GSH-Activated NIR Fluorescent Prodrug for Podophyllotoxin

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GSH-Activatable NIR Fluorescent Prodrug for Podophyllotoxin Delivery Yajing Liu, Shaojia Zhu, Kaizhi Gu, Zhiqian Guo, Xiaoyu Huang, Ming-Wei Wang, Hesham M. Amin, Wei-Hong Zhu, and Ping Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07091 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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ACS Applied Materials & Interfaces

GSH-Activatable NIR Fluorescent Prodrug for Podophyllotoxin Delivery Yajing Liu†#, Shaojia Zhu†#, Kaizhi Gu†, Zhiqian Guo†, Xiaoyu Huang‡*, Mingwei Wang⊥, Hesham M Amin§, Weihong Zhu†*, Ping Shi†* †

State Key Laboratory of Bioreactor Engineering, Key Laboratory for Advanced

Materials and Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China ‡

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345

Lingling Road, Shanghai 200032, P. R. China ⊥

Fudan University Shanghai Cancer Center, 270 Dongan Road, Shanghai 200032, P.

R. China §

Department of Hematopathology, The University of Texas MD Anderson Cancer

Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

#

These authors contributed equally to this work.

*To whom correspondence should be addressed. E-mail: [email protected](Shi P), East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China; [email protected] (Huang X), Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, P. R. China;

[email protected] (Zhu W), East China University of

Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China.

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ABSTRACT Theranostic prodrug therapy enables the targeted delivery of anticancer drugs with minimized adverse effects and real-time in situ monitoring of activation of the prodrugs. In this work, we report the synthesis and biological assessment of the near-infrared

(NIR)

prodrug

(mPEG-DSPE)-encapsulated

DCM-S-PPT nanoparticles.

and

its

amphiphilic

DCM-S-PPT

is

copolymer

composed

of

podophyllotoxin (PPT) as the anticancer moiety and a dicyanomethylene-4H-pyran (DCM) derivative as the NIR fluorescent reporter, which are linked by a thiol-specific cleavable disulfide bond. In vitro experiments indicated that DCM-S-PPT has low cytotoxicity and that glutathione (GSH) can activate DCM-S-PPT, resulting in PPT release and a concomitant significant enhancement in NIR fluorescence at 665 nm. After being intravenously injected into tumor-bearing nude mice, DCM-S-PPT exhibited

excellent

tumor-activatable

performance.

Furthermore,

we

have

demonstrated that mPEG-DSPE as a nanocarrier loaded with DCM-S-PPT (mPEG-DSPE/DCM-S-PPT) showed even greater tumor-targeted performance than DCM-S-PPT on account of the enhanced permeability and retention effect. Its tumor-targeting ability and specific drug release in tumors make DCM-S-PPT a promising prodrug that could provide a significant strategy for theranostic drug-delivery systems. Keywords: PPT; Prodrug; NIR fluorescent reporter; DCM-S-PPT; mPEG-DSPE

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INTRODUCTION Chemotherapy is a successful cancer therapy1 but a variety of challenges are associated with its use. Its nonselectivity and associated toxicity can cause severe damage to normal tissues and cells,2,3 resulting in the discontinuation of therapy before all cancer cells are eliminated. Podophyllotoxin (PPT) is a natural aryltetralin lignin isolated from the plant podophyllum. Recent studies have shown that PPT and its derivatives have significant antitumor and antiviral properties.4-6 PPT is used as the lead compound for the synthesis of well-known anticancer agents including etoposide and teniposide, which show good clinical effects against Wilms tumors, non-Hodgkin and other lymphomas, different types of genital tumors (carcinoma verrucosus, for example),

and lung cancer.7-9 Our group previously found that PPT has higher

antitumor activity and a broader antitumor spectrum than its derivatives.10 Its antineoplastic activities mainly attribute to prevent the assembly of tubulin into microtubules and induce apoptosis.11,12 However, PPT has been abandoned as a possible antitumor agent because of its water insolubility and adverse effects.13,14 In addition, it is difficult to monitor the delivery of a nonfluorescent compound such as PPT in vitro and in vivo.15 Therefore, developing a PPT theranostic prodrug that is able to monitor PPT delivery in real time and to reduce its adverse effects is of critical importance. The use of targeted drug-delivery systems (DDSs) to improve therapeutic efficacy and selectivity of chemotherapy drugs has been explored.16-19 The most common strategy is to use biomarker-targeting ligands, like aptamers, peptides, and antibodies, which can actively target specific tumor cells through selective interaction 3



