel nanomicelle, 66 (Figure 36), consisting of a PEG shell that could be shed and a poly(ε-benzyloxycarbonyl-lysine) core, linked by a disulfide bond.98 The nanoparticles of 66 encapsulated Dox and were taken up through the EPR effect. 5090


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Figure 38. Chemical structures of 68, PMA (x = 100 mol % and y = 0 mol %), and PMASH (x = 82 mol %, y = 18 mol %).

amine. The resulting films were swollen at physiological pH but remained intact; in contrast, films without the disulfide bonds were deconstructed at physiological pH. These capsules of 68 underwent disassembly upon the addition of DTT, releasing the model protein, transferrin, in the study. The preparation of a monodisperse, single-component degradable polymer capsule containing 69 (Figure 39) has been reported.101 The capsules consisted entirely of poly(methacrylic acid) (PMA) and were obtained by the sequential addition of thiolated poly(methacrylic acid) (PMASH) and poly(vinylpyrrolidone) (PVPON) to silica particles. After the thiol groups were oxidized to form bridging disulfide linkages in PMASH, the silica particles and PVPON were consecutively removed, leaving the PMA capsules as the sole component. Such disulfide linkages are certainly cleavable in the presence of physiological concentrations of glutathione. Caruso et al. has reported delivery of small interfering RNA (siRNA) using LbL microcapsules, which have superior payload capacity to smaller nanoparticles.102 The delivery of siRNA in this study is based on negatively charged nanometer-thin poly(methacrylic acid) films containing cross-linking disulfide bonds. Two systems, 70@siRNA and 71@siRNA microcapsules (Figure 40), with different loading methods, have been introduced: the system based on 70 is polycation-free, whereas the 71 version contains polylysine for complexation of siRNA in the microcapsule void. The siRNA in this study was targeted to survivin, a member of the inhibitor of apoptosis (IAP) family. Due to the high activity and abundance of survivin within human tumors, it has been regarded as a

Figure 36. Antitumor activity of redox-sensitive 66@DOX nanomicelles: (A) amphiphilic block copolymer with disulfide linkage, (B) PEG-shielded nanomicelle, (C) nanomicelle endocytosis into normal cells (low GSH), (D) accelerated endocytosis of nanomicelles into tumor cells with EPR effect, and (E) apoptosis of tumor cells.

hydrophobic drugs for delivery. A nanocapsule that allows better expression efficiency of plasmid DNA has been studied by Kataoka et al. The nanocapsule is composed of a block catiomer, PEG-SS-P[Asp(DET)] (67) (Figure 37), which forms a polyplex micelle with plasmid DNA molecules.99 Upon the addition of DTT, a reducing reagent, the disulfide bond is cleaved to release the DNA molecules. Nanocapsules of 67 exhibited improved gene transfection efficiency by 1−3 orders of magnitude, compared to corresponding nanocapsules that lack a cleavable unit. The surface of the nanoparticles itself may consist of interweaving disulfide cleavages. Caruso et al. investigated the use of hollow polymer capsules as drug delivery nanocapsules that are stable at physiological pH unless disintegrated by an external stimulus, a major challenge in drug delivery using hollow nanocapsules. These hollow polymer capsules, prepared in layer-by-layer (LbL) manner, consist of hydrogen-bonded multilayer thin films, containing thiol moieties cross-linked to the films.100 A capsule of 68 (Figure 38) was constructed using poly(vinylpyrrolidone) and poly(methacrylic acid) with cyst-

Figure 37. (a) Structure of 67 consisting of PEG and the Asp unit with a disulfide bond. (b) Schematic illustration of PEG−detachable polyplex micelle formation and PEG detachment in the reducing intracellular environment. 5091


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In a further advance in the development of hollow nanoparticles for drug delivery, Kim et al. developed a method for synthesizing hollow polymer capsules that obviates the need for a template structure to shape the core−shell structure and for core removal in order to form a hollow stimulus-responsive polymer nanocapsule.103 The polymer nanocapsules comprising 72, shown in Figure 41, are composed of CB[6] and

Figure 39. Preparation of disulfide-cross-linked PMA capsules (69) involves the assembly of PMASH/PVPON multilayers on the surface of template particles, conversion of the thiol groups into bridging disulfide linkages, removal of the core particle, and release of PVPON.

Figure 41. Synthesis of polymer nanocapsules of 72, with and without disulfide bridges.

disulfide bridges, enabling the release of the encapsulated drug in response to the reducing environment of the cytosol. In addition, this nontoxic nanoparticle has a cavity that can recognize and bind polyamines with a variety of functional moieties, such as targeting ligands and imaging probes, in a noncovalent manner. In this study, carboxyfluorescein (CF) was encapsulated to allow monitoring. Additionally, galactosetargeting units were introduced on the surface of the nanocapsule to facilitate entry into the cells through RME. As depicted in Figure 42, HepG2 hepatocellular carcinoma cells, which overexpress galactose receptors, were treated with these CF-encapsulating nanoparticles (CF@72@gal). When CF@ 72@gal was introduced into the cell media, a striking increase in the fluorescence signal was observed in the cells, indicating

