Co-delivery of doxorubicin and P-glycoprotein siRNA by multifunctional triblock copolymers for enhanced anticancer efficacy in breast cancer cells

Article abstract of DOI:10.1039/c5tb00031a

Combined treatment of chemotherapeutics and small interfering RNAs (siRNAs) is a promising therapy strategy for breast carcinoma via their synergetic effects. In this study, to improve the therapeutic effect of doxorubicin (DOX), novel triblock copolymers, folate/methoxy-poly(ethylene glycol)-block-poly(l-glutamate-hydrazide)-block-poly(N,N-dimethylaminopropyl methacrylamide) (FA/m-PEG-b-P(LG-Hyd)-b-PDMAPMA), were synthesized and used as a vehicle for the co-delivery of DOX and P-glycoprotein (P-gp) siRNA into breast cancer cells. The triblock copolymers were synthesized by a combination of ring-opening polymerization of γ-benzyl-l-glutamate-N-carboxyanhydride using cystamine-terminated heterotelechelic PEG derivatives possessing folate or methoxy end groups (FA/m-PEG-Cys) as initiators and reversible addition-fragmentation chain transfer polymerization of N,N-dimethylaminopropyl methacrylamide followed by hydrazinolysis. The successful synthesis of the copolymers was confirmed by ¹H NMR and gel permeation chromatography. DOX was covalently conjugated onto the poly(l-glutamate-hydrazide) blocks via a pH-labile hydrazone linkage, and the DOX-conjugated triblock copolymers could self-assemble into nanoparticles in aqueous solutions. P-glycoprotein (P-gp) siRNA was then bound to the cationic poly(N,N-dimethylaminopropyl methacrylamide) (PDMAPMA) blocks through an electrostatic interaction, resulting in the formation of spherical nanocomplexes with an average diameter of 196.8 nm and a zeta potential of +28.3 mV. The in vitro release behaviors of DOX and siRNA from the nanocomplexes were pH- and reduction-dependent, and the release rates were much faster under a reductive acidic condition (pH 5.0, glutathione: 10 mM) simulating the intracellular endo-lysosomal environment of cancer cells compared to physiological conditions. The fast payload release rates were closely related to both the glutathione-triggered detachment of PEG blocks from the nanocomplex surface and the pH-sensitive cleavage of hydrazone linkages. FA-decorated nanocomplexes showed higher cellular uptake efficiency and cytotoxicity against MCF-7 cells than FA-free nanocomplexes, as confirmed by confocal laser scanning microscopy, transmission electron microscopy, MTT and flow cytometry analyses. Our results demonstrated that the multifunctional triblock copolymer-mediated co-delivery of DOX and P-gp siRNA might be a new promising therapeutic strategy for breast cancer treatment. This journal is

Full text of DOI:10.1039/c5tb00031a

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Materials Chemistry B
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PAPER
Co-delivery of doxorubicin and P-glycoprotein
siRNA by multifunctional triblock copolymers for
enhanced anticancer efficacy in breast cancer
cells†
Cite this: DOI: 10.1039/c5tb00031a
a
a
Minghui Xu, Junmin Qian, Aili Suo,b Ning Cui,a Yu Yao,b Weijun Xu,a Ting Liua
*
and Hongjie Wanga
Combined treatment of chemotherapeutics and small interfering RNAs (siRNAs) is a promising therapy
strategy for breast carcinoma via their synergetic effects. In this study, to improve the therapeutic effect
of doxorubicin (DOX), novel triblock copolymers, folate/methoxy-poly(ethylene glycol)-block-poly(L-
glutamate-hydrazide)-block-poly(N,N-dimethylaminopropyl methacrylamide) (FA/m-PEG-b-P(LG-Hyd)-
b-PDMAPMA), were synthesized and used as a vehicle for the co-delivery of DOX and P-glycoprotein (P-
gp) siRNA into breast cancer cells. The triblock copolymers were synthesized by a combination of ring-
opening polymerization of g-benzyl-L-glutamate-N-carboxyanhydride using cystamine-terminated
heterotelechelic PEG derivatives possessing folate or methoxy end groups (FA/m-PEG-Cys) as initiators
and reversible addition–fragmentation chain transfer polymerization of N,N-dimethylaminopropyl
methacrylamide followed by hydrazinolysis. The successful synthesis of the copolymers was confirmed
by 1H NMR and gel permeation chromatography. DOX was covalently conjugated onto the poly-
(L-glutamate-hydrazide) blocks via a pH-labile hydrazone linkage, and the DOX-conjugated triblock
copolymers could self-assemble into nanoparticles in aqueous solutions. P-glycoprotein (P-gp) siRNA
was then bound to the cationic poly(N,N-dimethylaminopropyl methacrylamide) (PDMAPMA) blocks
through an electrostatic interaction, resulting in the formation of spherical nanocomplexes with an
average diameter of 196.8 nm and a zeta potential of +28.3 mV. The in vitro release behaviors of DOX
and siRNA from the nanocomplexes were pH- and reduction-dependent, and the release rates were
much faster under a reductive acidic condition (pH 5.0, glutathione: 10 mM) simulating the intracellular
endo-lysosomal environment of cancer cells compared to physiological conditions. The fast payload
release rates were closely related to both the glutathione-triggered detachment of PEG blocks from the
nanocomplex surface and the pH-sensitive cleavage of hydrazone linkages. FA-decorated
nanocomplexes showed higher cellular uptake efficiency and cytotoxicity against MCF-7 cells than FA-
free nanocomplexes, as confirmed by confocal laser scanning microscopy, transmission electron
microscopy, MTT and flow cytometry analyses. Our results demonstrated that the multifunctional
triblock copolymer-mediated co-delivery of DOX and P-gp siRNA might be a new promising therapeutic
strategy for breast cancer treatment.
