Disulfide-Based Multifunctional Conjugates for Targeted Theranostic

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Disulfide-Based Multifunctional Conjugates for Targeted Theranostic Drug Delivery Min Hee Lee,*,† Jonathan L. Sessler,*,‡ and Jong Seung Kim*,§ †

Department of Chemistry, Sookmyung Women’s University, Seoul 140-742, Korea Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712-1224, United States § Department of Chemistry, Korea University, Seoul 136-701, Korea ‡

CONSPECTUS: Theranostics, chemical entities designed to combine therapeutic effects and imaging capability within one molecular system, have received considerable attention in recent years. Much of this interest reflects the promise inherent in personalized medicine, including disease-targeted treatments for cancer patients. One important approach to realizing this latter promise involves the development of so-called theranostic conjugates, multicomponent constructs that selectively target cancer cells and deliver cytotoxic agents while producing a readily detectable signal that can be monitored both in vitro and in vivo. This requires the synthesis of relatively complex systems comprising imaging reporters, masked chemotherapeutic drugs, cleavable linkers, and cancer targeting ligands. Ideally, the cleavage process should take place within or near cancer cells and be activated by cellular components that are associated with cancer states or specifically expressed at a higher level in cancer cells. Among the cleavable linkers currently being explored for the construction of such localizing conjugates, disulfide bonds are particularly attractive. This is because disulfide bonds are stable in most blood pools but are efficiently cleaved by cellular thiols, including glutathione (GSH) and thioredoxin (Trx), which are generally found at elevated levels in tumors. When disulfide bonds are linked to fluorophores, changes in emission intensity or shifts in the emission maxima are typically seen upon cleavage as the result of perturbations to internal charge transfer (ICT) processes. In well-designed systems, this allows for facile imaging. In this Account, we summarize our recent studies involving disulfide-based fluorescent drug delivery conjugates, including preliminary tests of their biological utility in vitro and in vivo. To date, a variety of chemotherapeutic agents, such as doxorubicin, gemcitabine, and camptothecin, have been used to create disulfide-based conjugates, as have a number of fluorophores, including naphthalimide, coumarin, BODIPY, rhodol, and Cy7. The resulting theranostic core (drug−disulfide−fluorophore) can be further linked to any of several site-localizing entities, including galactose, folate, biotin, and the RGD (Arg-Gly-Asp) peptide sequence, to create systems with an intrinsic selectivity for cancer cells over normal cells. Site-specific cleavage by endogenous thiols serves to release the cytotoxic drug and produce an easy-to-monitor change in the fluorescence signature of the cell. On the basis of the results summarized in this Account, we propose that disulfide-based cancer-targeting theranostics may have a role to play in advancing drug discovery efforts, as well as improving our understanding of cellular uptake and drug release mechanisms.

1. INTRODUCTION In combination with surgery, chemotherapy involving the use of cytotoxic drugs remains a front line therapy for various cancers, including breast, prostate, colorectal, stomach, cervical, and certain lung cancers.1 Classic chemotherapy is often effective. However, it suffers from a number of limitations, including nonselectivity and high toxicity, which can be doselimiting or cause severe side effects. Enhancements in selectivity may allow the therapeutic efficacy of chemotherapeutics to be improved while minimizing side effects. Not surprisingly, therefore, targeted drug delivery systems have been extensively investigated.2−4 Typically, the targeted drug is linked to a guiding ligand with a cleavable linker, allowing conversion to the cytotoxic drug in the cells. In general, such systems can be activated by small biomolecules or enzymes that are specifically expressed at a higher level in cancer cells, resulting in a selective anticancer effect.4 More recently, so-called theranostics, © XXXX American Chemical Society

chemical constructs that allow for the selective targeting of a therapeutic agent while providing a concurrent means for detection, have begun to attract attention as a promising approach to enhancing the basic chemotherapeutic approach.5 In recent years, we have developed several multifunctional, disulfide-based conjugate systems that contain a fluorescent reporter, a cancer targeting ligand, and a chemotherapeutic drug. We have found that such systems can be used to provide a therapeutic effect while allowing drug uptake-related imaging at the subcellular level. In the targeted cells, the disulfide bonds are cleaved as the result of reaction by endogenous thiols, including glutathione (GSH) and thioredoxin (Trx), which are overexpressed in cancer cells.6 This cleavage, in turn, leads to release of the cytotoxic drug and produces an easy-to-monitor Received: September 3, 2015

