Article www.acsabm.org
Cite This: ACS Appl. Bio Mater. 2019, 2, 2567−2572
Photoactivatable BODIPY Platform: Light-Triggered Anticancer Drug Release and Fluorescence Monitoring Yul Jang,†,⊥ Tae-Il Kim,†,⊥ Hyunjin Kim,‡ Yongdoo Choi,*,‡ and Youngmi Kim*,† †
Department of Chemistry and Research Institute of Basic Sciences, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea ‡ National Cancer Center, 323 Ilsan-ro, Goyang-si, Gyeonggi-do 10408, Korea
Downloaded via BUFFALO STATE on July 20, 2019 at 12:49:07 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: We report a photoactivatable fluorophore that relies on the conversion of a dark meso-ester-BODIPY to an emissive meso-carboxylateBODIPY. The process is triggered by the photolysis of an aryl azide to an amine, which occurs in a high photochemical yield, and does not release toxic nitroso photoproducts. Its utility is demonstrated in platforms that simultaneously release upon irradiation both a bioactive molecule and an emissive dye, resulting in an approximate 1250-fold luminescence increase. KEYWORDS: caged dye, drug delivery, fluorescence monitoring, photoactivatable dye, photocontrolled release
■
that provide steric protection to the meso-carboxyl group.46 In the case of the p-azidobenzyl ester 1a, phototriggered reduction to the aniline initiates a 1,6-elimination that releases the highly emissive carboxylate salt 2 (Scheme 1).
INTRODUCTION Photoactivatable (“caged”) fluorescent dyes, which convert from a nonemissive to an emissive state upon light irradiation,1−5 serve as powerful tools in biological applications including sensors,6,7 super-resolution imaging microscopy,8,9 and caged biomolecules.10,11 Selective activation of these photoresponsive molecules allows for the microscopic study of biological systems in a temporally and spatially defined manner. Caged dye construction features an organic fluorophore (i.e., fluorescein,12−15 rhodamine,16−21 BODIPY,22−27 cyanine,28,29 and coumarin5,30 derivatives) that is caged with a photolabile group, among which o-nitrobenzyl derivatives are the most commonly used.31−35 Cleavage is accompanied by the modulation of photoinduced electron transfer (PeT),14,22−25 fluorescence resonance energy transfer (FRET),36 or excited state intramolecular proton transfer (ESIPT)37,38 processes to provide a more emissive photoproduct. The phototriggered reduction of electron-deficient aryl azides to aniline fluorophores39−42 and the ring opening of spirocyclic diazoketonecaged rhodamine detivatives43,44 have also been investigated as alternatives to the photocleavage of o-nitrobenzyl derivatives that do not result in the formation of potentially toxic nitroso photoproducts. In this work, we report an application of the aryl azide phototriggered reduction to BODIPY-based platforms that simultaneously release a bioactive molecule and an emissive dye upon photoactivation. Our design for fluorogenic activation was inspired by the remarkable enhancement in fluorescence emission that accompanies the conversion of a meso-ester group to the corresponding meso-carboxylate of 1,3,5,7tetramethyl-BODIPY dyes.45,46 As previously reported, the esters of tetramethyl BODIPY meso-carboxylates 1 are not luminescent and are also particularly resistant to background or enzymatic hydrolysis because of the flanking methyl substituents © 2019 American Chemical Society
■
EXPERIMENTAL SECTION
Materials. All reagents were of the highest commercial quality and used as received without further purification. All solvents were spectral grade unless otherwise noted. Anhydrous CH2Cl2, THF, CH3CN, and DMF were obtained as sure-seal bottles from Alfa Aesar. 4-
Scheme 1. Scheme Illustrating the Light-Induced Conversion of 1a to 2 and Chemical Structures of the meso-EsterBODIPY Derivatives 1a−c
Received: March 27, 2019 Accepted: May 9, 2019 Published: May 9, 2019 2567
DOI: 10.1021/acsabm.9b00259 ACS Appl. Bio Mater. 2019, 2, 2567−2572
Article
ACS Applied Bio Materials