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with the cell receptor on the cytomembrane surface.20-22 However, many cancer biomarkers are expressed in both normal and cancer cells,23 and the use of some biomarkers is impeded by the heterogeneity of tumors.24 It is well known that tumor microenvironments release different biological cues compared to physiologically normal tissues and cells.2,25 Therefore, a stimuli-responsive DDS,26 induced by the specific tumor microenvironment, becomes one of the research hotspots, which can significantly enhance the selectivity and efficacy of anticancer drugs. Among the components of the tumor microenvironment that can be selectively targeted, glutathione (GSH) has become a hot topic of research in recent years.27 The concentration of intracellular GSH in cancer cells is much higher than in normal cells and tissues.28-30 In addition, the GSH concentration is very low (20-40 μM) in plasma and other body fluids outside cells.31,32 To date, a number of GSH-responsive prodrugs containing disulfide bond have been reported to have potential therapeutic efficacy.27 For another, real-time monitoring of drug release inside the targeted tumors, especially in a noninvasive manner in vivo, is challenging and in its nascent stage.33,34 Most of the current antitumor drug candidates are intrinsically nonfluorescent,35 but theranostic prodrugs built with fluorophores as an optical reporter have exhibited the promising ability to monitor drug delivery because their release triggers fluorescence enhancement. So far, most of the fluorophores employed as reporters in theranostic prodrugs, such as xanthene,36 1,8-naphthalimide,37 and coumarin,38 have limited in vivo application owing to their short wavelength emissions and resultant poor tissue penetration. In contrast, near-infrared (NIR) fluorophores may be useful reporter 4


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subunits in theranostic prodrugs since NIR photons can deeply penetrate tissues and have low background interference.39-41 In this work, we report a novel prodrug, DCM-S-PPT, that uses a thiol-specific cleavable disulfide bond to link PPT, an activatable anticancer molecule, with the reporter dicyanomethylene-4H-pyran (DCM), an NIR fluorophore with high photostability and a large Stokes shift.42-44 As summarized in Scheme 1, when disulfide bond was cleaved by thiols like GSH,45 PPT is released and NIR fluorescence is simultaneously enhanced. So as to improve the stability of the prodrug under biological conditions and achieve enhanced permeability and retention ,46-48 which are important for in vivo application, the prodrug was encapsulated into the biodegradable

amphiphilic

phosphoethanolamine-N-[methoxy

copolymer (polyethylene

1,2-distearoyl-sn-glycero-3glycol)-2000]

(mPEG-DSPE)

micelle.49,50 Our novel theranostic prodrug (DCM-S-PPT) has several notable features: (i) significant enhancement of NIR fluorescence accompanied by GSH-activated PPT release ; (ii) reduced cytotoxicity due to PPT conjugation to fluorophore and lack of cytotoxicity in tissues not containing GSH; and (iii) excellent tumor-activatable performance, especially for its micelles. As the first NIR fluorescence-based prodrug of PPT, DCM-S-PPT is applied for in situ fluorescence tracking in vivo. These features make DCM-S-PPT a promising agent for treating and monitoring the progress of cancer therapy with high efficacy and reduced adverse effects. EXPERIMENTAL SECTION

