Cytosol-Specific Fluorogenic Reactions for Visualizing Intracellular

Jan 21, 2015 - *(S.L.) E-mail: [email protected]. ... Supramolecular aggregates of stimuli-responsive block copolymers are increasingly utilized as dru...
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Cytosol-Specific Fluorogenic Reactions for Visualizing Intracellular Disintegration of Responsive Polymeric Nanocarriers and Triggered Drug Release Yanyan Jiang, Guhuan Liu, Xiaorui Wang, Jinming Hu, Guoying Zhang, and Shiyong Liu* CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *

ABSTRACT: Supramolecular aggregates of stimuli-responsive block copolymers are increasingly utilized as drug nanocarriers. Although in situ tracking their triggered disintegration and drug release processes at the cellular level is highly desirable, it remains a considerable challenge. We report the fabrication of double hydrophilic block copolymers covalently conjugated with α,β-unsaturated ketone-caged coumarin functionalities in the thermoresponsive block. Upon thermo-induced micellization and cellular uptake, Michael addition reaction of unsaturated ketone moieties with thiol compounds (GSH and Cys) in the reductive subcellular compartments leads to micelleto-unimer transition. This is accompanied by concomitant fluorescence emission turn-on and triggered drug release, allowing for the process visualization.

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INTRODUCTION Polymeric assemblies of amphiphilic and double hydrophilic block copolymers (DHBCs) have been increasingly utilized as potent drug nanocarriers due to improved bioavailability and extended blood circulation, as compared to small molecule drugs.1 Though passive and active targeting strategies have been attempted to enhance drug accumulation at desired sites,2 the integration of stimuli-responsiveness into block copolymer (BCP) nanocarriers can further contribute to site-specific delivery of therapeutic/imaging agents.3 Appropriately designed responsive BCP assemblies exhibit microstructural rearrangement and inversion or even disassembly into unimers under external stimuli.4 Previously, a variety of pathologically relevant stimuli such as pH, temperature, enzymes, hypoxia, and redox milieu have been exploited to design bioresponsive polymeric nanocarriers.3d,5 Considering both the fundamental aspect and potential clinical applications of bioresponsive BCP nanocarriers, it is highly desirable to in situ track their intracellular transport and drug release triggered by structural transformations of responsive BCPs. This will also help optimize the design of polymeric nanocarriers and polymer−drug conjugates. Recently, biocleavable small molecule fluorophore-drug conjugates have been designed to achieve image-guided drug release.6 Although conventional dye-labeled polymeric nanocarriers can allow for the optical imaging of cellular events, the “always-on” nature precludes spatiotemporal quantification of nanocarrier transport, structural changes, and drug release processes.7 In this context, fluorescence-activatable processes in © 2015 American Chemical Society

response to external stimuli are more preferred due to low background signals. We then reasoned that if biostimuli-triggered BCP nanocarrier disintegration process is correlated with a fluorogenic event,8 then it should allow for the real-time imaging of structural changes and triggered release events. In addition, the reductive milieu of cytosol (glutathione, GSH ∼ 5−10 mM) has been frequently exploited to design disulfide-based bioresponsive nanocarriers.6d,7,9 Considering that GSH is a thiol-containing hydrophilic tripeptide, we herein attempt to use fluorogenic Michael addition reactions10 to actuate intracellular micelle-to-unimer transition and concomitantly monitor micellar disintegration and drug release processes (Scheme 1). Starting from DHBCs with the thermoresponsive block conjugated with initially nonfluorescent α,β-unsaturated ketone (PyCouMA), the cellular internalization of thermoinduced micelles is followed by endocytic vesicular trafficking; upon endosomal escape and entering into cytosol, Michael addition reactions of PyCouMA moieties with thiol compounds (GSH and Cys) will occur. This leads to fluorescence turn-on due to coumarin decaging,10a micelle disintegration due to the elevation of critical micellization temperatures (CMTs),11 and concomitant drug release. Received: November 26, 2014 Revised: December 31, 2014 Published: January 21, 2015 764

DOI: 10.1021/ma502389w Macromolecules 2015, 48, 764−774

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Scheme 1. Self-Assembly of Thiol-Reactive Thermoresponsive PEG-b-P(MEO2MA-co-EEO2MA-co-PyCouMA) DHBCs (P1− P4) and Intracellular Trafficking of Micellar Nanoparticlesa

a Upon cellular internalization, Michael addition reaction of α,β-unsaturated ketone moieties with thiols in the reductive cytosol milieu leads to micelle-to-unimer transition, accompanied with concommitant emission turn-on and release of physically encapsulated drugs.