Figure 40. Schematic representation of siRNA loading methods. 70@ siRNA microcapsules: (a) siRNA is adsorbed onto amine-functionalized silica particles, (b) LbL assembly of the PMASH film is performed around the siRNA-coated particles, and (c) the film is cross-linked and the core removed. 71@siRNA microcapsules: (i) mesoporous silica particles are infiltrated with the polycation PMASH−polylysine, (ii) LbL assembly of the PMASH film is performed around the polymer-filled particles, and (iii) following core removal, the siRNA is infiltrated into the particles and sequestered by the polycation core. Figure 42. Schematic representation of the noncovalent surface modification of the carboxyfluorescein (CF)-encapsulating polymer nanocapsule (CF@72) with Gal through host−guest interactions, RME, and the autonomous triggered release of the encapsulated CF into the cytosol.

relevant target for cancer treatment. In the cell test, these microcapsules containing the siRNA against IAP were delivered to PC3 prostate cancer cells, resulting in efficient inhibition of the expression of the antiapoptotic protein. 5092


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Figure 43. Schematic illustration for the synthesis of pH-sensing organic/inorganic hybrid mesoporous silica nanoparticles (MSN) coated with P(NAS-co-OEGMA-co-NaphMA) bushes, their cross-linking with cystamine, and the redox-responsive release of the embedded rhodamine B (RhB) dye.

Figure 44. (A) Functionalization of a FITC-encapsulated 74 nanoparticle. (B) Schematic representation for the release of Nile Red and FITC via different stimuli from 74-SS-FITC@Nile Red nanoparticle.

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Figure 45. 75 and 76 are cross-linked through imine bond formation to form N1. Cross-linking through disulfide bond formation generates N2. Disassembly of N2 through imine hydrolysis forms N3, and N4 is formed through disulfide reduction. Alternatively, disassembly is triggered through disulfide reduction to nanoparticle N5 followed by the hydrolysis of the imine cross-links to afford the polymeric component polymer chains N6. N3 was functionalized to afford the PEG-decorated N7, whose disassembly into N8 was triggered through the addition of TCEP at pH 5.5.

that upon polymer internalization into HepG2 cells, the disulfide bridges were degraded in the reducing intracellular environment, releasing the fluorophores. The reductive drug release mechanism based on the disulfide bond has been further exploited by interweaving it with other stimulus−response mechanisms for sophisticated and specific targeted drug release. Liu et al. reported the synthesis of an organic/inorganic hybrid mesoporous silica nanoparticle (MSN) capable of releasing its entrapped guest molecules upon redox stimuli (Figure 43).104 MSN 73 is synthesized using reversible addition−fragmentation chain transfer (RAFT) copolymerization of N-(acryloxy)succinimide (NAS), oligo(ethylene glycol)monomethyl ether methacrylate (OEGMA), and a 1,8-napthalimide-based pH-sensing monomer. Rhodamine B (RhB), a model molecule for drug in this study, was effectively loaded onto the P(NAS-co-OEGMA-co-NaphMA) MSN 73 via cross-linking to the surface of MSN by cystamine, yielding 73@RhB MSN. Addition of DTT to the complex results in cleavage of the disulfide linkage to open the blocked nanopores and release the embedded RhB. The RhB release rate depends on the concentration of DTT, demonstrating the potential of MSNs as controlled-release nanocarriers in cell and tissue imaging.

Boyer et al. studied the selective release of two hydrophobic guests within the nanoparticle, depending on the type of stimulus.105 They synthesized a nanoparticle, 74 (Figure 44), built from amphiphilic copolymers of the P(OEG-A) homopolymer chain that was extended with vinyl benzyl chloride (VBC) and pentafluorophenyl acrylate (PFP-A) comonomers. On substituting the chlorine atoms in the VBC units with sodium methanethiosulfonate, the copolymer was transformed to a copolymer chain with methanethiosulfonate (MTS) pendant functionality. Diverse functional groups with thiols can be introduced into this system by simple thiol/MTS exchange chemistry. The activated esters were cross-linked to form an acid-cleavable bond, to endow the core shell nanoparticle with pH-responsiveness (74). Molecules of Nile Red, a hydrophobic dye, and fluorescein isothiocyanate (FITC), a hydrophilic dye, were encapsulated, and the release of each fluorescent molecule was monitored. The authors found that the nanoparticle 74-SS-FITC@Nile Red could control the selective release of either a single dye or both dyes at once, by adopting different experimental stimuli. Stimulation with DTT, a reducing agent, allowed cleavage of the disulfide bond, releasing FITC, whereas an acidic environment induced the release of Nile Red only. Adopting both conditions (DTT with 5094


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Figure 46. (a) Amphiphilic ABA-type triblock copolymer (77) with a dual-stimuli-cleavable main chain for the middle hydrophobic block. (b) Schematic illustration of block copolymer micelles undergoing either fast or slow degradation as a result of either fast photoinduced or slow reduction-induced main chain cleavage.

Figure 47. Schematic representation of the newly synthesized disulfide cross-linker 78.