134.2, 130.0, 129.6, 129.5, 128.8, 128.0, 125.5, 121.4, 65.0, 29.7, 14.8, 12.8. HR-MS (ESI): calcd for C21H20N3O4F2BNa [M + Na]+, 450.1412; found, 450.1413. Synthesis of Compound 1a-Chl. To a stirred solution of 2 (50 mg, 0.17 mmol) and K2CO3 (35 mg, 0.26 mmol) in dry DMF (2 mL) at room temperature under a nitrogen atmosphere was added a solution of 6 (140 mg, 0.27 mmol) in dry DMF (1 mL). After stirring at room temperature for 3 h, the resulting mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The collected organic layer was dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel using progressively more polar, 100:1 to 5:1, hexanes/ethyl acetate as the mobile phase to afford 1a-Chl as an orange solid (72 mg, 57%). 1H NMR (CDCl3, 400 MHz): δ = 7.44 (s, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.04 (d, J = 8.0 Hz, 2H), 6.60 (d, J = 7.6 Hz, 2H), 6.03 (s, 2H), 5.35 (s, 2H), 5.07 (s, 2H), 3.68 (d, J = 6.0 Hz, 4H), 3.61 (d, J = 6.0 Hz, 4H), 2.52 (s, 8H), 2.37 (m, 2H), 2.00 (s, 6H), 1.92 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ = 173.1, 165.0, 157.8, 144.4, 141.0, 139.4, 131.2, 130.6, 130.4, 130.2, 129.7, 128.8, 127.8, 121.3, 118.5, 112.2, 67.7, 61.3, 53.6, 40.5, 33.9, 33.5, 26.7, 14.8, 12.8. HR-MS (ESI): calcd for C36H39N6O4F2BCl2Na [M + Na]+, 761.2369; found, 761.2364. HPLC Analysis of the Progress of Phototriggered Reactions. The photoreaction was performed by irradiating a solution of the compound (10 μM, 2 mL) in a quartz cuvette with a hand-held UV lamp (λirr = 365 nm, 5.7 mW/cm2) under aerobic conditions at 25 °C. After irradiation for the indicated time periods, a 20 μL aliquot of the reaction mixture was injected for analysis. Cell Culture. The HeLa cell line (human cervix) was obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% antibiotic− antimycotic in a humidified incubator containing 5% CO2 at 37 °C. In Vitro Phototoxicity Studies. HeLa cells were seeded at a density of 1 × 104 cells/well in a 96-well plate (ThermoFisher Scientific, MA, USA) and incubated for 24 h for cell attachment. The cells were irradiated with a UV lamp (365 nm, 3.2 mW/cm2) for 0, 10, and 20 min to evaluate light toxicity to the cells. Then, the cells were further incubated for 24 h, and the cell viability was measured using a CCK-8 assay kit (n = 8, Dojindo Laboratories, Mashikimachi, Kumamoto, Japan). In Vitro Cytotoxicity Studies. HeLa cells were seeded at a density of 1 × 104 cells/well in a 96-well plate and incubated for 24 h for cell attachment. Chlorambucil and 1a-Chl were dissolved, respectively, and diluted with cell culture medium at various concentrations. The cell culture medium was exchanged with fresh medium containing chlorambucil and 1a-Chl, and then the samples were incubated for 3 h. After the cells were washed 3 times with the cell culture medium, the cells were further incubated for 24 h. For the evaluation of light-induced anticancer drug release and subsequent therapeutic efficacy, HeLa cells incubated with 1a-Chl for 3 h were washed 3 times, irradiated with a UV lamp (365 nm, 3.2 mW/cm2) for 10 min, and then further incubated for 24 h. The cell viability was analyzed using a CCK-8 assay kit (n = 4 per group). Untreated control cells were used as a reference for 100% viable cells, while the cell culture medium served as the background. Fluorescence Microscopy Imaging. HeLa cells were seeded at a density of 1 × 105 cells/well in a 4-well Lab Tek-II chambered cover glass (ThermoFisher Scientific, MA, USA) and incubated for 24 h for cell attachment. HeLa cells were treated with 1a-Chl for 3 h at 10 μM concentration and then washed 3 times with cell culture medium. The cells were then irradiated for 10 min with a UV lamp (365 nm, 3.2 mW/ cm2), and the fluorescence images (λex = 450−490 nm, λem = 515−565 nm) of the cells were obtained using fluorescence microscopy (Axio Observer Z1, Germany). For comparison, the cells in another group were incubated with 1a-Chl for 3 h at 10 μM, and then fluorescence images of the cells were obtained without light irradiation. In addition, fluorescence images of the cells in the control group were obtained without treatment of both 1a-Chl and light. Statistical Analysis. The Student’s t-test was used to determine the significant difference between test groups. Data are expressed as mean ± standard deviation.