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Reagent Co. Ltd. and used as received. Dialysis tube (MWCO 3.5 and 14 kDa) was purchased from Shanghai Green Bird Technology Development Co., Ltd., China, and stored in 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution prior to use. Chloroquine diphosphate salt purchased from Sigma Life Sciences was dissolved in water at a concentration of 100 mM. 4-Chloro-7nitrobenzofurazan (NBD-Cl, 99%) was purchased from Alfa and used as received. All solvents were of analytical grade. PEG−Br macroinitiator was prepared according to literature procedures by the esterification reaction of PEG−OH with 2-bromoisobutyryl bromide in the presence of Et3N.13 4-(2-Acryloyloxyethylamino)-7- nitro-2,1,3benzoxadiazole (NBDAE) was synthesized according to literature procedures.14 Water used in this study was deionized with a Milli-QSP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ· cm. LysoTracker Green was purchased from Molecular Probes. Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s

EXPERIMENTAL SECTION

Materials. Di(ethylene glycol) monomethyl ether methacrylate (MEO2MA, Sigma-Aldrich) was passed through an alumina column to remove the inhibitor. 3-Azidopropyl methacrylate (AzPMA) was prepared by the esterification reaction of acryloyl chloride with 3azidoprppan-1-ol.12 All purified monomers were stored at −20 °C prior to use. 2-(2-Ethoxyethoxy)ethanol, propargyl alcohol, copper(I) bromide (CuBr), 2-bromoisobutyryl bromide, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 1,1,4,7,7pentamethyldiethylenetriamine (PMDETA), 2-pyridinecarboxaldehyde, and poly(ethylene glycol) monomethyl ether (PEG−OH, Mn = 2.0 kDa, Mw/Mn = 1.06) were purchased from Sigma-Aldrich and used as received. 1,3-Benzenediol, ethyl acetoacetoacetate, phosphorusoxychloride (POCl3), piperidine, triethylamine (Et3N), 2-(2ethoxyethoxy)ethanol, glutathione (GSH), and doxorubicin hydrochloride (DOX·HCl) were purchased from Sinopharm Chemical 765

DOI: 10.1021/ma502389w Macromolecules 2015, 48, 764−774

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Scheme 2. Synthetic Routes Employed for the Preparation of α,β-Unsaturated Ketone-Containing Precursor, (E)-7-(Prop-2-yn1-yloxy)-3-(3-(pyridin-2-yl)acryloyl)-2H-chromen-2-one (4), and PEG-b-P(MEO2MA-co-EEO2MA-co-AzPMA) and PEG-bP(MEO2MA-co-EEO2MA-co-PyCouMA) (P1−P4) Double Hydrophilic Diblock Copolymers (DHBCs)

modified Eagle’s medium (DMEM) were obtained from GIBCO and used as received. Sample Preparation. Synthetic schemes employed for the preparation of α,β-unsaturated ketone-containing precursor (4) and PEG-b-P(MEO 2 MA-co-EEO 2 MA-co-AzPMA) and PEG-b-P(MEO2MA-co-EEO2MA-co-PyCouMA) (P1-P4) DHBCs are shown in Scheme 2. Synthesis of 2-Hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde (2). To an ice-cooled solution (0−5 °C) of 1,3-benzenediol (44 g, 128 mmol) in acetonitrile (150 mL) were added dry DMF (12.6 g, 128 mmol) and freshly distilled dry POCl3 (22.6 g, 146 mmol) under magnetic stirring. The precipitated salts were filtered and washed twice with cold acetonitrile. Then water was added to salt residues and heated at 50 °C for 30 min; this is followed by crystallization upon cooling. The crystal was filtered, washed with cold deionized water, and then dried in a vacuum oven to afford 2,4-dihydroxybenzaldehyde (1) as a white crystal (34 g, yield: 61.6%). 1H NMR (CDCl3, δ, ppm, TMS, Figure S1a): 11.45 (s, 1 H), 9.75 (s, 1 H), 7.46 (d, 1 H), 6.48 (m, 1 H), 6.40 (d, 1 H), 5.63 (s, 1 H). A mixture of 2,4-dihydroxybenzaldehyde (10 g, 72.4 mmol), propargyl bromide (8.33 g, 70 mmol), and K2CO3 (19.35 g, 140 mmol) in acetone was heated at 50 °C. After overnight stirring, the

reaction mixture was allowed to cool to room temperature and subjected to filtration to remove salts. After evaporating all the solvents, the residues were dissolved in CH2Cl2, successively washed with water, dried over anhydrous MgSO4, filtered, and then concentrated on a rotary evaporator. The crude product was subjected to further purification by silica gel column chromatography using ethyl acetate and petroleum ether (v/v = 1:2) as the eluent, affording 2hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde (2) (3.2 g, yield: 25.9%) as a white powder. 1H NMR (CDCl3, δ, ppm, TMS, Figure S1b): 11.45 (s, 1 H), 9.75 (s, 1 H), 7.46 (d, 1 H), 6.61 (m, 1 H), 6.53 (d, 1 H), 4.75 (d, 2 H), 2.58 (m, 1 H). Synthesis of 3-acetyl-(E)-7-(prop-2-yn-1-yloxy)coumarin (3). Ethyl acetoacetate (1.43 g, 11 mmol) and 2 (1.72 g, 9.8 mmol) were dissolved in absolute EtOH (15 mL); piperidine (0.1 g) and AcOH (0.1 mL) were added as catalyst. The mixture was heated to reflux for 12 h, and then the reaction mixture was allowed to cool to room temperature. A bright yellow precipitate was formed, which was collected by suction filtration and washed with cold absolute EtOH. The crude product was further purified by recrystallization from absolute EtOH, yielding (E)-7-(prop-2-yn-1-yloxy)-3-acetyl-coumarin (3) (1.53 g, yield: 64.5%) as a bright yellow crystal. 1H NMR (CDCl3, 766