Figure 48. 79/PVPON thin film: under LbL polymer deposition conditions (a), cross-linked and at physiological pH (b), and under cellular reducing conditions (c).

endocytosis. Therefore, the orthogonal nature of the nanoparticle will confer great specificity in disassembly and in the delivery of the cargo drug. Zhao et al. reported the design of dual stimuli-responsive block copolymer (BCP) micelles.107 These micelles were composed of a new amphiphilic ABA-type triblock copolymer, 77 (Figure 46), in which the hydrophobic middle block consists of two different types of cleavable units, a disulfide group and an o-nitrobenzyl methyl ester group. The units can be cleaved by reducing agents and light, respectively. It is worth mentioning that either of the two stimulating conditions is enough to induce the cleavage and destruction of the micelle structure. The core of 77 can be degraded either by slow cleavage of the disulfide groups by a reducing agent, by rapid photocleavage of the o-nitrobenzyl methyl ester groups, or by a combination of the two stimuli. The fact that the nanoparticle is responsive to two stimuli provides the advantage of using a combination of the stimuli to generate on-demand release rate profiles.

acidic pH) resulted in the simultaneous release of both molecules. Fulton et al. investigated polymeric nanoparticles that can be disassembled by the simultaneous application of two stimuli: hydrolytic degradation by lowering pH and cleavage of disulfide cross-links by thiols.106 The chains of the nanoparticle consisted of the acrylamide-based linear copolymers 75 and 76, depicted in Figure 45, showing pyridyl disulfide appendages with either aldehyde or amine functional groups. The appendages were cross-linked through imine bond formation at pH 8.0 to form polymeric nanoparticles. These nanoparticles could be simultaneously disassembled by lowering the pH to 5.5 in the presence of a disulfide-reducing agent, tris(2carboxyethyl)phosphine (TCEP), which allowed the hydrolysis of imine cross-links and cleavage of the disulfide. It is notable that both stimuli are needed at the same time, and either stimulus alone is not sufficient to trigger the disassembly of the nanoparticles. Such dual responsiveness is beneficial for an endocytosis-mediated DDS, which will sequentially encounter the reducing environment in the cytosol and the acidic environments in late endosomes, the final products of 5095


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Figure 49. (a) Encapsulated DOX is released from 79 capsules after internalization of 79@DOX capsules by LIM1899 cells, as visualized by confocal laser scanning microscopy (CLSM), with nuclei counterstained with Hoechst 33342 (blue). (b) Enhanced cytotoxicity of 79@DOX capsules (compared to free DOX) on LIM1899 cells.

3.3. Disulfide-Based Hydrogel Carriers

50), was degraded into water-soluble polymers in the presence

108

In 2008, Galaev’s group produced a biodegradable supermacroporous (PHEMA) hydrogel, 78, as shown in Figure 47, composed of poly(ethylene glycol) diacrylate and a watersoluble disulfide cross-linker. The obtained hydrogel could be degraded into small pieces when the disulfide bond in the cross-linkers was subjected to reducing agents such as DTT; the disintegration rate depended on the cross-linking density in the hydrogels and the concentration of DTT, allowing finetuning of the degradation time from several hours to a fortnight. Caruso et al.109 investigated the parameters governing the formation and degradation of the polymer capsules using disulfide cross-linked PMA. They developed a new and efficient method to synthesize thiol-functionalized polymethacrylic acid, 79, and then to construct a polymer through the LbL deposition of 79 and PVPON on the silica particle templates, as depicted in Figure 48. 79 was cross-linked to form layers (linked through S−S bonds), and then PVPON and the templates were removed as in Figure 48b. The polymer capsules degraded inside the cells upon cleavage of the disulfide group by cellular GSH, as represented in Figure 48c. The thickness of the film and degradation rate of the capsules can be directly controlled by changing the amount of thiol-functionalized 79; the degradation rate was independent of thickness of the film. Further investigations have been carried out regarding the fate of LbL-assembled submicrometer-sized polymer hydrogel capsules (79) utilized as delivery carriers for Dox in living cells.110 The investigators found that the cellular uptake of the disulfide-stabilized 79 capsules by a human colon cancer cell line (LIM1899) was a time-dependent process and that the internalized capsules were distorted and accumulated in late endosomal or lysosomal compartments (Figure 49a); compared to the Dox-free version, the 79@DOX capsules exhibited 5000-fold enhanced cytotoxicity in cell viability studies (Figure 49b). A degradable hydrogel cross-linked with disulfide bonds, which was prepared through the Michael addition reaction between branched polyethylenimine (PEI) and the carbon− carbon double bonds of N,N′-bis(acryloyl)cystamine, has been reported by He’s group.111 The obtained hydrogel, 80 (Figure

of DTT; this can be utilized to initiate the release of encapsulated molecules (such as drugs) and to facilitate

Figure 50. Schematic representation of the degradation of a disulfide cross-linked hydrogel (80) in the presence of DTT. 5096


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Figure 51. Formation of cross-linked PEOS (81) hydrogel.