Received 7th January 2015
Accepted 27th January 2015
DOI: 10.1039/c5tb00031a
www.rsc.org/MaterialsB
However, the clinical application of most of the chemothera-
peutic drugs such as doxorubicin (DOX) in the treatment of
1. Introduction
patients with breast cancer has been limited by their poor water
solubility and stability, non-specic toxicity, and especially drug
resistance.2 It is well known that overexpression of drug trans-
porter proteins such as P-glycoprotein (P-gp) may play a key role
in regulating drug resistance.3 For example, P-gp overexpression
is observed in 40–50% of patients with breast cancer.4 Nowa-
days, the development of strategies to deliver chemotherapeutic
drugs and to simultaneously inhibit the P-gp function or down-
regulate the expression of P-gp has become one of the hot
research topics in the eld of breast cancer chemotherapy.5
Breast carcinoma is a leading cause of cancer-related death in
women worldwide, and chemotherapy remains one of the most
effective approaches for treatment of breast cancer in clinics.1
aState Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University,
Xi'an 710049, China. E-mail: jmqian@mail.xjtu.edu.cn; Fax: +86 29 82663453; Tel:
+86 29 82668614
bDepartment of Medical Oncology, First Affiliated Hospital of Medical School, Xi'an
Jiaotong University, Xi'an 710061, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5tb00031a
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Recent studies have demonstrated that the co-delivery of che- by the high intracellular concentration of GSH (2–10 mM) which
motherapeutical agents and inhibitors of the drug transporter is much higher than the extracellular one (2–20 mM).25
proteins such as verapamil6 and depharantine7 using nano-
The aim of the present study was to develop novel
particles may sensitize tumor cells to chemotherapy. These multifunctional triblock copolymers, folate/methoxy poly(ethy-
results suggest that the sensitization of cancer cells by inhibi- lene glycol)-block-poly(L-glutamate-hydrazide)-block-poly(N,N-
tors of efflux transporters is not only cell specic but also drug dimethylaminopropyl methacrylamide) (FA/m-PEG-b-P(LG-
specic.8
Hyd)-b-PDMAPMA), for the co-delivery of DOX and therapeutic
Small interfering RNA (siRNA) has the ability to disrupt P-gp siRNA to improve the anti-tumor efficiency of DOX on
cellular pathways by knocking down the targeted genes and human breast cancer MCF-7 cells via their synergetic effect. The
siRNA interference is regarded as a potential therapeutic option triblock copolymers were synthesized by a combination of ring-
in cancer treatment.9 However, the major challenges facing the opening polymerization (ROP) and reversible addition–frag-
therapeutic uses of siRNA include rapid clearance from the mentation chain transfer (RAFT) polymerization. Folate, a high-
systemic circulation, low selectivity for tumor tissue and poor affinity ligand for folate receptors, was introduced to achieve the
cellular uptake, which may signicantly decrease the effective- active targeting to breast carcinoma cells. The triblock copoly-
ness of siRNA therapy.10 Hence, an efficient delivery vehicle is mers could chemically conjugate DOX via an acid-labile
essential for the applications of siRNA interference to overcome hydrazone linkage to the hydrazide groups of the P(LG-Hyd)
these limitations. Numerous natural and synthetic polymers blocks and simultaneously condense P-gp siRNA with their
are preferred as non-viral delivery vehicles for siRNA over cationic PDMAPMA blocks through an electrostatic interaction.
viral vectors since they are safer and less toxic.11 In the past The resulting nanocomplexes were expected to be stable under
decade, cationic polymers that have been widely investigated as physiological conditions, be selectively taken up by MCF-7 cells
siRNA carriers include polyethylenimine,12–14 poly-L-lysine,15 via folate receptor-mediated endocytosis, and rapidly release
poly(amido amine) dendrimer16 and chitosan.17 In addition, payloads in the intracellular reductive acidic microenviron-
poly(N,N-dimethylaminopropyl methacrylamide) (PDMAPMA), ment, thereby improving the antitumor efficacy of DOX.
