Reversible core-crosslinked nanocarriers with pH-modulated targeting and redox-controlled drug release for overcoming drug resistance

Article abstract of DOI:10.1039/c7tb01504f

Herein, a pH and redox dual-sensitive core-crosslinked targeting nanocarrier was prepared and used for co-delivery of doxorubicin (DOX) and tariquidar (TQR). The nanocarrier not only had excellent stability but also prevented the leakage of the drug in the normal physiological environment efficiently. Meanwhile, the targeting function of nanocarriers could also be suppressed in the normal physiological environment, protecting nanocarriers from being captured by RAW264.7 cells. Under mild acidic conditions, the targeting function was regained, leading to an effective tumor cell uptake of the nanocarrier. Furthermore, reduction-responsive drug release would occur in the cytoplasm due to the collapse of the reduction-sensitive crosslinked structure in the nanocarrier. By means of ligand-receptor mediated endocytosis and TQR-mediated glycoprotein (P-gp) inhibition, the IC₅₀ value of DOX to MCF-7/ADR cells reduced from more than 100 μg mL^-1 to 8.55 μg mL^-1, exhibiting great potential in overcoming drug resistance.

Full text of DOI:10.1039/c7tb01504f

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ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
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ARTICLE
Dual Drug-Loaded Reversible Core-Crosslinked nanocarriers with
pH-Modulated Targeting and Redox-Controlled Drug Release for
Overcoming Drug Resistance
Received 00th January 20xx,
Dan Zhao, Shujie Ma, Xiaoqing Yi, Sixue Cheng, Renxi Zhuo, Feng Li*
Herein, a pH and redox dual-sensitive core-crosslinked targeting nanocarrier was prepared and used for co-delivery of
doxorubicin (DOX) and tariquidar (TQR). The nanocarrier not only owned an excellent stability but also prevented the
leakage of drug in normal physiological environment efficiently. Meanwhile, the targeting function of nanocarriers could
also be suppressed in normal physiological environment, protecting the nanocarriers from being captured by RAW264.7
cells. Under the mild acidic condition, the targeting function was regained, leading to an effective tumor cell uptake of the
nanocarrier. Furthermore, reduction-responsive drug release would occur in cytoplasm due to the collapse of reduction-
sensitive crosslinked structure in the nanocarrier. By means of the ligand-receptor mediated endocytosis and TQR-
mediated glycoprotein (P-gp) inhibition, the IC50 value of DOX to MCF-7/ADR cells reduced from more than 100 μg/mL to
8.55 μg/mL, exhibiting a great potential in overcoming drug resistance.
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
nanoparticles has
a great potential in overcoming MDR by
Introduction
endosomal transportation, thus evading the recognition of Pꢀgp and
reducing the drug efflux11,12. In our previous studies, we prepared a
smart nanocarrier in which the targeting function of the nanocarrier
could be hidden in normal physiological environment but recovered
under tumor acidic condition13. The smart drugꢀloaded nanoparticles
were expected to enhance the specific enrichment of
chemotherapeutics and further increase the toxicity to drug resistant
cells by pHꢀactivated targeting.
Doxorubicin (DOX) is a commonly used chemotherapy drug in the
clinic as it shows an excellent antitumor activity by inserting into
DNA1,2. However, the undesirable biodistribution and poor solubility
of DOX may lead to lifeꢀthreaten cardiac toxicity. Moreover, its low
drug bioavailability and limited efficacy may also cause multidrug
resistance (MDR) that severely restricts the utilization of
chemotherapeutic drugs and is a key determinant for treatment
failure in cancer chemotherapy3ꢀ5. A major mechanism responsible
for the acquired MDR is the increased expression of Pꢀglycoprotein
(Pꢀgp), an ATPꢀbinding cassette (ABC) protein that can effectively
reduce the intracellular drug concentration by increasing the energyꢀ
dependent drug efflux6,7. Meanwhile, Pꢀgp also exists naturally in
many normal tissues, such as liver, kidney and brain, playing a key
role in preventing the entry of poisons into the vital organs or
regulating the absorption and excretion of drugs8ꢀ10. Therefore, direct
use of Pꢀgp inhibitor, i.e. tariquidar (TQR), may cause a serious
systemic toxicity. The emergence of smart nanoparticles may
provide a promise to solve these problems, as they can help the
drugs to accumulate into cancer cells efficiently by passive or active
targeting effect. In addition, it has been reported that targeted
Nevertheless, the application of our drugꢀloaded nanocarrier in
cancer therapy also faces other limitations. The primary one was
their premature disintegration during systematic circulation induced
by the infinite dilution of body fluids14,15. One simple and effective
strategy to overcome this problem was the crosslinking of
nanocarriers by which the stability of nanocarriers could be greatly
improved. It was reported that stimuliꢀresponsive crosslinked
micelles not only kept superior stability upon dilution but also
released drugs rapidly under stimulations at target site16,17. Many
stimuli like pH18, light19,20 and redox potential21ꢀ23, have been
introduced to manage the degradation of crosslinked structure and
drug release. Among these internal or external stimuli, glutathione
(GSH) has attracted great attentions because the concentration of
GSH in cancer cells is 2ꢀ10 mM, which is 50ꢀ1000 fold to that in
extracellular milieu or body fluids, inducing the rapid cleavage of
reductionꢀsensitive crosslinked structure in tumor cells. For example,
Wang and coworkers24 prepared redoxꢀsensitive shell crosslinked
micelles that could hold their structure in several different
environments but be destructed in the presence of GSH. In addition,
our group also reported a redoxꢀresponsive core crosslinked micelle
for intracellular drug delivery25. Even with 200 fold dilution, the
crosslinked micelles still showed excellent stability and prevented
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previous
report13.
1ꢀ(3,5ꢀBis(trifloromethyl)phenyl)ꢀ3ꢀ
drug leakage efficiently. While internalized into cancer cells, the
micelles could collapse rapidly.
cyclohexylthiourea (TU) was synthesized following the procedure
described in the literature26. Ethyl chloroformate was distilled before
use. Doxorubicin hydrochloride (DOX•HCl) was purchased from
Zhejiang Hisun Pharmaceutical Co., Ltd. (China). Tariquidar (TQR)
was purchased from Melone Pharmaceutical Co., Ltd. (China).