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Table 1. Molecular Parameters of DHBCs Covalently Conjugated with α,β-Unsaturated Ketone-Caged Coumarin Moieties in the Thermoresponsive Block entry

samples

Mn (kDa)a

Mw/Mna

Mn (kDa)b

CMT/°C (GSH−)c

CMT/°C (GSH+)c,d

P1 P2 P3 P4 P2-NBD

PEO45-b-P(MEO2MA0.9-co-PyCMA0.1)42 PEO45-b-P(MEO2MA0.81-co-EEO2MA0.09-co-PyCMA0.1)40 PEO45-b-P(MEO2MA0.71-co-EEO2MA0.19-co-PyCMA0.1)43 PEO45-b-P(MEO2MA0.65-co-EEO2MA0.25-co-PyCMA0.1)45 PEO45-b-P(MEO2MA0.81-co-EEO2MA0.09-co-PyCMA0.1-NBD)40

10.3 9.4 10.7 10.4 9.5

1.17 1.21 1.23 1.19 1.18

10.9 10.6 11.3 11.8 10.6

36 32 27 20 32

∼90 84 75 69 84

a Determined by GPC using THF as eluent at a flow rate of 1.0 mL/min. bCalculated from 1H NMR analysis. cDetermined by temperaturedependent optical transmittance at a wavelength of 600 nm, the CMT was defined as the temperature at which ∼1% decrease in the optical transmittance can be discerned. dCMT determined after treating with 10 mM GSH.

δ, ppm, TMS, Figure S1c): 8.49 (s, 1 H), 7.57 (d, 1 H), 6.97 (m, 2 H), 4.80 (d, 2 H), 2.71 (s, 3 H), 2.61 (t, 1 H). Synthesis of 3-(3-(pyridin-2-yl)acryloyl)-(E)-7-(prop-2-yn-1-yloxy)coumarin (4). 3 (1.4 g, 5.8 mmol) and 2-pyridinecarboxaldehyde (1.24 g, 11.6 mmol) were dissolved in EtOH/CH3CN (40 mL, 1:1 v/ v), then 0.4 g piperidine were added as a catalyst. The mixture was heated to reflux for 24 h, and the solvent was removed under reduced pressure. The crude product was purified by recrystallization from absolute EtOH, affording 3-(3-(pyridin- 2-yl)acryloyl)-(E)-7-(prop-2yn-1-yloxy)coumarin (4) as a yellow powder (0.77 g, yield: 40%). 1H NMR (CDCl3, δ, ppm, TMS, Figure S 1d): 8.68 (d, 1 H), 8.56 (s, 1 H), 8.30 (d, 1 H), 7.80 (d, 1H), 7.70 (m, 1 H), 7.59 (m, 2H), 7.28 (m, 1 H), 6.97 (m, 2 H), 4.80 (d, 2 H), 2.61 (t, 1 H). 13C NMR (CDCl3, δ, ppm TMS, Figure S2a): 186.8, 162.9, 159.3, 157.5, 153.5, 150.3, 148.3, 143.1, 136.7, 131.4, 127.8, 124.6, 124.3, 121.9, 114.3, 112.8, 101.7, 56.4. HR-MS: m/z calcd for C20H13NO4, 331.09; found, 332.09119 [M + H]+ (Figure S2b). Synthesis of 2-(2-ethoxyethoxy)ethyl Methacrylate (EEO2MA). 2(2-Ethoxyethoxy)ethanol (13.4 g, 0.1 mol) and triethylamine (12.1 g, 0.12 mol) were dissolved in anhydrous CH2Cl2 (100 mL), then methacryloyl chloride (11.4 g, 0.11 mol) was slowly added under magnetic stirring in an ice-bath; the mixture was stirred overnight at room temperature. After that, triethylamine hydrochloride was removed, and the solution was washed with sodium bicarbonate solution and saline, dried over anhydrous MgSO4, filtered, and concentrated on a rotary evaporator. The crude product was subjected to further purification by silica gel column chromatography, affording EEO2MA as a colorless liquid (15.2 g, yield: 75%). 1H NMR (CDCl3, δ, ppm, TMS): 6.13 (m, 1 H), 5.57 (m, 1 H), 4.31 (m, 2H), 3.80−3.46 (m, 8 H), 1.95 (m, 3 H), 1.21 (t, 3 H). Synthesis of PEO45-b-P(MEO2MA0.9-co-AzPMA0.1)42 and PEO45-bP(MEO2MA0.9-co- PyCouMA0.1)42 Diblock Copolymers (P1). Typical procedures employed for the ATRP synthesis of thermoresponsive diblock copolymers, PEO45-b-P(MEO2MA0.9-co-AzPMA0.1)42, are as follows. Into a reaction tube equipped with a magnetic stirring bar were charged with MEO2MA (2.0 g, 10.6 mmol), AzPMA (180 mg, 1.065 mmol), PEG45−Br (468 mg, 0.213 mmol), PMDETA (37 mg, 0.213 mmol), and isopropanol (2.5 mL). The reaction tube was carefully degassed by three freeze−pump−thaw cycles, CuBr (30 mg, 0.213 mmol) was added into the tube and degassed once more and then the tube was sealed under vacuum. After thermostatting at 35 °C in an oil bath and stirring for 6 h, the reaction tube was quenched into liquid nitrogen, opened, and diluted with tetrahydrofuran, and then the reaction mixture was stirred under air for 4 h. The solution obtained after stirring was subjected to passing through flash column (silica gel, THF) to remove the cooper catalyst, then precipitated into an excess of diethyl ether. The above dissolution−precipitation cycle was repeated for three times. PEO45-b-P(MEO2MA0.9-co- AzPMA0.1)42 was obtained as a colorless liquid (1.8 g, yield: 67.9%). The thiol-reactive double hydrophilic copolymers were prepared by the click reaction of PEO45-b-P(MEO2MA0.9-co-AzPMA0.1)42 with 4 in the presence of CuBr and PMDETA. In a typical procedure, into a reaction tube equipped with a magnetic stirring bar were charged PEO45-b-P(MEO2MA0.9-co-AzPMA0.1)42 (500 mg, 0.05 mmol), 4 (83 mg, 0.25 mmol), CuBr (36 mg, 0.25 mmol), PMDETA (43 mg, 0.25 mmol), and DMF (3 mL). The reaction tube was carefully degassed by