a cationic polymer, has also received a lot of attention as an The chemical structures, self-assembling behaviors, siRNA-
innovative carrier in the gene and siRNA delivery community binding abilities and drug release of PEG-b-P(LG-Hyd-DOX)-b-
due to its relatively low toxicity and high buffer capacity.18,19 PDMAPMA copolymers were analyzed in detail. The in vitro
Although these cationic polymers can interact electrostatically cellular uptake and cytotoxicity of the nanocomplexes were
with negatively charged siRNA, the resultant net positively- evaluated using confocal laser scanning microscopy (CLSM),
charged siRNA/polycation complexes may interact with anionic transmission electron microscopy (TEM), MTT and ow
serum proteins and cause aggregation or decomplexation, cytometry assays. The preliminary in vitro results indicated the
signicantly reducing or ablating siRNA efficacy. So far, the great potential of the multifunctional triblock copolymers as a
PEGylation of nanocarriers has been extensively studied as an carrier for the co-delivery of DOX and siRNA to improve the anti-
effective method for improving the stability of siRNA/polycation tumor efficiency for breast cancer treatment.
complexes and reducing toxicity of delivery systems.20 However,
the stealthy PEG shell is intensely expected to be detached from
the carrier at the disease site because it would hamper the
cellular uptake kinetics and intracellular transport of the
carrier.21
2. Experimental
2.1 Materials
Folic acid (FA), 3,30-dithiodipropionic acid, pyrene, triethyl-
Until now, stimuli-responsive vehicles have been extensively amine (TEA), N,N0-dicyclohexylcarbodiimide (DCC), 4-(N,N-
studied for the controlled release of drugs owing to their dimethylamino)pyridine (DMAP), anhydrous hydrazine, para-
responses to the intracellular pH, temperature, enzymes and formaldehyde, 2-(4-amidinophenyl)-6-indolecarbamidine dihy-
reducing substances of tumor cells.22 The hydrazone bond, a drochloride (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
typical acid-sensitive linkage, has been extensively used in the tetrazolium bromide (MTT), poly(ethylene glycol) (HO-PEG-OH)
conjugation of drugs to carriers. It has been reported by Gong's and methoxy poly(ethylene glycol) (mPEG-OH) (molecular
research group that DOX was linked to the poly(L-glutamate- weight: 2 kDa) were purchased from Sigma-Aldrich (Shanghai,
hydrazide) (P(LG-Hyd)) by a hydrazone linker.23 By this linkage China). g-Benzyl-L-glutamate (BLG) and N,N-dimethylamino-
design, the nanoparticles could stably preserve drugs under propyl methacrylamide (DMAPMA) were purchased from
physiological conditions (pH 7.4) and selectively and rapidly Aladdin Chemistry Co., Ltd. (Shanghai, China), and DMAPMA
release them by sensing the intracellular pH decrease in endo- was distilled from calcium hydride under reduced pressure
somes and lysosomes (pH 5–6). Nowadays, the disulde linkage, prior to use. Doxorubicin hydrochloride (DOX$HCl) was
which may be prone to rapid cleavage through thiol-disulde purchased from Hisun Pharmaceutical Co., Ltd. (Zhejiang,
exchange reactions with intracellular reducing molecules China). All other reagents and solvents were of analytical
especially glutathione (GSH), has been employed to develop grade and purchased from Sinopharm Chemical Reagent Co.,
reduction-sensitive nanocarriers.24 For example, it was reported Ltd. (Shanghai, China). Tetrahydrofuran (THF), toluene, diethyl
that PEGylated nanovehicles containing disulde linkages ether, dichloromethane (DCM), 1,4-dioxane, N,N-dime-
could markedly enhance drug release at cytoplasm. This is thylformamide (DMF), n-hexane and dimethyl sulfoxide
attributed to the reductive cleavage of disulde linkages caused (DMSO) were dried by reuxing over sodium wire or calcium
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ꢀ
hydride and distilled before use. g-Benzyl-L-glutamate-N-car- The mixture was magnetically stirred at 100 C for 48 h. Aer
boxyanhydride (BLG-NCA) was synthesized according to our the solvent was eliminated by distillation, an intermediate
previously reported method26 and puried by recrystallization product BIED-functional homotelechelic PEG derivative was
from a mixture of THF and n-hexane (1 : 3, v/v). RAFT obtained by precipitating in anhydrous diethyl ether. The PEG
agent S-1-dodecyl-S0-(a,a0-dimethyl-a00-acetic acid)trithiocar- derivative was dissolved in distilled water, stirred at 60 ꢀC for 6 h
bonate (DDAT) was synthesized according to the reported and lyophilized, and Cys-PEG-Cys was obtained with a yield of
procedure (mp 60–61 ꢀC).27,28 1H NMR (CDCl3, Bruker 400 MHz, 92.6%. mPEG-Cys was also synthesized using the same proce-
dppm): 0.87 (t, 3H), 1.25–1.38 (m, 18H), 1.68–1.73 (m, 8H), 3.28 dure (yield 91.8%).
