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Dyes and Pigments

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Molecular theranostic based on esterase-mediated drug activation forhepatocellular carcinoma

Amit Sharmaa,1, Eun-Joong Kimb,1, Seokgyu Munc, Myung Sun Jia, Bong Geun Chungd,∗∗,Jong Seung Kima,∗

a Department of Chemistry, Korea University, Seoul, 02841, South Koreab Research Center, Sogang University, Seoul, 04107, South Koreac Department of Biomedical Engineering, Sogang University, Seoul, 04107, South KoreadDepartment of Mechanical Engineering, Sogang University, Seoul, 04107, South Korea

A R T I C L E I N F O

Keywords:CancerHepatocellular carcinomaTheranosticTargeted therapyDrug delivery system

A B S T R A C T

In past few years, cancer specific targeted drug formulations have shown some hope of improving the ther-apeutics with minimized associated side effects of chemotherapy. However, the benefits has proven modest dueto lack of systems that can be assessed easily from parent drugs while maintaining the same features of cancerassociated prodrug activation. We developed a small molecule-based, carboxylesterase responsive theranostic,EDOX, with tumor-specific enzymatic activation for targeted delivery of the anticancer drug, Doxorubicin (Dox),to hepatocellular carcinoma (HCC) cells. Easy synthetic access, physiological stability, tumor-selective activa-tion, and sustained drug release make EDOX an excellent candidate for use as a next-generation drug deliverysystem for HCC.

1. Introduction

Cancer includes a range of diseases related to the dysregulatedgrowth of malignant cells, often with the ability to invade distant tis-sues and organs [1]. According to an estimate by the World HealthOrganization (WHO), cancer-related mortality has witnessed a steepincrease in the last decades and over 13.1 million cancer-related annualdeaths are expected by 2030 [2]. In the last few years, a better un-derstanding of cancer biology, as well as improved diagnostic andtherapeutic tools, have led to a noticeable decrease in mortality rates[3]. Currently, available cancer treatments include surgical interven-tion, radiation therapy, photodynamic therapy, chemotherapy or acombination of these options [4–7]. Most conventional chemother-apeutics interfere with DNA synthesis and mitosis, causing the death ofproliferating cancer cells [8]. However, these agents are usually non-selective and ultimately result in the damage of healthy cells and tis-sues, with severe undesired side effects including hair loss, appetiteloss, and nausea. It has been calculated that the side effects of che-motherapeutics strongly contribute to high mortality in cancer patients[9].

Hepatocellular carcinoma (HCC), the fifth most frequently

diagnosed cancer type worldwide, is second cause of cancer-associateddeath in males and the sixth in females [10,11]. Due to the long latencyof HCC, most patients are already in advanced stages of disease at thetime of first diagnosis, and chemotherapy is the only available option. Itwas observed that chemotherapeutic agents accumulating in the tumormay only be 5%–10% of the dosage reaching normal organs [12]. Dueto such inefficient delivery, the concentration of active drugs at theHCC site is too low to be effective. Additionally, intrinsic and acquiredmultidrug resistance (MDR) rates are high in HCC, resulting in cancerreoccurrence with poor survival and overall life quality [13]. Thus, newstrategies to improve HCC response to treatment are strongly desired.

Carboxylesterase (CE) is an enzyme involved in the hydrolytic me-tabolism of various drugs including carboxylic acid esters, amides, andthioesters. There are two main types of human carboxylesterases, hCE1,and hCE2, with distinct roles in the hydrolysis of specific drugs [14,15].Particularly, CE is known to be highly expressed in the liver and com-monly related to several liver-associated diseases, including hepaticsteatosis, hyperlipidemia, and HCC [16–20].

In past few years, extensive efforts have been made to pursue ver-satile drug carriers such as small molecules, nanoparticles, polymers,inorganic materials, polymeric hydrogels, lipids and

https://doi.org/10.1016/j.dyepig.2018.12.026Received 24 October 2018; Received in revised form 20 November 2018; Accepted 15 December 2018

∗ Corresponding author.∗∗ Corresponding author.E-mail addresses: bchung@sogang.ac.kr (B.G. Chung), jongskim@korea.ac.kr (J.S. Kim).