Bovine serum albumin (BSA), Dubelcco’s Modified Eagle’s
Medium (DMEM), penicillinꢀstreptomycin, trypsin, phosphateꢀ
buffered saline (PBS), fetal bovine serum (FBS), Hoechst 33342 and
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT)
were purchased from GIBCO Invitrogen Corporation.
Herein, we designed a nanocarrier with enhanced performance based
on our previous work (Scheme 1), which still retained the advantage
of our former nanocarrier, i.e., selfꢀhidden but pHꢀreversed targeting.
On the other hand, the nanocarrier was endowed with other
performances in antiꢀdilution, improved drugꢀloading capacity and
controlled drug release by introducing a redox sensitive coreꢀ
crosslinking via DTTꢀcatalyzed disulfide exchange reaction. When
the nanocarrier was in the normal physiological environment, it
could avoid the recognition of macrophages and suppress the
premature drug leakage. While in the mimic tumor
microenvironment, the active targeting function of the nanocarrier
would regained, improving the selective recognition and uptake of
tumor cells. After internalization, the crosslinked structure of the
nanocarrier was destroyed in cytoplasm, resulting in the drug release
within tumor cells. Further, coꢀdelivery a small amount of Pꢀgp
inhibitor TQR with DOX could lead to a significant inhibition of
drug resistance.
Synthesis of materials
2-(Bromomethyl)-2-methylpropane-1,3–diol. According to the
report27, 1.19 g (11.3 mmol) 3ꢀmethylꢀ3ꢀoxetanemethanol was
dissolved into dried THF at 0 °C, then 4.8 mL HBr (40%) was added
dropwise. After 4 h, 10 mL H2O was added into the mixed solution.
The diluted solution was extracted with diethyl ether and then the
organic phase was dried over MgSO4. Finally, a white solid was
obtained by the elimination of organic solvent. Yield: 2.0 g (93.7%).
1H NMR (CDCl3, 300 MHz) δ: 3.66 (dd, 4H), 3.54 (s, 4H), 0.93 (s,
3H). 13C NMR (CDCl3, 75 MHz) δ: 67.99, 40.56, 39.14, 18.29.
2-(Azidomethyl)-2-methylpropane-1,3-diol. The similar synthesis
procedure of 2ꢀ(azidomethyl)ꢀ2ꢀmethylpropaneꢀ1,3ꢀdiol was used28.
0.45 g (2.7 mmol) KI and 5.3 g (81.9 mmol) NaN3 were added into
the THF solution of 5.0
g (27.3 mmol) 2ꢀ(bromomethyl)ꢀ2ꢀ
methylpropaneꢀ1,3ꢀdiol, and then the solution was kept stirring for
48 h at 80 °C. After the reaction, the mixture was partitioned
between 150 mL ethyl acetate and 150 mL H2O. The aqueous phase
was extracted with ethyl acetate. The combined organic phase was
dried with MgSO4. After the removal of organic solvent, the residue
was recrystallized from diethyl ether to obtain a white solid. Yield:
3.46 g (87.3%). 1H NMR (CDCl3, 300 MHz): δ: 3.66 (dd, 4H), 3.54
(s, 4H), 0.93 (s, 3H). 13C NMR (CDCl3, 75 MHz) δ: 67.99, 55.48,
39.14, 18.29.
5-(Azidomethyl)-5-methyl-1,3-dioxan-2-one (AMPC). To the
solution of dried THF with 2.0 g (13.8 mmol) 2ꢀ(azidomethyl)ꢀ2ꢀ
methylpropaneꢀ1,3ꢀdiol and 2.25 g (20.7 mmol) ethyl chloroformate
at 0 °C, a solution of dried THF with 4.4 mL Et3N was added
dropwise. The mixed solution was further stirred for 4 h at 0 °C and
then filtered. The filtrate was concentrated and the residue was
recrystallized from THF to obtain a tabular crystal. Yield: 2.18 g
(91.5%). 1H NMR (CDCl3, 300 MHz): δ: 4.28ꢀ4.11 (dd, 4H), 3.38 (s,
2H), 1.12 (s, 3H).
Scheme 1. The possible mechanism of pH-reversed targeting and
GSH triggered drug release to overcome drug resistance.
5-(1,2-Dithiolan-3-yl)-N-(prop-2-yn-1-yl)pentanamide
Experimental
(PTA). To the solution of dried CH2Cl2 with 0.5 g (2.4 mmol)
thioctic acid, 0.34 g (2.9 mmol) NHS and 0.59 g (2.9 mmol)
DCC, a solution of 2ꢀpropargylamine (0.16 g, 2.9 mmol) in
CH2Cl2 was added dropwise. After reaction at room
temperature for 24 h, the solution was filtered and the filtrate
was diluted by 100 mL CH2Cl2 and washed with saturated
brine. A crude product was obtained by the removal of solvent
Materials
NꢀHydroxysuccinimide (NHS), propargyl alcohol, 3ꢀmethylꢀ3ꢀ
oxetanemethanol, 4ꢀ(dimethylamino) pyridine (DMAP), succinic
anhydride and N, N’ꢀdicyclohexylcarbodiimide (DCC) and
hydrobromic acid (40%, SCRC) were obtained from Sinopharm
Chemical Reagent Co., Ltd. 2ꢀPropargylamine, 2,2'ꢀbipyridine and
CuBr were obtained from Aladdinꢀreagent (China). 1,5ꢀ
Diaza(5,4,0)undecꢀ5ꢀene (DBU) was obtained from Energy
Chemical (China). Thioctic acid and DLꢀdithiothreitol (DTT) were
obtained from SigmaꢀAldrich. PBAꢀPEGꢀCD, PEGꢀCD, Adꢀ
lys(Diol)ꢀN3 and AlkynylꢀPCL were synthesized according to our
and
further
purified
by
column
chromatography
(CH2Cl2/methanol: 10/1) to obtain a yellow solid. Yield: 0.513
1
g (87.8%). H NMR (CDCl3, 300 MHz): δ: 5.68 (s, 1H), 4.07
(m, 2H), 3.59ꢀ3.55 (m, 1H), 3.20ꢀ3.11 (m, 2H), 2.49ꢀ2.43 (m,
2 | J. Name., 2012, 00, 1-3
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1H), 2.24ꢀ2.22 (m, 3H), 1.94ꢀ1.88 (m, 1H), 1.70ꢀ1.65 (m, 4H), The preparation of PBAꢀPEGꢀCD/Adꢀlys(Diol)ꢀPCLꢀTA
1.58ꢀ1.37 (m, 2H).