(t, 2H).
FA was covalently linked to one end-group of Cys-PEG-Cys by
Targeting human P-gp siRNA (sense: 50-GAA ACC AAC UGU carbodiimide reaction. In a typical reaction, a mixture of FA,
CAG UGU AdTdT-30, anti-sense: 50-UAC ACU GAC AGU UGG DCC and DMAP with a molar ratio of 1 : 1.1 : 1 was dissolved in
UUU CdTdT-30) and carboxyuorescein-tagged siRNA (FAM- 30 mL of DMSO, and then a solution of Cys-PEG-Cys (2 g, 1 eq.)
siRNA) were supplied by Shanghai GenePharma Co., Ltd. in 20 mL of DMSO was added. The reaction mixture was stirred
(Shanghai, China). Dulbecco's modied Eagle medium for a further 24 h at room temperature in the dark. The solution
(DMEM), fetal bovine serum (FBS), penicillin–streptomycin, was precipitated in diethyl ether, and the collected solid was re-
0.25% (w/v) trypsine–0.03% (w/v) EDTA solution and DNase/ dissolved in DCM, washed with distilled water and re-precipi-
RNase–free water were purchased from Invitrogen. The Annexin tated in ice-cold diethyl ether, obtaining FA-PEG-Cys (yield
V-FITC apoptosis detection kit from 7-Sea Biotech Co., Ltd. 89.6%).
(Shanghai, China) was used according to its protocol. Human
2.2.2 Synthesis of FA/m-poly(ethylene glycol)-block-poly(b-
breast cancer MCF-7 cells were obtained from the Medical benzyl L-glutamate) (PEG-b-PBLG) diblock copolymers. Diblock
Center of Xi'an Jiaotong University (China) and maintained in a copolymer mPEG-b-PBLG was synthesized by ROP of BLG-NCA
DMEM medium containing 10% FBS, penicillin (100 U mLꢁ1
)
using mPEG-Cys as the macromolecular initiator. Briey, BLG-
and streptomycin (100 mg mLꢁ1) at 37 ꢀC in a humidied NCA (8 g, 32.13 mmol) was dissolved in 70 mL of DMF in a 250
incubator with 5% CO2.
mL ask under stirring in a nitrogen atmosphere, and then a
solution of mPEG-Cys (4.0 g, 2.0 mmol) in 10 mL of DMF was
ꢀ
added. Thereaer, the ask was placed in an oil bath at 40 C.
2.2 Synthesis of FA/m-PEG-b-PBLG-b-PDMAPMA triblock
copolymers
Aer 48 h of reaction, the mixture was poured into a 10-fold
ꢀ
volume of cold diethyl ether (ꢁ20 C), and the precipitate was
2.2.1 Synthesis of a-folate-u-cystamine heterotelechelic collected by ltration, washed with diethyl ether and dried in a
poly(ethylene glycol) (FA-PEG-Cys). FA-PEG-Cys was synthesized vacuum, obtaining the mPEG-b-PBLG copolymer (yield 88.7%).
as follows. Cystamine-functional homotelechelic PEG (Cys-PEG- The FA-PEG-b-PBLG copolymer was also synthesized using the
Cys) was rstly synthesized by reacting HO-PEG-OH with same protocol (yield 90.4%).