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Materials. PPT (purity = 98%) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. GSH (purity = 98%) was purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). DSPE-PEG (purity = 99%) was purchased from Avanti Lipids (Shanghai, China). All other reagents and solvents were purchased commercially and in analytical grade. Synthesis of DCM-S. DIEA (1.5 g, 11.7 mmol) was added dropwise to a mixture of DCM-NH2 (225 mg, 0.72 mmol), triphosgene (860 mg, 2.9 mmol), and dry toluene (35 mL) under an argon atmosphere at room temperature. The resulting solution was refluxed under argon protection for 3 h. After any unreacted phosgene gas was removed by flushing with argon gas, a solution of 2, 2’-dithiodiethanol (1.2 g, 90%, 7.2 mmol) in anhydrous CH2Cl2/THF (1:1, 15 mL) was added to the mixture, and the reaction mixture was stirred overnight at room temperature. After removing the solvent under reduced pressure, the crude product was purified by silica gel chromatography using ethyl acetate/PE (v/v, 1:1) as the eluent to produce DCM-S as a yellow solid (96 mg) with 27% yield. 1H NMR (400 MHz, CDCl3, ppm): δ 8.91 (d, J = 8.4 Hz, 1H, phenyl-H), 7.75 (t, 1H, phenyl-H), 7.61–7.44 (m, 8H, phenyl-H and alkene–H), 6.85 (s, 1H, phenyl-H), 6.73 (d, J = 16.0 Hz, 1H, alkene–H), 5.55 (s, 1H, NH), 4.48 (t, 2H, –O–CH2), 3.93 (t, 2H, OH–CH2), 3.02 (t, 2H, –O–CH2–CH2), 2.95 (t, 2H, –OH–CH2–CH2). 13C NMR (100 MHz, CDCl3, ppm): δ 157.62, 152.86, 152.33, 139.85, 138.24, 134.63, 129.85, 129.09, 125.96, 125.84, 118.75, 118.58, 117.86, 117.39, 116.87, 115.80, 106.63, 63.35, 60.41, 41.62, 37.52. Mass spectrometry (ESI

6


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negative ion mode for [M - H]-): Calcd. for C25H20N3O4S2, 490.0895; found, 490.0906. Synthesis of DCM-S-PPT. A mixture of DMAP (122 mg, 1.0 mmol), PPT (50 mg, 0.1 mmol), and triphosgene (20 mg, 0.07 mmol) in 10 mL of anhydrous chloroform was stirred under argon protection at room temperature until the reaction solution was clear and transparent. Then, a solution of DCM derivative (50 mg, 0.1 mmol) in anhydrous chloroform was added under ice bath condition. The mixture was stirred overnight at room temperature. After the solvent was removed under reduced pressure, the residue was then purified by silica gel chromatography (dichloromethane/ethyl acetate/PE 1:0.8:1.2, v/v/v) to produce DCM-S-PPT as a pale yellow solid with 11% yield. 1H NMR (400 MHz, CDCl3, ppm): δ = 2.91-2.93 (m, 2 H), 2.99-3.05 (m, 4 H, -CH2SSCH2-), 3.75 (s, 6 H, -OCH3), 3.80 (s, 3 H, -OCH3), 4.22 (t, J = 9.2 Hz, 1 H), 4.43-4.51 (m, 6 H, -CH2CH2SS CH2CH2-), 4.60 (d, J = 3.6 Hz, 1 H), 5.76 (d, J = 8.4 Hz, 1 H), 5.99 (s, 2 H), 6.39 (t, 2 H), 6.55 (s, 1 H), 6.71 (d, J = 16 Hz, 1 H, alkene-H), 6.83 (s, 1 H), 6.88 (s, 1 H), 7.15 (s, 1 H), 7.43-7.58 (m, 6 H, phenyl-H), 7.74 (t, J = 7.6 Hz, 1 H, phenyl-H), 8.90 (d, J = 8.0 Hz, 1 H, phenyl-H) (Figure S1).

