A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic

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A GSH-responsive NIR Theranostic Prodrug for Cancer Therapy and Imaging Fanpeng Kong, Ziye Liang, Dongrui Luan, Xiaojun Liu, Kehua Xu, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01135 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Analytical Chemistry

A GSH-responsive NIR Theranostic Prodrug for Cancer Therapy and Imaging Fanpeng Kong, Ziye Liang, Dongrui Luan, Xiaojun Liu, Kehua Xu* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China. ABSTRACT: To reduce the side effects of chemotherapy, nontoxic prodrugs activated by the tumor microenvironment are urgently required for use in cancer treatment. In this work, we developed prodrug 4 for tumor-targeting treatment and imaging of the anticancer drug release in vivo. Taking advantage of the high GSH concentration in cancer cells, the disulfide bond in prodrug 4 was cleaved, resulting in the release of an active anticancer drug and an NIR fluorescence dye turn-on. Furthermore, contrast to the free anti-cancer drug, the prodrug exhibited higher cytotoxicity to hepatoma cells than that to normal HL-7702 cells. Thus, prodrug 4 is a promising platform for specific tumor-activatable drug delivery system due to its favorable features of in situ and in vivo monitoring of the drug release and therapeutic efficacy.

Cancer is one of the most life-threatening diseases in the world. Although various approaches for cancer therapy have been developed, such as gene therapy,1-2 photodynamic therapy,3 photothermal ablation4 and radiation therapy,5-6 chemotherapy remains a front line therapy for cancer in combination with surgery. However, the main challenge of cancer chemotherapy is the selective elimination of tumor cells without affecting normal tissues. In an ideal case, drugs would be applied in vivo at exactly the minimally needed therapeutic concentration and would precisely target cancer cells. To achieve this goal, the use of theranostic systems,7-12 which contain an inactive anticancer drug and an imaging reagent, has received attention as a promising cancer chemotherapeutic approach due to its imaging capability and selective elimination of cancer cells. In these systems, masked anticancer drugs and imaging reagents are conjugated by tumor-sensitive linkers. In the tumor microenvironments, such as those with low pH,13 high ROS level,14 high expressed enzymes15-16 and high GSH concentration,17 the breaking of linkers is followed by the release of the anticancer drugs and the activation of the imaging reagents. In theranostics, fluorophores are usually used as imaging reagents for monitoring the release of the drugs. However, fluorophores used for theranostic prodrugs, such as coumarin,18 BODIPY,19 1, 8-naphthalimide20 and xanthene,21-22 suffer from short wavelength emission that is not suitable for monitoring the drug release in vivo. Near-infrared (NIR) fluorophores with characteristics such as deep tissue penetration, minimal damage to the biological samples and low background interference23-25 are ideal candidate fluorophores for theranostics prodrug systems. To date, very few theranostics prodrug systems using NIR fluorophores have been reported. Kim et al. developed a theranostic prodrug bearing the Cy7 dye as a NIR fluorescence reporter.26 Zhu et al. described dicyanomethylene-4H-pyran (DCM)-based theranostic prodrug, DCM-S-

CPT, for monitoring cancer treatment in vivo.27 However, the capability of tracking drug releases in vivo in these studies is limited by poor photostability, low quantum yield, or water solubility. Thus, there is an urgent need for monitoring drug releases in vivo; NIR fluorophores, which has excellent properties, is one way to monitor drug releases in vivo. Compared with traditional cyanine dyes, merocyanine dyes28-31 showed unique properties of photostability and high fluorescence quantum yield that are favorable for improving the quality of the image in vivo. Although merocyanine dyes have been employed as signal reporters of various probes,32-39 they have rarely been used in theranostics prodrug systems to date. Herein, we describe a novel NIR theranostic prodrug 4 for cancer treatment obtained by conjugating a merocyanine dye and an anticancer drug camptothecin (CPT) 40-41 via disulfide linker. The disulfide bond in the prodrug 4 can be cleaved by a high concentration of glutathione (GSH) in cancer cells,42-45 resulting in the anticancer drug release and NIR fluorescence turn-on. Surprisingly, compared with free CPT, 4 showed effective pharmacodynamic effects in cancer cells and low cytotoxicity to normal cells. This cancer cell-targeting ability make 4 a promising prodrug platform with high efficacy and reduced side-effects. EXPERIMENTAL SECTION Materials and Instruments Cy7.Cl and compound 1 was synthesized in our laboratory46-48. Bis (2-hydroxyethyl) disulfide was purchased from Alfa Aesar Chemical Ltd. (Tianjin, China). N-(4-acerylphenyl)malemide (NEM), glutathione (GSH), L-cysteine (L-cys), LHomocysteine (Hcy), ascorbic acid (Vc) , N-acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) were all purchased

