Cytosol-specific fluorogenic reactions for visualizing intracellular disintegration of responsive polymeric nanocarriers and triggered drug release

Article abstract of DOI:10.1021/ma502389w

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 ?±,?2-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 micelle-to-unimer transition. This is accompanied by concomitant fluorescence emission turn-on and triggered drug release, allowing for the process visualization.

Full text of DOI:10.1021/ma502389w

Article
pubs.acs.org/Macromolecules
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 micelle-
to-unimer transition. This is accompanied by concomitant fluorescence emission turn-on
and triggered drug release, allowing for the process visualization.
response to external stimuli are more preferred due to low
background signals.
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 rearrange-
ment 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. Re-
cently, biocleavable small molecule fluorophore-drug con-
jugates have been designed to achieve image-guided drug
release.6 Although conventional dye-labeled polymeric nano-
carriers 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
We then reasoned that if biostimuli-triggered BCP nano-
carrier 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 thermo-
induced 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
© XXXX American Chemical Society
A
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Scheme 1. Self-Assembly of Thiol-Reactive Thermoresponsive PEG-b-P(MEO2MA-co-EEO2MA-co-PyCouMA) DHBCs (P1−
a
P4) and Intracellular Trafficking of Micellar Nanoparticles
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.
Reagent Co. Ltd. and used as received. Dialysis tube (MWCO 3.5 and
EXPERIMENTAL SECTION
■
14 kDa) was purchased from Shanghai Green Bird Technology
Materials. Di(ethylene glycol) monomethyl ether methacrylate
Development Co., Ltd., China, and stored in 1 mM ethyl-
(MEO2MA, Sigma-Aldrich) was passed through an alumina column to
enediaminetetraacetic acid (EDTA) aqueous solution prior to use.
remove the inhibitor. 3-Azidopropyl methacrylate (AzPMA) was
Chloroquine diphosphate salt purchased from Sigma Life Sciences was
dissolved in water at a concentration of 100 mM. 4-Chloro-7-
nitrobenzofurazan (NBD-Cl, 99%) was purchased from Alfa and used
prepared by the esterification reaction of acryloyl chloride with 3-
azidoprppan-1-ol.12 All purified monomers were stored at −20 °C
prior to use. 2-(2-Ethoxyethoxy)ethanol, propargyl alcohol, copper(I)
as received. All solvents were of analytical grade. PEG−Br macro-
initiator 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-2,1,3-
benzoxadiazole (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
bromide (CuBr), 2-bromoisobutyryl bromide, 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 1,1,4,7,7-
pentamethyldiethylenetriamine (PMDETA), 2-pyridinecarboxalde-
hyde, 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, phosphor-
usoxychloride (POCl3), piperidine, triethylamine (Et3N), 2-(2-
ethoxyethoxy)ethanol, glutathione (GSH), and doxorubicin hydro-
chloride (DOX·HCl) were purchased from Sinopharm Chemical
B
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Scheme 2. Synthetic Routes Employed for the Preparation of α,β-Unsaturated Ketone-Containing Precursor, (E)-7-(Prop-2-yn-
1-yloxy)-3-(3-(pyridin-2-yl)acryloyl)-2H-chromen-2-one (4), and PEG-b-P(MEO2MA-co-EEO2MA-co-AzPMA) and PEG-b-
P(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.