bis(b-isocyanatoethyl)disulde (BIED) followed by hydrolysis in
2.2.3 Synthesis of FA/m-PEG-b-PBLG-b-PDMAPMA triblock
distilled water. BIED as a pale yellow liquid was synthesized copolymers by RAFT polymerization. To synthesize the triblock
according to the reported procedure.29,30 In detail, 3,30-dithio- copolymers by RAFT polymerization of DMAPMA, RAFT agent
dipropionic acid (10 g, 47.6 mmol) and ethanol (22.1 g, DDAT was attached to the amine end groups of FA/m-PEG-b-
480 mmol) were dissolved in toluene containing p-toluene- PBLG copolymers via carbodiimide reaction to form macro-
sulfonic acid (4%), and the reaction mixture was heated and RAFT agents. In brief, a solution of DDAT (2.73 g, 7.5 mmol),
reuxed for 5 h using a Dean–Stark trap to produce dithiodi- DCC (3.09 g, 15 mmol) and DMAP (0.183 g, 1.5 mmol) in 80 mL
propionate diethyl (yield 94%). Dithiodipropionate diethyl of DCM was introduced into a ask containing the anhydrous
(10 g, 37.5 mmol) and hydrazine hydrate (12 g, 0.24 mol) were mPEG-b-PBLG or FA-PEG-b-PBLG copolymer (1.5 mmol) under a
then dissolved in ethanol (20 mL) and the mixture was reuxed nitrogen atmosphere. The amidation reaction proceeded for
under stirring for 4 h followed by recrystallization from a water– 48 h under stirring at room temperature. The resulting macro-
ethanol mixture (1/3, v/v), yielding dithiodipropionate dihy- RAFT agents (mPEG-b-PBLG-DDAT or FA-PEG-b-PBLG-DDAT)
drazide (68%). Dithiodipropionate dihydrazide (13 g, 54 mmol) were recovered by precipitation from cold diethyl ether. Aer
was dissolved in a HCl solution (2 mol Lꢁ1, 62 mL) and then the ltration, the product was dried under vacuum at 40 ꢀC, and the
solution was cooled in an ice bath and stirred. Aer the addition yield was 90%. The mPEG-b-PBLG-b-PDMAPMA triblock
of sodium nitrite solution (7.6 g, 10 mL water) and benzene, the copolymer was synthesized by RAFT polymerization of
organic phase was separated and incubated at 70 ꢀC for 30 min. DMAPMA in the presence of the mPEG-b-PBLG-DDAT macro-
Aer the evaporation of benzene, BIED was obtained as a pale RAFT agent. In a typical synthetic example, under a nitrogen
yellow liquid (yield 53%). 1H NMR (CDCl3, dppm): 2.91 (t, 2H, –S– atmosphere, DMAPMA (2.554 g, 15 mmol), mPEG-b-PBLG-
CH2–), 3.66 (t, 2H, NCO–CH2–).
DDAT (3 g, 0.5 mmol), AIBN (8.21 mg, 0.05 mmol) and 19 mL of
BIED-functional homotelechelic PEG was synthesized as 1,4-dioxane were added into a 50 mL Schlenk ask. The ask
ꢀ
follows. Briey, in a 100 mL round-bottom ask equipped with a was sealed and placed into an oil bath thermostatted at 60 C.
reux condenser, anhydrous HO-PEG-OH (2.5 g, 1.25 mmol), Aer the reaction proceeded with magnetic stirring for 48 h, the
BIED (3 g, 15 mmol) and the dibutyltin dilaurate catalyst were resulting triblock copolymer was isolated by precipita-
dissolved in 50 mL of toluene under the protection of nitrogen. tion in cold diethyl ether, ltration and drying in a vacuum. The
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FA-PEG-b-PBLG-b-PDMAPMA triblock copolymer was also consisted of CH3OH and H2O (7 : 3, v/v) with a ow rate of
synthesized with this process. Yield: 72–80%.
0.8 mL minꢁ1. The UV detection wavelength was 490 nm.
2.5 Preparation and characterization of DOX-NPs
2.3 Synthesis of FA/m-PEG-b-P(LG-hydrazone(Hyd)-DOX)-b-
PDMAPMA triblock copolymers
The mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA copolymer (10 mg)
was added to 10 mL of H2O, and the suspension system was
sonicated for 10 min using a probe-type ultrasonicator (JY 92-
2D, Ningbo Scientz Biotechnology Co., Ltd., China) at 100 W in
an ice bath to obtain the DOX-NPs. The size distribution and
zeta potential of the DOX-NPs were determined by dynamic
light scattering (DLS) using a Malvern Zetasizer Nano-ZS90
instrument (Malvern instruments, UK). The DLS measurements
were performed at a xed scattering angle of 90ꢀ. The
morphology and size of the DOX-NPs were observed using a
transmission electron microscope (JEM-2000CX, JEOL, Japan).
To obtain tumor-targeted DOX-NPs (FA-DOX-NPs), a mixture of
mPEG-b-P(LG-Hyd)-b-PDMAPMA and FA-PEG-b-P(LG-Hyd)-b-
PDMAPMA copolymers with a weight ratio of 19 : 1 was adopted
during the preparation of nanoparticles.
The critical micelle concentration (CMC) of the mPEG-b-
P(LG-Hyd-DOX)-b-PDMAPMA copolymer was determined using
pyrene as a uorescence probe. The concentration of the
copolymer varied from 5 ꢂ 10ꢁ5 to 0.125 mg mLꢁ1 and the
concentration of pyrene was xed at 6 ꢂ 10ꢁ7 mol Lꢁ1. The
uorescence spectra were recorded using a uorescence spec-
trometer (LS55, Perkin Elmer, USA) with an excitation wave-
length of 330 nm. The emission uorescence was monitored at
373 and 384 nm. The CMC was estimated as the cross-point
when extrapolating the intensity ratio (I373/I384) at low and high
concentration regions.
The mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA copolymer was
synthesized using a two-step reaction procedure. In the rst
step, the benzyl groups of the mPEG-b-PBLG-b-PDMAPMA
copolymer were substituted with hydrazide groups by reacting 1
g of the mPEG-b-PBLG-b-PDMAPMA copolymer with 150 mg of
anhydrous hydrazine in 50 mL of DMF at 40 ꢀC for 24 h.