Dimethylaminopyridine (DMAP) and glutathione (GSH) were obtained from Alfa Aesar. 2,4-Dimethylpyrrole was obtained from Acros Organics (Geel, Belgium). 2-Nitrobenzyl alcohol was obtained from TCI (Tokyo, Japan). Chlorambucil, bovine serum albumin (BSA), homocysteine, esterase from porcine liver (PLE, EC 3.1.1.1, 17 U/mg), trypsin from porcine pancreas (EC 3.4.21.4, 1000−2000 U/ mg), and lipase from porcine pancreas (PPL, EC3.1.1.3, 100−500 U/ mg) were obtained from Aldrich (Saint Louis, MO). Hydrogen peroxide (H2O2) and sodium hypochlorite (NaOCl) were obatained from Samchun Chemicals (Korea). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Welgene (Korea). Flash column chromatography was performed using silica gel (38−75 μm), which was supplied from Qingdao Meigao Chem. Co., Ltd. (Chengyang, China). Aqueous solutions were freshly prepared with deionized water from a water purification system (Younglin Corp. Korea). General Methods, Instrumentation, and Measurements. Synthetic manipulations that required an inert atmosphere (where noted) were carried out under nitrogen using standard Schlenk techniques. NMR (1H and 13C) spectra were recorded on a Bruker 400 MHz spectrometer or JEOL 400 MHz spectrometer. The 1H and 13C chemical shifts were reported as δ in units of parts per million (ppm), referenced to the residual solvent resonances. Splitting patterns are denoted as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). High-resolution electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained at the Korean National Center for InterUniversity Research. UV−vis absorption spectra were obtained on a SCINCO S-3100 spectrophotometer. Fluorescence measurements were recorded on a Hitachi F-7000 fluorescence spectrophotometer using quartz cuvettes with a path length of 1 cm. Fluorescence quantum yields were determined by standard methods, using fluorescein (ΦFL = 0.95 in 0.1 N NaOH) as a standard. Particle size was measured by dynamic light scattering (DLS) using a Malvern particle analyzer ZEN1690. The photoactivation studies were performed by continuous irradiation of each solution using a hand-held UV lamp (VL-4.LC, Vilber, France; λirr = 365 nm, 5.7 mW/cm2) and photoreactor with blue lamps (LZC-420, Luzchem, Canada, λirr = 420 nm, 6.5 mW/cm2) as irradiation sources. Synthesis of Compound 1a. To a solution of compound 2 (25.7 mg, 0.09 mmol)46 and 1,8-diazabicyclo[5.4.0]undec-7-ene (26.8 mg, 0.18 mmol) in dry THF (2.5 mL) at room temperature under a nitrogen atmosphere was added p-azidobenzyl bromide (33.6 mg, 0.16 mmol) in dry THF (0.5 mL). After the mixture was stirred at room temperature for 12 h, the reaction solvent was removed under reduced pressure. The crude mixture was diluted with CH2Cl2 (100 mL) and washed twice with a saturated NH4Cl solution (100 mL × 2). The collected organic layers were dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel using progressively more polar 100:1 to 20:1 hexanes/ethyl acetate as the mobile phase to afford compound 1a as a red solid (6 mg, 16%). 1H NMR (CDCl3, 400 MHz): δ = 7.43 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 6.04 (s, 2H), 5.35 (s, 2H), 2.52 (s, 6H), 2.01 (s, 6H). 13C NMR (CDCl3, 100 MHz): δ = 165.2, 157.8, 141.0, 131.0, 130.6, 130.3, 121.2, 119.3, 67.9, 29.7, 14.8, 12.7. HR-MS (ESI): calcd for C21H20N5O2F2BNa [M + Na]+, 446.1573; found, 446.1576. Synthesis of Compound 1b. To a solution of 2 (10 mg, 0.034 mmol) and K2CO3 (7 mg, 0.051 mmol) in dry DMF (1 mL) at room temperature under a nitrogen atmosphere was added 2-nitrobenzyl bromide (22 mg, 0.103 mmol). After stirring at room temperature for 2 h, the resulting mixture was diluted with CH2Cl2 (50 mL) and washed with water (50 mL). The organic layer was dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel using progressively more polar, 50:1 to 8:1, hexanes/ethyl acetate as the mobile phase to afford compound 1b as an orange solid (9.8 mg, 67%). 1 H NMR (CDCl3, 400 MHz): δ = 8.20 (d, J = 8.0 Hz, 1H), 7.71 (m, 2H), 7.56 (m, 1H), 6.07 (s, 2H), 5.81 (s, 2H), 2.54 (s, 6H), 2.09 (s, 6H). 13C NMR (CDCl3, 100 MHz): δ = 164.6, 158.0, 147.4, 141.1, 2568
DOI: 10.1021/acsabm.9b00259 ACS Appl. Bio Mater. 2019, 2, 2567−2572
Article
ACS Applied Bio Materials
■
RESULTS AND DISCUSSION Synthesis and Photophysical Properties of Compounds. BODIPY esters 1a−c and the carboxylate 2 were prepared according to minor modifications of literature procedures (Scheme S1).46 In ethanol, a good solvent for each of these derivatives, esters 1a−1c all showed similar spectroscopic properties with absorption (λabs) and emission (λem) maxima at ca. 511 nm and ca. 530 nm, respectively, and very low emission quantum yields (1a−1c, ΦF ≤ 0.01; Table S1). By contrast, the absorption and emission maxima of carboxylate 2 are blue-shifted (λabs= 493 nm, λem= 508 nm), relative to 1a−1c, and its quantum yield (ΦF = 0.7) is approximately 70-fold greater. Photoconversion Properties of Compounds 1a−1c in EtOH. Irradiation of dilute ethanol solutions of 1a (10 μM, 25 °C) with a low power UV lamp (λirr = 365 nm, 5.7 mW/cm2) resulted in the instant growth of blue-shifted absorption and emission bands at λabs 493 nm and λem 508 nm (Figure 1),