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from Sigma-Aldrich Co. Ltd. Triphosgene, 3-Nitrophenol, 4nitrophenyl-chloroformate, (S)-(+)-Camptothecin(CPT), and DL-Dithiothreitol were obtained from Aladdin Chemical Company (Shanghai, China). N,N-Dimethylformamide (DMF), Triethylamine, Dichloromethane, Methanol, SnCl2-H2O, Hydrochloric Acid, Sodium hydroxide, Sodium sulfate, pyridine, tetrahydrofuran, 4-dimethylaminopyridine, Ether, Aluminum oxide were purchased from Sinopharm Chemical Reagent. Co. Ltd. (Shanghai, China). Mito-Tracker Green was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The silica gel for the ﬂash chromatography was purchased from Qingdao Haiyang Chemical Co. (China). Sartorius ultrapure water (18.2 MΩ cm) was purified with Sartorius Arium 611 VF system (Sartorius AG, Germany). The solvents were used after appropriate distillation or purification. High-resolution mass spectral analyses were carried out on Bruker maXis ultra-high resolution-TOF MS system. 1H NMR and 13C NMR spectra were taken on Bruker Advance 300 MHz and 400 MHz spectrometer (Bruker, Germany). The fluorescence spectra measurements were performed with FLS920 Edinburgh fluorescence spectrometer. Absorption spectra were recorded on UV-1700 UV−vis spectrophotometer (Shimadzu, Japan). All pH measurements were performed with a pH-3c digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glasscalomel electrode. The MTT assay was measured with a microplate reader (RT 6000, Rayto, United States). The fluorescence imaging studies were performed with a Leica DMI6000 fluorescence microscope (Leica Co., Ltd., Germany). The in vivo fluorescence imaging was carried out using an IVIS LuminaⅢ in vivo imaging system. Synthesis of compound 2 To a solution of compound 1 (0.37 g, 0.5 mmol) in MeOH were added SnCl2·H2O (2.25 g, 10 mmol) and hydrochloric acid (3 mL). The mixture was stirred at 70 °C under Ar over night. Methanol was evaporated, and the residue was separated in CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over anhydrous sodium sulfate and evaporated. The crude product was purified via chromatography (silica gel) eluting with CH2Cl2: MeOH (20:1) to afford 0.11 g (42 %) of the compound 2. 1H NMR (400 MHz, CDCl3 ): δ 1.48 (s, 3H), 1.77 (s, 6H), 1.94 (s, 2H), 2.65 (s, 2H), 2.75 (s, 2H), 3.48 (s, 2H), 4.08 (s, 1H), 5.90 (d, J = 16Hz, 1H), 6.68 (s, 2H), 6.92 (s, 1H), 7.06 (d, J = 8Hz, 1H), 7.14 (s, 2H), 7.39-7.41 (m, 3H), 8.53 (d, J = 16Hz, 1H). 13 C NMR (100 MHz, CDCl3 ): δ 12.5, 24.2, 28.3, 49.5, 79.7, 97.6, 100.1, 111.7, 113.3, 114.5, 115.2, 122.7, 129.1, 130.3, 139.1, 141.4, 142.0, 155.7, 162.9, 173.1. HR MS [M-I] +: m/z calcd. 397.2274, found 397.2282. Synthesis of compound 3 To a solution of the 4-nitrophenyl chloroformate (0.1g, 0.5 mmol) in THF (1 mL) was added dropwise the mixture of compound 2 (26.2 mg, 0.05 mmol) and pyridine (20 µL) in dry CH2Cl2 (4 mL). The reaction mixture was stirred at 0 °C for 2 h. Then, the above mixture was added dropwise into a solution of bis (2-hydroxyethyl) disulfide (100µL) and pyridine (1 mL) in THF (3 mL), the mixture was stirred room temperature under Ar for 12 h. The solvents were evaporated, and the residue was separated in CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried and evaporated. The crude product was purified via chromatography (neutral alumina) eluting with CH2Cl2:

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MeOH (100:0 then 20:1) to afford 11.6 mg (33 %) of the compound 3. 1H NMR (400 MHz, CDCl3 ): δ 1.54 (s, 3H), 1.63 (s, 6H), 1.90 (s, 2H), 2.65 (s, 2H), 2.73 (s, 2H), 3.03 (s, 4H), 3.93 (s, 2H), 4.39 (s, 4H), 5.09 (s, 1H), 6.26 (s, 1H), 7.25-7.37 (m, 7H), 7.92 (s, 1H), 8.07 (s, 1H), 8.63 (s, 2H), 10.75 (s, 1H). 13C NMR (100 MHz, CDCl3 ): δ12.6, 20.3, 28.2, 38.1, 41.0, 50.6, 60.8, 62.45, 76.7, 77.0, 77.4, 105.1, 11.7, 116.6, 122.8, 129.0, 135.2, 141.0, 153.8, 162.6, 176.3. HR MS [M-I] +: m/z calcd 577.2189, found 577.2188. Synthesis of compound 4 The solution of camptothecin (83.6 mg, 0.24 mmol) in CH2Cl2 (10 mL) were added DMAP (61.1 mg, 0.5 mmol) and triphosgene (23.7 mg, 0.08 mmol). The reaction mixture was stirred at room temperature for 15 min. Then a solution of the compound 3 (70.4 mg, 0.1 mmol) in CH2Cl2 (20 mL) was slowly added into above mixture. The mixture was stirred at room temperature over night, poured into diethyl ether. The crude product produced was filtered and purified via chromatography (neutral alumina) eluting with CH2Cl2: MeOH (100:0 then 20:1) to afford 73.8 mg (68%) of the compound 4. 1H NMR (400 MHz, CDCl3 ): δ 1.54 (s, 3H), 1.63 (s, 6H), 1.90 (s, 2H), 2.65 (s, 2H), 2.73 (s, 2H), 3.03 (s, 4H), 3.93 (s, 2H), 4.39 (s, 4H), 5.09 (s, 1H), 6.26 (s, 1H), 7.25-7.37 (m, 7H), 7.92 (s, 1H), 8.07 (s, 1H), 8.63 (s, 2H), 10.75 (s, 1H). 13C NMR (100 MHz, CDCl3 ): δ12.6, 20.3, 28.2, 38.1, 41.0, 50.6, 60.8, 62.45, 76.7, 77.0, 77.4, 105.1, 11.7, 116.6, 122.8, 129.0, 135.2, 141.0, 153.8, 162.6, 176.3. HR MS [M-I]+:m/z calcd 951.3092, found 951.3092. RESULTS AND DISCUSSION Scheme 1. Synthesis of prodrug 4

Conditions: (a) m-Nitrophenol, Et3N, DMF, RT. (b) SnCl2, HCl, MeOH, 70 °C. (c) 4-Nitrophenyl chloroformate, bis-(2hydroxyethyl)disulfide, pyridine, THF (d) CPT, DMAP, triphosgene, CH2Cl2. The synthesis of prodrug 4 is depicted in Scheme 1. The merocyanine dye was synthesized from Cy7.Cl following the previously reported procedures.28-31 Compound 2 was reacted with triphosgene, followed by treatment with 2, 2’dithiodiethanol to afford compound 3. Finally, prodrug 4 was obtained by coupling of CPT with compound 3 in the presence of triphosgene. The overall chemical structures of the prodrug and the intermediate compounds were confirmed by 1H NMR, 13 C NMR and HRMS (Supporting Information).


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Scheme 2. Proposed CPT Release Mechanism of the Activatable Prodrug 4

The proposed CPT release mechanism of the activatable prodrug 4 is shown in Scheme 2. In the presence of GSH, the disulfide bond of prodrug 4 is cleaved. Then, the intermediates participate in the intramolecular cyclization44-45 and release the NIR dye and the active drug CPT. In the high resolution mass spectrometry (MS) spectra, the intrinsic peak (951.3097) of prodrug 4 disappeared, and new peaks at 397.2273 (corresponding to dye 2) and 349.1177 (corresponding to anti-cancer drug CPT) were observed upon interaction of 40 equiv of GSH with prodrug 4 (Figure. S1). These results are fully consistent with our proposed drug release mechanism.