three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 35 °C in an oil bath and stirring for 24 h, the reaction tube was quenched into liquid nitrogen, opened, and diluted with tetrahydrofuran, then stirred under air for 4 h. The solution obtained after stirring was subjected to passing through flash column (silica gel, THF) to remove the salt, then precipitated into an excess of diethyl ether. The above dissolution−precipitation cycle was repeated for three times. P1 was obtained as a yellow powder (432 mg, 74% yield). According to the similar protocols, thermoresponsive DHBCs, P2, P2-NBD, P3, and P4, with varying ratios of MEO2MA and EEO2MA were also synthesized. Their structural parameters are summarized in Table 1. Synthesis of PEO45-b-P(EEO2MA-co-NBD)42 (P2-NBD). Typical procedures employed for the ATRP synthesis of PEO 45 -bP(EEO2MA-co-NBD)42 diblock copolymer are as follows. Into a reaction tube equipped with a magnetic stirring bar were charged with EEO2MA (2.1 g, 10.6 mmol), NBDAE (5.6 mg, 0.02 mmol), PEG45− Br (468 mg, 0.213 mmol), PMDETA (37 mg, 0.213 mmol), and 2propanol (2.5 mL). The reaction tube was carefully degassed by three freeze−pump−thaw cycles, CuBr (30 mg, 0.213 mmol) was added into the tube and degassed once more and then the tube was sealed under vacuum. After thermostating at 35 °C in an oil bath and stirring for 6 h, the reaction tube was quenched into liquid nitrogen, opened, and diluted with tetrahydrofuran, and then the reaction mixture was stirred under air for 4 h. The solution mixture was subjected to passing through a silica gel column to remove the copper catalyst, then precipitated into an excess of diethyl ether. The above dissolution− precipitation cycle was repeated for three times. PEO45-b-P(EEO2MAco-NBD)42 was obtained as a viscous liquid (1.7 g, yield: 66.2%). GPC analysis revealed an Mn of 9.2 kDa and an Mw/Mn of 1.13. Characterization. All nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV300 NMR spectrometer (resonance frequency of 300 MHz for 1H) operated in the Fourier transform mode. CDCl3 was used as the solvent. Molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC) equipped with Waters 1515 pump and Waters 2414 differential refractive index detector (set at 30 °C), employing a series of two linear Styragel columns (HR2 and HR4) at an oven temperature of 45 °C. The eluent was THF at a flow rate of 1.0 mL/ min. A series of six polystyrene standards with molecular weights ranging from 800 to 400 000 g mol−1 were used for calibration. Surface tension was conducted by JK99B tensiometer with a platinum plate. Dynamic light scattering (DLS) measurements were conducted on Malvern Zetasizer Nano ZS. All data were averaged over three measurements. All samples were filtered through 0.45 μm Millipore Acrodisc-12 filters to remove dust. Transmission electron microscopy (TEM) observations were conducted on a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV. The sample for TEM observations was prepared by placing 10 μL of micellar solution (0.5 g L−1) on copper grids coated with thin films of Formvar and carbon successively. Confocal laser scanning microscopy (CLSM) images were acquired using a Leica TCS SP5 microscope. HPLC analysis was performed with a Shimadzu HPLC system, equipped with a LC-20AP binary pump, a SPD-20A UV−vis detector, and a Symmetry C18 column. All UV−vis spectra were acquired on a Unico UV/vis 767