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 2-
hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde (2) (3.2 g, yield: 25.9%)
Sample Preparation. Synthetic schemes employed for the
preparation of α,β-unsaturated ketone-containing precursor (4) and
PEG-b-P(MEO2MA-co-EEO2MA-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 powder. H 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,
1
(1) as a white crystal (34 g, yield: 61.6%). H 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
C
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Table 1. Molecular Parameters of DHBCs Covalently Conjugated with α,β-Unsaturated Ketone-Caged Coumarin Moieties in
the Thermoresponsive Block
a
a
b
c
cd
,
entry
P1
samples
Mn (kDa)
Mw/Mn
Mn (kDa)
CMT/°C (GSH−) CMT/°C (GSH+)
PEO45-b-P(MEO2MA0.9-co-PyCMA0.1
)
42
10.3
9.4
1.17
1.21
1.23
1.19
1.18
10.9
10.6
11.3
11.8
36
32
27
20
∼90
84
P2
PEO45-b-P(MEO2MA0.81-co-EEO2MA0.09-co-PyCMA0.1
PEO45-b-P(MEO2MA0.71-co-EEO2MA0.19-co-PyCMA0.1
PEO45-b-P(MEO2MA0.65-co-EEO2MA0.25-co-PyCMA0.1
)
40
)
43
)
45
P3
10.7
10.4
75
P4
69
P2-NBD
PEO45-b-P(MEO2MA0.81-co-EEO2MA0.09-co-PyCMA0.1-NBD)40
9.5
10.6
32
84
a
b
c
1
Determined by GPC using THF as eluent at a flow rate of 1.0 mL/min. Calculated from H NMR analysis. Determined by temperature-
dependent optical transmittance at a wavelength of 600 nm, the CMT was defined as the temperature at which ∼1% decrease in the optical
d
transmittance can be discerned. CMT 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).
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 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-2-
1
yn-1-yloxy)coumarin (4) as a yellow powder (0.77 g, yield: 40%). H
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(EEO2MA-co-NBD)42 (P2-NBD). Typical
procedures employed for the ATRP synthesis of PEO45-b-
P(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 2-
propanol (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(EEO2MA-
co-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
Synthesis of PEO45-b-P(MEO2MA0.9-co-AzPMA0.1 42
)
and PEO45-b-
P(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
1
frequency of 300 MHz for H) 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
D
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
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
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).
temperature of 37.0
∼0.012 g/L.
Reactivity of 4 and P2 toward Thiols. The reactivity of small
0.2 °C. CMC of P2 was determined to be
1
molecule 4 was studied by H NMR and HPLC methods using 2-
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
mercaptoethanol 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 H 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 trans-
illumination, 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 encapsula-
tion efficiency (EE %) was calculated as
1
colocalization ratio % = [pixelscolocalization/pixelsP2] × 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 experi-
ments, A549 cells were treated with DMEM containing 0.4 g/L P2-
NBD 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.
EE% = [W − Wunloaded]/W × 100%
total
total
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
The loading content (LC %) was calculated as
LC% = [Wloaded]/[W + Wloaded] × 100%
polymer
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. DOX-
loaded 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.
E
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 1. (a) Hydrodynamic diameter distribution, f(Dh), recorded for the micellar dispersion of PEG-b-P(MEO2MA0.81-co-EEO2MA0.09-co-
PyCouMA0.1)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
1H 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
1
RESULTS AND DISCUSSION
(Figure 1b), UV−vis absorbance (Figures 2a,b), and H 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-to-
unimer 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 1-
mercaptopropane, 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.
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-b-
P(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 summar-
ized in Table 1. Temperature- dependent turbidity measure-
ments 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 intensity-
average 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).
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 measure-
ments 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
Next, the reactivity of α,β-unsaturated ketone functionality in
4 toward thiol compounds was examined. The addition
F
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
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.
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-mercap-
toethanol 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 prom-
inent 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
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 hydro-
philic 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 cytosol-
specific. 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
G
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
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).
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 (<20%). Meanwhile,
an abrupt blue channel emission increase was observed at
extended incubation time (6 and 12 h; Figures 3 and S12b). In
comparison, when cells were pretreated with N-ethylmaleimide,
which acts as a scavenger for intracellular thiol compounds, the
blue channel emission prominently decreased after 12 h
coincubation (Figure S13). This suggested that thiol- involved
Michael addition reactions are solely responsible for cytosolic
milieu-specific emission turn-on.
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.
Because coumarin moieties in P2 micelles remained in the
caged nonfluorescent state within acidic endolysosomes, it was
difficult to locate their exact distribution before endosomal
escape. We then synthesized NBD-labeled P2 (P2-NBD) to
further verify the cytosolic milieu-specific nature of emission
turn-on and micellar disassembly for P2 micelles. The green
To evaluate the drug loading and controlled release profile of
P2 micellar nanoparticles, they were loaded with hydrophobic
H
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 4. (a) CLSM images recorded for A549 cells after coincubation at 37 °C with P2- NBD micellar solution (0.4 g/L) for 1, 6, and 12 h. Late
endosomes and lysosomes were stained with LysoTracker Red (red). The blue and green channel fluorescence emissions were originated from
decaged PyCouMA and NBD moieties, respectively. (b and c) Colocalization ratio analysis (b) between green channel fluorescence from NBD
moieties and red channel fluorescence of stained late endosomes/lysosomes (LysoTracker Red), and (c) between blue channel fluorescence from
decaged PyCouMA moieties and red channel fluorescence of stained late endosomes/lysosomes (LysoTracker Red).