(PBAꢀTA) and PEGꢀCD/Adꢀlys(Diol)ꢀPCLꢀTA (PEGꢀTA)
conjugates was carried out according to our previous report13.
To a solution of DMF with 12.8 mg (2.0 mmol) PBAꢀPEGꢀCD
(or 12.4 mg (2.0 mmol) PEGꢀCD), 2 mL DMF solution of 4.1
mg (2.0 mmol) Adꢀlys(Diol)ꢀPCLꢀTA was added dropwise.
After stirred under Ar condition for 24 h at 35 °C, the mixed
solution was poured into a dialysis bag (MWCO = 3500 Da),
and dialyzed against with DI water, then filtered. The filtrate
was freezeꢀdried to obtain the conjugates.
5-(1,2-Dithiolan-3-yl)-N-(1-((5-methyl-2-oxo-1,3-dioxan-5-
yl)methyl)-1-H-1,2,3-triazol-4-yl)pentanamide
(AMPC-
PTA). By means of a click reaction between AMPC and PTA,
AMPCꢀPTA was obtained. Briefly, 0.45 g (2.6 mmol) AMPC
and 0.64 g (2.6 mmol) PTA were dissolved in 2 mL of dried
CH2Cl2. After three freezeꢀpumpꢀthaw cycles, 0.037 g (0.26
mmol) CuBr and 0.082 g (0.26 mmol) 2,2'ꢀbipyridine were
added. After reaction for 72 h at 35 °C under Ar atmosphere, a
yellow crystal of AMPCꢀPTA was obtained by purification of Determination of the critical aggregation concentration
column chromatography (CH2Cl2/methanol: 10/1) and further (CAC)
1
recrystallization from diethyl ether. Yield: 0.61 g (56.3%). H
The critical aggregation concentration (CAC) of the conjugates
NMR (CDCl3, 300 MHz): δ: 7.59 (s, 1H), 6.16 (s, 1H), 4.52ꢀ
4.29 (dd, 4H), 4.05 (s, 2H), 3.83 (s, 2H), 3.56 (m, 1H), 3.16 (m,
fluorescence probe. Briefly, to the solution of PEGꢀP(AMPCꢀgꢀ
2H), 2.44 (m, 1H), 2.24ꢀ2.18 (m, 2H), 1.89 (m, ) 3.56 (m, 1H),
3.20ꢀ3.11 (m, 2H), 2.49ꢀ2.43 (m, 1H), 2.21 (m, 3H), 1.89 (m,
gradient, pyrene was added and its concentration was fixed at
6×10ꢀ7 mol/L. The fluorescence spectra of the solutions were
1H), 1.67 (m, 4H), 1.45 (m, 2H), 0.96 (s, 3H).
and PEGꢀP(AMPCꢀgꢀPTA) was determined using pyrene as a
PTA), PBAꢀTA or PEGꢀTA with a certain concentration
PEG-P(AMPC-g-PTA).
PEGꢀP(AMPCꢀgꢀPTA)
was recorded by a FLS920 fluorescence spectrometer. The
synthesized by a ringꢀopening polymerization (ROP) of excitation wavelength was set as 334 nm. The fluorescence
AMPCꢀPTA in dried DCM by using DBU and TU as catalysts emissions from 340 to 500 nm were recorded. The CMC was
and mPEG as initiator. Briefly, 0.1 g (0.02 mmol) mPEG, 0.08 determined as the crossꢀpoint when extrapolating the intensity
g (0.2 mmol) AMPCꢀPTA, 1.86 ꢁL (0.012 mmol) DBU and ratio I394/I378 at the concentration regions.
4.13 mg (0.012 mmol) TU were added into a Schlenk flask
under Ar atmosphere, and then 2 mL of dried DCM was added.
After three freezeꢀpumpꢀthaw cycles, the mixture was stirred in
an oil bath at 35 °C for 24 h. After that, the solvent was
method. Briefly, to a solution of DMF with 200 mg PEGꢀ
removed by rotary evaporation and the residue was redissolved
into DMSO and dialyzed against deionized water for 48 h
Preparation and characterization of crosslinked (CL) mixed
micelles
Firstly, uncrosslinked (UCL) micelles were prepared by dialysis
P(AMPCꢀgꢀPTA) and 22 mg PBAꢀTA or PEGꢀTA, DI water
was added dropwise. The mixture was dialyzed against DI
(cutoff Mw 3500).
A
white solid was obtained by
water to remove DMF for 12 h at room temperature.
Afterwards, the solution was filtered and freezeꢀdried to obtain
the UCL mixed micelles. The CL mixed micelles were obtained
via DTTꢀcatalyzed disulfide exchange reaction by further
introducing excess DTT into UCL micellar solution: 100 mg
UCL PEGꢀP(AMPCꢀgꢀPTA)/PBAꢀTA or PEGꢀP(AMPCꢀgꢀ
PTA)/PEGꢀTA was dissolved in 50 mL DI water, then 11.6 mg
DTT was added and the mixture was stirred under Ar
atmosphere for 24 h, and then poured into a dialysis bag
(MWCO = 3500 Da) to dialyzed against with DI water for 48 h.
After lyophilization, the freezeꢀdrying powder of crosslinked
lyophilization. Yield: 0.11 g (59.3%).
Alkynyl-PCL-TA. To the solution of dried CH2Cl2 with 0.5 g
(0.38 mmol) alkynylꢀPCL, 0.095 g (0.46 mmol) thioctic acid
and 0.052 g (0.46 mmol) NHS, a CH2Cl2 solution of 0.095 g
(0.46 mmol) DCC was added dropwise. After 48 h under Ar
atmosphere, the solution was filtered and the filtrate was
concentrated by rotary evaporation. The residue was poured
into excess methanol. The white precipitate was collected and
1
dried under vacuum. Yield: 0.49 g (85.5%). H NMR (CDCl3,
300 MHz): δ: 4.62 (s, 2H), 4.01 (m, 30H), 3.52ꢀ3.44 (m, 1H),
3.12ꢀ3.04 (m, 2H), 2.42 (s, 1H), 2.31 (s, 1H), 2.24ꢀ2.22 (m,
30H), 1.87 (m, 1H), 1.62ꢀ1.55 (m, 60H), 1.42ꢀ1.35 (m, 4H),
1.33ꢀ1.28 (m, 32H).