13

C NMR

(100 MHz, CDCl3, ppm): δ = 36.99, 37.59, 38.46, 43.73, 45.51, 56.18, 60.76, 62.40, 62.96, 66.24, 71.20, 101.71, 106.60, 107.16, 108.00, 109.74, 115.81, 116.89, 117.35, 117.84, 118.60, 118.74, 125.81, 125.96, 127.47, 129.07, 129.79, 132.44, 134.61, 134.64, 137.11, 138.25, 139.98, 147.72, 148.41, 152.33, 152.66, 152.73, 152.84, 155.36, 157.64, 173.46 (Figure S2). Mass spectrometry (ESI positive ion mode for [M + Na]+): Calcd. for C48H41N3O13S2Na: 954.1979; found: 954.1979 (Figure S3). 7



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Characterization. 1H and

13

C NMR spectra in CDCl3 or DMSO-d6 were acquired

using a Bruker Avance III 400 MHz instrument (Bruker Biospin, CA, USA), and tetramethylsilane (TMS) was used as the internal standard. A Waters LCT Premier XE spectrometer (Waters, MA, USA) was used to measure high-resolution mass spectra. UV/vis spectra and fluorescence spectra were detected with a Varian Cary 500 spectrophotometer and a Varian Cary Eclipse (1 cm quartz cell), respectively (Varian, CA, USA). The SEM images were measured using a JEOL JSM-6360 scanning electron microscope (JEOL, Tokyo, Japan). DLS experiments were carried out with a Nicomp TM 380 ZLS zeta-potential/particle sizer (PSS Nicomp, Santa Barbara, CA, USA). Confocal laser scanning images were acquired using an inverted fluorescence microscope (Nikon, Nikon, Tokyo, Japan). Flow cytometric analysis was carried out with a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). In Vitro GSH Response Experiments. 10 µM of DCM-S-PPT in DMSO/PBS solvent (1/1, v/v, pH7.4) was selected as a final solution for the measurements. The final solution was incubated with the desired concentration of reducing agent for 1 h at 37 °C, and then the UV/vis and fluorescence spectra were measured with a Varian Cary 500 spectrophotometer and a Varian Cary Eclipse , respectively. Kinetic Studies. The kinetic studies were followed by measuring the fluorescence spectra after mixing DCM-S-PPT and GSH in a 3 mL quartz cuvette. The reaction was performed under excess GSH at 37℃, expecting 100% conversion of DCM-S-PPT to DCM-NH2. And the reaction constant of DCM-S-PPT reaction with 8


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Cys and Hcy was measured according to the same protocol. The reaction constant was determined according to the Pseudo-First Order Equation: Ln{(Fmax-Ft)/Fmax} = -kt. Where Ft and Fmax are the fluorescence intensities at 665 nm at times t and the maxima values when the reaction reached the conversion of 100%. The k is the reaction constant. Cell Culture. The human epithelioid cervical carcinoma cell line HeLa and normal embryonic kidney 293T cell line, were purchased from the Institute of Cell Biology (Shanghai, China). Cells were all propagated in T-75 flasks cultured in RPMI-1640 medium

or

DMEM

medium

(GIBCO/Invitrogen,

Camarillo,

CA,

USA),

supplemented with 10% fetal bovine serum (FBS) (Biological Industry, Kibbutz Beit Haemek, Israel) and 1% penicillin–streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin; Solarbio Life Science, Beijing, China) at 37 ℃ under a humidified 5% CO2 atmosphere. Measurement of Intracellular GSH Levels. To measure the intracellular glutathione (GSH) concentration, human cell lines including ovarian cancer SKOV3, neuroblastoma

SH-SY-5Y,

epithelioid

cervical

carcinoma

HeLa,

hepatoma

SMMC-7721, hepatocyte QSG-7701, embryonic kidney 293T, and mouse fibroblasts L929 were cultured and prepared. The endogenous GSH from cell extract can react with DTNB (5,5-dithio-bis-[2-nitrobenzenic acid]) to form the coloured GSH-DTNB conjugate, which can be determined by the change in absorbance at 412 nm. All data were normalized by milligrams of protein.