the expected byproduct of the 1,6-elimination that led to the formation of 2. HPLC and ESI-MS analyses further confirmed that the release of 2 is responsible for the fluorescence emission increase at 508 nm during irradiation of 1a in aq EtOH and that it occurs alongside the release of the p-aminobenzyl alcohol and p-(ethoxymethyl)aniline as the major solvolysis byproducts (Figures S23 and S25). These results support the proposed mechanism for the photoconversion of 1a into 2 (Scheme 1). Carboxylate 2 is released in a high photochemical yield (88% by HPLC; Figure 2b and Figure S23) upon irradiation for 2 h in EtOH. The quantum yield (ΦP) of the 1a → 2 photoconversion process was determined to be 0.54%, which is similar to those of other azide-to-amine photoprocesses,39 and corresponds to a photoconversion efficiency (ε × Φp) of 15 M−1 cm−1. No decomposition of 2 was observed upon UV irradiation (Figures S21 and S22), indicating its photostability under the conditions relevant to photorelease applications. The photoconversion can also be triggered, albeit more slowly, with visible light (λirr = 420 nm, 6.5 mW/cm2; Figure S12). The phototriggered release of 2 can also be accomplished in biologically relevant aqueous buffer solutions (10 mM phosphate buffer, pH 7.4, 1% DMSO; Figure S13) instead of EtOH. Importantly, the formation of 2 was accompanied by a ca. 1430-fold (F/F0) increase in fluorescence intensity within 1 h of irradiation (Figure 1b inset). By comparison, photocleavage of the o-nitrobenzyl derivative 1b under identical conditions was considerably slower (Figure 1b, inset; Figures S14 and S16) and only lead to a ca. 40-fold (F/F0 at 503 nm) fluorescence enhancement. Meanwhile, no spectral changes were discerned upon irradiation of the control benzyl ester 1c (Figure 1b, inset; Figures S15 and S16) or when 1a was incubated for the same period of time in the dark (Figure S8). Design of Dye−Drug Conjugate 1a-Chl and LightTriggered Anticancer Drug Release in Aqueous Solution. The phototriggered release of 2 from 1 through photoreduction and 1,6-elimination was combined with a 1,4-elimination to enable the simultaneous release of both fluorophore 2 and a biologically active payload. In doing so, the drug release can be monitored in real time using fluorescence microscopy. In the proof-of-concept photorelease platform 1a-Chl (Schemes S2 and S3), the photoactivatable fluorophore 1a is linked via an ester linkage to the anticancer drug chlorambucil (Figure 3a). The simultaneous phototriggered release from 1a-Chl was carried out in aqueous phosphate buffer (10 mM, pH 7.4, 1% DMSO). In this solvent, the hydrophobic dye−drug conjugate 1a-Chl is virtually nonemissive (ΦF = 0.002) and is present as suspended colloidal particles with an average diameter of 121 ± 15 nm according to DLS analysis (Figure S7). Upon irradiation with a low power UV lamp (λirr = 365 nm, 5.7 mW/cm2), the release of 2 in solution becomes apparent with the growth of its characteristic absorption and emission peaks at 495 and 510 nm, respectively (Figure 3b and Figure S17). Photolysis of 1a-Chl for 1 h resulted in an ∼1250-fold fluorescence enhancement (F/ F0) at 510 nm. The release of fluorescent 2 under UV irradiation is readily visible, as shown in Figure 3b, inset. In the dark, the background release of 2 from the dye−drug conjugate 1a-Chl was not detected upon incubation for 1 h in aqueous buffered solutions (pH 7.4 and pH 9), in cell culture media (DMEM with 10% FBS, pH 7.3), in the presence of hydrolytic enzymes (esterase, trypsin, lipase, pH 7.4) or else in the presence of reactive oxygen species (HOCl/OCl−, H2O2, •OH) (Figure 3c and Figure S20). Thiols such as NaSH have been reported to induce the reduction of aryl azides to anilines.47 However, the release of fluorophore 2 from the dye−drug
Figure 1. Absorption (a) and fluorescence emission (b) spectra of 1a (10 μM) in EtOH upon irradiation with a UV lamp (λirr = 365 nm, 5.7 mW/cm2) at 25 °C. Irradiation times: 0, 1, 3, 5, 10, 20, 30, 40, 50, 60 min. The absorption and emission (λex = 460 nm) spectra of 1a, before (red lines) and after 1 h irradiation (blue lines), are highlighted. Inset: kinetic curves at λabs 493 nm and λabs 511 nm for 1a (a) and at λem 508 nm for 1a and 1c and λem 503 nm for 1b (b) under light irradiation.
corresponding to those of 2. 1H NMR analyses of 1a (1 mM) in DMSO-d6 showed its conversion to the fluorophore 2 upon irradiation with UV light (λirr = 365 nm; Figure 2a). Moreover, the formation of 2 coincided with that of p-aminobenzyl alcohol,
Figure 2. (a) Partial 1H NMR study of 1a (1 mM) in DMSO-d6 before (1) and after 365 nm photoirradiation for 1 h (2) and 4 h (3) at 25 °C, compared to authentic samples of 2 (4) and p-aminobenzyl alcohol (5). (b) Kinetic curves of the depletion of 1a and the release of 2 under light irradiation (λirr = 365 nm, 5.7 mW/cm2) at 25 °C as a function of irradiation time by HPLC analysis (initial concentration of 1a = 10 μM). 2569
DOI: 10.1021/acsabm.9b00259 ACS Appl. Bio Mater. 2019, 2, 2567−2572
Article
ACS Applied Bio Materials
Figure 4. (a) HPLC monitoring of the progress of the release of fluorophore 2 and chlorambucil from 1a-Chl (10 μM) in phosphate buffer (10 mM, pH 7.4, 1% DMSO) under light irradiation with a handheld UV lamp (λirr = 365 nm, 5.7 mW/cm2) for 2 h at 25 °C. Inset: quantitative monitoring of the release process of fluorophore 2 and chlorambucil and the depletion process of 1a-Chl, respectively, as a function of irradiation time (initial concentration of 1a-Chl = 10 μM). (b) Progress for the photoregulated release of the drug from 1a-Chl (10 μM) under alternating light (15 min) and dark (15 min) conditions by HPLC analysis. Arrows indicate the beginning of light irradiation (hν) and the ending of light irradiation (dark).