1 These authors contributed equally.

Dyes and Pigments 163 (2019) 628–633

Available online 17 December 20180143-7208/ © 2018 Elsevier Ltd. All rights reserved.

T

biomacromolecular motifs for the selective delivery of chemother-apeutic agents at tumor sites [21–28]. These systems may result inimproved drug specificity and reduced side effects, minimizing multi-drug resistance (MDR). Although several strategies for passive drugtargeting have been developed, only a few of them have made their wayinto clinical trials [29]. Recently, active targeting strategies in nano-medicine have shown interesting results, albeit physiological barrierssuch as tumor heterogeneity, deep penetration, hypoxia, and en-dosomal escape are still major hurdles to their optimal therapeuticapplication [30,31]. From a biopharmaceutical standpoint, the idealsystem should preserve the drug from undesired chemical or enzymaticdegradation for a sufficient time to allow for payload delivery at thedesired target site. Small molecule-based systems hold the potential toovercome the above-mentioned drawbacks as they exhibit several ad-vantages over nano-based systems, including active targeting, easiersynthetic access, and deep tumor penetration [21,24].

In this study, we have developed a carboxylesterase responsive bi-functional drug delivery system, EDOX, for delivery of an anticancerdrug, doxorubicin (Dox), and tested its effects on various cancer celllines. Our preliminary results showed that EDOX was preferentiallyactivated by the intracellular carboxylesterase of HCC-derived cells, incomparison with normal fibroblasts and other cancer cell lines. Fig. 1summarizes the overall chemical design strategy.

2. Materials and procedures

2.1. General information and materials

All chemicals and solvents used for synthetic purposes were pur-chased from Sigma-Aldrich, Alfa, or TCI-Korea. Moisture sensitive re-actions were generally carried out under a blanket of argon gas. NMRspectral analyses were performed on Bruker NMR (500MHz for 1H,125MHz for 13C) instruments at room temperature using CDCl3, MeOD,and DMSO‑d6 as the solvents with tetramethylsilane (TMS) as the in-ternal standard. Chemical shifts (δ) are recorded in parts per million(ppm) and coupling constants are given in Hz. High-resolution massspectra were recorded on an Ion Spec Hi-Res ESI mass spectrometer.HPLC analyses were performed on a YL9101S (YL-Clarity) instrumentequipped with a reverse-phase (RP) column (C18, 5mm, Waters).Eluent used in the analysis was a water-acetonitrile (ACN) gradient(acetonitrile from 5% to 85%; 0–30min) with a flow rate of 1.0 mL/minand UV–Vis detector (475 nm).

2.2. Preliminary solution and interference studies

A stock solution of EDOX (1 mM) was prepared in the DMSO solu-tion. The working solution (10uM) of EDOX was prepared by diluting inphosphate saline buffered (PBS, pH 7.4, 37 °C). The carboxylesterase(Sigma Aldrich, E2884-1KU) was used in this study for preliminarysolution tests. For interference studies, all the stock solution of bio-analytes were prepared in PBS and used in concentrations as mentioned

in studies.

2.3. Cell culture, fluorescence imaging, and flow cytometry analysis

The HepG2 and HT-29 were maintained in RPMI1640 medium(Gibco, Invitrogen. Co., Carlsbad, CA, USA) while HeLa, MCF7, andNIH3T3 cells were cultured with the Dulbecco's Modified Eagle Medium(DMEM), Gibco, supplemented with fetal bovine serum (10% FBS,Gibco) and penicillin/streptomycin (1%, Gibco) in an incubator (5%CO2, 37 °C). To screen for the most proper cancer cell line response toEDOX among various cells, we observed the fluorescence in EDOX-treated cells using confocal laser scanning microscopy (CLSM) and theflow cytometry. All cells were seeded at 1×105 cells/mL suspension inμ-slide, 8 well (ibidi, Munich) and incubated for 12 h at 37 °C. Next,5 μM of EDOX was added to cells for 2 h, followed by washing in PBS.After fixing with 4% paraformaldehyde for 30min at room tempera-ture, the cells were stained by fluorophore Alexa Fluor 488 phalloidin(Invitrogen, Molecular Probe, Eugene, OR, USA) for 20min and 4′ 6-diamidino-2-phenylindole (DAPI) for 5min. The fluorescent cell imageswere obtained by a CLSM (LSM 710, Carl Zeiss, Germany). In addition,the cellular fluorescence was analyzed by flow cytometry (FACS, BDBiosciences, USA) for quantitative evaluation of the fluorescence inEDOX-treated cells, all cells were incubated in 12-well plate at a densityof 4× 105/well and treated with 5 μM EDOX for 2 h. After washingwith PBS 3 times, the harvested cells were examined by the flow cy-tometry. The histogram plots were performed using the FlowJo soft-ware (TreStar, Switxìzerland).

