Photoactivatable BODIPY Platform: Light-Triggered Anticancer Drug

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A Photoactivatable BODIPY Platform: Light-Triggered Anticancer Drug Release and Fluorescence Monitoring Yul Jang, Tae-Il Kim, Hyunjin Kim, Yongdoo Choi, and Youngmi Kim ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00259 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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ACS Applied Bio Materials

A 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 ‡Molecular Imaging & Therapy Branch, National Cancer Center, 323 Ilsan-ro, Goyang-si, Gyeonggi-do, 10408, Korea

KEYWORDS: caged dye, drug delivery, fluorescence monitoring, photoactivatable dye, photocontrolled release

ABSTRACT: We report a photoactivatable fluorophore that relies on the conversion of a dark meso-ester-BODIPY to an emissive meso-carboxylate-BODIPY. The process is triggered by the photolysis of an aryl azide to an amine, which occurs in 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 a ca. 1250-fold luminescence increase.

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 photo-responsive 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 coumarin,5,30 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 photo-triggered reduction of electron-deficient aryl azides to aniline fluorophores,39-42 and the ring opening of spirocyclic diazoketone-caged rhodamine detivatives43,44 have also been investigated as alternatives to the photocleavage of onitrobenzyl derivatives that do not result in the formation of potentially toxic nitroso photoproducts. In this paper, we report an application of the aryl azide photo-triggered 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 1,3,5,7-tetramethyl-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 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). Scheme 1. Scheme illustrating the light-induced conversion of 1a to 2, and chemical structures of the meso-esterBODIPY derivatives 1a-c. h N3 O

N

O

O

NH2

O

O

N

N

F

F

O

h N F

B 1a

N

N

F

F

B

B

N F

Non Emissive

NH2 1,6-elimination

O

N F

OR

B

R =

N3

O NO2

N

N

F 1a

1b

1c

F

O

B 2

N F

Highly Emissive

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-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

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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 m), 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 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 matrixassisted 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 F7000 fluorescence spectrophotometer using quartz cuvettes with a path length of 1 cm. Fluorescence quantum yields were determined by standard methods, using fluorescein (FL = 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; irr = 365 nm, 5.7 mW/cm2) and photoreactor with blue-lamps (LZC420, Luzchem, Canada, irr = 420 nm, 6.5 mW/cm2) as irradiation sources. Synthesis of compound 1a. To a solution of compound 246 (25.7 mg, 0.09 mmol) 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 pazidobenzyl bromide (33.6 mg, 0.16 mmol) in dry THF (0.5 mL). After stirring at room temperature for 12 hours, the reaction solvent was removed under reduced pressure. The crude mixture was diulted with CH2Cl2 (100 mL), and washed twice with 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.

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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 hours, 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 propgressively 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%). 1H 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, 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 hours, 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 photo-triggered reactions. Photoreaction was performed by irradiating a solution of compound (10 M, 2 mL) in quartz cuvette with a hand-held UV lamp (irr = 365 nm, 5.7 mW/cm2) under aerobic conditions at 25 C. After irradiation for the indicated time periods, a 20 L aliquot of the reaction mixture was injected for anaylsis. Cell culture. 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 hours 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 hours and cell viability was measured using CCK-8 assay kit (n = 8, Dojindo Laboratories, Mashikimachi, Kumamoto, Japan).


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ACS Applied Bio Materials 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 hours for cell attachment. Chlorambucil and 1a-Chl were dissolved, respectively, and diluted with cell culture medium at various concentrations. Cell culture medium was exchanged with fresh ones containing chlorambucil and 1a-Chl, and then incubated for 3 hours. After washing the cells 3 times with cell culture medium, the cells were further incubated for 24 hours. For the evaluation of light-induced anticancer drug release and subsequent therapeutic efficacy, HeLa cells incubated with 1aChl for 3 hours were washed 3 times, irradiated with a UV lamp (365 nm, 3.2 mW/cm2) for 10 minutes, and then further incubated for 24 hours. 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 hours for cell attachment. HeLa cells were treated with 1a-Chl for 3 hours at 10 M concentration, then washed 3 times with cell culture medium. The cells were then irradiated for 10 minutes with a UV lamp (365 nm, 3.2 mW/cm2) and fluorescence images (ex = 450-490 nm, em = 515-565 nm) of the cells were obtained using fluorescence microscopy (Axio Observer Z1, Germany). For comparison, the cells in the another group were incubated with 1a-Chl for 3 hours at 10 M, 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. Student’s t-test was used to determine significant difference between test groups. Data are expressed as mean  standard deviation.

spectra of 1a, before (red lines) and after 1 h irradiation (blue lines) are highlighted. Inset: kinetic curves at abs 493 nm and abs 511 nm for 1a (a) and at em 508 nm for 1a and 1c, em 503 nm for 1b (b) under light irradiation.

Photoconversion properties of compounds 1a-1c in EtOH. Irradiation of dilute ethanol solutions of 1a (10 M, 25 C) with a low power UV lamp (irr = 365 nm, 5.7 mW/cm2) resulted in the instant growth of blue-shifted absorption and emission bands at abs 493 nm and em 508 nm (Figure 1), 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 (irr = 365 nm; Figure 2a). Moreover, the formation of 2 coincided with that of p-aminobenzyl alcohol, the expected by-product 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 by-products (Figures S23 and S25). These results support the proposed mechanism for the photoconversion of 1a into 2 (Scheme 1).