Figure 52. Schematic presentation of the polymer nanoparticles (83@Nile-Red@TAT-peptide or FITC) and the release of the capsulated molecules.

with pyridine disulfide (PDS)-derived thioethyl methacrylates for cross-linkable functionality. A random copolymer, 82, containing the oligoethyleneglycol methacrylate and the PDSderived methacrylate, was prepared by RAFT polymerization. Cross-linked particles (83) were synthesized from 82 by adding DTT in proportion to the number of PDS functionalities in the polymer. Nile Red or other hydrophobic molecules can be incorporated into the gel by noncovalent (hydrophobic) interactions. The surface of the gel was functionalized with thiol-modified TAT peptide or FITC under mild conditions. The encapsulated molecules (83@Nile-Red@TAT-peptide or FITC) can be released into cells upon degradation of the nanogel caused by the intracellular reducing conditions. The same group also synthesized a highly stable nanoscopic vehicle, 84, depicted in Figure 53, based on a random copolymer, oligoethyleneglycol (OEG), and PDS, where the latter provides side-chain functionalities.114 Through noncovalent interactions, the nanogel encapsulated guest mole-

removal of empty cargo molecules. This disulfide cross-linked hydrogel, 80, played a critical role in accelerating the release of its coated drugs, and the drug release rate could be finely controlled by changing the cross-linking density of the hydrogel. Park and colleagues112 developed a biodegradable crosslinked polyethylene oxide sulfide (PEOS, 81) hydrogel (Figure 51), for the controlled release of a model drug. The hydrogel was obtained through disulfide cross-linking of PEOS (81), which exhibited a high dependence on temperature, necessitating efficient cross-linking at 40−50 °C. The obtained 81 hydrogel can be selectively degraded in response to high GSH concentrations. This is a promising candidate for use as an injectable biogel precursor, as confirmed by in situ gel formation in mice. As shown in Figure 52, Thayumanavan et al.113 reported simple, emulsion-free preparation of a biocompatible polymeric nanogel, 83, via intra/intermolecular disulfide bond formation 5097


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the stars in a facile manner (reducing reagents or acidic environments). This kind of architecture can be useful in the development of prodrugs, in biological imaging, and in other applications. Yang and co-workers117 adopted a disulfide bond as a cleavable linker to control molecular self-assembly, and the formation of small molecular (SM) hydrogel composed of 88 (Nap-GFFYE-CS-EERGD) (Figure 56a) was utilized as the precursor of the SM hydrogelator with several features: (1) The Nap-GFFY sequence guaranteed the formation of the gels after disulfide bond cleavage because derivatives of this had been demonstrated to be “supergelators”; (2) the CS moiety had a disulfide bond that could be cleaved by reducing agents such as TCEP, DTT, and GSH; (3) the EERGD sequence ensured the formation of clear solutions of 88 in aqueous solution. This method can yield homogeneous SM hydrogels that are useful in tissue engineering. Additionally, it offers a convenient way to develop SM hydrogels for cellular applications because the hydrophilic peptide on 87 can be replaced with a cellpenetration peptide or other specific targeting peptides. Subsequently, Yang et al.118 introduced a SM hydrogel, 89 (Figure 56b), that can rapidly respond to β-cyclodextrin (βCD) or β-CD derivatives, based on an adamantine peptide for recovery of the postcultured cells. The gel was formed by disulfide bond reduction by DTT or GSH; a gel-to-clear solution transition was achieved by the addition of β-CD derivatives. Mouse fibroblast 3T3 cells were shown to attach and grow well at the surface of the SM hydrogels, and the 3T3 cells postculture were also recoverable from gels as well as clear solutions upon the interaction between adamantine and β-CD; this process did not cause obvious effects on the behavior of 3T3 cells. Lyon’s group119 synthesized a thermoresponsive and degradable hydrogel, composed of poly(N-isopropylmethacrylamide) (pNIPMAm) and the disulfide-based cross-linker N,N′bis(acryloyl)cystamine (BAC), via a redox-initiated, aqueous precipitation polymerization approach. The use of the disulfidebased initiator BAC as the radical source during polymerization leads to a reduction in the temperature needed to conduct the synthesis. The obtained nanogel was redox-responsive, and the intraparticle disulfide bond can be cleaved in the presence of a reducing agent such as DTT to form particles bearing thiols; these thiols can also be transferred into interparticle disulfide

Figure 53. Schematic representation of the preparation of biodegradable 84 nanogels.

cules, which could be released in response to a redox trigger, such as GSH, thereby minimizing any undesired loss of guest molecules before cell entry. The tunability of guest molecules was also demonstrated through an in vitro FRET experiment. These nanogels, with different cross-linking densities, played a key role in the stable encapsulation of hydrophobic drug (such as DOX) molecules and high cell-uptake efficiencies. Wu and Liu et al.115 reported a new method to generate loose and compact in situ biodegradable hydrogels (85, shown in Figure 54), through the polymerization of N-aminoethylpiperazine (AEPZ) and 2,2′-diacrylicamidodisulfide (BAC) and functionalization with polyethylene glycol (PEGNH2) under very mild conditions. A pH-responsive thiol− disulfide exchange was employed to manipulate hydrogel formation in situ; this can be readily utilized to tailor the structure and properties of the hydrogel. Syrett et al.116 reported the development of an acid- and disulfide-degradable microgel star polymer, 86 (Figure 55). The gel was composed of a degradable core generated through RAFT polymerization of poly(polyethyleneglycol methyl ether methacrylate) and poly(PEGMA300), with 4-cyanopentanonic acid dithiobenzoate (CPDB) as the RAFT agent and AIBN as the initiator. An acid (ketal) and disulfide were employed as biodegradable cross-linkers, which could be functionalized via both thiol−ene and thiol−pyridyl disulfide exchange reactions. This arm can be released upon the degradation of the core of

Figure 54. Structures of 85 hydrogels. 5098


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Figure 55. General schemes for the synthesis and functionalization of the 86 microgel.