PEGꢀP(AMPCꢀgꢀPTA)/PBAꢀTA
or
PEGꢀP(AMPCꢀgꢀ
PTA)/PEGꢀTA mixed micelles were obtained.
The particle size of the crosslinked mixed micelles was
determined by dynamic light scattering (DLS) on NanoꢀZSZEN
3600 (Malvern, UK) instrument. The characterization of
morphology was performed with a FEI Tecnai G2 20 TWIN
instrument operated at an acceleration voltage of 200 KV.
Ad-lys(Diol)-PCL-TA. 91 mg (0.06 mmol) alkynylꢀPCLꢀTA
and 50.0 mg (0.088 mmol) Adꢀlys(Diol)ꢀN3 were added into a
Schlenk flask with 2 mL dried DCM. After three freezeꢀpumpꢀ
thaw cycles, 15.2 mg (0.088 mmol) PMDETA and 12.6 mg
(0.088 mmol) CuBr were added. After 72 h at 35 °C under Ar
atmosphere, the mixed solution was poured into excess cold
Drug loading and redox-triggered in vitro release
methanol for precipitation of Adꢀlys(Diol)ꢀPCLꢀTA. Yield: The drug loaded CL mixed micelles were prepared by dialysis
0.11 g (88.7%).
method similar to that of blank CL micelles. Briefly, 15 mg
DOXꢂHCl, 15 ꢁL Et3N and 1.5 mL solution of DMSO with
TQR (1 mg/mL) was added into 5 mL DMSO, and the solution
was stirring in the dark for 4 h. Then 100 mg UCL micelle, 11.6
mg DTT and 100 mL DI water were added in turn. After
Preparation of PBA-PEG-CD/Ad-lys(Diol)-PCL-TA (PBA-
TA)
and
PEG-CD/Ad-lys(Diol)-PCL-TA
(PEG-TA)
conjugates
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stirring under Ar condition for 12 h, the mixture was dialyzed replaced by fresh medium, and 20 ꢁL of MTT (5 mg/mL)
against with DI water, then filtered by a 0.45 ꢁm filter and the solution was added carefully. After 4 h, the medium was moved
filtrate was freezeꢀdried to get the drug loaded CL mixed out and 150 ꢁL DMSO was added to dissolve the purple
micelles.
crystals. The optical density (OD) at 570 nm of the plate was
recorded by a microplate reader (Multiskan GO, Thermo
Fisher). The in vitro cell viability can be calculated by the
following equation:
Fluorescence emission spectroscopy was utilized to determine
the drug loading content (DLC) and drug encapsulation
efficiency (DEE). The excitation wavelength was set as 488 nm
and the emission wavelength was 560 nm. The DLC and DEE Cell viability (%) = (OD570sample) / (OD570control) × 100%.
were calculated by the following equations.
In vitro cellular uptake
DLC% = (weight of the drug in micelles) / (weight of the drug
loaded micelles) × 100%
DEE% = (weight of the drug in micelles) / (weight of the medium containing 2.5 × 105 MCFꢀ7/ADR cells was placed on
In vitro cellular uptake was observed by confocal laser
scanning microscopy (CLSM, Nikon C1ꢀsi). Briefly, 1 mL
feeding drug) × 100%.
35 mm cell culture dishes. After the cells were incubated at 37
°C for 24 h, the fresh medium containing drug loaded micelles
or free DOX was added to replace the cultured medium and
incubated for another 4 h. Then the medium was moved out and
the cells were washed by PBS for three times. After that, the
cells were fixed with 4 wt% formaldehyde in PBS for 15 min
and the nuclei were stained with Hoechst 33342 at room
temperature. The fluorescence was examined under excitation
at 405 nm for Hoechst 33342 and 488 nm for DOX.
The in vitro DOX release of drug loaded CL micelles was
studied at 37 °C in PBS (0.01M, pH 7.4) or PBS (0.01M, pH
7.4) containing 10 mM DTT. 3 mL freshly prepared drugꢀ
loaded micellar solution was added into a dialysis tube
(MWCO = 3500 Da), and then immersed into 30 mL of
corresponding PBS solution and shaken at 37 °C. At the
predetermined time intervals, 3 mL of the PBS solution was
replaced by equal volume of fresh PBS solution. The releasing
dose of DOX was measured by fluorescence emission Flow cytometry analysis
spectroscopy.
RAW264.7 and MCFꢀ7/ADR cells were seeded into a 6ꢀwell
Hemolysis assay
plate, respectively. After the cells were incubated at 37 °C for
24 h, the fresh medium containing sample was added to replace
the medium and coꢀincubated for another 4 h. Then the medium
was moved out and the cells were washed by PBS for three
times. After that, the cells were harvested by using trypsin
solution. The cells were analyzed by FACSCalibur flow
cytometer (Becton Dickinson) after centrifuge (1500 rpm for 3
min) and resuspended in PBS solution.
To a heparinꢀcoated tube containing 1 mL fresh mice blood, 2
mL of PBS was added, and the tube was centrifuged at 3000
rpm for 5 min to isolate the red blood cells. The red blood cells
were washed by PBS solution for three times, and then diluted
by PBS solution to give a 2% blood cells dispersion. 0.5 mL of
PBS solution of blank CL micelles (2 mg/mL) was placed in a
tube containing 0.5 mL of diluted blood cells dispersion. PBS
solution and water with equal volume were used as negative
control and positive control, respectively. All the tubes were
incubated at 37 °C for 1 h, and then the mixed solution was
centrifuged at 3000 rpm for 5 min. The supernatant was
collected to record the absorbance at 545 nm using UVꢀvis
spectroscopy. The hemolysis rate of the red blood was
calculated as follows:
Results and Discussion
Synthesis and characterization of monomer AMPC-PTA
Disulfideꢀcontaining cyclic carbonate AMPCꢀPTA was
synthesized by a fourꢀstep reaction shown in Figure 1. Firstly,
3ꢀmethylꢀ3ꢀoxetanemethanol was reacted with HBr at 0 °C for
4 h to obtain 2ꢀ(bromomethyl)ꢀ2ꢀmethylpropaneꢀ1,3ꢀdiol. Then
the diol compound was treated with NaN3 at 80 °C for 2 days to
obtain 2ꢀ(azidomethyl)ꢀ2ꢀmethylpropaneꢀ1,3ꢀdiol. Afterwards,
a cyclization reaction between the azidoꢀdiol compound and
ethyl chloroformate was carried out to synthesize a cyclic
carbonate with pendent azido (denoted as AMPC). Finally,
through a click reaction between AMPC and 5ꢀ(1,2ꢀdithiolanꢀ3ꢀ
yl)ꢀNꢀ(propꢀ2ꢀynꢀ1ꢀyl) pentanamide (PTA), a functionalized
cyclic aliphatic carbonate monomer AMPCꢀPTA with pendent
Hemolysis rate = [OD (materials) – OD (negative control)] /
[OD (positive control) – OD (negative control)] × 100%
where OD (materials), OD (negative control) and OD (positive
control) are the optical density (OD) values in the presence of
CL mixed micelles, PBS and water, respectively.