Thereaer, the solution was dialyzed against 0.25% ammonia
solution for 48 h followed by freeze-drying, producing the
mPEG-b-P(LG-Hyd)-b-PDMAPMA copolymer (yield 93%). In the
second step, 40 mg of the mPEG-b-P(LG-Hyd)-b-PDMAPMA
copolymer was dissolved in 10 mL of DMF, and an excess
amount of DOX$HCl (10 mg) was added together with TEA. The
mixture solution was stirred at room temperature for 24 h and
then dialyzed against phosphate buffered saline (PBS, 2 mM,
pH 7.4) until no further color change was observed. The solu-
tion was protected from light all the time. The mPEG-b-P(LG-
Hyd-DOX)-b-PDMAPMA copolymer was recovered aer lyophi-
lization. The FA-PEG-b-P(LG-Hyd-DOX)-b-PDMAPMA copolymer
was also obtained using the above method. The amount of free
drug in PBS was determined by uorescence spectrophotometry
using a standard calibration curve (0.01–2 mg mLꢁ1). Drug
loading content (DLC) and drug loading efficiency (DLE) were
calculated according to formulae (1) and (2), respectively.
weight of loaded drug
DLCð%Þ ¼
DLEð%Þ ¼
ꢂ 100
ꢂ 100
(1)
weight of polymer
2.6 Gel retardation assay
weight of loaded drug
weight of drug in feed
(2)
The siRNA binding ability of the DOX-NPs was assessed by
agarose gel electrophoresis. A series of DOX-NPs and siRNA
complexes (nanocomplexes) were prepared at different N/P
ratios (0.25, 0.5, 1, 2, 4, 8, 16 and 32). Electrophoresis was
carried out on 1% agarose gel at a constant current of 80 V for
30 min in TAE buffer solution (40 mM Tris–HCl, 1 v/v% acetic
acid, and 1 mM EDTA). The retardation of the nanocomplexes
was visualized by staining with ethidium bromide. The nal
siRNA amount was 0.2 mg per well. The size distribution and
zeta potential of the nanocomplexes at different N/P ratios were
measured. These data were used to determine the threshold N/P
ratio for subsequent experiments. To evaluate the inuence of
DOX loading, the gel electrophoresis assay of the mPEG-b-P(LG-
Hyd)-b-PDMAPMA triblock copolymer was also performed.
2.4 Characterization
1H NMR spectra were obtained on a Bruker spectrometer
(400 MHz) at 25 ꢀC using DMSO-d6 or CDCl3 as the solvent and
tetramethylsilane as the internal reference. Chemical shis
were reported in ppm. Fourier transform infrared spectroscopy
(FTIR) spectra were recorded on a Nicolet AVATAR 360 spec-
trometer using the KBr disc method. Gel permeation chroma-
tography (GPC) measurements were performed using a Waters
1515 GPC instrument equipped with a Waters 1515 isocratic
high-performance liquid chromatography (HPLC) pump, a
Waters 2414 refractive index detector, a Waters 717 plus auto-
sampler and a set of MZ-Gel SD plus columns (300 ꢂ 8.0 mm).
2.7 DOX or siRNA release from the nanocomplexes
DMF containing 0.05 mol Lꢁ1 LiBr was used as the mobile The release kinetics of DOX and siRNA from the nanocomplexes
phase at a ow rate of 0.8 mL minꢁ1 through the columns at were investigated using the dialysis method. Briey, the nano-
40 ꢀC. A series of polystyrene standards were used to generate a complexes (5 mg) were dispersed in 3 mL of PBS (pH 7.4), and
GPC calibration curve. HPLC analysis was carried out on a many such dispersed solutions were prepared and divided
Shimadzu LC-10AVP HPLC system and used to determine the evenly into two groups. To determine the in vitro release proles
form of DOX molecules existing in the mPEG-b-P(LG-Hyd-DOX)- of DOX and siRNA from the nanocomplexes, the two groups of
b-PDMAPMA nanoparticles (DOX-NPs). The applied column was dispersed solutions were sealed in dialysis tubes with molecular
Shim-pack VP-ODS (150 ꢂ 4.6 mm, 5 mm). The mobile phase weight cut-off of 3.5 kDa and 50 kDa, respectively. Each dialysis
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tube was immersed in 47 mL of PBS (pH 5.0 or 7.4) with or without internalized by cells. Trypsinized cells were harvested, xed with
10 mM glutathione and gently shaken at 160 rpm. The amounts of 2.5% glutaraldehyde along with 4% paraformaldehyde for 1 h,
released DOX and FAM-siRNA were monitored for 6 days by post-xed with 1% osmium tetroxide for 1 h, and rinsed with
measuring the uorescence intensities of DOX and FAM-siRNA at distilled water. The xed cells were dehydrated with a graded
an excitation/emission wavelength at 480/590 nm and an excita- series of ethanol solutions (40, 60, 80, 90, and 100%) and
tion/emission wavelength at 488/520 nm, respectively. At pre- embedded in epoxy resin. Ultrathin sections (50–100 nm) were
determined intervals, 2 mL aliquots were withdrawn and replaced sliced by ultramicrotome, stained with 2% uranyl acetate and lead
by the same volume of PBS. The amounts of released payloads citrate solution, and then imaged on a Hitachi H-600 trans-
were determined by uorescence spectrophotometry using a mission electron microscope at an acceleration voltage of 75 kV.