Figure 56. Structures and optical images of the precursors of the small molecular hydrogels 88 and 89.

Compared with the behavior of thermally initiated particles, these nanogels retained a cross-linked network following disulfide cleavage due to uncontrolled chain-branching and

bonds in the presence of oxidants (e.g., NaIO4), as depicted in Figure 57. The resultant particles were completely eroded in response to reducing conditions or thiol competition. 5099


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Figure 57. Reversible redox-responsive transformation of the hydrogel.

self-cross-linking side reactions. These particles swell upon BAC cross-link scission and present reactive thiols. This pendant thiol functionality has been demonstrated to be useful for the conjugation of thiol-reactive probes and in reversible network formation by assembling particles cross-linked by disulfide linkages. Sun and Wang et al.120 reported the synthesis and characterization of a new 3D hydrogel, 90 in Figure 58, based on poly(amic acid) (PAA), which has applications in tissue regeneration. Hydrogel 90 contains two kinds of

cleavable bonds (amide bonds and disulfide bonds) that can be used to adjust its degradation rate. Different ratios of cystamine and amino-terminated polyethylene glycol (ATPEG800) were used to cross-link aliphatic PAA, a kind of hydrophilic multifunctional prepolymer that contains amide groups in its backbone and pendant reactive groups. The introduction of ATPEG800 into the hydrogel reduces the interfacial free energy and improves the PAA molecule number and biocompatibility. The gel was shown to be degraded in the presence of a reducing agent such as DTT and finally disintegrated, and the degradation time correlated with the molar ratio of cystamine and ATPEG800, as well as the crosslinked density. 3.4. Disulfide-Based Inorganic Carriers

In 2005, Dai and co-workers121 constructed a “smart” nanomaterial, 91@SWNTs, as depicted in Figure 59, with high potential for gene and protein delivery. Various biological molecules have been attached to phospholipid-functionalized single-walled carbon nanotubes (SWNTs) via cleavable disulfide linkages. The material was composed of three parts: SWNTs, phospholipids, and DNA or RNA containing a thiol group (X; SH-DNA or SH-SiRNA) and a six-carbon-long spacer; the latter two moieties were conjugated via a cleavable disulfide group, and then the obtained molecule were assembled to the carbon nanotube surface to afford the final nanomaterial. The cleavable disulfide linkage enabled the controlled intracellular release of drugs or genes from the SWNT surface. This nanomaterial exhibited much higher delivery efficiency of siRNA and more potent RNA interference

Figure 58. Chemical structure of the 90 hydrogel. 5100


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Figure 59. Schemes of SWNT functionalization by a thiolated biological molecule X with (91) and without (92) disulfide bonds.

Figure 60. Functionalization of 93@MWNT@QD−steptavidin−AFM tips: QDot−streptavidin was attached to the MWNT surface through a linker containing a disulfide bond.

organelles without the need for a carrier solvent and with no apparent damage. Yu and colleagues123 devised a nanoscale mechanochemical method utilizing a membrane-penetrating nanoneedle to deliver fluorescent quantum dots (QDs) into living cells. It consists of four components: a gold layer, an NH2-terminated selfassembly monolayer (SAM), sulfo-NHS−S−S−biotin (sulfoNHS attached to biotin via a cleavable disulfide subunit, 94), and streptavidin-coated QDs. All these components were conjugated together to afford the nanoneedle, 94@Au@ QDot−steptavidin (Figure 61). The monodispersed QDs were selectively delivered into the cytoplasm, and the nucleus of living cells and the processes inside the cells were monitored. This provided an unconventional strategy to practice biological investigation in living cells with high spatial and temporal precision with molecular resolution.

functionality than the widely used conventional transfection agent lipofectamine. Chen and Bertozziet et al.122 developed a nanoscale cell injector system (nanoinjector) based on carbon nanotubes to deliver cargo. The nanoinjector, 93@MWNT@QDot−steptavidin, shown in Figure 60, was constructed of two parts: a functionalized multiwall carbon nanotube (MWNT) and a specific recognizing unit. The former was functionalized with a protein-coated QDot−steptavidin cargo and the latter contained a hydrophobic pyrene and biotin moiety attached via a cleavable disulfide linkage and an atomic force microscope (AFM) tip. The functionalized carbon tube was tethered to the AFM tip to afford a retractable nanoneedle injector controlled by AFM. The disulfide linkage was reduced inside the cells, resulting in the release of the cargo. This system can selectively deliver a discrete, small amount of molecules into cells or 5101


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(PAH-2) and negatively charged poly(acrylic acid) (PAA), followed by the removal of cleavable disulfide-containing polycations after incubation in 1 mM DTT solution (Figure 64).126 The thickness of the original multilayered films decreased with increasing incubation time. The irreversible formation of nanopores with sizes ranging from 50 to 120 nm was confirmed by AFM measurements, and the pores were stable in buffer solution at pH values ranging from 7.4 to 1.6. This study may provide a general cleavable polycation template for the construction of non-cross-linked porous multilayered films from any kind of polycation/polyanion, and either weak or strong polyelectrolytes. A disulfide-bond-containing gemini surfactant, bis[N,Ndimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide] disulfide (96, Figure 65), was converted to N,Ndimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide by GSH through the cleavage of the intramolecular S− S bond of 96 in free-standing monolayers on an air−water interface, in supported monolayers immobilized on solid surfaces, and in vesicles.127 Both in monolayers and in vesicles, reductive cleavage of the disulfide bond of 96 could be obtained by GSH. The resulting monomers detached from the supported monolayers, leading to a loss of DNA affinity of the surface. In giant vesicles containing 96, microinjection of GSH caused disruption of the vesicle within approximately 30 s.