In vitro cell cytotoxicity
In vitro cell cytotoxicity of drug loaded micelles and free DOX
was investigated by MTT assay in RAW264.7 and MCFꢀ
7/ADR cells and the cytotoxicity of blank micelles were
determined on MCFꢀ7/ADR cells. Briefly, 100 ꢁL medium
containing 3500 cells were seeded into each well of a 96ꢀwell
plate. After incubation at 37 °C with 5% CO2 for 24 h, the
medium was moved out and 100 ꢁL fresh medium containing
samples with various concentrations was added, and further
incubated for 48 h. After that, the cultured medium was
1
dithiolane was obtained. The H NMR spectra of AMPC and
AMPCꢀPTA are shown in Figure S1. Not only the characteristic
peaks of AMPC and PTA components were clearly detected,
but also a new peak attributed to the proton of triazole group
appeared at δ 7.59. Moreover, the integral ratio of methylene at
δ 4.04 and methyl at δ 0.96 was 2:3, indicating the successful
synthesis of AMPCꢀPTA.
4 | J. Name., 2012, 00, 1-3
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The CAC values of PEGꢀP(AMPCꢀgꢀPTA), PBAꢀTA and PEGꢀ
TA were firstly determined by fluorescence spectroscopy using
pyrene as a probe in PBS (0.01 M, pH 7.4) at room
temperature. PEGꢀP(AMPCꢀgꢀPTA) possessed a CAC value at
23.2 mg/L, while the CAC values of amphiphilic conjugates of
PBAꢀTA and PEGꢀTA were 24.5 mg/L and 21.4 mg/L,
respectively.
Figure 1. The synthesis route of AMPC-PTA.
Synthesis and characterization of amphiphilic copolymer
PEG-P(AMPC-g-PTA) and amphiphilic conjugates PBA-
TA and PEG-TA
Figure 3. The size and distribution of UCL PBA-TA/PEG-
P(AMPC-g-PTA), CL PBA-TA/PEG-P(AMPC-g-PTA), UCL PEG-
The reductionꢀsensitive amphiphilic block copolymer PEGꢀ
P(AMPCꢀgꢀPTA) was prepared by
a
ringꢀopening
TA/PEG-P(AMPC-g-PTA),
CL
PEG-TA/PEG-P(AMPC-g-PTA)
polymerization (ROP) of AMPCꢀPTA in dried dichloromethane
at 35 °C for 48 h with mPEG (Mn: 5000) as an initiator and
DBU, TU as catalysts. The chemical structure of the copolymer
micelles (A) and the size change of UCL PBA-TA/PEG-P(AMPC-
g-PTA) and CL PBA-TA/PEG-P(AMPC-g-PTA) micelles treated
with 100-fold or 1000-fold dilution (B) determined by DLS.
1
was characterized by H NMR spectrum. As shown in Figure
Table 1. The size distribution of mixed micelles determined by DLS
and the corresponding drug loading content (DLC) and drug
encapsulation efficiency (DEE) of these micelles determined by
fluorescence intensity.
2A, the characteristic signals of PEG segment and poly(AMPCꢀ
gꢀPTA) segment were detected clearly. Bases on the integral
ratio of signals at δ 3.62 (methylene protons of PEG) and δ 0.98
(methyl protons of poly(AMPCꢀgꢀPTA)), the numberꢀaverage
molecular weight (Mn) of PEGꢀP(AMPCꢀgꢀPTA) was
calculated as 7200 and the corresponding polymerization
degree of AMPCꢀPTA was 5.
Size
(nm)
205.3±4.
4
DLC
(%)
DEE
(%)
Sample
PDI
UCL PBAꢀTA/PEGꢀ
P(AMPCꢀgꢀPTA)
UCL PEGꢀTA/PEGꢀ
P(AMPCꢀgꢀPTA)
0.093±0.052
0.078±0.02
0.113±0.017
0.095±0.033
0.165±0.009
195.2±3.
7
CL PBAꢀTA/PEGꢀP(AMPCꢀ 176.3±0.
gꢀPTA) 11
CL PEGꢀTA/PEGꢀP(AMPCꢀ 169.1±0.
gꢀPTA)
6
CL PBAꢀTA/PEGꢀP(AMPCꢀ
gꢀPTA)/DOX/TQR
187.7±1
1.1
8.95
74.58
83.5
CL PEGꢀTA/PEGꢀP(AMPCꢀ 179.7±2.
Figure 2. The structure of PEG-P(AMPC-g-PTA) (A) and Ad-
lys(Diol)-PCL-TA (B) determined by 1H NMR spectrum (300
MHz).
0.199±0.007 10.02
gꢀPTA)/DOX/TQR
CL PBAꢀTA/PEGꢀP(AMPCꢀ 180.7±1.
gꢀPTA)/DOX
CL PEGꢀTA/PEGꢀP(AMPCꢀ 174.5±6.
gꢀPTA)/DOX
5
0.17±0.04
9.94
9.43
82.83
78.58
3
Moreover, through an esterification reaction and a followed
click reaction, Adꢀlys(Diol)ꢀPCLꢀTA with an end group of
dithiolane was easily synthesized. As shown in Figure 2B, the
characteristic peaks of PCL, TA, Adꢀlys(Diol) as well as
triazole proton at δ 7.69 were all found. Furthermore, the peak
at δ 3.64 assignable to the methylene protons adjacent to
terminal hydroxyl group of PCL disappeared after reaction, also
indicating the successful modification of thioctic acid group at
the end of PCL segment. Finally, by virtue of hostꢀguest
interaction between Adꢀlys(Diol)ꢀPCLꢀTA and PBAꢀPEGꢀ
CD/PEGꢀCD, amphiphilic polymeric conjugates, i.e., PBAꢀ
PEGꢀCD/Adꢀlys(Diol)ꢀPCLꢀTA and PEGꢀCD/Adꢀlys(Diol)ꢀ
PCLꢀTA (denoted as PBAꢀTA and PEGꢀTA, respectively) were
obtained.