Nile red as a model drug and the controlled release profile was
evaluated. Upon addition of 10 mM GSH, the fluorescence of
Nile red decreased gradually within ∼140 min (Figure S15a),
which is in accord with GSH-triggered disassembly of micellar
nanoparticles (Figure 2). Interestingly, the controlled release
behavior could also be discerned by naked eyes under both
visible light and UV irradiation (Figures S15b and S15c). A
colorimetric transition from reddish to colorless and a
fluorescence transition from red to blue were observed due
to Nile red release and fluorescence turn-on of caged coumarin
moieties. The concomitant thiol-triggered guest molecule
release and dramatic emission turn-on endows this system
with the unique capability of in situ fluorogenically monitoring
intracellular nanocarrier disintegration and drug release
processes.
By using doxorubicin (DOX) as a model therapeutic drug,
we further explored GSH- responsive drug release and
intracellular emission turn-on and disassembly for P2 micelles.
The DOX loading content was determined to be ∼10.1 w/w%.
Notably, DOX release at pH 7.4 was quite slow without GSH
or at low GSH levels (i.e., 10 μM) and less than ∼10% drug
release was observed within ∼150 min. Dramatically enhanced
drug release was achieved at relatively high GSH levels, i.e.,
∼73.5% and 92.6% DOX release at 5 mM and 10 mM GSH,
respectively (Figure 5a). The above difference can be safely
ascribed to the GSH-regulated micellar disintegration process
(Figure 2), as further evidenced by GSH-dependent emission
changes recorded for Nile red-loaded micelles (Figure S15).
Figure S16 shows time- dependent evolution of both
fluorescence emission intensity and drug release profiles for
DOX-loaded P2 micelles upon incubating with GSH at varying
concentrations. When the cumulative DOX release contents
were plotted against normalized emission intensities, it is
intriguing to note that at GSH concentrations of 5 and 10 mM,
which is quite comparable to that in the cytosol milieu, the
correlation is almost linear (Figure 6). These results further
confirmed that the current system can monitor the micellar
disintegration and triggered drug release processes via thiol-
actuated fluorogenic events (Scheme 1). Previously, Boyer and
Davis et al.17 employed fluorescence lifetime microscopy
(FLIM) technique to in situ monitor intracellular drug release.
It is worthy of noting that the strategy of synergistic drug
release/emission turn-on in this work is unique considering that
it can report the fate and evolution of both encapsulated drugs
and delivery nanocarriers.
In vitro cytotoxicity evaluation against Hela cells revealed that
DOX-loaded P2 micelles were more cytotoxic and efficient
than free DOX (Figure 5b); whereas over 80% cancer cells
were killed at an equivalent DOX concentration of ∼4 μg/mL.
Finally, the intracellular delivery and release of DOX were then
monitored. As shown in Figure 5c, the blue channel intensity
ascribing to activated coumarin moieties continuously increased
I
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 5. (a) In vitro drug release profile recorded for DOX-loaded P2 micelles at varying GSH levels (pH 7.4 buffer; 37 °C). (b) MTT-based
cytotoxicity assay against Hela cells after coincubating with free DOX or DOX-loaded P2 micellar solution. (c) CLSM images recorded for A549
cells after coincubation at 37 °C with DOX-loaded P2 micellar solution for 1, 6, and 12 h. Late endosomes and lysosomes were stained with
LysoTracker Green (green channel). The red and blue channel fluorescence emissions were originated from DOX and decaged PyCouMA moieties,
respectively.