DOI: 10.1021/ma502389w Macromolecules 2015, 48, 764−774

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In Vitro Cytotoxicity Assay. Hela cells were chosen as to evaluate the in vitro cytotoxicity of DOX loaded micelles via the MTT assay. Hela cells were first cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine’ serum (FBS), penicillin (100 units per mL), and streptomycin (100 μg per mL) at 37 °C in a CO2/air (5:95) incubator for 2 days. For cytotoxicity assay, Hela cells were seeded into 96-well plates at a density of 5000 cells per well. Afterward, the cells were incubated for 24 h, then DMEM was replaced with fresh media, and the cells were treated with DOX-loaded or blank micellar solution at varying concentrations, the treated cells were incubated in a humidified environment with 5% CO2 at 37 °C for 48 h. MTT reagent in 20 μL PBS, 5 mg/mL was then added. The cells were further incubated for 4 h at 37 °C. The medium in each well was then removed and replaced by 180 μL DMSO. The plate was gently agitated for 15 min before the absorbance at 570 nm was recorded by a microplate reader (Thermo Fisher). Each experiment condition was done in quadruplicate, and the data are shown as the mean value plus a standard deviation (±SD). Cellular Uptake of Micelles Observed with Confocal Laser Scanning Microscopy (CLSM). The intracellular distribution of micelles was determined using CLSM. A549 cells were plated onto Petri dishes at a density of 1 × 106 cells per dish and cultured in Dulbecco’s modified Eagle medium (DMEM) supplement with 10% fetal bovine serum (FBS), penicillin (100 units per mL), and streptomycin (100 μg mL−1) for 24 h at 37 °C in CO2/air (5:95). The prepared P2 micellar solution was added at a final concentration of 0.4 g/L. After incubating for ∼1, 4, 6, and 12 h, cells were washed with PBS buffer for three times. The images were taken on the microscope. Late endosomes and lysosomes were stained with LysoTracker Green at 200 nM after incubation with cells for 30 min before imaging and excited using a 488 nm laser, and the emission wavelength was read from 515 to 560 nm and expressed as green. The blue fluorescence of activated coumarin moieties in P2 was observed using a 405 nm laser with the emission channel set at 420−470 nm. The internalization kinetics was estimated by quantifying the blue fluorescence intensity from fluorescence micrographs. The intracellular distribution of P2 was quantitatively evaluated by calculating the colocalization ratio of blue fluorescence pixels with LysoTracker Green pixels. Colocalization ratio was quantified as

2802PCS spectrophotometer. Concerning temperature- dependent turbidimetry, the optical transmittance of aqueous solutions at a wavelength of 600 nm was acquired on a Unico UV/vis 2802PCS spectrophotometer. A thermostatically controlled cuvette was employed and the heating rate was 0.2 °C min−1. The CMT was defined as the temperature corresponding to ∼1% decrease of optical transmittance. Fluorescence spectra were recorded on F-4600 (Hitachi) spectrofluorometer. The slit widths were both set at 5 nm for excitation and emission. Determination of Critical Micelle Concentration (CMC). The CMC of P2 was determined by surface tensiometry. Equilibrium surface tensions were measured using a JK99B tensiometer with a platinum plate. The measuring accuracy of the device as reported by the manufacturer is ±0.1 mN/m. The reported surface tension values were averaged from four to five successive measurements at a temperature of 37.0 ± 0.2 °C. CMC of P2 was determined to be ∼0.012 g/L. Reactivity of 4 and P2 toward Thiols. The reactivity of small molecule 4 was studied by 1H NMR and HPLC methods using 2mercaptoethanol as model thiol compound (Figure S6a and Figure S6b). 1H NMR results suggested the disapperance of resonance signals characteristic of unsatuated double bond and HPLC results revealed a shorter elution volume after 2-mercaptoethanol treatment, indicating the complete Michael addition reactions between small mocleule 4 and 2-mercaptoethanol (UV traces monitored at λ = 365 nm). The reactivity of P2 was also examined by 1H NMR analysis (Figure S7), exhibiting a similar reaction tendency as compared to small molecule 4. Agarose Gel Retardation Assay. The solution of P2 (20 μL, 0.01 g/L), before and after treatment with 10 mM GSH, was mixed with 3 μL loading buffer and then loaded to agarose gel. The polymers were electrophoresed on a 0.9% (w/v) agarose gel in Tris-boric acid-EDTA buffer at 90 V for 150 min at 25 °C. The bands were visualized by immersing the gel into 10 mM GSH solution and excited by UV transillumination, and then photographed by the gel-imaging system. Fabrication of Diblock Copolymer Micelles. A 10 mg sample of P2 was dissolved in DMSO (1 mL) at 40 °C under stirring, then water (9 mL) was slowly added to the solution using a syringe pump within 1 h, the mixture was dialyzed (MWCO ∼ 14 kDa) against pH 7.4 PBS buffer at 40 °C to remove DMSO. Preparation of Micelles and DOX Encapsulation. A 10 mg sample of P2 and 1.5 mg doxorubicin hydrochloride were dissolved in DMSO (1 mL) at 40 °C under stirring, then TEA (10 μL) was added to the solution and water (9 mL) was slowly added to the solution using a syringe pump within 1 h, the mixture was dialyzed (MWCO 14 kDa) against pH 7.4 PBS buffer at 40 °C for 12 h to remove DMSO, TEA and unloaded DOX. The DOX-loading content, defined as the weight percentage of DOX in freeze-dried micelles, was quantified by UV−vis analysis using a UV−vis spectrophotometer. The encapsulation efficiency (EE %) was calculated as