Figure1. (a) Fluorescence changes of prodrug 4 after treatment with increasing concentrations of GSH (0-100 equiv). (b) Changes in fluorescence intensity at 702 nm as a function of GSH concentration. Each spectrum was acquired 1 h after exposure to GSH at room temperature in HEPES buffer (pH = 7.4) with λex = 660 nm. To verify that GSH was able to cleave the disulfide bond of the prodrug 4 and consequently, activate the NIR fluorophore, the fluorescence spectra of 4, upon addition of 0-100 equiv of GSH, were investigated under optimum conditions (Figure.S2S4). As seen in Figure 1a, the fluorescence at 702 nm gradually increased upon a stepwise addition of GSH and reached saturation upon an addition of 40 equiv of GSH. The fluorescence intensity increased by approximately five times and shows good linearity with the concentration of GSH over a wide range (0-0.7 mM). This observation indicated that the disulfide linker of the prodrug 4 can be effectively cleaved by GSH. Therefore, the high concentration of GSH in cancer cells may lead to the release of CPT and the NIR fluorescence turnon.

Figure 2. (a) Fluorescence response of prodrug 4 (17.5 µM) toward GSH, DTT, Cys, Hcy, other amino acids (Trp, Lys, Met, Glu, Thr, Pro) and metal ions, 1 mM for each. (b) Fluorescence intensity at 702 nm of 4 with and without GSH as a function of pH. Each spectrum was acquired 1 h after exposure to GSH (40 equiv.) at room temperature in HEPES buffer (pH = 7.4) with λex = 660 nm. To evaluate the possible application of 4 in real biological systems, the fluorescence response of 4 toward other biologically relevant analytes, such as amino acids and metal ions, was investigated. As shown in Figure 2a, no significant changes in fluorescence intensity were observed upon addition of the nonthiol amino acids and metal ions to prodrug 4. Although, cysteine (Cys), dithiothreitol (DTT), and homocysteine (Hcy) were found to cause a fluorescence response of 4 similar to that of GSH, the potential interference of these compounds can be neglected because of their relatively low concentrations in the biologically systems. The pH effect on the GSH-induced fluorescence changes of 4 was also investigated. As shown in Figure 2b, prodrug 4 remained stable and emits a weak fluorescence within 5.5-8.0 pH range. After treatment by 40 equiv of GSH, the fluorescence at 702 nm was activated and reached saturation in the 6.7-8.0 pH range. The above results indicated that prodrug 4 can be applied as a GSH-induced theranostics system under physiological pH conditions with high selectivity over potential interference by various biological compounds.

Figure 3 Cellular fluorescence images of prodrug 4-treated HepG2 and HL-7702 cells. (a) HepG2 cells pretreated by NEM (A), HL-7702 cells (B), HepG2 cells were incubated with prodrug 4 (C), HepG2 cells were pretreated with GSH followed incubated with prodrug 4 (D). (b) Quantitative analysis of fluorescence intensity in cells incubated with prodrug 4. (c) Confocal microscopic images of colocalized experiment in HepG2 cells. Fluorescence images of HepG2 cells contained with mitochondria tracker (E), fluorescence images of HepG2 cells contained with 4 (17.5 µM) for 1 h (F). Overlay of merged images of E and F (G). Fluorescence intensity profile of regions of interest (green line in G) across the lines (H). The photostability of NIR fluorophores in the theranostic prodrug is crucial for practical application in bioimaging. The photo-stability of prodrug 4 treated by GSH was investigated by time-sequential scanning of living cells. After 500 s of continuous irradiation with a 633 nm laser, no obvious changes



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were observed in fluorescence brightness (Figure. S5), indicating that the merocyanine fluorophore can be used as an excellent NIR imaging agent in this theranostic prodrug system. Then, the cellular uptake and intracellular localization of prodrug 4 were investigated by confocal laser scanning microscopy. In the HepG2 cells, a red fluorescence (dye 2 released from prodrug 4) was observed after 1 h of incubation at 37 °C because prodrug 4 was activated sufficiently by the high concentration of GSH in cancer cells (Figures 3a, C). To verify the activation function of GSH for prodrug 4, extra 1 mM GSH was added to HepG2 cells, and enhanced fluorescence was observed (Figures 3a, D). HepG2 cells treated by Nethylmaleimide (NEM, a thiols scavenging agent) and normal HL-7702 cells were also incubated for 1 h with prodrug 4 for an examination of the effect of GSH on the bioactivity of 4. Very weak fluorescence signals were observed by confocal microscopy (Figures 3a, A, B), due to low GSH contents in these cells. The similar results were obtained in the parallel experiments carried out in MCF-7 cells (Figure. S6). All of these results indicated that the high concentration of GSH in cancer cells can effectively activate prodrug 4, while the turnon NIR fluorescence can also be used to monitor the drug release process in cancer cells. Furthermore, mitochondria localization of activated prodrug 4 triggered by GSH was investigated using a selective fluorescent marker for mitochondria (Mito-tracker Green). As shown in Figure 3c, the fluorescence signal that can be ascribed to 4 colocalized well with the Mitotracker (Pearson’s correlation coefficient, ρ = 0.82). Because the NIR fluorescence dye released from the prodrug 4 was located in the mitochondria due to the positive charge found in its structure.