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DOI: 10.1021/acs.accounts.5b00406 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research fluorescent change. In our view, this combination of attributes makes disulfide-based multifunctional conjugates particularly attractive as targeted theranostics. In principle, their use may allow for more precise monitoring of dosage levels while helping to improve our understanding of various cellular uptake and release mechanisms relevant to drug delivery and chemotherapeutic design. In this Account, we summarize our efforts to prepare and characterize disulfide-based theranostics.

Also, detailed are the results of initial tests designed to evaluate their biological utility in vitro and in vivo.

2. DESIGN OF TARGETED FLUORESCENT DRUG DELIVERY CONJUGATES Targeted, fluorescent drug delivery conjugates that allow targeted therapeutic release and imaging must, in our estimation, contain the following elements: (i) fluorescent reporters, (ii) masked chemotherapeutic agents, (iii) cleavable linkers, and (iv) cancer targeting ligands. To date, hydrolysis of esters, hydrazone linkers, disulfide exchange, hypoxia-based activation, photolysis, thermolysis, etc. have been used to effect conjugate cleavage.7 Among the cleavable linkers currently used for drug conjugate construction, disulfide bonds are of particular interest. Disulfide bonds are known to be stable in blood pools but be efficiently cleaved by cellular thiols, including GSH and Trx. Many biological thiols are present at elevated levels in tumors. For instance, it has been reported that the GSH levels in cancer cells can be >1000 times those in blood plasma.8 Moreover, when disulfide bonds are linked to fluorophores displaying internal charge transfer (ICT)-based emission bands, an easy-to-monitor fluorescent change (emission off−on or emission wavelength shift) typically results upon cellular thiol-triggered disulfide cleavage. ICT-based fluorophores that have proved attractive for conjugate-based sensor development include naphthalimide, coumarin, BODIPY, rhodol, and Cy7. A variety of chemotherapeutic agents, including doxorubicin, camptothecin, paclitaxel, gemcitabine, and cisplatin, can be linked to a fluorophore through a disulfide bond. The tumor targeting capability of the basic conjugated theranostic (drug−disulfide−fluorophore constructs) may be enhanced by attaching to specific site-localizing entities (“ligands” in biological parlance), such as folate, biotin, galactose, and peptide sequences such as RGD (Arg-GlyAsp), which display intrinsic selectivity for cancer cells over normal cells due to their genetic signatures or because of an overexpression of specific receptors on certain tumor cells.

Figure 1. Design elements underlying disulfide-based, targeted, fluorescent drug delivery conjugates. As conceived, the conjugates are expected to enter the cell selectively as the result of receptormediated endocytosis. Upon target-specific internalization, these conjugates will undergo thiol-triggered disulfide bond cleavage to provide a fluorescent signal change while releasing the cytotoxic drug.

Figure 2. Cleavage reaction of the disulfide bond in 1 (or 2) by GSH that is expected to occur under physiological conditions. Reproduced with permission from ref 9. Copyright 2012 American Chemical Society. B


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Figure 3. Fluorescence response (a) and CPT release (b) for 1 in the absence and presence of GSH. All data were obtained at 37 °C in PBS (phosphate-buffered saline solution, pH 7.4) containing 16% (v/v) DMSO. Reproduced with permission from ref 9. Copyright 2012 American Chemical Society.

Figure 4. Confocal microscopic images of U87 and C6 cells with 1 recorded at 0, 30, and 60 min following addition of the proposed theranostic. The bottom panels show an overlay of the fluorescence image with a phase contrast image. Fluorescence images were obtained under excitation at 458 nm using a long-path (>505 nm) emission filter. Reproduced with permission from ref 9. Copyright 2012 American Chemical Society.

Figure 5. Cell viability after treatment with different concentrations of 1 (a) and 2 (b). MTT assays were performed in U87 cells for 48 h. Reproduced with permission from ref 9. Copyright 2012 American Chemical Society.