standard calibration curve (0.01–2 mg mLꢁ1 for DOX and 1–200 nM
for FAM-siRNA). All measurements were done in triplicate.
2.11 In vitro cytotoxicity measurements
The in vitro cytotoxicities of the mPEG-b-P(LG-Hyd)-b-
PDMAPMA triblock copolymer, DOX-NPs, nanocomplexes and
FA-nanocomplexes were evaluated by the MTT assay using MCF-
7 cells. Briey, MCF-7 cells (approximately 3 ꢂ 103 per well)
were plated in 96-well plates in 200 mL of culture medium and
incubated at 37 ꢀC for 24 h. 200 mL of culture media containing
different concentrations of the formulations (equivalent DOX
concentration in the range of 0.032 to 6.4 mg mLꢁ1) were
exchanged to each of the 3 wells. Aer 24 h or 48 h of culture, 20
mL of MTT (5 mg mLꢁ1) was added to each well and the plates
were incubated for another 4 h. The resulting formazan product
was dissolved with 150 mL DMSO, and the absorbance at 490 nm
was read on a Microplate Spectrophotometer (Bio-Rad 60, USA).
Viability was calculated by comparing with the blank control
using the formula (test/control) ꢂ 100%.
2.8 Flow cytometry analysis
Two-color ow cytometry was used to determine the cellular
uptake of FAM-siRNA and DOX formulated in the nano-
complexes. Briey, MCF-7 cells were seeded into 6-well plates
with 2 ꢂ 105 per well and incubated at 37 ꢀC until 70%
conuence was reached, and then FA-decorated nanocomplexes
(FA-nanocomplexes) containing DOX (6 mg mLꢁ1, equivalent
concentration of DOX) and FAM-siRNA (200 nM) were added,
using the mPEG-b-P(LG-Hyd)-b-PDMAPMA copolymer, DOX-
NPs and nanocomplexes as controls. Aer 4 h of incubation, the
medium was aspirated. The cells were rinsed with cold PBS
three times, trypsinized, washed with cold PBS, and nally
examined on the Becton Dickson Facs Canto II ow cytometer
(BD Biosciences, USA). The uorescence intensities of DOX and
FAM-siRNA were analyzed on a ow cytometry by exciting at
488 nm and FL1 band-pass emission (581 ꢃ 21 nm) for the red
DOX or FL2 band-pass emission (530 ꢃ 20 nm) for the green
FAM-siRNA. FlowJo soware was used to analyze the data.
2.12 In vitro apoptosis detection
Annexin V and propidium iodide (PI) were used to assess the
levels of apoptosis induced by different formulations. Briey,
MCF-7 cells were cultured with the mPEG-b-P(LG-Hyd)-b-
PDMAPMA triblock copolymer, free DOX, DOX-NPs, nano-
complexes, or FA-nanocomplexes (containing 3 mg mLꢁ1 DOX
and 100 nM siRNA) for 48 h. A minimum of 5 ꢂ 105 cells was
harvested and stained using the Annexin V-FITC apoptosis
detection kit according to the manufacturer's protocol prior to
ow cytometry measurements. Data analysis was performed by
FlowJo soware. Early apoptotic cells with exposed phosphati-
dylserine but intact cell membranes bound to Annexin V-FITC
but excluded PI. Cells in necrotic or late apoptotic stages were
positive with both Annexin V-FITC and PI.
2.9 CLSM observation
CLSM was used to evaluate the cellular uptake and intracellular
distribution of different formulations in MCF-7 cells. MCF-7
cells (5 ꢂ 104) were seeded into CLSM dishes and incubated at
37 ꢀC for 24 h. The nanocomplexes and FA-nanocomplexes
(containing 3 mg mLꢁ1 DOX and 100 nM siRNA) were added
separately to CLSM dishes. Aer incubating for different time
intervals (1, 4, and 24 h), the medium was discarded, and then
the cells were rinsed with PBS three times, xed with 4%
paraformaldehyde for 20 min, stained with 150 mL of DAPI
solution for 5 min and washed with PBS again. The samples
were examined and photographed using a confocal laser scan-
ning microscope (Olympus Fluoview FV1000, Japan) equipped
with 60ꢂ oil-immersion lenses. Both DOX and FAM-siRNA were
excited at 488 nm, and their uorescence signals were collected
with 580–610 nm and 500–530 nm band-pass lters, respec-
tively. DAPI was excited at 340 nm, and the uorescence signal
was detected at 480–500 nm.