Figure 4. Cell viability of 4 and free CPT at different concentrations on HepG2 cells (a) and HL-7702 cells (b). All compounds were incubated with cells for 24 h, and the cell viability observed via MTT assay. We then investigated the ability of the prodrug 4 to selectively target cancer cells by measuring its cytotoxicity to both HepG2 and HL-7702 cells. As shown in Figure 4a and figure S7, the cytotoxicity of the prodrug 4 to HepG2 cells and MCF7 cells is similar to that of the free CPT because the high concentration of GSH in the tumor cells can effectively activate the prodrug. On the other hand, 4 showed less cytotoxicity in the normal HL-7702 cells even at high concentrations, whereas free CPT was highly toxic (Figure 4b). These results indicated the prodrug 4 can selectively kill cancer cells with negligible side effects for normal cells.

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Figure 5. In vivo imaging of tumor-bearing mice at various time (0 h, 0.5 h, 1 h) after orthotopic injection of the prodrug 4 (5 mg / kg). The drug release of the prodrug 4 in tumor-bearing mice was investigated by in vivo fluorescence imaging. The nude mice bearing H22 xenografts were injected in the tumor site with the prodrug 4 at a dose of 5 mg / kg , and in vivo images were obtained at various times. As shown in Figure 5, NIR fluorescence intensity representing the release of the anticancer drug was clearly observed at 0.5 h after the injection of the prodrug 4, the NIR fluorescence then increased in time-dependent manner. These results indicated that prodrug 4 can be used as an ideal theranostics prodrug in vivo. To assess whether drug release for the prodrug 4 occur in tumor-affected tissues, H22 tumor-bearing mice were treated with 4 and saline by intravenous injection (at a CPT equivalent dose of 5 mg / kg). As shown in figure 6a, the obvious fluorescence was seen in the tumor region after treated by prodrug 4, which indicated the drug released effectively in the tumor region. In contrast, negligible signals were obtained in the saline-treated mice (figure 6b). Furthermore, in fluorescence images of the internal organs after anatomy, the strong fluorescence was observed in the tumor of mice treated by prodrug 4 (figure 6c). And much weaker fluorescences were seen in lung, heart, liver, kidney, spleen and stomach. The results indicated prominent tumor-targeting ability of prodrug 4. Meanwhile, in the control experiments, no obvious fluorescences were observed in the tumor or other internal organs (figure 6d). The tumor-targeting ability make 4 a promising prodrug to achieve high efficacy and reduced side-effects.


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Figure 6. In vivo imaging of tumor-bearing mice at various time after intravenous injection of prodrug 4 (13.65 mg / kg) (a) and saline (b). Fluorescence images of the internal organs after anatomy for prodrug 4 (c) and saline (d). CONCLUSIONS In summary, by conjugating the anti-cancer drug CPT and NIR merocyanine fluorophores via a disulfide bond, we developed a novel GSH-responsive NIR theranostics prodrug 4. The prodrug can be effectively activated by the high concentration of GSH in cancer cells or in tumor-bearing mice and gave rise to an enhanced NIR fluorescence. More importantly, the prodrug 4 showed high anti-cancer activity to cancer cells and lower side effect to normal cells compared to the free CPT drug. Therefore, the prodrug 4 provides a promising platform for specific tumor-activatable drug delivery system, which can be easily monitored by cellular and tissue fluorescence imaging.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The 1H NMR, 13C NMR and HRMS spectra of compound 2, 3 and prodrug 4 and other materials.

AUTHOR INFORMATION

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Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21390411, 21535004, 21227005, 21275092, 21575081 and 21405098).

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