2.4. Anti-proliferation assay and western blotting

Anti-cancer effects of EDOXwere evaluated in HepG2, HT-29, HeLa,MCF-7 and NIH3T3 cells by using MTT assays and western blot analysis.All cells were seeded into each well of 96 well plates at 1×104 cells perwell, and then, they were treated with various concentrations of EDOXfor 48 h, followed by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide (MTT, Roche Diagnostics GmbH, Germany)colorimetric analysis. After replacing the culture media, MTT reagentwas added into each well and incubated for an additional 4 h. Followingthe treatment of solubilization buffer (Roche Diagnostics) to each well,the absorbance of formazan product produced by MTT was measured at570 nm using a microplate reader (EL800, Bio-Tek Instruments,Winooski, USA). For western blot analysis, HepG2 cells were cultured ina 6 well plate and treated with 5 μM of EDOX for 12 h. Then, theharvested cells were lysed with cell lysis solution (GenDEPOT, TX, USA)including protease inhibitor cocktail solution (Xpert Protease InhibitorCocktail Solution, GenDEPOT). Total protein was quantified by usingthe BCA protein assay, and the equal amount (50 μg) of protein wasseparated by 10% (w/v) SDS-polyacrylamide gel electrophoresis. Afterthe proteins were transferred to polyvinylidene fluoride (PVDF) mem-brane, they were blocked by 5% skimmed milk for 1 h. The membraneswere incubated with the following antibodies: anti-PARP-1 (1:1000;abcam, UK), anti-Caspase-3 (1:1000; Cell Signaling Technology, USA),and β-actin (Santa Cruz, USA). Then, The HRP-conjugated secondaryantibodies were incubated for 1 h at room temperature. After washing,the membrane was incubated with ECL reagent (AmershamBiosciences, UK) as the substrate, and then expose the X-ray film in thedark room.

3. Results and discussion

3.1. Design, synthesis, and characterization of EDOX

To prevent any undesired chemical or enzymatic degradation of theprodrug conjugate, intermediate 1 (Scheme S1) was chosen for EDOXsynthesis. On the basis of the difference in electron donor ability be-tween amides and carbamates (weak donors, Hammett constant,Fig. 1. Schematic representation of the EDOX conjugate.

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σp=0.00) and amines (strong donors, σp=−0.66), we predicted ahigher stability and a more efficient drug release profile of this con-jugate upon enzymatic hydrolysis [31]. Moreover, esterase-mediatedhydrolysis of aromatic amides is well-documented in the literature[32,33]. Additionally, the presence of a covalent bond between the Doxamino group and the chemical linker may attenuate the drug action,until unmasking takes place [24,25,34,35]. The synthetic route forEDOX is shown in Scheme S1. First, we evaluated drug stability andactivation mechanism. Upon incubation of EDOX in phosphate salinebuffered (PBS, pH 7.4, 37 °C) for 4 h, no degradation was observed.EDOX showed an intrinsic weak fluorescence emission at 560 nm(Fig. 2A). Likewise, EDOX exhibited similar kind of stability in culturemedia up to 12 h (Fig. S1). However, exposure to esterase (3 U/mL) ledto cleavage of the aniline amide bond, followed by self-immolation via1,6-elimination and release of the active drug, Dox. This phenomenonwas accompanied by a concomitant enhancement of the emission bandat 560 nm, corresponding to free Dox. EDOX activation was furtherconfirmed by combined fluorescence experiments and HPLC analysis.Exposure of EDOX to esterase resulted in a time-dependent cumulativerelease of active Dox up to 4 h (Fig. 2B). We reasoned that fluorescencerestoration at 560 nm could be a turn-on response potentially suitablefor the diagnostic assessment of drug distribution. Under the detectionconditions specified in the supplementary information, the reverse-phase HPLC chromatograms showed a peak at retention time of 2060 scorresponding to EDOX (Fig. 2C). Notably, the Dox standard peaks at1975 s, indicating that EDOX is more hydrophobic than Dox itself. Thisproperty may result particularly convenient in the design of self-as-sembled nanomedicine systems based on PEGylation, for the optimi-zation of drug pharmacokinetics (PK) [36]. Upon carboxylesterase ac-tion, EDOX was exclusively converted to active Dox within 4 h by aproposed route shown in Fig. 2D. Possible interference with EDOX re-sponse of various bioanalytes including amino acids, biomolecules, andenzymes was also tested. None of these molecules induced significantEDOX fluorescence change, that was solely activated by esterase, in-dicating that the esterase-EDOX interaction was highly specific. (Fig.S2).