The underlying postulate is that the selective internalization of the targeted theranostic conjugate by tumor cells will then be facilitated through receptor-mediated endocytosis. Once taken up, the conjugate will undergo thiol-mediated disulfide cleavage. This will release the delivered cytotoxic chemotherapeutic while providing a concomitant change in fluorescence. This designbased approach to theranostic drug development is illustrated in Figure 1. It is discussed further below in the context of specific examples.

Disulfide bond cleavage leads to intramolecular cyclization followed by cleavage of the neighboring carbamate bond. This, in turn, releases CPT and produces a fluorescent signal change. To confirm that CPT release occurs concurrent with the observed change in fluorescence, the reaction of 1 with GSH was monitored using HPLC and fluorimetric time-dependent analyses (Figure 3). Upon the addition of GSH to a solution of 1, CPT release was found to correlate with the increase in fluorescence intensity at 535 nm. In the absence of GSH, a control solution containing just 1 exhibited neither CPT release nor a fluorescence increase at 535 nm. The concurrent nature of the processes led us to propose that studies with conjugate 1 might provide useful information as to when, where, and how a pharmaceutically active agent (CPT in this case) may be delivered and released into cells using the basic targeting theranostic strategy shown in Figure 1.9 To confirm the targeted therapeutic effect of 1, U87 and C6 cells were subjected to confocal microscopic analysis (Figure 4). U87 and C6 cells were used because the expression level of

3. TARGETED THERANOSTIC CONJUGATES FOR CAMPTOTHECIN DELIVERY We recently reported conjugate 1.9 In accord with the design approach embodied in Figure 1, this system contains a disulfide linker as a cleavable release subunit, a naphthalimide part as a fluorescent reporter,10 a cyclic RGD peptide as a cancer targeting element,11 and a tethered camptothecin (CPT) moiety as an antitumor inhibitor of topoisomerase I.12 As shown in Figure 2, under physiological conditions, the disulfide linker of 1 is cleaved by cellular thiols, such as GSH, which is the most abundant thiol in the U87 and C6 cells used for these tests. C


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Figure 6. (a) Thiol-mediated cleavable conjugates. (b) Schematic illustration of intracellular drug release of the conjugates by thiols. Reproduced with permission from ref 15. Copyright 2014 Wiley-VCH.

αvβ3 integrin in U87 cells is much higher than in C6 cells.13 The cyclic RGD peptide unit in 1 can be recognized and internalized by αvβ3 integrin, a well-known tumor-associated receptor.11 When U87 cells were incubated with 1, a strong green fluorescent image was observed, whereas only a weak fluorescent image was observed when C6 cells were treated with 1. In contrast, analogue 2, lacking an RGD moiety, exhibited strong fluorescence in U87 and C6 cells. These results were taken as evidence that 1 enters preferentially to U87 cells in comparison with C6 cells via RGD-dependent integrin-receptor-mediated endocytosis. To investigate the cellular location of CPT release from 1, we performed colocalization studies using organelle-selective fluorescent markers. In U87 cells, CPT was released from 1 within the endoplasmic reticulum (ER), whereas CPT was released from analogue 2 in the mitochondria. On this basis, we suggested that cleavage of the disulfide bond in 1 by thiol occurs in the ER, leading to release of CPT. Presumably, the free CPT then diffuses into the nucleus and inhibits topoisomerase I.14 The therapeutic efficacy of 1 was also evaluated using a standard MTT assay (Figure 5). Analogue 2, without the RGD unit, was tested for comparison. In U87 cells, treatment with 1.0 μM of 1 decreased cell viability up to 49% within 48 h; however, treatment with 1.0 μM of 2 produced a lower degree of cytotoxicity (76% cell viability). These results provide experimental support for the suggestion that cellular uptake of

Figure 7. (a) Absorption and (b) fluorescence spectra of 5 recorded in the absence and presence of GSH. (c) Changes in fluorescence following exposure to 5 at different GSH concentrations (0−80 equiv). (d) Fluorescence spectra of 5 recorded in the presence of GSH as a function of pH. The excitation wavelength was 510 nm. (e) Cellular uptake of 5 and 5a by HeLa cells was measured by FACS analysis. (f) Confocal fluorescent images of HeLa, A549, and NIH3T3 cells incubated with 5 in PBS. Nuclear counter-staining using DRAQ5 (red). Images were obtained using excitation wavelengths of 510 and 630 nm with emission recorded over the 540−600 and 680−750 nm spectral regions for the green and red color images, respectively. Reproduced with permission from ref 15. Copyright 2014 WileyVCH.