Figure 61. (a) Schematic of nanoneedle delivery of QDs (94@Au@ QDot−steptavidin) into living cells. (b) Procedure for functionalization of the nanoneedle (94@Au) and surface attachment of QDs.

4. APPLICATION OF DISULFIDE CLEAVAGE IN OTHER FIELDS

4.2. Fabrication of Nanotubes

A nanoporous alumina template membrane was used to fabricate hollow, uniform, biodegradable chitosan nano-testtubes for applications in drug delivery (Figure 66).128 These chitosan nano-test-tubes were cross-linked using a disulfidecleavable cross-linker, DTBP, before being removed from the alumina template membrane. The pore walls were first modified with a monolayer of silica (A), which was then reacted with polyethylene glycol silane (B). The surface layer of silica and polyethylene glycol was removed using argon plasma etching (C). Chitosan or carboxymethyl cellulose was then solution-cast into the template and cross-linked with either dimethyl 3,3′-dithiobispropionimidate or epichlorohydrin (D). The alumina template was removed by dissolving in acid (E) to liberate the biodegradable tubes. The tubes were degraded upon exposure to either lysozyme or sulfhydryl-containing reducing reagents. The dimensions were easily manipulated on the basis of the dimensions of the pores in the alumina template membrane, with lengths ranging from 100 nm to 1.2 μm and diameters ranging from 70 to 100 nm. This system may allow for a mechanism for intracellular drug delivery, as the tubes should degrade in the presence of intracellular GSH.

4.1. Fabrication of Nanoporous Materials

Block copolymers have been used to form self-assembled nanostructures. Thayumanavan’s group demonstrated the generation of nanoporous structures by selective degradation of one of the blocks after assembly using block copolymers,124 based upon their previously developed synthetic methodology for a wide variety of block copolymers that are cleaved under mild conditions.125 The diblock copolymer, polystyrene-blockpoly(ethylene oxide), 95, was synthesized by RAFT polymerization of styrene using a PEO-based macroinitiator containing a disulfide bond (Figure 62). The copolymer can be broken under mild conditions, leaving behind a chemically active functional group, resulting in a nanoporous thin film with a high degree of lateral order, where chemical functionalization inside the pores is considerably limited. In this way, wellordered polymer−gold composite nanostructures could be generated by a mild etching process and pore functionalization using disulfide chemistry (Figure 63). Porous films were fabricated from nonporous Lbl multilayers composed of a blend of positively charged disulfide-containing polyamidoamine (PAH-1) and poly(allylamine hydrochoride)

Figure 62. Synthesis of PS-ss-PEO (95) block copolymer. 5102


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Figure 63. Structure of 95 and schematic representation of the preparation of a nanoporous thin film and gold-coated nanopore.

Figure 64. Procedure used to produce porous polymer film from (PAH-1 + PAH-2)/PAA multilayered film. First, a polycation-adsorbed quartz substrate is subjected to repeated adsorption of PAA. Then, PAH-1 is degraded in DTT solution to disrupt the S−S bonds. Finally, PAH-1 and excess PAA are released, and the films are rearranged to form pores.

Figure 65. Reductive cleavage of the gemini surfactant 96 by GSH.

A rigid duplex cyclodextrin, 98, composed of two αcyclodextrin macrocycles connected by two disulfide bonds in the “transannular” [C6(I), C6(IV)] positions was prepared from partially debenzylated α-cyclodextrin 97 (Figure 67).129 Its ability to bind α,ω-alkanediols (C9−C14) and 1-alkanols (C9 and C10) was analyzed by isothermal titration calorimetry in aqueous solutions. It was found that the doubly bridged dimer exhibits higher binding affinity toward the series of α,ω-

Figure 66. Template synthesis of biodegradable nano-test-tubes.

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defined molecular layer in spite of the involvement of highly reactive radicals in the first step. The resulting thiophenolmodified materials can self-assemble on Au surfaces, which may turn out to be favorable for the development of nanoscale devices. 4.4. Synthesis of Redox-Sensitive Macrocyles