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Encapsulation of DCM-S-PPT into Nanoparticles. mPEG-DSPE (3.0 mg) was rapidly dissolved in distilled water (9 mL) with vigorous sonication for 30 min. DCM-S-PPT (2×10-2 mol/L, 53.7 µL) in DMSO solution was then rapidly added into the mixture under continuous sonication for another 30 min. Unpackaged DCM-S-PPT and DMSO were removed by dialysis against distilled water over 48 h. Then the aqueous solution was filtered through a polyvinylidene fluoride syringe-driven filter (0.22-µm). The resulting mPEG-DSPE/DCM-S-PPT micellar solution was stored at 4℃. The size distribution and morphology of the assembled micelles were measured by DLS and SEM. Then, the concentration of DCM-S-PPT in the micellar solution was estimated from the absorbance at 450 nm, in the UV/vis spectra, after extracting the DCM-S-PPT from the micellar solution into DMSO. In Vitro Cytotoxicity Assay. The cell cytotoxicities of free PPT, DCM-S-PPT, and mPEG-DSPE /DCM-S-PPT in both cancer and normal cell lines were determined by the Cell Counting Kit-8 (Dojindo, Tokyo, Japan). Cells (1×104 cells/well) were plated in 96-well plates in DMEM medium with 10% FBS (0.1 ml) and allowed to culture for 12 h. Then cells were treated with the desired concentrations of free PPT, DCM-S-PPT, and mPEG-DSPE/DCM-S-PPT (PPT and DCM-S-PPT were dissolved in DMSO and diluted in PBS, mPEG-DSPE/DCM-S-PPT was dissolved in PBS). After incubation for 24 h or 48 h, absorbance at 450 nm was measured with a Tecan GENios Pro multifunction reader (Tecan Group Ltd., Maennedorf, Switzerland). The relative cell viability was calculated by the equation: cell viability (%) = (ODtreated/ODcontrol) ×100%. 10


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In Vitro Cellular Imaging. The HeLa cells and 293T cells at 1×105 cells/well were seeded onto glass-bottom petri dishes with complete medium (1.5 mL) for 12 h. Then the cells were exposed to desired concentrations of DCM-S-PPT for 3 h. PBS (pH 7.4) was used to washed cells for three times to clean the background. 4% paraformaldehyde was added at room temperature for 20 min. The fixed cells were rinsed with PBS (pH 7.4) twice. The images were then photographed by using a Nikon A1R confocal laser scanning microscope with 488 nm as the excitation wavelength and 570-650 nm as the emission wavelength. GSH-Dependent Intracellular Fluorescent Release by Flow Cytometry Assay. The HeLa cells and 293T cells at 2×105 cells/well were seeded onto six-well plates with 2 mL complete medium and cultured overnight. The cells were then treated with the designed concentrations of DCM-S-PPT at 37℃ for 3 h or 6 h . After washed with PBS twice, the cells were harvested using 0.05% (w/v) trypsin/0.02% (w/v) EDTA. The collected cells were resuspended in PBS (500µL) after washed with cold PBS twice. Each sample was measured with flow cytometry (FACSCalibur) within 1 h (λex = 488 nm, 680/20 nm bandpass filter). Establishment of Mouse Xenograft Model. All animal experiments were approved by the regional animal committee and were in accordance with international guidelines on the ethical use of laboratory animals. The 6- to 8-week-old BALB/c nude mice were obtained from Shanghai Slac Laboratory Animal Company. They were maintained under standard conditions with food and water ad libitum. Prior to experiment, all of the animals were acclimatized to the new environment for at least 2 11