3.2. Cellular uptake and activation of EDOX

Inspired by these findings, we next evaluated EDOX activity withvarious cell lines, including cells derived from human hepatocyte car-cinoma (HepG2), human colorectal adenocarcinoma (HT-29), humancervix adenocarcinoma (HeLa), human breast adenocarcinoma (MCF7),and mouse embryo fibroblasts (NIH3T3). To assess EDOX activation bycellular carboxylesterase, confocal laser scanning microscopy (CLSM)was employed for the detection of intracellular fluorescence arisingfrom Dox. As shown in Fig. 3, only HCC-derived HepG2 cells showed asignificant fluorescence enhancement, corresponding to active Dox,upon treatment with 5 μM EDOX for 2 h. These results were also con-firmed by flow cytometry analysis (Fig. S3) and indicated that EDOXinternalization and/or activation did not occur (or occurred at negli-gible rates) in HT-29, HeLa, MCF7 and NIH3T3 cells as its quenchedfluorescence emission remain intact. Consistently, previous studieshave reported that human carboxylesterase is mainly expressed in theliver and is highly detectable in HepG2 cell lysates by western blottinganalysis [37–39]. Moreover, carboxylesterase activity has been de-tected in HepG2 cells by highly specific fluorescent reporter assays[19]. To evaluate the dynamics of the EDOX-cell interaction, we mea-sured the time-dependent fluorescence enhancement in HepG2 cellsexposed to the conjugate (Fig. S4). A significant enhancement influorescent emission was observed within 30min and reached satura-tion in 24 h. Furthermore, we observed that, upon EDOX enzymaticcleavage, active Dox was initially localized in the cytosol and succes-sively translocated to the nucleus over an incubation time of 36 h (Fig.S4). Collectively, these findings suggested that EDOX hydrolysis re-quires the presence of elevated carboxylesterase activity and, therefore,the complex is preferentially activated by the HCC-derived HepG2 cells,resulting in fluorescence enhancement.

3.3. Cell viability of EDOX towards various cancer cell lines and apoptosismechanism

Next, we evaluated the anticancer activity of EDOX in different celllines by using a tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-

Figure 2. (A) Fluorescence intensity response of EDOX (10 μM) upon incubation with esterase (3 U/mL) in phosphate buffered solution (PBS) at 37 °C (Excitationwavelength= 470 nm). (B) Time-dependent fluorescence enhancement was observed upon incubation of EDOX with esterase (3 U/mL) in PBS at 37 °C. The changein fluorescence intensity is directly related to the release of active Dox from EDOX. (C) Reverse phase high-performance liquid chromatography (RP-HPLC) curves ofEDOX in the presence of esterase at 37 °C for 0 h, 1 h, and 4 h, and Dox standard. (D) Proposed mode of EDOX activation upon exposure to esterase.

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diphenyltetrazolium bromide (MTT) assay. Cell viability was measuredafter a 48 h incubation with different concentrations of EDOX (Fig. 4A).EDOX exerted a HepG2 cell-specific anticancer activity, even at con-centrations below 10 μM, and was remarkably effective up to 50 μM,whereas no significant impact on the viability of the other cell lines wasobserved in this range of concentrations. The results of the MTT assaywere in agreement with those obtained by confocal microscopy.