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, the esters 1a-1c all showed similar spectroscopic properties, with absorption (abs) and emission (em) maxima at ca. 511 nm and ca. 530 nm, respectively, and very low emission quantum yields (1a-1c: F  0.01; Table S1). By contrast, the absorption and emission maxima of carboxylate 2 are blue-shifted (abs = 493 nm, em = 508 nm), relative to 1a1c, and its quantum yield (F 0.7) is approximately 70-fold greater.

Figure 1. Absorption (a) and fluorescence emission (b) spectra of 1a (10 M) in EtOH upon irradiation with UV lamp (irr = 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 (ex = 460 nm)

Figure 2. (a) Partial 1H-NMR study of 1a (1 mM) in DMSO-d6 before (1) and after 365 nm photo-irradiation for 1 h (2) and 4 h (3) at 25 ° C, compared to authentic samples of 2 (4) and paminobenzyl alcohol (5). (b) Kinetic curves of the depletion of 1a and the release of 2 under light irradiation (irr = 365 nm, 5.7 mW/cm2) at 25 °C as a function of irradiation time by HPLC analysis (Initial concentration of 1a: 10 M).

Carboxylate 2 is released in high photochemical yield (88% by HPLC; Figure 2b and Figure S23) upon irradiation for 2 h in EtOH. The quantum yield (P) 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 (  p) of 15 M-1cm-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 (irr = 420 nm, 6.5 mW/cm2; Figure S12). The photo-triggered 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 and 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 light-triggered anticancer drug release in aqueous solution. The phototriggered release of 2 from 1 through photoreduction and 1,6elimination was combined with a 1,4-elimination to enable the simultaneous release of both fluorophore 2 and a biologically active payload. In doing so, drug release can be monitored in real time using fluorescence microscopy. In the proof-ofconcept photorelease platform 1a-Chl (Schemes S2-S3), the photoactivatable fluorophore 1a is linked via an ester linkage to the anticancer drug chlorambucil (Figure 3a).

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 M) as a function of UV irradiation times (0, 1, 3, 5, 10, 20, 30, 40, 50, 60 min). Sample was irradiated with a hand-held UV lamp (irr = 365 nm, 5.7 mW/cm2) at 25 °C. Emission (ex = 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 em = 510 nm of 1a-Chl (10 M) 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 (irr = 365 nm, 5.7 mW/cm2) at 25 °C. [Esterase] = [Trypsin] = [Lipase] = 10 U/mL. [Others] = 1 mM.

Simultaneous photo-triggered 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 non-emissive (F: 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 (irr = 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 nm and 510 nm, respectively (Figures 3b and S17). Photolysis of 1a-Chl for 1 h resulted in a ca. 1250-fold fluorescence enhancement (F/F0) at 510 nm. The release of

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fluorescent 2 under UV irradiation is readily visible, as shown in Figure 3b inset. In the dark, 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) (Figures 3c and S20). Thiols such as NaSH have been reported to induce the reduction of aryl azides to anilines.47 However, release of fluorophore 2 from the dye-drug conjugate 1a-Chl was not observed in the dark upon incubation with various reducing biothiols (NaSH, GSH, Cys, HCy; Figures 3c and 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.

Figure 4. (a) HPLC monitoring of the progress of the release of fluorophore 2 and chlorambucil from 1a-Chl (10 M) in phosphate buffer (10 mM, pH 7.4, 1% DMSO) under light irradiation with a hand-held UV lamp (irr = 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 M) (b) Progress for the photoregulated release of drug from 1a-Chl (10 M) under alternating light (15 min) and dark (15 min) conditions by HPLC analysis. Arrows indicate the beginning of light irradiation (h) and the ending of light irradiation (dark).

The progress of 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 1a-Chl (10 μM) for 2 h released 5.17 M 2 and 4.93 M chlorambucil, the latter concentration being sufficient to elicit a cellular response. The identity of the released drug was confirmed by 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 (p) of the photoconversion for drugconjugate 1a-Chl in aqueous buffer was determined to be p= 0.1%. These value is comparable to the efficiencies recorded


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ACS Applied Bio Materials 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 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 indicated that 1a-Chl is resistant to background hydrolysis in the dark, as neither fluorophore 2 nor the drug was released during incubation in aqueous buffer for a period of 24 h (Figure S11).

cells treated with chlorambucil alone (IC50 = 430 M, 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 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 photo-triggered release of chlorambucil in living cells from 1a-Chl 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 (irr = 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 M after 24 h (Figure S28).

CONCLUSIONS

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 light-triggered drug release, 1a-Chl-treated HeLa cells received a light irradiation for 10 min with a hand-held UV lamp (irr = 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 M) 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 (ex = 450-490 nm, em = 515–565 nm). Scale bar: 20 m.

In vitro anticancer cytotoxicity by light-triggered dyedrug 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 M 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

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.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthetic details and characterization of compounds Additional spectroscopic data, and photoactivation studies

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y. Choi). *E-mail: [email protected] (Y.Kim).

Author Contributions ⊥Y.J.

and T.-I.K. contributed equally to this work.



Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This research was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIP) (NRF-2017M3D9A1073769, NRF-2018R1D1A1B07044894, and NRF-2015R1A5A1008958).

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Photoactivatable BODIPY Platform: Light-Triggered Anticancer Drug

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