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d. To establish the tumor-bearing mouse model, approximately 5 × 106 HeLa human cervical adenocarcinoma cells in 0.1 mL PBS were injected in the right axillas of the mice. In Vivo and Ex Vivo Fluorescence Imaging. When the tumors grew up to 10 mm in diameter, the mice were treated with a single intravenous injection via the tail vein of either DCM-S-PPT or mPEG-DSPE/DCM-S-PPT at a PPT-equivalent dose of 1 mg/kg and a single intratumoral injection of DCM-S-PPT at a PPT dose of 0.5 mg/kg with muscle tissue injection in the same mouse as a control, and each group consists of three mice. Whole body optical imaging was taken 0-24 h after prodrug injection using a PerkinElmer IVIS Lumina Kinetic Series III imaging system (PerkinElmer, MA, USA). After injection, the mice were sacrificed at 24 h. The grafted tumor tissues and major organs, including kidney, lung, spleen, liver, and heart, were excised and washed with 0.9% saline. The optical images of the organs and tissues were taken using a PE in vivo Professional Imaging System as described above. Statistical Analysis. Data were presented as mean ± standard errors. A student’s t-test was used to determine the statistical significance of differences between experiments and control groups. P values < 0.05 were considered significant. RESULTS AND DISCUSSION Design and Synthesis of the Prodrug. According to Scheme S1, the prodrug DCM-S-PPT was synthesized. The intermediate compounds DCM and DCM-NH2 were synthesized by previously reported methods.42 Next, in the presence of N, N-diisopropylethylamine (DIEA), DCM-NH2 was reacted with triphosgene and then 12


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with 2, 2’-dithiodiethanol to obtain DCM-S. Next, DCM-S was reacted with chloroformate of PPT under mild conditions to get the target prodrug DCM-S-PPT .51 1

H and

13

C NMR and high resolution mass spectrometer (HRMS) was used to

precisely characterize the structure of DCM-S-PPT. The stability of DCM-S-PPT under biological conditions is critically important for in vivo application. However, the synthesized prodrug DCM-S-PPT was aggregated in PBS (pH 7.4) (Figure S4), limiting its biological applications. Thus, the prodrug was encapsulated into a biodegradable amphiphilic copolymer mPEG-DSPE micelle so as to improve its solubility in PBS and achieve EPR. As shown in Figure S4, the resulting mPEG-DSPE/DCM-S-PPT exhibited superb stability in PBS (Scheme S2). As shown in Figure S4A, the micelles were clearly distinguished and had a well-monodispersed spherical shape. The DLS results showed that the monodispersed particle size was at an average of 90 nm (Figure S4B), consistent with the SEM observation. Finally, the concentration of DCM-S-PPT in micellar solution was accurately measured based on the standard curve of DCM-S-PPT (Figure S5). GSH-Activatable Properties in Vitro. To determine whether the disulfide bond that covalently links the DCM and PPT moieties in DCM-S-PPT is cleavable by thiol-containing molecules,27,52 the absorption and fluorescence spectral changes of prodrug DCM-S-PPT after activation by GSH were detected. DCM-S-PPT exhibited a strong absorption band at 450 nm and a weak emission centered at 665 nm in DMSO/PBS solution (50/50, v/v, pH7.4, 10 mM) (Figure 1). Both the colorimetric and fluorescence spectroscopies of the prodrug DCM-S-PPT showed significant 13



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changes upon treatment with 2.5 mM GSH (Figure 1A). The absorption peak moved to 493 nm, with a 43-nm red-shift. Simultaneously, the color of the solution transformed from canary yellow to pink. Moreover, upon excitation at 465 nm, an obvious NIR fluorescence at 665 nm was strongly rocketed round 4.5-time. The absorption and emission spectra of DCM-S-PPT treated with GSH in DMSO/PBS solution were identical to those of DCM-NH2 under the same conditions (Figure S6). In addition, when the concentration of GSH was gradually increased (0−20 equiv), the fluorescence intensity of the solution at 665 nm increased significantly in a linear manner (Figure 1B). The data showed that the disulfide bond of DCM-S-PPT can be cleaved by GSH in a dose-dependent manner. Next, we investigated whether the enhanced fluorescence from DCM-S-PPT in Figure 1 resulted in PPT release. As shown in Figure S7, the fluorescence centered at 325 nm (-the PPT moiety) gradually recovered upon treatment with increased concentrations of