0.169±0.005
8
The UCL mixed micelles, i.e. UCL PBAꢀTA/PEGꢀP(AMPCꢀgꢀ
PTA) and UCL PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA), were readily
prepared by mixing PBAꢀTA or PEGꢀTA with PEGꢀP(AMPCꢀ
gꢀPTA) via dialysis method, while the core crosslinked
micelles, that is, CL PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA) and CL
PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA), were prepared by a disulfide
exchange reaction via introducing DTT into the solution of
corresponding UCL micelles. The size and distribution of these
mixed micelles were investigated by DLS and TEM,
respectively. As shown in Figure 3A and Table 1, both UCL
mixed micelles possessed an average size of ~200 nm with a
relatively low polydispersity (PDI) of 0.08ꢀ0.09, while the
Preparation and characterization of the mixed micelles
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hydrodynamic sizes of CL PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA) size of CL micelles was not obviously affected after 100ꢀ or
and CL PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA) were 176.3 nm (PDI: 1000ꢀfold dilution. However, the size of UCL PBAꢀTA/PEGꢀ
0.113) and 169.1 nm (PDI: 0.095), respectively. The particle P(AMPCꢀgꢀPTA) increased significantly upon dilution,
size were significantly decreased after crosslinking, because of implying the disassembly of UCL micelles. This result
the formation of a more compacted core in CL micelles. On the indicated that the CL mixed micelles has an excellent
other hand, the TEM images revealed that all these micelles performance in antiꢀdilution and maintaining its structure.
showed a wellꢀdefined spherical morphology with a clear
boundary and had a uniform size distribution of approximate 40
circulation, the size of CL micelles incubated with PBS solution
nm (Figure 4AꢀD).
To further evaluate the possible stability of CL micelles in the
containing 5 % BSA or 150 mM NaCl was also determined by
DLS. As depicted in Figure S2, the size of CL micelles changed
negligibly and the PDI values were still around 0.1 after 192 h
of incubation. The results indicated that the CL mixed micelles
of PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA) could not only possess
good stability in a mimic body environment but also still hide
targeting function of PBA groups effectively. Otherwise, the
interaction between PBA and BSA might result in the
aggregation of CL PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA) micelles.
Herein the interaction between PBA groups and cisꢀdiol groups
was also detected by steadyꢀstate fluorescence quenching
measurement. The precursor molecule, that is, PBAꢀPEGꢀ
Alkynyl (PPA), was chosen to accomplish this detection,
because the light scattering of PBAꢀTA conjugates influenced
the results. As shown in Figure S3, at pH 7.4, the fluorescence
of PPA solution became weakened as the introduction of
serinol. When the pH value of this mixed solution was adjusted
to 6.5, the fluorescence recovered. Accompanied by the
addition of sialic acid (SA), the fluorescence intensity of mixed
solution decreased again. Therefore, PBA groups of PBAꢀTA
conjugates were proven to form stable complex with serinol at
pH 7.4, thereby decreasing the undesirable contact of PBA
groups in normal physiological environment. While in mild
acidic environment, PBA groups became free and could be
recognized by SA, which was consistent with our previous
study13.
In vitro drug loading and redox triggered drug release
The CL micelles loaded one or two drugs were prepared by the
dialysis method. By quantitative analysis using fluorescence
emission spectroscopy, the drug loading content (DLC) and
drug encapsulation efficiency (DEE) were calculated (Table 1).
The DOX loading content of CL PEGꢀTA/PEGꢀP(AMPCꢀgꢀ
PTA)/DOX, CL PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX, CL
PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX/TQR and CL PBAꢀ
TA/PEGꢀP(AMPCꢀgꢀPTA)/DOX/TQR were 9.43%, 9.94%,
10.02%, and 8.95%, and the corresponding encapsulation
efficiency of these micelles were 78.58%, 82.83%, 83.5% and
74.58%, respectively. To further improve Pꢀgp inhibitory
effect, a small amount of TQR (10 wt% of DOX) was coꢀ
loaded with DOX. However, the amount of TQR was too low
to accurately measure its DLC and DEE by HPLC or UV
analysis. According to our experience, the encapsulation
efficiency increased with the decrease in the drugꢀfed amount.
Therefore, we supposed that the DEE of TQR was higher than
Figure 4. The TEM images of (A) UCL PEG-TA/PEG-P(AMPC-g-
PTA), (B) UCL PBA-TA/PEG-P(AMPC-g-PTA), (C) CL PEG-TA/PEG-
P(AMPC-g-PTA) (D) CL PBA-TA/PEG-P(AMPC-g-PTA), (E) CL PEG-
TA/PEG-P(AMPC-g-PTA)/DOX, (F) CL PBA-TA/PEG-P(AMPC-g-
PTA)/DOX, (G) CL PEG-TA/PEG-P(AMPC-g-PTA)/DOX/TQR, and
(H) CL PBA-TA/PEG-P(AMPC-g-PTA)/DOX/TQR micelles.
The stability under multiple dilution was a very important
property that was closely related to the stability in the
that of DOX. The morphology and size distribution of these
drugꢀloaded CL micelles were determined by TEM and DLS.
an example, the size of both CL and UCL micelles under the
The TEM images (Figure 4EꢀF) showed that they still
circulation. Herein, taking PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA) as
dilution was measured by DLS. As shown in Figure 3B, the
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maintained spherical structure and uniform size distribution. macrophages at pH 7.4. It meant that the targeting ligands were
The hydrodynamic sizes of CL PBAꢀTA/PEGꢀP(AMPCꢀgꢀ hided effectively and the hydrophilic PEG shell could inhibit
PTA)/DOX, CL PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX, CL the recognition of the nanocarrier by macrophages in normal
PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX/TQR and CL PEGꢀ physiological environment.
TA/PEGꢀP(AMPCꢀgꢀPTA)/DOX/TQR were 180.7±1.3 nm,
174.5±6.8 nm, 187.7±11.1 nm and 179.7±2.5 nm, respectively,
which were larger than that of corresponding blank CL micelles
(Figure S4).