colocalization ratio % = [pixelscolocalization /pixels P2] × 100% where pixelscolocalization represents the number of P2 pixels colocalizing with LysoTracker Green, and pixelsP2 represents the number of all the P2 pixels in cells. Endosomal Escape Analysis. For endosomal escaping experiments, A549 cells were treated with DMEM containing 0.4 g/L P2NBD for 5 h at 37 °C and replaced with fresh DMEM containing 100 μM chloroquine and incubated for another 1 h before microscopy imaging. The blue fluorescence of activated coumarin moieties was observed using the 405 nm laser with the emission channel set at 420− 470 nm. NBD fluorescence emission was observed using a 488 nm laser with the emission channel set at 515−560 nm. Cellular Uptake of DOX-Loaded P2 Micelles Observed with Confocal Laser Scanning Microscopy (CLSM). A549 cells were plated onto Petri dishes at a density of 1 × 106 cells per dish and cultured in Dulbecco’s modified Eagle medium (DMEM) supplement with 10% fetal bovine serum (FBS), penicillin (100 units per mL), and streptomycin (100 μg mL−1) for 24 h at 37 °C in CO2/air (5:95). DOX-loaded P2 micellar solution was added to the Petri dishes at a final concentration of 0.2 g/L. After incubated for 1, 6, and 12 h, cells were washed with PBS for three times. The images were taken on the microscope. Coumarin, the blue fluorescence of P2 from coumarin moieties was observed using a 405 nm laser with the emission channel set to be 420−470 nm. Fluorescence of DOX was observed using a 592 nm laser with the emission channel set to be 620−700 nm. Late endosomes and lysosomes were stained with LysoTracker Green at 200 nM after incubation with cells for 30 min before imaging and observed using a 488 nm laser, and the emission wavelength was read from 515 to 560 nm and expressed as green. The internalization kinetics was estimated by quantifying the blue and red fluorescence

EE% = [Wtotal − Wunloaded]/Wtotal × 100% The loading content (LC %) was calculated as LC% = [Wloaded]/[Wpolymer + Wloaded] × 100% where Wtotal, Wunloaded, Wloaded, and Wpolymer refer to the weights of drug used, unloaded drug, drug encapsulated by micelles, and diblock copolymer, respectively. In Vitro DOX Release from Self-Assembled Micelles. DOXloaded micellar solution (0.5 g/L) was dispersed in 0.1 mol·L−1 phosphate buffer (pH 7.4 with 10 mM GSH) and transferred to a dialysis tube (MWCO: 3.5 kDa) immersed in the same buffered media at 37 °C, at selected time intervals, aliquots of the external medium were withdrawn and replaced with the same volume of fresh buffer solution, the concentration of DOX was calculated based on the absorbance intensity of DOX at 482.5 nm by UV−vis analysis. The cumulative amount of released drug was calculated, and the percentages of drug release from micelles were plotted against time. 768

DOI: 10.1021/ma502389w Macromolecules 2015, 48, 764−774

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Figure 1. (a) Hydrodynamic diameter distribution, f(Dh), recorded for the micellar dispersion of PEG-b-P(MEO2MA0.81-co-EEO2MA0.09-coPyCouMA0.1)40 (P2, 0.2 g/L, [PyCouMA] = 75 μM) at 37 °C. (b) Agarose gel electrophoresis of P2 (left) before and (right) after treating with 10 mM GSH; after electrophoresis, the gel layer was further stained with GSH; the image was taken under UV lamp. (c and d) TEM images of P2 micelles (c) before and (d) after treating with 10 mM GSH. intensity from fluorescence micrographs. The intracellular distribution of P2/DOX was quantitatively evaluated by calculating the colocalization ratio of blue/red fluorescence pixels with LysoTracker Green pixels.