GSH,, suggesting that the PPT moiety was probably released from

DCM-S-PPT . Furthermore, the expected release of PPT was confirmed by ESI-MS analysis. When DCM-S-PPT was treated with 20 equiv of GSH

for 1 h at 37 °C,

peaks of 413.21 ([PPT-H]) and 310.17 ([DCM-NH2-H]) simultaneously arose in the HRMS ( Figure S8). These results led us to determine that the active PPT was released from the prodrug DCM-S-PPT with the treatment of GSH through a two-step reaction in which the S-S bond is cleaved followed by intramolecular nucleophilic substitution, accompanied by the generation of the NIR reporter DCM-NH2 (Scheme 1). The emission spectrum of DCM-NH2 is in the NIR region (650−900 nm), and is 14


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suitable for in vivo imaging due to its deep penetration ability and low background autofluorescence.39-41 To further apply DCM-S-PPT to biosystems, it is crucial to investigate the possible interference of other related biological molecules, including amino acids (cysteine (Cys), homocysteine (Hcy) and etc.) and abundant metal ions (K+, Ca2+, Na+ and Mg2+). As shown in Figure 2, spectroscopic changes in DCM-S-PPT treated with Cys or Hcy could be observed similar to those exposed to GSH. These results were expected because the thiol-containing structures of Cys and Hcy are identical to that of GSH.53-55 However, there were no obvious spectroscopic changes observedupon exposure to other amino acids without thiol, K+, Ca2+, Na+ and Mg2+. So, the disulfide bond cleavage in DCM-S-PPT was selective for the thiol species. According to the Figure S6, the reaction constants of DCM-S-PPT reaction with GSH, Cys, and Hcy are 0.027 min-1, 0.015 min-1, and 0.016 min-1, respectively (Figure S9). In fact, compared to the high concentration of GSH in the cytoplasm (1−15 mM), concentrations of Cys and Hcy were as low as to be neglected.27,56 Therefore, disulfide-linked DCM-S-PPT is suitable for further application in biosystems in vitro and in vivo.

15



Scheme 1. Proposed PPT Release Mechanism of the Activatable Prodrug by Treatment of GSH.

0.3

A

DCM-S-PPT DCM-S-PPT with GSH

0.2

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

GSH 0.1

0.0 300

400

500

600

Wavelength (nm)

16


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500

B

400

F.I. at 665nm

Fluorescence Intensity (a.u.)

500

400

300 200 100

300 0 0

20

40

60

80

100

Equivalent of GSH

250 eq 200 GSH 100 0 0 600

700

800

900

Wavelength (nm) Figure 1. Absorption (A) and fluorescence (B) spectra changes of DCM-S-PPT (10 µM) before and after activated by GSH in DMSO/PBS solution. Inset (A): Color changes in solution of DCM-S-PPT upon treatment with GSH. Inset (B): fluorescence intensity at 665 nm (I665 nm, λex = 465 nm) of DCM-S-PPT. After addition of GSH at 37 °C for 1 h, aach point was recorded ,.

500

Fluorescence Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GSH, Cys, Hcy 400

300

K+, Ca2+, Na+, Mg 2+ and other amino acids

200

100

0 600

700

800

900

Wavelength (nm) Figure 2. Fluorescence intensity of DCM-S-PPT (10 µM) after treated with GSH, Cys, Hcy, other amino acids without thiol (Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Phe, Pro, Ser, Thr, Tro, Tyr, Val (2.5 mM)), K+, Ca2+, Na+, and Mg2+. Each spectrum was recorded after treated with GSH at 37 °C for 1 h, λex = 465 nm.