Figure 3. (a) Scheme illustrating the light-induced release of 2 and chlorambucil from 1a-Chl. (b) Fluorescence emission spectral changes of 1a-Chl (10 μM) as a function of UV irradiation times (0, 1, 3, 5, 10, 20, 30, 40, 50, 60 min). The sample was irradiated with a hand-held UV lamp (λirr = 365 nm, 5.7 mW/cm2) at 25 °C. Emission (λex = 460 nm) spectra before (red line) and after 1 h irradiation (blue line) are highlighted. Inset: photographs of 1a-Chl before (A) and after (B) irradiation for 1 h. (c) Fluorescence intensity at λem = 510 nm of 1a-Chl (10 μM) upon incubation with various biological species in phosphate buffer (10 mM, pH 7.4, 1% DMSO, 37 °C) in the dark for 1 h, in comparison with light irradiation for 10 min (λirr = 365 nm, 5.7 mW/ cm2) at 25 °C. [esterase] = [trypsin] = [lipase] = 10 U/mL; [others] = 1 mM.
indicated that 1a-Chl is resistant to background hydrolysis in the dark, as neither fluorophore 2 nor the drug was released during incubation in an aqueous buffer for a period of 24 h (Figure S11). In Vitro Anticancer Cytotoxicity by the LightTriggered Dye−Drug Conjugate 1a-Chl. Light-triggered uncaging experiments of dye−drug conjugate 1a-Chl were carried out in living cells. The cytotoxicity of 1a-Chl was first quantified by a CCK-8 assay using HeLa cells with and without light irradiation (Figure 5b). An IC50 value of 131 μM was found for 1a-Chl-loaded cells in the dark and of 18 μM upon illumination with a UV lamp (365 nm, 3.2 mW/cm2, 10 min). Both values are greater than the cytotoxicity observed in the cells treated with chlorambucil alone (IC50 = 430 μM, Figure 5a), suggesting that endocytosis of 1a-Chl is more efficient than that of the free drug in these cancer cells. Indeed, illumination of cells loaded with 1a-Chl for 10 min led to a significantly decreased survival. These results indicate that the dye−drug conjugate may act as a delivery vehicle for chlorambucil with an enhanced cell permeability, combined with a light-triggered release of deadly intracellular concentrations of the free drug. No significant cell death was observed when the cells were simply irradiated without dye−drug loading for the same period of time (Figure S27), indicating the absence of light toxicity. Fluorescence Imaging by the Light-Triggered Release of Emissive Dye from 1a-Chl in Living Cells. The phototriggered release of chlorambucil in living cells from 1aChl could be tracked by fluorescence microscope imaging of the coincident release of fluorophore 2. As established in the cytotoxicity experiment, 1a-Chl effectively penetrates into HeLa cells, but the cells incubated with 1a-Chl (10 μM) in the dark for 3 h only exhibit basal levels of intracellular fluorescence (Figure 5c, middle). In contrast, the 1a-Chl-loaded cells showed bright fluorescence within 10 min of UV irradiation (λirr = 365 nm, 3.2 mW/cm2), as seen in Figure 5c, indicating that 1a-Chl was uncaged to release fluorophore 2 within the cells. Additionally, cell viability assays showed that fluorophore 2 had no cytotoxic effect on the cells up to 150 μM after 24 h (Figure S28).
conjugate 1a-Chl was not observed in the dark upon incubation with various reducing biothiols (NaSH, GSH, Cys, HCy; Figure 3c and Figure S20). The lack of reactivity of 1a-Chl toward these thiols may be a consequence of their presence as suspended colloidal aggregates in aqueous buffer, which would suppress the reactivity of the aryl azide group. Overall, 1a-Chl was found to be chemically stable under assay conditions and resistant to potential chemical interferences, ensuring that the release of fluorophore 2 is only achieved upon the photochemical activation of the aryl azide group. The progress of the photoreaction of the caged drug conjugate was further monitored by HPLC analysis (Figure 4a). Under UV irradiation, the dye−drug conjugate (10 μM) was steadily decomposed with a concomitant release of both fluorophore 2 and the drug payload (chlorambucil). The released concentrations of 2 and the drug increased in parallel as a function of irradiation time (Figure 4a, inset). For instance, photolysis of 1aChl (10 μM) for 2 h released 5.17 μM 2 and 4.93 μM chlorambucil, the latter concentration being sufficient to elicit a cellular response. The identity of the released drug was confirmed by an independent ESI-MS experiment, establishing the simultaneous release of both 2 (m/z 293.1 [M + H]+) and chlorambucil (m/z 304.1 [M + H]+) from the corresponding dye−drug conjugate upon irradiation (Figure S26). The quantum yield (Φp) of the photoconversion for drugconjugate 1a-Chl in aqueous buffer was determined to be 0.1%. This value is comparable to the efficiencies recorded for dye− drug photorelease platforms based on the photolysis of B−O bonds in BODIPY-phenols.23 Photoregulated drug release was also demonstrated by monitoring the drug release from 1a-Chl under alternating exposure to light and dark conditions. The drug release only proceeded under UV light and ceased when the irradiation was interrupted, allowing for precise control over the drug release (Figure 4b). Importantly, HPLC analysis 2570
DOI: 10.1021/acsabm.9b00259 ACS Appl. Bio Mater. 2019, 2, 2567−2572
Article
ACS Applied Bio Materials
■
Synthetic details and characterization of compounds, additional spectroscopic data, and photoactivation studies (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Youngmi Kim: 0000-0003-2189-8342 Author Contributions ⊥
Y.J. and T.-I.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIP) (NRF-2017M3D9A1073769, NRF2018R1D1A1B07044894, and NRF-2015R1A5A1008958).