micellar disintegration triggered by extensive Michael addition
reactions at neutral pH and high thiol levels. Notably, after 12 h
incubation, DOX fluorescence mainly located within the
cellular nucleus and exhibited an overall 3.3-fold increase in
emission intensities (Figures 5c and S17b). To verify that
intracellular DOX release is due to cytosol-specific fluorogenic
reactions associated with micellar disassembly, we further
synthesized NBD-labeled thiol-unreactive PEO45-b-P-
(EEO2MA-co-NBD)42 dibilock copolymer (see Supporting
Information for details). Upon coincubation with A549 cells
for 12 h in the presence of DOX-loaded PEO45-b- P(EEO2MA-
co-NBD)42 micelles, colocalization of NBD-labeled copolymer
Figure 6. Evolution of cumulative DOX release contents as a function
matrix and DOX was observed and no nucleus entry can be
of normalized emission intensities recorded for DOX-loaded P2
apparently discerned (Figure S19), which is in stark contrast to
micelles upon incubating with GSH at concentrations of 5 and 10 mM,
P2-NBD micelles.
respectively (pH 7.4 buffer; 37 °C).
CONCLUSIONS
■
in the entire incubation duration. In comparison to the
In summary, thiol-reactive thermoresponsive DHBCs labeled
with α,β-unsaturated ketone moieties were synthesized. They
self-assemble into micelles consisting of thiol-reactive cores and
hydrophilic PEG coronas at temperatures higher than the
CMT. Micellar nanocarriers could be solely triggered to
disintegrate in the presence of GSH at neutral pH by virtue
of thiol-mediated Michael addition reactions under the specific
cytosolic milieu following cellular uptake; notably, this process
negligible blue emission at 1 h incubation, quantitative analysis
revealed ∼60-fold increase after 12 h incubation (Figure S17a).
Additionally, the colocalization between DOX red channel and
LysoTracker green channel prominently decreased from
∼77.4% at 1 h incubation to 18.6% at ∼12 h incubation
duration (Figure S18), suggesting that most DOX-loaded
micelles have escaped from acidic organelles and entered into
the cytosol, where effective DOX release occurred due to
J
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
H.; Canton, I.; MacNeil, S.; Du, J.; Armes, S. P.; Ryan, A. J.; Lewis, A.
L.; Battaglia, G. Adv. Mater. 2007, 19, 4238. (j) Jia, Z. F.; Liu, J. Q.;
Boyer, C.; Davis, T. P.; Bulmus, V. Biomacromolecules 2009, 10, 3253−
3258. (k) Rao, J.; Hottinger, C.; Khan, A. J. Am. Chem. Soc. 2014, 136,
5872−5875. (l) Liu, G.; Hu, J.; Zhang, G.; Liu, S. Bioconjugate Chem.
2015, DOI: 10.1021/bc500548r. (m) Hu, X.; Liu, G.; Li, Y.; Wang, X.;
Liu, S. J. Am. Chem. Soc. 2015, DOI: 10.1021/ja5105848. (n) Li, Y. M.;
Yu, H. S.; Qian, Y. F.; Hu, J. M.; Liu, S. Y. Adv. Mater. 2014, 26, 6734−
6741.
(6) (a) Bhuniya, S.; Lee, M. H.; Jeon, H. M.; Han, J. H.; Lee, J. H.;
Park, N.; Maiti, S.; Kang, C.; Kim, J. S. Chem. Commun. 2013, 49,
7141−7143. (b) Lee, M. H.; Han, J. H.; Kwon, P. S.; Bhuniya, S.; Kim,
J. Y.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2012, 134,
1316−1322. (c) Lee, M. H.; Kim, J. Y.; Han, J. H.; Bhuniya, S.; Sessler,
J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2012, 134, 12668−12674.
(d) Lee, M. H.; Yang, Z.; Lim, C. W.; Lee, Y. H.; Dongbang, S.; Kang,
C.; Kim, J. S. Chem. Rev. 2013, 113, 5071−5109. (e) Santra, S.;
Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. J. Am. Chem. Soc. 2011,
133, 16680−16688. (f) Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.;
James, T. D.; Tian, H.; Zhu, W. J. Am. Chem. Soc. 2014, 136, 3579−
3588. (g) Yuan, Y. Y.; Kwok, R. T. K.; Feng, G. X.; Liang, J.; Geng, J.