1 in U87 cells occurs via integrin-mediated endocytosis. Conjugate 2, lacking an RGD moiety, is not transported by D


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Figure 8. Ex vivo biodistribution of conjugates 5 and 6 and evidence of in vivo therapeutic efficacy (xenograft mouse model; HeLa cells) in the case of conjugate 5. Reproduced with permission from ref 15. Copyright 2014 Wiley-VCH.

Figure 9. Proposed thiol-mediated disulfide cleavage reaction of 7.

exhibited very weak absorption and emission bands, which were centered at 510 and 544 nm, respectively (Figures 7a,b); however, upon addition of GSH, the absorption and emission intensities were enhanced by approximately 25- and 32-fold, respectively. The enhancement in the fluorescence intensity at 544 nm reached saturation upon the addition of approximately 80 mol equiv of GSH (Figure 7c). These findings are fully consistent with the design expectations that the disulfide bond of 5 would be cleaved by thiols and give rise to an increase in the fluorescence intensity. The pH dependence of the GSH-induced fluorescence enhancement was also tested. Conjugate 5 produced a negligible increase in fluorescence over the pH range of 3.2 to 8.6. In contrast, a significant increase in fluorescence was seen when 5 was treated with GSH over the 3.2 to 8.6 pH range (Figure 7d). This led us to propose that conjugate 5 would

integrin-mediated endocytosis and is less active than 1 in U87 cells. Taken together, the results obtained with conjugate 1 provide support for the suggestion that the theranostic approach illustrated in Figure 1 can provide small molecule constructs that both mediate a therapeutic effect and allow drug uptake-related imaging information to be obtained. In a separate study designed to exploit the paradigm embodied in Figure 1, we developed conjugate 5. This system incorporates a piperazine−rhodol moiety as a signaling unit, biotin as a cancer-targeting subunit, and a SN-38 subunit. SN-38 is one of the most potent anticancer drugs in the CPT class.15 In this case, and in analogy to what was proven for 1 above, we were able to demonstrate that disulfide cleavage of 5 led to targeted release of SN-38 while engendering a strong enhancement in the fluorescence intensity (Figure 6). Phosphatebuffered saline (PBS) solutions of 5 containing 25% DMSO E