■
Figure 5. (a) Cell viability of HeLa cells treated with chlorambucil alone. (b) Cell viability of HeLa cells treated with 1a-Chl without (−) and with (+) light irradiation. HeLa cells were treated with chlorambucil (a) or 1a-Chl (b) for 3 h at various concentrations, and after washing, the cells were further incubated for 24 h. For the lighttriggered drug release, 1a-Chl-treated HeLa cells received light irradiation for 10 min with a hand-held UV lamp (λirr = 365 nm, 3.2 mW/cm2). *P < 0.05. (c) Fluorescence images of 1a-Chl-loaded HeLa cells without (−) and with (+) light irradiation. Left: unstained control. HeLa cells were incubated with 1a-Chl (10 μM) for 3 h, washed, and imaged before (middle) and after (right) irradiation by a hand-held UV lamp (365 nm, 3.2 mW/cm2, 10 min). Bright-field (top) and fluorescence (bottom) images (λex = 450−490 nm, λem = 515−565 nm). Scale bar: 20 μm.
■
CONCLUSIONS In summary, we report the design of a photoactivatable fluorophore 1a, based on the conversion of a meso-esterBODIPY to a meso-carboxylate-BODIPY with a remarkable fluorescence difference between the caged and uncaged state. The current system can be a useful addition to the chemical tool box of available photoactivatable fluorophores that do not result in the formation of potentially toxic nitroso photoproducts. We validated its utility as a versatile photoactivatable platform for bioactive molecules, by attaching an anticancer drug chlorambucil (i.e., 1a-Chl). Light activation of 1a-Chl in aqueous media enabled the simultaneous release of both a drug and an emissive dye with a controlled release profile, allowing for the real-time monitoring of a light-triggered release of the drug in living cells.
■
REFERENCES
(1) Pelliccioli, A. P.; Wirz, J. Photoremovable protecting groups: reaction mechanisms and applications. Photochem. Photobiol. Sci. 2002, 1, 441−458. (2) Puliti, D.; Warther, D.; Orange, C.; Specht, A.; Goeldner, M. Small photoactivatable molecules for controlled fluorescence activation in living cells. Bioorg. Med. Chem. 2011, 19, 1023−1029. (3) Raymo, F. M. Photoactivatable synthetic dyes for fluorescence imaging at the nanoscale. J. Phys. Chem. Lett. 2012, 3, 2379−2385. (4) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A. Light-controlled tools. Angew. Chem., Int. Ed. 2012, 51, 8446−8476. (5) Li, W.-H.; Zheng, G. Photoactivatable fluorophores and techniques for biological imaging applications. Photochem. Photobiol. Sci. 2012, 11, 460−471. (6) Grenier, V.; Walker, A. S.; Miller, E. W. A small-molecule photoactivatable optical sensor of transmembrane potential. J. Am. Chem. Soc. 2015, 137, 10894−10897. (7) Goldberg, J. M.; Wang, F.; Sessler, C. D.; Vogler, N. W.; Zhang, D. Y.; Loucks, W. H.; Tzounopoulos, T.; Lippard, S. J. Photoactivatable sensors for detecting mobile zinc. J. Am. Chem. Soc. 2018, 140, 2020− 2023. (8) Chozinski, T. J.; Gagnon, L. A.; Vaughan, J. C. Twinkle, twinkle little star: Photoswitchable fluorophores for super resolution imaging. FEBS Lett. 2014, 588, 3603−3612. (9) Li, H.; Vaughan, J. C. Switchable fluorophores for single-molecule localization microscopy. Chem. Rev. 2018, 118, 9412−9454. (10) Maurel, D.; Banala, S.; Laroche, T.; Johnsson, K. Photoactivatable and photoconvertible fluorescent probes for protein labeling. ACS Chem. Biol. 2010, 5, 507−516. (11) Hauke, S.; von Appen, A.; Quidwai, T.; Ries, J.; Wombacher, R. Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells. Chem. Sci. 2017, 8, 559−566. (12) Krafft, G. A.; Sutton, W. R.; Cummings, R. T. Photoactivable fluorophores. 3. Synthesis and photoactivation of fluorogenic difunctionalized fluoresceins. J. Am. Chem. Soc. 1988, 110, 301−303. (13) Tang, X.; Dmochowski, I. J. Phototriggering of caged fluorescent oligodeoxynucleotides. Org. Lett. 2005, 7, 279−282. (14) Kobayashi, T.; Urano, Y.; Kamiya, M.; Ueno, T.; Kojima, H.; Nagano, T. Highly activatable and rapidly releasable caged fluorescein derivatives. J. Am. Chem. Soc. 2007, 129, 6696−6697. (15) Furukawa, K.; Abe, H.; Tsuneda, S.; Ito, Y. Photoactivatable fluorescein derivatives with azidomethyl caging groups for tracing