reaction between 4 and 2-mercaptoethanol was confirmed by 1 H NMR and HPLC analysis (Figure S6). Moreover, the reactivity of α,β- unsaturated ketone moieties with thiol compounds remained unaffected when covalently attached to polymer chains, as evidenced by agarose gel electrophoresis (Figure 1b), UV−vis absorbance (Figures 2a,b), and 1H NMR (Figure S7). For P1−P4, Michael addition reactions with GSH all led to considerable elevation of CMTs (Table 1), which should be ascribed to the highly hydrophilic and charged nature of GSH.15 Thus, at a temperature between initial CMT and CMT after GSH addition, themo-induced micelles will undergo disintegration. Indeed, GSH addition induced micelle-tounimer transition for P2 at 37 °C was verified by TEM and DLS (Figures 1d and 2c) and discernible by direct visual checking (inset in Figure 2b). Comparably, P4 possessed a CMT of ∼20 °C; upon GSH addition, the CMT increased to ∼69 °C (Table 1). Thus, at intermediate temperatures, GSH addition led to micellar disintegration (Figure S8). In sharp contrast, when GSH was replaced with hydrophobic 1mercaptopropane, although Michael addition reactions still occurred, neither scattered light intensity nor micellar sizes exhibit any appreciable changes (Figure S9), implying that micellar disassembly did not take place. Previously, small molecule dyes caged with α,β-unsaturated ketone moieties have been utilized to design fluorogenic probes for thiols;10 thus, GSH addition-triggered disassembly process of DHBC micelles should also be associated with the emission turn-on event. This was confirmed by fluorescence measurements upon addition GSH into thermo-induced P2 micellar solution. Prominently increased emission was observed within ∼2 h after GSH addition at pH 7.4, exhibiting a cumulative ∼33-fold increase (Figure 2d). Note that there existed

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RESULTS AND DISCUSSION α,β-Unsaturated ketone conjugated with alkynyl functionality and caged coumarin (4, Scheme 2) was synthesized at first (Figures S1 and S2). Next, a series of DHBCs, PEG-bP(MEO2MA-co-EEO2MA-co-PyCouMA) (P1-P4), with the thermoresponsive block covalently modified with 4 was synthesized by combining atom transfer radical polymerization (ATRP) and click protocols (Figures S3−S5), where PEG, MEO2MA, and EEO2MA are poly(ethylene glycol), di(ethylene glycol) methyl ether methacrylate, and di(ethylene glycol) ethyl ether methacrylate, respectively. The structural parameters of DHBCs with varying MEO2MA/EEO2MA ratios are summarized in Table 1. Temperature- dependent turbidity measurements exhibited a constant decrease of CMTs with increasing EEO2MA fractions (Table 1), which should be ascribed to the relatively hydrophobic nature of EEO2MA compared to MEO2MA. Taking P2 as an example, its thermo-induced micellization in aqueous media can be visualized by the naked eye from the characteristic colloidal bluish tinge (inset in Figure 1a). Dynamic light scattering (DLS) revealed an intensityaverage hydrodynamic diameter, ⟨Dh⟩, of ∼125 nm and polydispersity index of 0.075 at 37 °C (Figure 1a), which is in accord with its CMT (∼32 °C). TEM analysis also revealed the presence of spherical nanopaticles for P2 at 37 °C (Figure 1c). Next, the reactivity of α,β-unsaturated ketone functionality in 4 toward thiol compounds was examined. The addition 769

DOI: 10.1021/ma502389w Macromolecules 2015, 48, 764−774

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Macromolecules

Figure 2. (a) Evolution of UV−vis spectra, (b) time-dependent absorbance at 700 nm, and (c) hydrodynamic diameter distributions recorded for P2 micellar solution upon addition of 10 mM GSH at 37 °C and pH 7.4. (d and e) Evolution of fluorescence emission spectra (λex = 370 nm) recorded for P2 micellar solution (37 °C) upon addition of 10 mM GSH (10 mM) at (d) pH 7.4 and (e) pH 5.5; the insets show time-dependent changes in emission intensities (λem = 425 nm). (f) Time-dependent scattered light intensity recorded for P2 micellar solution (0.2 g/L, 37 °C) upon addition of 10 μM, 5 mM, and 10 mM GSH.