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5 would suffice to direct the conjugate to a specific cancer cell type. Here, we used HeLa and A549 cells as biotin-receptorpositive cell lines and NIH3T3 cells as a biotin-receptornegative control cell line.17,18 First, the cellular uptake of 5a and its analogue 6a, lacking a biotin moiety, was assessed using fluorescence-activated cell sorting (FACS) using these three cell lines (Figures 7e,f). In the case of 5a, uptake by the HeLa cell lines was 3-fold greater than that in the NIH3T3 cell lines. However, precursor 6a showed similar uptake in each of the tested cell lines. To confirm that this difference reflected active biotinreceptor-mediated endocytosis in the case of 5a, the biotinreceptor-positive A549 and HeLa cells were then preincubated with an excess of free biotin prior to treatment with 5a. This was expected to prevent cellular uptake of 5a via the expressed biotin receptors. In accord with these expectations, confocal fluorescence microscopic analyses revealed that, in the presence of biotin, the number of fluorescent cells was lower. Decreased cellular uptake of 5a by biotin receptors following incubation with an excess of biotin was also demonstrated using FACS analysis. Considered in aggregate, these results were taken as evidence that the observed cellular uptake of 5 occurs via receptor-mediated endocytosis. The therapeutic efficacy of 5 was evaluated in HeLa, A549, and NIH3T3 cells using MTT assays. Conjugate 5 reduced the proliferation of HeLa and A549 cells in a dose-dependent manner. Thus, we concluded that the biotin moiety present in 5 serves to target the conjugate to biotin-receptor-overexpressing A549 and HeLa cells. Following disulfide cleavage, an antiproliferative effect is seen, presumably as the result of the release of SN-38 inside the cell. However, prodrug 6 did not exhibit target-specific antiproliferative activity in tumor cells overexpressing the biotin receptor. Next, we evaluated the targeting efficacy of 5 in a mouse xenograft model (Figure 8). HeLa tumor-bearing mice were prepared and injected with 5 (2.5 mg/kg) intravenously. When subject to ex vivo imaging, a strong fluorescent signal was observed in the tissues bearing the implanted HeLa cancer cells. However, only weak fluorescent signals were observed in the other organs such as the liver, kidney, lung, and spleen. Under similar conditions, precursor 6 did not display a tumor-specific effect. The in vivo therapeutic efficacy of 5 was then assessed. This was done by monitoring the changes in solid tumor volume using HeLa cell mice xenografts. In these experiments, conjugate 5 was administered to the mice at a dose of 125 μg/kg every other day (for a total of five doses) via tail vein injection. The tumor volume of the group treated with 5 was significantly reduced in comparison with that of the control group treated with saline only (Figure 8d). These findings were taken as evidence that the cleavable theranostic 5 not only exhibits specific tumor targeting in vitro and in vivo but also (1) delivers SN-38 inside the tumor cells, (2) allows fluorescent imaging of cancerous lesions, and (3) displays antiproliferative activity in a xenograft tumor model.

Figure 10. (a) Fluorescence microscopic images of A549 and WI38 cells incubated with 7. The left and right panels display confocal fluorescence and phase contrast images, respectively. (b) Colocalization experiment for 7 in A549 cells using lysosome-specific and ER-specific trackers. Cell images were attained using excitation at 750 nm, a band path (blue image, 350−440 nm; green image, 550− 600 nm; red image, 650−690 nm), and emission filters, respectively. (c) Cell viability of A549 cells following treatment with 7 and 7a. The cells were treated with each compound for 72 h, after which time cell viability was measured via MTT assay. Reproduced with permission from ref 19. Copyright 2013 American Chemical Society.

4. TARGETED THERANOSTIC CONJUGATES FOR GEMCITABINE DELIVERY To test further the utility of biotin targeting in the context of theranostic delivery, we developed the gemcitabine−coumarin− biotin conjugate 7. This multicomponent system contains a disulfide bond as a cleavable linker, a coumarin moiety as a signaling unit, gemcitabine (GMC) as a chemotherapeutic

undergo thiol-mediated cleavage in biological milieus independent of differences in cellular pH. Biotin molecules and their conjugates are taken up preferentially by cancer cells that express the biotin receptor at a high level.16 Therefore, we tested whether the biotin subunit in F


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Figure 11. Proposed disulfide cleavage reaction of 8 with thiols under physiological conditions.

agent, and biotin as the cancer-guiding subunit.19 As illustrated in Figure 9, conjugate 7 can undergo a disulfide cleavage by thiols, releasing both a pharmaceutically active GMC molecule and an emitting fluorophore. The thiol-induced cleavage of 7 was confirmed by UV/vis absorption and fluorescence spectroscopy, as well as mass analysis. The targeted therapeutic efficacy of 7 was evaluated in biotin-receptor-positive A549 cells and biotin-receptor-negative WI38 cells.8,16 Confocal microscopic experiments were performed using two-photon excitation at 750 nm. As seen in the cell images displayed in Figure 10a, a strong fluorescence signal was seen when the A549 cells were incubated with 7. In contrast, no fluorescence was monitored in the case of the WI38 cells. These results are consistent with the notion that conjugate 7 is taken up efficiently by A549 cells but not by the biotin-receptor-negative WI38 cells. To gain insight into the cellular thiol-triggered cleavage, confocal microscopy was performed using A549 cells in the presence of N-ethylmaleimide (NEM), a reagent that reacts readily with biological thiols.20 The fluorescence intensity of 7 in A549 cells diminished as the concentration of NEM was increased, as expected for a system whose fluorescence signature is attributable to thiol-mediated cleavage. The site of intracellular cleavage of 7 was then investigated by fluorescence-based colocalization experiments using fluorescent lysosome- and ER-selective trackers. As shown in Figure 10b, the green fluorescence of 7 colocalized well with the red fluorescence of the lysosome-specific tracker. However, this fluorescence was not localized with that of an ER-specific tracker. It is noteworthy that thiol-triggered disulfide cleavage of 7 occurs in the lysosome, whereupon GMC is released to diffuse into the nucleus. Once in the nucleus, GMC is known to replace cytidine during DNA replication, leading to the generation of faulty nucleosides. This results in apoptosis.21 We also tested the anticancer effects of 7 using MTT assays in A549 and WI38 cells. For comparison, separate groups of A549 and WI38 cells were exposed to a system that lacks biotin, namely, precursor 7a. The assay revealed that 7 was a more potent antiproliferative drug than 7a in A549 cells, as inferred from a comparison of cell viability data recorded following exposure to 7 and 7a at a concentration of 1.0 μM (Figure 10c). However, 7 and 7a displayed similar potency in WI38 cells,