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00259. 2571
DOI: 10.1021/acsabm.9b00259 ACS Appl. Bio Mater. 2019, 2, 2567−2572
Article
ACS Applied Bio Materials oligonucleotides in living human cells. Org. Biomol. Chem. 2010, 8, 2309−2311. (16) Gee, K. R.; Weinberg, E. S.; Kozlowski, D. J. Caged Q-rhodamine dextran: a new photoactivated fluorescent tracer. Bioorg. Med. Chem. Lett. 2001, 11, 2181−2183. (17) Fölling, J.; Belov, V.; Kunetsky, R.; Medda, R.; Schönle, A.; Egner, A.; Eggeling, C.; Bossi, M.; Hell, S. W. Photochromic rhodamines provide nanoscopy with optical sectioning. Angew. Chem., Int. Ed. 2007, 46, 6266−6270. (18) Banala, S.; Maurel, D.; Manley, S.; Johnsson, K. A caged, localizable rhodamine derivative for superresolution microscopy. ACS Chem. Biol. 2012, 7, 289−293. (19) Lee, M. K.; Rai, P.; Williams, J.; Twieg, R. J.; Moerner, W. E. Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy. J. Am. Chem. Soc. 2014, 136, 14003− 14006. (20) Grimm, J. B.; Klein, T.; Kopek, B. G.; Shtengel, G.; Hess, H. F.; Sauer, M.; Lavis, L. D. Synthesis of a far red photoactivatable silicon containing rhodamine for super resolution microscopy. Angew. Chem., Int. Ed. 2016, 55, 1723−1727. (21) He, H.; Ye, Z.; Zheng, Y.; Xu, X.; Guo, C.; Xiao, Y.; Yang, W.; Qian, X.; Yang, Y. Super-resolution imaging of lysosomes with a nitroso-caged rhodamine. Chem. Commun. 2018, 54, 2842−2845. (22) Kobayashi, T.; Komatsu, T.; Kamiya, M.; Campos, C.; GonzalezGaitan, M.; Terai, T.; Hanaoka, K.; Nagano, T.; Urano, Y. Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins. J. Am. Chem. Soc. 2012, 134, 11153−11160. (23) Umeda, N.; Takahashi, H.; Kamiya, M.; Ueno, T.; Komatsu, T.; Terai, T.; Hanaoka, K.; Nagano, T.; Urano, Y. Boron dipyrromethene as a fluorescent caging group for single-photon uncaging with longwavelength visible light. ACS Chem. Biol. 2014, 9, 2242−2246. (24) Goswami, P. P.; Syed, A.; Beck, C. L.; Albright, T. R.; Mahoney, K. M.; Unash, R.; Smith, E. A.; Winter, A. H. BODIPY-derived photoremovable protecting groups unmasked with green light. J. Am. Chem. Soc. 2015, 137, 3783−3786. (25) Peterson, J. A.; Wijesooriya, C.; Gehrmann, E. J.; Mahoney, K. M.; Goswami, P. P.; Albright, T. R.; Syed, A.; Dutton, A. S.; Smith, E. A.; Winter, A. H. Family of BODIPY photocages cleaved by single photons of visible/near-infrared light. J. Am. Chem. Soc. 2018, 140, 7343−7346. (26) Wijesooriya, C. S.; Peterson, J. A.; Shrestha, P.; Gehrmann, E. J.; Winter, A. H.; Smith, E. A. A photoactivatable BODIPY probe for localization-based super-resolution cellular imaging. Angew. Chem., Int. Ed. 2018, 57, 12685−12689. (27) Kand, D.; Pizarro, L.; Angel, I.; Avni, A.; Friedmann-Morvinski, D.; Weinstain, R. Organelle-targeted BODIPY photocages: visiblelight-mediated subcellular photorelease. Angew. Chem., Int. Ed. 2019, 58, 4659−4663. (28) Gorka, A. P.; Nani, R. R.; Zhu, J.; Mackem, S.; Schnermann, M. J. A near-IR uncaging strategy based on cyanine photochemistry. J. Am. Chem. Soc. 2014, 136, 14153−14159. (29) Klötzner, D. P.; Klehs, K.; Heilemann, M.; Heckel, A. A new photoactivatable near-infrared-emitting QCy7 fluorophore for singlemolecule super-resolution microscopy. Chem. Commun. 2017, 53, 9874−9877. (30) Zhao, Y. R.; Zheng, Q.; Dakin, K.; Xu, K.; Martinez, L. M.; Li, W. H. New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications. J. Am. Chem. Soc. 2004, 126, 4653−4663. (31) Hagen, V.; Dekowski, B.; Kotzur, N.; Lechler, R.; Wiesner, B.; Briand, B.; Beyermann, M. {7 [Bis(carboxymethyl)amino]-coumarin 4 yl}methoxycarbonyl derivatives for photorelease of carboxylic acids, alcohols/phenols, thioalcohols/thiophenols, and amines. Chem. - Eur. J. 2008, 14, 1621−1627. (32) Lin, Q.; Huang, Q.; Li, C.; Bao, C.; Liu, Z.; Li, F.; Zhu, L. Anticancer drug release from a mesoporous silica based nanophotocage regulated by either a one- or two-photon process. J. Am. Chem. Soc. 2010, 132, 10645−10647.