L.; Tang, B.; Liu, B. Chem. Commun. 2014, 50, 295−297.
(7) Hu, X. L.; Hu, J. M.; Tian, J.; Ge, Z. S.; Zhang, G. Y.; Luo, K. F.;
Liu, S. Y. J. Am. Chem. Soc. 2013, 135, 17617−17629.
is accompanied by dramatic fluorescence emission turn-on. The
cytosol-specific fluorogenic nature confers this system with the
advantage of in situ monitoring micellar transport/disintegra-
tion and triggered drug release processes, which represents a
potent strategy for designing next-generation image-guided
drug delivery nanocarriers.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR, FT-IR, GPC, plots of temperature
dependence, mass spectra, fluorescence spectra, CLSM images,
and additional characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(S.L.) E-mail: sliu@ustc.edu.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the National Natural Scientific
Foundation of China (NNSFC) Project (21274137,
51273190, 5147315 and 51033005) and the Specialized
Research Fund for the Doctoral Program of Higher Education
(SRFDP, 20123402130010) is gratefully acknowledged.
(8) (a) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666−
676. (b) Yusop, R. M.; Unciti-Broceta, A.; Johansson, E. M. V.;
Sanchez-Martin, R. M.; Bradley, M. Nat. Chem. 2011, 3, 239−243.
(9) (a) Dai, J.; Lin, S. D.; Cheng, D.; Zou, S. Y.; Shuai, X. T. Angew.
Chem., Int. Ed. 2011, 50, 9404−9408. (b) Kim, H.; Kim, S.; Park, C.;
Lee, H.; Park, H. J.; Kim, C. Adv. Mater. 2010, 22, 4280. (c) Liu, J. Y.;
Pang, Y.; Huang, W.; Huang, X. H.; Meng, L. L.; Zhu, X. Y.; Zhou, Y.
F.; Yan, D. Y. Biomacromolecules 2011, 12, 1567−1577. (d) Ryu, J. H.;
Roy, R.; Ventura, J.; Thayumanavan, S. Langmuir 2010, 26, 7086−
7092. (e) Sun, H. L.; Guo, B. N.; Li, X. Q.; Cheng, R.; Meng, F. H.;
Liu, H. Y.; Zhong, Z. Y. Biomacromolecules 2010, 11, 848−854.
(10) (a) Lin, W. Y.; Yuan, L.; Cao, Z. M.; Feng, Y. M.; Long, L. L.
Chem.Eur. J. 2009, 15, 5096−5103. (b) Yi, L.; Li, H. Y.; Sun, L.; Liu,
L. L.; Zhang, C. H.; Xi, Z. Angew. Chem., Int. Ed. 2009, 48, 4034−4037.
(11) Jiang, X.; Lavender, C. A.; Woodcock, J. W.; Zhao, B.
Macromolecules 2008, 41, 2632−2643.
REFERENCES
■
(1) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347−360.
(2) (a) Farokhzad, O. C.; Langer, R. ACS Nano 2009, 3, 16−20.
(b) Peer, D.; Karp, J. M.; Hong, S.; FarokHzad, O. C.; Margalit, R.;
Langer, R. Nat. Nanotechnol. 2007, 2, 751−760.
(3) (a) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Angew. Chem.,
Int. Ed. 2003, 42, 4640−4643. (b) Elsabahy, M.; Wooley, K. L. Chem.
Soc. Rev. 2012, 41, 2545−2561. (c) Kakizawa, Y.; Kataoka, K. Adv.
Drug Delivery Rev. 2002, 54, 203−222. (d) Zhuang, J. M.; Chacko, R.;
Torres, D. F. A.; Wang, H.; Thayumanavan, S. ACS Macro Lett. 2014,
3, 1−5.
(12) Sumerlin, B. S.; Tsarevsky, N. V.; Louche, G.; Lee, R. Y.;
Matyjaszewski, K. Macromolecules 2005, 38, 7540−7545.
(4) (a) Wang, X. R.; Liu, G. H.; Hu, J. M.; Zhang, G. Y.; Liu, S. Y.
Angew. Chem., Int. Ed. 2014, 53, 3138−3142. (b) Wang, X. R.; Hu, J.