which lack the biotin receptor. These results provide strong support for the notion that the biotin subunit in 7 serves to target the conjugate to specific tumor cells. Conjugate 7 is also of fundamental interest as a system that produces a therapeutic effect while allowing cellular uptake to be readily monitored at the subcellular level by two-photon confocal microscopic imaging. In a similar vein, we reported a GMC−biotin conjugate containing a near-IR fluorescing BODIPY fluorophore (8).22 As depicted in Figure 11, intracellular thiol-induced disulfide cleavage of 8 led to release of the active GMC as well as an enhanced fluorescence emission feature centered at 720 nm. The theranostic potential of 8 was evaluated in both A549 cells and WI38 cells. Confocal microscopic imaging experiments revealed that conjugate 8 enters preferentially into the biotinreceptor-overexpressed A549 cells. This conjugate also produces a fluorescence signal and mediates an anticancer effect as a result of thiol-induced release of active GMC. On the basis of fluorescence-based colocalization experiments, it was concluded that 8 localizes initially in the ER. Following disulfide cleavage, free GMC is released into the ER. Over time, GMC diffuses into the nucleus, where it acts as a pro-apoptotic agent. A reporter that emits a fluorescence in the near-infrared (NIR) region is potentially useful for real-time monitoring of drug delivery in vivo. NIR radiation (650−900 nm) is capable of deep tissue penetration, which enhances microscopic imaging. Radiation in this spectral region is also energetically weaker than that in the visible or UV, reducing the potential for tissue damage. Moreover, NIR emission is less affected by autofluorescence from tissues.23 Recently, we developed a new theranostic conjugate (9) bearing the Cy7 dye, which emits light in the NIR region.24 Conjugate 9 contains a folate unit as a cancer targeting group, a Cy7 unit as a NIR fluorescent reporter, and GMC as an anticancer drug (Figure 12). Upon addition of GSH to a solution of 9, the absorption band at 794 nm decreased, while a new peak appears at 630 nm. The fluorescence intensity of the emission band at 735 nm was also enhanced by 42-fold under conditions of disulfide cleavage (Figure 13). Conjugate 9 was tested with folate-receptor-positive and -negative cell types, namely, the KB and A549 cell lines, respectively.25 As seen in Figure 14a, KB cells exhibited a strong G


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Figure 12. Proposed disulfide cleavage reaction of 9 with thiols under physiological conditions.