Level of Intracellular GSH. We measured the glutathione (GSH) concentration of some cell lines from different organs. The result indicated that the most cancer cell 17



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lines contained higher GSH than noncancerous cell lines (Figure S10, ). According to the result of measurement, the cancer cell lines HeLa and the noncancerous cell line 293T were chosen and used in our following studies. GSH-Activatable Properties in Living Cells. Our excellent in vitro results showing active drug release and concomitant NIR fluorescence enhancement in DCM-S-PPT (Scheme 1) led us to investigate whether DCM-S-PPT works in live cells. A low concentration of DCM-S-PPT (300 nM) was incubated with HeLa and 293T cells for 3 h at 37°C and their fluorescence from DCM-NH2 (red) was monitored with confocal laser scanning microscopy (Figure 3). Compared to 293T, the cancer cell line HeLa showed much higher fluorescence from DCM-NH2, indicating that more DCM-NH2 and PPT were released from DCM-S-PPT because of the higher endogenous GSH concentration in HeLa cells.

Figure 3. Confocal laser scanning microscopy images of HeLa and 293T cells incubated with DCM-S-PPT at 37oC, each group had brightfield (left), fluorescence (center) and merge (right) channels.

In addition, flow cytometry analysis was performed to verify the performance of 18


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DCM-S-PPT in HeLa and 293T cells (Figure 4). A negative control without any treatment was used to rule out autofluorescence. After incubation with DCM-S-PPT (300 nM) for 3h or 6h, analyses of the two cell lines exhibited DCM-NH2 fluorescence intensity consistent with the results of confocal laser scanning imaging. The uptake ratios of DCM-S-PPT at 3h and 6h, respectively, were 5.65% and 22.8% for HeLa cells, while those were 1.14% to 4.29% for 293T cells respectively. By contrast, the fluorescence intensities of DCM-NH2 in 293T cells are much lower than those in HeLa cells ( ~5-fold, P< 0.05).

These results provide further evidence for

the conclusion that DCM-S-PPT can be quickly enter broken down into DCM-NH2 and PPT by high concentration of GSH in cancer cells, and that the fluorescence of DCM-NH2 can be applied for real-time monitoring of the drug release process in cancer cells.

Figure 4. Flow cytometric analyses of intracellular fluorescence intensity from DCM-S-PPT in HeLa (A) and 293T (B) cells after 3, 6 h.

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In order to offer direct evidence of thiol-induced disulfide bond cleavage, we measured the fluorescence of DCM-S-PPT in HeLa cells in the presence of GSH and N-ethylmalemide (NEM), a strong thiol-reacting agent (Figure S11). We again observed significant fluorescence intensity of DCM-S-PPT in HeLa cells upon treatment with 2.5 mM GSH. However, treatment with NEM (2.5 mM) reversed the GSH-induced

fluorescence

enhancement,

confirming

that

the

fluorescence

enhancement of DCM-S-PPT is specifically triggered by intracellular thiols. Cytotoxicity Evaluation. We next carried out cell viability experiments to investigate the cytotoxicity profile of DCM-S-PPT. The cytotoxicity of free PPT, DCM-S-PPT, and

its

nanomicelles

mPEG-DSPE/DCM-S-PPT

against

both

cancer

and

noncancerous cell lines was evaluated using the Cell Counting Kit 8 (Figure 5). Similar as the parent compound PPT, DCM-S-PPT showed significant antitumor activity against HeLa cells. The cells exhibited significant growth inhibition, and cell viability decreased along with the increased concentration of DCM-S-PPT. It is obvious that DCM-S-PPT could release PPT sufficiently by the high GSH concentration in cancer cells. Moreover, mPEG-DSPE/DCM-S-PPT cytotoxicity was the same as that of DCM-S-PPT against HeLa, indicating that encapsulation of DCM-S-PPT into mPEG-DSPE micelles did not affect the cleavage of the disulfilde linkage. Furthermore, at the same drug concentration, the parent compound PPT exhibited significant cytotoxicity toward the human normal cells 293T, while DCM-S-PPT, and mPEG-DSPE/DCM-S-PPT showed much lower cytotoxicity (P

GSH-Activated NIR Fluorescent Prodrug for Podophyllotoxin

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