M.; Zhang, G. Y.; Liu, S. Y. J. Am. Chem. Soc. 2014, 136, 9890−9893.
(c) Liu, G. H.; Wang, X. R.; Hu, J. M.; Zhang, G. Y.; Liu, S. Y. J. Am.
Chem. Soc. 2014, 136, 7492−7497. (d) Ghosh, S.; Irvin, K.;
Thayumanavan, S. Langmuir 2007, 23, 7916−7919. (e) Lee, H. I.;
Wu, W.; Oh, J. K.; Mueller, L.; Sherwood, G.; Peteanu, L.;
Kowalewski, T.; Matyjaszewski, K. Angew. Chem., Int. Ed. 2007, 46,
2453−2457. (f) Schopf, E.; Sankaranarayanan, J.; Chan, M. N.;
Mattrey, R.; Almutairi, A. Mol. Pharmaceutics 2012, 9, 1911−1918.
(g) Hu, J. M.; Zhang, G. Y.; Ge, Z. S.; Liu, S. Y. Prog. Polym. Sci. 2014,
39, 1096−1143. (h) Hu, J. M.; Liu, S. Y. Acc. Chem. Res. 2014, 47,
2084−2095. (i) Li, Y. M.; Hu, X. L.; Tian, S. D.; Li, Y.; Zhang, G. Q.;
Zhang, G. Y.; Liu, S. Y. Biomaterials 2014, 35, 1618−1626. (j) Hu, X.
L.; Li, Y.; Liu, T.; Zhang, G. Y.; Liu, S. Y. Sci. ChinaChem. 2014, 57,
615−623. (k) Hu, J. M.; Liu, G. H.; Wang, C.; Liu, T.; Zhang, G. Y.;
Liu, S. Y. Biomacromolecules 2014, 15, 4293−4301. (l) Liu, T.; Zhang,
Y. F.; Liu, S. Y. Chin. J. Polym. Sci. 2013, 31, 924−937.
(13) Ishizu, K.; Makino, M.; Uchida, S. Macromol. Rapid Commun.
2007, 28, 882−887.
(14) Uchiyama, S.; Matsumura, Y.; de Silva, A. P.; Iwai, K. Anal.
Chem. 2003, 75, 5926−5935.
(15) Hong, R.; Han, G.; Fernandez, J. M.; Kim, B. J.; Forbes, N. S.;
Rotello, V. M. J. Am. Chem. Soc. 2006, 128, 1078−1079.
(16) Moskaug, J. O.; Sandvig, K.; Olsnes, S. J. Biol. Chem. 1987, 262,
10339−10345.
(17) Duong, H. T. T.; Hughes, F.; Sagnella, S.; Kavallaris, M.;
Macmillan, A.; Whan, R.; Hook, J.; Davis, T. P.; Boyer, C. Mol.
Pharmaceutics 2012, 9, 3046−3061.
(5) (a) Ge, Z. S.; Liu, S. Y. Chem. Soc. Rev. 2013, 42, 7289−7325.
(b) Gillies, E. R.; Frechet, J. M. J. Bioconjugate Chem. 2005, 16, 361−
368. (c) Hu, J. M.; Zhang, G. Q.; Liu, S. Y. Chem. Soc. Rev. 2012, 41,
5933−5949. (d) Ma, N.; Li, Y.; Xu, H. P.; Wang, Z. Q.; Zhang, X. J.
Am. Chem. Soc. 2010, 132, 442−443. (e) Roy, D.; Cambre, J. N.;
Sumerlin, B. S. Prog. Polym. Sci. 2010, 35, 278−301. (f) Schattling, P.;
Jochum, F. D.; Theato, P. Polym. Chem. 2014, 5, 25−36. (g) Zhuang, J.
M.; Gordon, M. R.; Ventura, J.; Li, L. Y.; Thayumanavan, S. Chem. Soc.
Rev. 2013, 42, 7421−7435. (h) Agut, W.; Brulet, A.; Schatz, C.; Taton,
D.; Lecommandoux, S. Langmuir 2010, 26, 10546−10554. (i) Lomas,
K
DOI: 10.1021/ma502389w
Macromolecules XXXX, XXX, XXX−XXX