Figure 13. Absorption (a) and fluorescence (b) spectra of 9 (4.0 μM) in the presence of different concentrations of GSH in DMSO−PBS solution (10 mM, pH = 7.4) (50:50, v/v) at room temperature (λex = 615 nm). Plots of A630/A794 (c) and I735 (d) as a function of GSH concentration. All data were attained after the samples were allowed to incubate for 2 h at room temperature. Reproduced with permission from ref 24. Copyright 2013 American Chemical Society.

fluorescence within 20 min upon incubation of 9, whereas A549 cells displayed little or no fluorescence. Based on these results, we concluded that 9 is preferentially taken up by KB cells, possibly via folate-mediated uptake mechanisms. To obtain insight into the thiol-mediated disulfide cleavage leading to the observed fluorescence enhancement in KB cells, confocal images of 9 were obtained in the presence of NEM, a reagent that reacts well with thiols as noted above.20 The fluorescence signal of 9 in the cells diminished with increasing concentration of NEM, as would be expected for a scenario wherein cleavage of 9 is largely mediated by cellular thiols. To investigate the cellular location characteristics of GMC following release from 9, fluorescence-based colocalization experiments were performed. It was found that the fluorescence of 9 overlapped with that of an ER-specific tracker. On this basis, it is considered reasonable that disulfide cleavage and GMC release occurs in the ER, leading to apoptosis.

The therapeutic effect of conjugate 9 was evaluated by MTT assays using both the KB and A549 cell lines. As seen in Figure 14, conjugate 9 was taken up preferentially by KB cells. On the other hand, a parallel experiment using 9a, a control system without a folate unit, revealed high cytotoxicity but no selectivity with regard to cell type. The relatively greater cytotoxicity seen for 9 in the case of the KB tumor cells was considered to reflect the high expression level of the folate receptor on such cells. The results shown in Figure 14a,b are consistent with the design strategy, wherein the folate moiety serves to guide 9 to folate-receptor-positive cells. Following receptor-mediated endocytosis and drug delivery, disulfide cleavage then results in GMC-induced apoptosis. The tumor targeting ability of 9 was also examined after it was injected intravenously into xenograft tumors in mice. The mice bearing KB and A549 tumor cells on the right and left H


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Figure 14. (a) Confocal microscopic images of KB and A549 cells incubated with 9 at different time points (0, 5, 10, and 20 min). Confocal microscopic images were obtained at an excitation wavelength of 633 nm and a band path of 650−750 nm. Scale bars = 20 μm. (b) MTT assay of 9 and 9a in the KB and A459 cells. The cells were treated with each compound for 72 h. Reproduced with permission from ref 24. Copyright 2013 American Chemical Society.

basis, we conclude that conjugate 9 represents a potentially useful theranostic that exhibits specific tumor targeting in vivo in that it successfully delivers GMC into the KB cancer cells in live mice.

sides of the back, respectively, were prepared and 0.1 mM/kg (DMSO/PBS = 50:50) 9 was injected into the mice via the tail vein. As exhibited in Figure 15, a fluorescence signal was observed only in tumor tissues bearing the KB cells. On this I


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Figure 15. Fluorescence images of tumor tissues from mice bearing KB and A549 tumors 24 h after injection of 0.1 mM/kg 9. Reproduced with permission from ref 24. Copyright 2013 American Chemical Society.

5. TARGETED THERANOSTIC CONJUGATES FOR PEPTIDE DELIVERY Several peptide-based antitumor drugs display target selectivity with few side effects.2,26 However, pharmaceutical application of most peptides is limited by poor oral bioavailability, metabolic instability, and notoriously low membrane permeability. Peptide drug delivery systems (PDDS) may provide a means of circumventing these limitations.27 We recently reported a fluorescent PDDS, theranostic 10,28 for which we used the Holliday junction inhibitor peptide 2 (KWWCRW) as a model peptide drug because of its antimicrobial and anticancer activity.29 The Holliday junction (HJ) is a transient, four-stranded DNA structure formed during homologous recombination repair of DNA damage.30 The HJ inhibitor peptide 2 contains a cysteine residue, by which it is conjugated to a biotin−naphthalimide subunit via a disulfide linker to create theranostic 10 as depicted in Figure 16. To confirm the targeted therapeutic effect of the biotin moiety in 10, biotin-receptor-positive HepG2 cells and biotinreceptor-negative WI38 cells were exposed to the conjugate. As can be seen in Figure 17a, theranostic 10 was selectively taken up by HepG2 cells and produced enhanced intracellular fluorescence. Fluorescence-based colocalization experiments revealed that the fluorescence of 10 mirrored that of an ERspecific tracker. On this basis, we propose that theranostic 10 enters into biotin-receptor-positive cells selectively via biotinmediated endocytosis, whereupon it releases its peptide drug within the ER. This leads to enhanced fluorescence, as well as an anticancer effect. Evidence that conjugate 10 mediates an antiproliferative effect came from standard MTT assays. Upon incubation of HepG2 cells with the HJ inhibitor peptide 2 (0−300 μM), 90% cell viability was observed even at the highest tested concentration. Presumably, this reflects the known poor penetrating ability of the peptide (Figure 17b). However, when HepG2 cells were treated with 10, cell viability