Although EDOX-induced cytotoxicity was lower than that exerted byfree Dox under these conditions, its cell specificity could help minimizeDox-related side effects (Fig. S5). Moreover, the comparison of 50%growth inhibitory concentration (IC50) values exhibited that EDOX-treated HepG 2 cells presented more potent therapeutic effects thanother cell lines (Table S1). To further characterize EDOX effects, theexpression of apoptosis-related proteins was evaluated by western blot

Fig. 3. Confocal laser scanning microscopy (CLSM) images of intracellular fluorescence of EDOX in various cell lines. HepG2, HT-29, HeLa, MCF7, and NIH3T3 cellswere incubated with EDOX (5 μM) for 2 h, fixed with paraformaldehyde (4%), and counterstained with the DAPI (blue) and Alexa Fluor 488-Phalloidin (green) tovisualize nucleus and F-actin, respectively. CLSM images of EDOX (red) were obtained by using an excitation wavelength of 488 nm and an emission wavelength560–590 nm. Scale bars: 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. Anticancer effects of EDOX in various cell lines. (A)HepG2, HT-29, HeLa, MCF7, and NIH3T3 cells were treatedwith EDOX at various concentrations for 48 h. Then, cellviability was determined by MTT assay. (B) Western blotanalysis in Dox- and EDOX-treated HepG2 cells. Cells weretreated with Dox or EDOX (5 μM) for 12 h. Apoptosis wasassessed by western blot using anti-PARP-1 and anti-Caspase-3 monoclonal antibodies, and a β-actin antibody asa loading control.

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in EDOX-treated HepG2 cells. PARP-1 (poly (ADP-ribose) polymerase-1) plays an important role in the recovery of DNA-damaged cells. Thelevel of cleaved PARP-1 is one of the most important markers ofapoptosis and it was reported to be increased after Dox treatment[40–42]. Similarly, the expression level of activated caspase-3, a mi-tochondrial apoptosis-associated protein, is known to be upregulated inDox-treated cells [43]. We found that the expression of both PARP-1and cleaved caspase-3 was strongly enhanced in HepG2 cells upontreatment with Dox or 5 μM EDOX (Fig. 4B). The different ability of Doxand EDOX to induce the expression of apoptotic proteins in HepG2 cellsat 12 h might be due to the additional time required for completion ofDox release upon cleavage by carboxylesterase. Overall, our findingsshowed that EDOX was specifically activated by HepG2 carbox-ylesterase and this led to cell death by the induction of apoptosis.

3.4. Esterase mediated activation of EDOX in HCC cells

Finally, to clearly establish the relationship between EDOX activa-tion and endogenous carboxylesterase, we verified whether EDOXfluorescence enhancement could be prevented by the inhibition ofcarboxylesterase activity [44]. Therefore, HepG2 cells were pretreatedfor 12 h with bis-(4-nitrophenyl)phosphate (BNPP), a carboxylesteraseinhibitor, followed by treatment with 10 μM EDOX for an additional2 h. As shown in Fig. 5, EDOX response was nearly abolished in BNPP-pretreated HepG2 cells, further supporting the key role of carbox-ylesterase in EDOX activation.

4. Conclusion

In conclusion, using the acetamide-chemodrug conjugate, EDOX,we have developed a bifunctional system allowing, on the one hand, forsustained, HCC-specific drug release and, on the other, for real-timemonitoring of prodrug activation and drug distribution. EDOX testingin various cancer cell lines revealed a preferential cytotoxic effect onHCC-derived cells. Thus, we propose EDOX, a stimuli-responsive pro-drug conjugate, as a suitable chemotherapeutic option for HCC.

Acknowledgment

This work was supported by the National Research Foundation ofKorea (CRI 2018R1A3B1052702, J.S.K; 2017R1D1A1B04033453,E.J.K). This research was also supported by BioNano Health-GuardResearch Center funded by the Ministry of Science and ICT(MSIT) ofKorea as Global Frontier Project (Grant number H-

GUARD_2013M3A6B2078950 (2014M3A6B2060302)).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.dyepig.2018.12.026.

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