Figure 5. In vitro DOX release from CL PEG-TA/PEG-P(AMPC-g-
PTA)/DOX and PBA-TA/PEG-P(AMPC-g-PTA)/DOX at pH 7.4 in
the presence of 10 mM DTT or not.
The prevention of drug leakage under normal condition and the
effect of reductive environment on in vitro drug release of CL
micelles were also studied. Herein, PBS solution containing 10
mM DTT was selected to simulate the reductive environment in
the cytoplasm. As shown in Figure 5, in the absence of DTT,
the release rate was relatively low and only about 25% of DOX
was released from DOXꢀloaded CL micelles within 48 h. This
results implied that the CL mixed micelles were really stable to
suppress the leakage of drugs in normal physiological
environment and they were probably appropriate for long time
lasting in blood circulation. In contrast, when the CL mixed
micelles existed in a reductive environment, the release of drug
became significantly accelerated and complete. After 48 h, the
cumulative release amount of DOX reached 80%. This
phenomenon may be associated with the damage of
crosslinking structure induced by the disulfide exchange
reaction in reductive environment. In addition, there was no
obviously difference in the drug release behavior of CL
micelles with or without targeting ligands.
Figure 6. Confocal images of MCF-7/ADR cells incubated with
free DOX, PEG-TA/PEG-P(AMPC-g-PTA)/DOX, PBA-TA/PEG-
P(AMPC-g-PTA)/DOX, PEG-TA/PEG-P(AMPC-g-PTA)/DOX/TQR
and PBA-TA/PEG-P(AMPC-g-PTA)/DOX/TQR at pH 6.5 for 4 h.
Blue Hoechst 33342 fluorescence and red DOX fluorescence
were recorded under excitation at 405 nm and 488 nm,
respectively. (B) The flow cytometry analysis of MCF-7/ADR
cells treated with (a) blank medium, (b) free DOX, (c) PEG-
TA/PEG-P(AMPC-g-PTA)/DOX, (d) PBA-TA/PEG-P(AMPC-g-
In vitro cellular uptake
PTA)/DOX, (e) PEG-TA/PEG-P(AMPC-g-PTA)/DOX/TQR and (f)
PBA-TA/PEG-P(AMPC-g-PTA)/DOX/TQR at pH 6.5 for 4 h.
The effective targeting and drug delivery to tumor cells can be
hampered by the recognition and endocytosis of macrophages.
Therefore, the internalization of our nanocarrier in
macrophages was firstly studied on RAW264.7 cells by flow
cytometry. As shown in Figure S5A, after 4 h incubation, the
mean fluorescence intensity (MFI) of the cells incubated with
free DOX was two times to that treated with PBAꢀTA/PEGꢀ
P(AMPCꢀgꢀPTA)/DOX incubated at pH 7.4, implying that this
smart targeting drug delivery system was ‘invisible’ to the
To investigate the in vitro cellular uptake of drugꢀloaded CL
micelles by drug resistant tumor cells, the endocytosis of free
Dox, PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX, PBAꢀTA/PEGꢀ
P(AMPCꢀgꢀPTA)/DOX,
PTA)/DOX/TQR
PEGꢀTA/PEGꢀP(AMPCꢀgꢀ
PBAꢀTA/PEGꢀP(AMPCꢀgꢀ
and
PTA)/DOX/TQR by MCFꢀ7/ADR cells was qualitatively and
quantitatively detected by CLSM and flow cytometry,
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respectively. The results are shown in Figure 6. After 4 h of could efficiently evade from the capture of macrophages and
incubation, the cells treated with free DOX showed the lowest further reduce the side effect of chemotherapeutics.
fluorescence intensity, revealing that free DOX cannot be
concentrated into the drug resistant cells efficiently. This
phenomenon may be attributed to the excellent recognition and
efflux function of Pꢀgp. When free DOX crosses the cell
membrane through diffusion, it is easily identified and captured
by the efflux pump overexpressed on the membrane, resulting
in the reduced concentration of DOX in cells. Nevertheless,
when the drugs were loaded in a nanocarrier, no matter whether
targeting ligands existed or not, an enhanced DOX fluorescence
was detected, suggesting that the nanocarrier could improve the
intracellular concentration of drug via endosome pathway that
could evade the recognition of Pꢀgp and avoid the drug efflux.
Furthermore, compared with the single DOXꢀloaded micelles,
dualꢀdrug loaded micelles exhibited a stronger intracellular
DOX fluorescence intensity. In other words, coꢀencapsulation
of TQR was helpful for the enrichment of DOX in MCFꢀ
7/ADR cells. The results indicated that the Pꢀgp inhibitor could
enhance the drug accumulation by inhibiting the drug efflux in
drug resistant cells. Moreover, both single and dualꢀdrug loaded
micelles modified with targeting ligands really exhibited
stronger fluorescence intensity than nontargeted micelles,
because of the specific interaction between the reꢀexposed PBA
groups on the micelles and the SA moieties overꢀexpressed on
the surface of cancer cells under the stimulation of acidic tumor
environment. The result of flow cytometry was also consistent
with that of CLSM. As expected, the dualꢀdrug loaded smart
Figure 7. (A) The cytotoxicity of blank PBA-TA/PEG-P(AMPC-g-
PTA) and PEG-TA/PEG-P (AMPC-g-PTA) micelles in MCF-F/ADR
cells after incubation for 48 h. (B) The cell viability of MCF-
7/ADR cells in the presence of free DOX, PEG-TA/PEG-P(AMPC-
g-PTA)/DOX, PBA-TA/PEG-P(AMPC-g-PTA)/DOX, PEG-TA/PEG-
P(AMPC-g-PTA)/DOX/TQR
and
PBA-TA/PEG-P(AMPC-g-
PTA)/DOX/TQR after incubation at pH 6.5 for 48 h. Data
displayed as mean ± SD; n= 3.
The inhibitory effect of free DOX and drugꢀloaded micelles on
MCFꢀ7/ADR cells was also investigated by MTT assay. As
shown in Figure 7B, all the samples showed a doseꢀdependent
cytotoxicity, and the drugꢀloaded micelles exhibited stronger
cytotoxicity than free DOX, which was consistent with the
results of in vitro cellular uptake. The IC50 values of free DOX,
PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX,
PBAꢀTA/PEGꢀ
P(AMPCꢀgꢀPTA)/DOX,
PTA)/DOX/TQR
PEGꢀTA/PEGꢀP(AMPCꢀgꢀ
PBAꢀTA/PEGꢀP(AMPCꢀgꢀ
and
targeting
micelles,
i.e.