Figure 16. Proposed disulfide cleavage reaction of 10 with thiols.

decreased as a function of the concentration, reaching 55% when the cells were exposed to 10 at a concentration of 300 μM. The anticancer effect of 10 was more potent than that of HJ inhibitor peptide 2. These results were taken as evidence that the fluorescence-based peptide delivery theranostic 10 might allow both improved peptide delivery and enhanced therapeutic targeting and fluorescence-based uptake monitoring. J


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Accounts of Chemical Research *E-mail: [email protected] (J. S. Kim). Funding

This work was supported by the Sookmyung Women’s University Research Grants (No. 1-1503-0152, L.M.H.), the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning in Korea (CRI Project No. 2009-0081566, J.S.K.), the U.S. National Institutes of Health (CA 68682, J.L.S.), and the Robert A. Welch Foundation (F-1018, J.L.S.). Notes

The authors declare no competing financial interest. Biographies Prof Min Hee Lee was born in Icheon, Korea, in 1983. She received her Ph.D. from Korea University in 2012 under the supervision of Prof. Jong Seung Kim. After postdoctoral work at The University of Texas at Austin (Department of Chemistry, Prof. Jonathan L. Sessler), she began her academic career at Sookmyung Women’s University in 2015. Her research interests are focused on the development of novel fluorescence-based smart molecules involved in sensing and imaging of bioactive species and their applications in drug delivery systems. Prof Jonathan L. Sessler was born in Urbana, Illinois, USA, in 1956. He received his Ph.D. from Stanford University in 1982. After postdoctoral work with Profs. Jean-Marie Lehn and Iwao Tabushi, he began his academic career at The University of Texas at Austin in 1984, where he now holds the position of Pettit Centennial Chair. He is a cofounder of Pharmacyclics, Inc., a company that was recently acquired by AbbVie for $21B. His research interests include cancer drug development, ion recognition, supramolecular chemistry, sensing, expanded porphyrins, and electron transfer. Prof Jong Seung Kim was born in Daejon, Korea in 1963. He received his Ph.D. from the Department of Chemistry and Biochemistry at Texas Tech University. After a 1-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 moved to the Department of Chemistry at Korea University in Seoul as a professor. His research interests include host−guest chemistry, organic sensors, and small molecule based theranostic prodrugs.

Figure 17. (a) Confocal microscopic images of HepG2 and WI38 cells treated with 10. (b) Cell viability assay for 10 and HJ inhibitor peptide 2 in HepG2cells. Reproduced with permission from ref 28. Copyright 2014 Royal Society of Chemistry.

6. CONCLUSION AND FUTURE PROSPECTS Theranostic systems have received considerable attention in recent years because they could be used for therapeutic and diagnostic purposes in cancer treatment. One appealing approach is the development of disulfide-based multifunctional conjugates composed of a fluorescent reporter, a cancer targeting ligand, and a chemotherapeutic drug. As inferred from spectroscopic analyses, confocal microscopic imaging studies, and MTT assays, we believe that the disulfide linked conjugates discussed in this Account are selectively internalized by specific tumor cells and undergo endogenous thiolpromoted disulfide bond cleavage to release a cytotoxic drug and produce a detectable fluorescence signal. This approach appears viable both in vitro and in vivo. The present theranostic strategy can likely be generalized further to achieve specific therapeutic effects while providing drug uptake-related imaging that may allow for more precise monitoring of dosage levels, as well as an improved understanding of cellular uptake and release mechanisms.

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Corresponding Authors

*E-mail: [email protected] (M. H. Lee). *E-mail: [email protected] (J. L. Sessler). K


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