(33) Patchornik, A.; Amit, B.; Woodward, R. B. Photosensitive protecting groups. J. Am. Chem. Soc. 1970, 92, 6333−6335. (34) Bochet, C. G. Photolabile protecting groups and linkers. J. Chem. Soc. Perkin trans. 2002, 1, 125−142. (35) Klán, P.; Śolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy. Chem. Rev. 2013, 113, 119−191. (36) Zheng, G.; Guo, Y. M.; Li, W. H. Photoactivatable and water soluble FRET dyes with high uncaging cross section. J. Am. Chem. Soc. 2007, 129, 10616−10617. (37) Zhou, L. Y.; Zhang, X. B.; Lv, Y. F.; Yang, C.; Lu, D. Q.; Wu, Y.; Chen, Z.; Liu, Q. L.; Tan, W. H. Localizable and photoactivatable fluorophore for spatiotemporal two-photon bioimaging. Anal. Chem. 2015, 87, 5626−5631. (38) Barman, S.; Mukhopadhyay, S. K.; Biswas, S.; Nandi, S.; Gangopadhyay, M.; Dey, S.; Anoop, A.; Singh, N. D. P. A phydroxyphenacyl-benzothiazole-chlorambucil conjugate as a real-timemonitoring drug-delivery system assisted by excited-state intramolecular proton transfer. Angew. Chem., Int. Ed. 2016, 55, 4194−4198. (39) Lord, S. J.; Conley, N. R.; Lee, H.-I. D.; Samuel, R.; Liu, N.; Twieg, R. J.; Moerner, W. E. A photoactivatable push−pull fluorophore for single-molecule imaging in live cells. J. Am. Chem. Soc. 2008, 130, 9204−9205. (40) Lord, S. J.; Conley, N. R.; Lee, H.-I. D.; Nishimura, S. Y.; Pomerantz, A. K.; Willets, K. A.; Lu, Z.; Wang, H.; Liu, N.; Samuel, R.; Weber, R.; Semyonov, A.; He, M.; Twieg, R. J.; Moerner, W. E. DCDHF fluorophores for single molecule imaging in cells. ChemPhysChem 2009, 10, 55−65. (41) Lord, S. J.; Lee, H. D.; Samuel, R.; Weber, R.; Liu, N.; Conley, N. R.; Thompson, M. A.; Twieg, R. J.; Moerner, W. E. Azido push−pull fluorogens photoactivate to produce bright fluorescent labels. J. Phys. Chem. B 2010, 114, 14157−14167. (42) Lee, H. D.; Lord, S. J.; Iwanaga, S.; Zhan, K.; Xie, H.; Williams, J. C.; Wang, H.; Bowman, G. R.; Goley, E. D.; Shapiro, L.; Twieg, R. J.; Rao, J.; Moerner, W. E. Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores. J. Am. Chem. Soc. 2010, 132, 15099−15101. (43) Belov, V. N.; Wurm, C. A.; Boyarskiy, V. P.; Jakobs, S.; Hell, S. W. Rhodamines NN: a novel class of caged fluorescent dyes. Angew. Chem., Int. Ed. 2010, 49, 3520−3523. (44) Belov, V. N.; Mitronova, G. Y.; Bossi, M. L.; Boyarskiy, V. P.; Hebisch, E.; Geisler, C.; Kolmakov, K.; Wurm, C. A.; Willig, K. I.; Hell, S. W. Masked rhodamine dyes of five principal colors revealed by photolysis of a 2-diazo-1-indanone caging group: synthesis, photophysics, and light microscopy applications. Chem. - Eur. J. 2014, 20, 13162−13173. (45) Kim, S.; Bouffard, J.; Kim, Y. Tailoring the solid-state fluorescence emission of BODIPY dyes by meso substitution. Chem. Eur. J. 2015, 21, 17459−17465. (46) Kim, S.; Kim, H.; Choi, Y.; Kim, Y. A new strategy for fluorogenic esterase probes displaying low levels of non specific hydrolysis. Chem. Eur. J. 2015, 21, 9645−9649. (47) For fluorescent H2S probes based on the reduction of aryl azides to amines, see: Lin, V. S.; Chen, W.; Xian, M.; Chang, C. J. Chemical probes for molecular imaging and detection of hydrogen sulfide and reactive sulfur species in biological systems. Chem. Soc. Rev. 2015, 44, 4596−4618 and references therein. .
2572
DOI: 10.1021/acsabm.9b00259 ACS Appl. Bio Mater. 2019, 2, 2567−2572