A redox-mediated interconversible pair of monobenzo-21crown-7 (101, Figure 71) analogues with a dithiol group at the α- and ω-positions (101red) and a disulfide bond in the ring (101ox) has been introduced.134 The oxidation process (101red → 101ox) is remarkably subject to the metal template effect, where the presence of Cs+ increases the formation of 101ox (70%) and cyclic dimer (28%), without the production of polymeric material (MW > 2000), which is the major product in its absence. This is due to the fact that the rate constants for the oxidation of 101red by 3-methyllumiflavin are significantly enhanced with increasing concentrations of Rb+ and Cs+. In ion transport across a liquid (CHCl3) membrane, 101ox carried Cs+ 6.2 times faster than 101red. Thus, it was demonstrated that the rate of Cs+ transport could be regulated by the interconversion between 101red and 101ox in the membrane phase. Dipyridyl disulfide has been introduced to lanthanide complexes based on bis(amides) of diethylenetriaminepentaacetic acid to allow them to selectively react with thiolfunctionalized complexes to form heterometallic lanthanide macrocycles. 135 N,N″-Bis[p-thiophenyl(aminocarbonyl)]diethylenetriamine-N,N′,N″-triacetic acid (H3Lx) and its dipyridyl sulfide derivative (H3Ly) can generate complex lanthanide metal ions. A lanthanide-coordinated complex composed of H3Lx and H3Ly can undergo a disulfide exchange reaction to form a new bimetallic photoactive molecule (EuTbLx2) (Figure 72). The EuTbLx2 macrocycle displays two-color emission with a weak energy-transfer process operating between the lanthanide centers. The assembly of mononuclear lanthanide complexes to binuclear systems may be an interesting example of the development of multimodal and dual-color probes.

Figure 67. Structures of 97 and 98.

alkanediols than the corresponding singly bridged analogue, by about 2 orders of magnitude in K (M−1) or 3.1−3.3 kcal/mol in ΔGo, the enhancement being due to enthalpic factors. A duplex α-cyclodextrin triply bridged with disulfide linkages130 was synthesized by template-free oxidative dimerization. Its binding ability to alkanediols was similar to that of the doubly bridged cyclodextrin duplex. An organic nanotube based on β-cyclodextrin (100) has also been synthesized using seven disulfide linkages between two modified cyclodextrins (99) that substitute all the primary hydroxyl groups of a β-cyclodextrin.131 A deep and rigid hydrophobic channel with a size of more than 1.5 nm is found in the molecule; however, an inclusion body example was not presented. 4.3. Functionalization of Inorganic Surfaces

Diazonium salts of diaryl disulfides132 have been explored for constructing a monolayer or near-monolayer of molecules on conducting surfaces by the degradation of a multilayer film formed via the reduction of aryldiazonium salts (Figure 69). Otherwise, grafting of surfaces employing aryldiazonium salts frequently results in the formation of intertwined multilayers in a polymerization reaction that is difficult to control. Due to the cleavable disulfide group, the subsequent degradation of the polymeric network may allow the formation of a thin reactive molecular layer and the produced thiophenolates (ArS−) may be reversibly oxidized to disulfides to establish a covalently attached ArSSAr/ArS− redox pair at the surface. Similarly, diphenyl sulfide has been adopted in a diazonium salt-based strategy to form a covalently attached multilayer on the surface of single-walled carbon nanotubes (SWCNTs) or multiwalled carbon nanotubes (MWCNTs) (Figure 70).133 The interlayer S−S bonds are subsequently degraded under reducing conditions to produce essentially a single layer of thiophenols (or closely related derivatives) on the nanotube surface in a one-pot procedure. This “formation−degradation” modification approach allows the generation of a thin, well-

5. CONCLUSIONS Remarkable progress has been made in the design and synthesis of various disulfide cleavage chemosensors and their biological applications over the past several years. In this review, we have covered recent exciting research developments in the use of disulfide-based compounds as chemosensors (thiol and metallic ion recognition), prodrugs, hydrogels, and nanocarriers due to the biodegradation of these materials in the presence of thiols or in reducing milieus. Disulfide groups have been extensively incorporated into fluorophores to afford chromogenic and

Figure 68. Structures of 99 and 100. 5104


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Figure 69. Reductive cleavage of diaryl disulfide units, ArSSAr, in multilayers.

Figure 70. Functionalization of MWCNT or SWCNT by the “formation−degradation” approach, including pristine CNTs and multilayered (A) and degraded (B) materials.

disulfide groups or encapsulating them into organic polymer hydrogels, allowing the delivery of these drug molecules to the target position and their smooth release from the prodrug or delivery carrier to enhance their cytotoxicity; this can in turn improve the EPR of the drugs. Disulfide-based compounds have also been successfully utilized in functionalization of nanoparticles or carbon nanotubes and fabrication of various materials, making it easy to modify the surface and control the morphology of the material; all these applications have attracted extensive research interest. During the past decades, exciting progress has also been made in disulfide-based cleavage systems in thiol detection and biological applications. We then noticed that disulfide-bondcontaining organic-based DDS and nanomaterial fabrication can be easily accessed by most chemists because of their easyto-follow synthesis. All thiol-containing compounds can degrade the disulfide bond, resulting in signal changes of fluorescent reporters, but further efforts need to be made to improve the selectivity of these systems for different thiols. On the other hand, most drug molecules are encapsulated into carriers mainly by noncovalent (hydrophobic) interactions; therefore, an excellent delivery system should be designed and synthesized to increase payload efficiency and avoid the loss of

Figure 71. Redox-mediated interconversion of monobenzo-21-crown7 (101) as a redox switch.

fluorogenic sensors with various mechanisms. The breakdown of the disulfide bond upon attack by specific thiols can cause marked changes in the fluorescent signals of the probes with high sensitivity and rapid response. The disulfide bond can also be cleaved to detect some metal ions such as Hg2+ and Cu+. In biological systems, the contrasting GSH concentrations inside cells and in plasma are the prerequisite for a disulfide-based cleavage system. The cytotoxicity of drugs can be largely decreased by attaching them to specific fluorophores via 5105


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Figure 72. Structures of H3Lx, H3Ly, and EuTbLx2.