PBAꢀTA/PEGꢀP(AMPCꢀgꢀ
PTA)/DOX/TQR after 48 h of incubation at pH 6.5 were
calculated to be >100, 33.19, 22.45, 13.44 and 8.55 ꢁg/mL,
respectively. The result might be associated with the
overexpressed efflux pump and transmembrane pathway. As we
know, the cell internalization of free DOX is mainly dependent
on diffusion, which is vulnerable to Pꢀgp on the cell membrane
in this process. For drug resistant cells, free DOX was easily be
pumped out, thereby reducing the intracellular drug
concentration. On the contrary, the loaded DOX in micelles
was mainly transported into cells by endocytosis, which could
effectively avoid Pꢀgpꢀmediatd drug efflux. The improved
inhibitory effect of PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX and
PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA)/DOX on MCFꢀ7/ADR cells
might mainly result from the stealth endocytosis of
nanocarriers29,30. On the other hand, the coꢀdelivery of TQR
could further inhibit the function of Pꢀgp, contributing to the
accumulation of drugs and thus improving the cytotoxicity of
DOXꢀloaded micelles.
PTA)/DOX/TQR, showed the strongest fluorescence intensity
of DOX in MCFꢀ7/ADR cells, which was 6 times higher than
the result of free DOX. All the results indicated that PBAꢀ
TA/PEGꢀP(AMPCꢀgꢀPTA)/DOX/TQR showed an excellent
potential of delivering chemotherapeutics into MCFꢀ7/ADR
cells.
In vitro cellular uptake
The biocompatibility was one of the most important factors to
assess the potential application of materials in biomedical field.
In this study, cytotoxicity and hemolysis were used to evaluate
the biocompatibility of materials. When the MCFꢀ7/ADR cells
were treated with both PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA) and
PBAꢀTA/PEGꢀP(AMPCꢀgꢀPTA) micelles at concentration
ranging from 7.81ꢀ1000 ꢁg/mL for 48 h, the cell viabilities
were always above 85% (Figure 7A), indicating that both blank
micelles had negligible toxicity to MCFꢀ7/ADR cells.
Moreover, even at the concentration of 1 mg/mL, the hemolysis
ratio of both PEGꢀTA/PEGꢀP(AMPCꢀgꢀPTA) and PBAꢀ
TA/PEGꢀP(AMPCꢀgꢀPTA) was below 5% (Figure S6),
suggesting that these mixed micelles possessed good blood
compatibility and were suitable for intravenous injection.
Conclusions
pH and redox dualꢀsensitive coreꢀcrosslinked mixed micelles
were prepared and utilized for coꢀdelivery of DOX and TQR.
The multifunctional micelles showed an excellent stability in
mimic blood stream and could prevent the leakage of drug in
the normal physiological environment efficiently. The targeting
function was also suppressed in the normal physiological
environment and regained under mild tumor acidic condition,
The cytotoxicity of free DOX and PBAꢀTA/PEGꢀP(AMPCꢀgꢀ
PTA)/DOX to RAW264.7 cells was measured at pH 7.4 to
determine the side effect of drugꢀloaded micelles and free DOX
to macrophages. As shown in Figure S5B, compared with free
DOX, the drugꢀloaded micelles showed a relative low toxicity
to RAW264.7 cells, which indicated that this smart CL micelles
8 | J. Name., 2012, 00, 1-3
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ARTICLE
22 H. Y. Wen, H. Q. Dong, J. Liu, A. J. Shen, Y. Y. Li, D. L.
Shi, J. Mater. Chem. B, 2016, 4, 7859.
23 W. J. Wei, X. J. Zhang, X. F. Chen, M. J. Zhou, R. R. Xu, X.
H. Zhang, Nanoscale, 2016, 8, 8118.
24 K. Wang, G. F. Luo, Y. Liu, C. Li, S. X. Cheng, R. X. Zhuo,
X. Z. Zhang, Polym. Chem., 2012, 3, 1084.
which could protect the micelles from being captured by
macrophages. Combined the ligandꢀreceptor mediated
endocytosis with TQRꢀmediated Pꢀgp inhibition, this
multifunctional smart targeting coꢀdelivery system showed a
great potential in overcoming drug resistance.
25 X. Q. Yi, D. Zhao, Q. Zhang, J. Q. Xu, G. D. Yuan, R. X.
Zhuo, F. Li, Nanotechnology, 2017, 28, 085603.
26 C. B. Tripathi, S. Mukherje, J. Org. Chem., 2012, 77, 1592.
27 W. Chen, Y. Zou, J. N. Jia, F. H. Meng, R. Cheng, C. Deng,
J. Feijen, Z. Y. Zhong, Macromolecules, 2013, 46, 699.
28 T. Naolou, K. Busse, J. Kressler, Biomacromolecules, 2010,
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Acknowledgements
This work was supported by National Natural Science
Foundation of China (21374082 and 51533006).
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Journal of Materials Chemistry B
Page 10 of 11
View Article Online
DOI: 10.1039/C7TB01504F
Table of Contents
Dual Drug-Loaded Reversible Core-Crosslinked Nanoaggregates
Endowed with pH-Modulated Targeting and Redox-Controlled Drug
Release for Overcoming Drug Resistance
Dan Zhao, Shujie Ma, Xiaoqing Yi, Sixue Cheng, Renxi Zhuo, Feng Li*
Key Laboratory of Biomedical Polymers of Ministry of Education & College of Chemistry&
Molecular Science, Wuhan University, Wuhan 430072, China
E-mail address: lfsj2004@hotmail.com; lifeng@whu.edu.cn (F. Li),
Herein, a pH and redox dual-sensitive core-crosslinked targeting nanocarrier was prepared and

Page 11 of 11
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C7TB01504F
used for co-delivery of DOX and TQR. By means of the ligand-receptor mediated endocytosis and
TQR-mediated P-gp inhibition, the IC50 value of DOX to MCF-7/ADR cells reduced from more than
100 μg/mL to 8.55 μg/mL, exhibiting a great potential in overcoming drug resistance.