micellar disassembly can be discerned; whereas at [GSH] = 5 or 10 mM, which is typical of the cytosolic milieu, prominent emission enhancement was observed. In addition, we further verified that P2 micelles are also stable in human serum (1 g/L) for at least 24 h, as evidenced from the almost negligible emission changes compared to the blank control (Scheme 1). The above results led to the conclusion that to switch on the fluorescence emission of DHBC micelles, all the following requirements must be fulfilled at the same time: micellar disassembly; extensive Michael addition reaction with hydrophilic thiol compounds; high thiol levels and neutral pH. We then reasoned that when P1−P4 micelles are utilized as drug nanocarriers, the concomitant micellar disintegration, drug release, and emission turn-on events are actually cytosolspecific. This endows the system with the feature of spatiotemporal accuracy of image-guided delivery. During blood circulation and cellular uptake into acidic organelles, the low thiol level and acidic pH will, respectively, prohibit micellar disassembly and emission turn-on; whereas in the cytosol, all of these prerequisites are satisfied (Scheme 1). When P2 micelles were coincubated with cells, we could not observe blue emission originated from activated coumarin moieties for the first 4 h (Figure 3); this is quite reasonable considering the acidic milieu in endolysosomes and the poor reactivity16 (Figure 2e). Upon extending the incubation

negligible emission increase when subjected to Michael addition reaction with 1-mercaptopropane, which should be due to the self-quenching effect of decaged coumarin fluorophores within hydrophobic micellar cores (Figure S9b). We also utilized 2-mercaptoethanol as a hydrophilic thiol compound to actuate Michael addition reaction with P2. It was found that the fluorescence emission cannot be switched on either due to the persistence of micellar state. This can be ascribed to insufficient hydrophilicity endowed by 2-mercaptoethanol and less prominent shift in CMTs. The above results implied that only when both Michael addition reaction and micelle-to-unimer transition occurred, can we observe prominent fluorescence emission turn-on. Considering that the endocytic transport of micellar nanoparticles are typically associated with acidic organelles such as early/late endosomes and lysosomes before escaping into the cytosol, the Michael addition reaction of DHBC micelles with GSH was examined at pH 5.5. It was found that even after 1 h, negligible emission increase can be discerned (Figure 2e), which should be ascribed to the pH-dependent nature of thiol−ene addition reaction (Figure 2e).16 Even at pH 7.4, the reaction kinetics are highly dependent on thiol concentrations, as evidenced from both DLS and fluorescence measurements (Figure 2f and Figures S10−S11). At low GSH levels (0 or 10 μM), neither fluorescence emission turn-on nor 770

DOI: 10.1021/ma502389w Macromolecules 2015, 48, 764−774

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Macromolecules

Figure 3. CLSM images recorded for A549 cells after coincubation at 37 °C with P2 micellar solution (0.4 g/L) for 1, 4, 6, and 12 h. Late endosomes and lysosomes were stained with LysoTracker Green (green). The blue channel fluorescence emission was originated from PyCouMA moieties upon decaging via reaction with intracellular thiol-containing compounds (GSH or Cys).

fluorescence of NBD is always on, which can serve as a fluorescent marker for tracking. As shown in Figure 4a, by incubating P2-NBD micelles with A549 cells for 1 h, there is almost no blue emission from activated coumarin moieties due to the acidic milieu in endolysosomes and poor Michael addition reactivity; however, intense blue emission from decaged coumarin moieties can be observed after 6 or 12 h coincubation. The colocalization ratio between blue emission from activated coumarin moieties and red emission of LysoTracker Red remained low (Figure 4c). On the other hand, the green fluorescence from NBD moieties was observed at all incubation durations (1, 6, and 12 h) and colocalization ratio between LysoTracker red and NBD green channel decreased from 62.5% at 1 h incubation to 30.1% at 12 h incubation duration (Figure 4b). The decrease of colocalization ratio between green and red channels indicated partial endosomal escape of P2-NBD micelles. Besides, upon coincubation in the presence of chloroquine, a well-known endosome disrupting agent, uniform distribution of both blue emission of activated coumarin moieties and green emission of NBD residues throughout the whole cytoplasm, and complete overlap between these two channels can be clearly observed (Figure S14). This further indicated that endosomal escape into the cytosol is a prerequisite for the emission activation of caged coumarin residues in micellar nanoparticles. To evaluate the drug loading and controlled release profile of P2 micellar nanoparticles, they were loaded with hydrophobic

duration to 6 h, blue emission could be obviously discerned, which was further enhanced upon 12 h incubation. The incubation time-dependent blue emission changes suggested endosomal escape of micellar nanoparticles into cytosol, followed by effective Michael addition with thiol compounds (GSH) at neutral pH, micelle-to-unimer transition, and concomitant fluorescence turn-on. This series of spatiotemporal events was further confirmed by fluorescence colocalization studies, revealing no apparent overlap between the green emission of LysoTracker green and blue emission of activated coumarin moieties (Figure 3 and Figure S12a) for all incubation durations (1, 4, 6, and 12 h), and the green/blue channel colocalization ratios remained low (