Biographies

drug molecules before reaching the target location. Most functional compounds, such as drugs and other delivery cargos, do not possess a disulfide bond, so it is critical to rationally incorporate the disulfide group into the delivery system to ensure that the drug is released at the right time and location, allowing real-time monitoring of the delivery and release of the drug inside live bodies. In addition to that, an ideal molecular sensor based on disulfide bond cleavage or molecular probes with a disulfide bond for organic-based DDS should be completely soluble in water media and have good biocompatibility and cell penetration efficiency for in vitro and in vivo biological application. In most fluorogenic sensors with a

Min Hee Lee was born in Suwon, Korea, in 1983. She joined Prof.

number of various systems, including prodrugs, nanocapsules,

Jong Seung Kim’s lab and is involved in the design and synthesis of

hydrogels, and hybrid nanoparticles, it is strongly recom-

molecular probes to detect specific metal ions or biospecies via

mended to synthesize the switch-on type or clear-cut

fluorescence and visual color changes. Her research publications

ratiometric changeable fluorescence probes with which we

include 30 reviewed papers and 6 patents so far. She received her

can undoubtedly see the physical property changes in their

Ph.D. from the Department of Chemistry at Korea University in 2012.

fluorescence. In these contexts, a number of chemists are still actively involved in this promising project; hence, the most efficient and advanced molecular probe with a disulfide bond system, including DDS, covering all above-mentioned problems could be discovered in the near future. Thereby, we believe that the present review of the disulfide cleavage reactions in chemical sensing, DDS, and nanomaterial fabrication is of great impact on the scientific community involved in crossdisciplinary fields such as bioanalytical, organic, and pharmaceutical chemistry. Zhigang Yang was born in 1981 in Hubei province, China, and

AUTHOR INFORMATION

received his Ph.D. in Applied Chemistry from Dalian University of

Corresponding Author

Technology (Dalian, China) in 2011 under the supervision of Prof.

*C.K.: e-mail, [email protected]. J.S.K.: e-mail, jongskim@ korea.ac.kr; fax, 82-2-3290-3121.

Xiaojun Peng. He is presently working as a postdoctoral fellow in the group of Prof. Jong Seung Kim at Korea University. His researching

Notes

interests mainly focus on the design and synthesis of new fluorescent

The authors declare no competing financial interest.

systems to be utilized in drug delivery. 5106


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Chulhun Kang received a M.S. in Organic Chemistry from Department of Chemistry at Seoul National University and a Ph.D. in Biochemistry from the Department of Biochemistry and Biophysics at Iowa State University. Since 1997, he has been at a faculty member at Kyung Hee University and is currently a Professor in the Department of Medical Science. To date, he has authored 50 scientific publications and 10 domestic and international patents in the fields of organic chemistry, protein chemistry, and biology.

Choon Woo Lim received his Ph.D. in Organic Chemistry at Seoul National University under the supervision of Prof. Jong-In Hong. He worked as a postdoctoral fellow with Prof. Yeon-Gyu Yu at KIST (2002−2003) and with Prof. David N. Reinhoudt in the Supramolecular Chemistry and Technology group at the University of Twente (2003−2005). After working for Samsung Mobile Display as Senior Researcher (2006−2010), he joined Kyung Hee University as a Research Assistant Professor.

Jong Seung Kim was born in Daejon, Korea, in 1963. He received a Ph.D. from the Department of Chemistry and Biochemistry at Texas Tech University. After a one-year postdoctoral fellowship at the University of Houston, he joined the faculty at Konyang University in 1994 and transferred to Dankook University in 2003. In 2007, he became Professor in the Department of Chemistry at Korea University in Seoul. To date, he has authored 290 scientific publications and 30 domestic and international patents.

Yun Hak Lee was born in Seoul, Korea, in 1986. He received B.S. degree in 2011 from Department of Chemistry at Sejong University in Seoul, South Korea. He is as a graduate student in the group of Prof. Jong Seung Kim at Korea University.

ACKNOWLEDGMENTS This work was supported by the CRI project (20120000243) (J.S.K.) and by the Basic Science Research Program (2012R1A1A2006259) (C.K.) of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. REFERENCES (1) Singh, R.; Whitesides, G. M. J. Am. Chem. Soc. 1990, 112, 1190. (2) Singh, R.; Whitesides, G. M. J. Am. Chem. Soc. 1990, 112, 6304. (3) Mulhearn, D. C.; Bachrach, S. M. J. Am. Chem. Soc. 1996, 118, 9415. (4) Bachrach, S. M.; Hayes, J. M.; Dao, T.; Mynar, J. L. Theor. Chem. Acc. 2002, 107, 266. (5) Bachrach, S. M.; Woody, J. T.; Mulhearn, D. C. J. Org. Chem. 2002, 67, 8983. (6) Hogg, P. J. Trends Biochem. Sci. 2003, 28, 210.

Sun Dongbang received her B.S. degree from Department of Chemistry at Korea University. She is now currently working toward her Master’s Degree in Organic Synthesis at Korea University under the guidance of Prof. Jong Seung Kim. Her main research theme is the design and synthesis of fluorescent reporters for drug delivery systems. 5107


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