2.13 Statistical analysis
All experiments were conducted three times independently with
triplicates, and the results were presented as average values ꢃ
standard deviation. Signicant differences were evaluated using
the Student's t-test. Values of p < 0.05 were considered to be
statistically signicant.
3. Results and discussion
2.10 Cellular uptake studies by TEM
3.1 Synthesis of FA/mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA
triblock copolymers
nanocomplexes or FA-nanocomplexes (containing 3 mg mLꢁ1 To co-deliver DOX and P-gp siRNA to MCF-7 breast cancer cells,
DOX and 100 nM siRNA), the cells were thoroughly washed with novel multifunctional triblock copolymers FA/mPEG-b-P(LG-
PBS for three times to eliminate the nanoparticles that were not Hyd)-b-PDMAPMA were designed and synthesized, as shown in
MCF-7 cells (4 ꢂ 105 per well) were seeded into 6-well plate and
incubated at 37 ꢀC for 24 h. Aer 4 h of incubation with the
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Fig. 1 (A) Synthesis route of FA/m-PEG-b-P(LG-Hyd-DOX)-b-PDMAPMA triblock copolymers. (B) Schematic illustrations of the formation of
nanocomplexes and their endocytosis by MCF-7 cells.
Fig. 1. The triblock copolymers were synthesized by a combi-
The synthesized FA-PEG-Cys, mPEG-b-PBLG, mPEG-b-PBLG-
nation of ROP, RAFT polymerization and hydrazinolysis b-PDMAPMA and mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA copoly-
(Fig. 1A). Firstly, two telechelic PEG derivatives (mPEG-Cys and mers were characterized using 1H NMR, and the results are
1
Cys-PEG-Cys) were synthesized by reacting BIED with mPEG-OH shown in Fig. 2. As observed in the H NMR spectrum of FA-
or HO-PEG-OH, followed by hydrolysis in distilled water. Cys- PEG-Cys (Fig. 2A), the successful synthesis of FA-PEG-Cys was
PEG-Cys was used to react with FA to produce FA-PEG-Cys conrmed by the appearance of both the proton signals at 6.7–
through the carbodiimide reaction between one primary amine 8.7, 5.5, 4.5, 1.8 ppm corresponding to the aromatic protons of
group of Cys-PEG-Cys and the carboxyl group of FA. Secondly, FA and the characteristic signal at 3.6 ppm ascribed to the
1
FA/m-PEG-b-PBLG diblock copolymers were synthesized by the methylene protons of PEG. As indicated by the H NMR spec-
ROP of BLG-NCA using FA-PEG-Cys or mPEG-Cys as the initi- trum of the mPEG-b-PBLG copolymer (Fig. 2B), the chemical
ator, respectively. Thirdly, to generate FA/m-PEG-b-PBLG-b- shis at 7.3 and 5.1 ppm were ascribed to the protons of benzyl
PDMAPMA triblock copolymers by the RAFT polymerization of and methylene groups in the side chains of PBLG, respectively.
DMAPMA, two macro-RAFT agents (FA-PEG-b-PBLG-RAFT and The proton signals at 1.9–2.2 ppm corresponded to the (–CH2–
mPEG-b-PBLG-RAFT) were synthesized through the carbodii- CH2–COO–) moieties of BLG units.33 The chemical shi at 3.6
mide reaction between the DDAT RAFT agent and mPEG-b- ppm was due to the methylene protons of the oxyethylene units
PBLG or FA-PEG-b-PBLG copolymers. The RAFT polymerization of PEG. The number average molecular weight (Mn) of the
of DMAPMA was performed in 1,4-dioxane using AIBN as an mPEG-b-PBLG diblock copolymer was calculated from Fig. 2B
initiator in the presence of either of the two macro-RAFT agents. by integrating the characteristic peaks according to eqn (3) and
Finally, the pendant benzyl protecting groups of the PBLG (4).34 The calculated Mn was 6820 Da which was consistent
blocks of the two triblock copolymers were converted into with that (8770 g molꢁ1) determined by GPC (Fig. 3A). These
hydrazide groups via the hydrazinolysis reaction, producing FA/ results demonstrated the formation of the mPEG-b-PBLG
m-PEG-b-P(LG-Hyd)-b-PDMAPMA triblock copolymers. The two diblock copolymer.
triblock copolymers have the following advantages. The PEG
46 ꢂ Ab
m ¼
(3)
(4)
blocks have good water solubility, low toxicity and immunoge-
nicity, and they have extensively been used in drug delivery
systems.31 The P(LG-Hyd) blocks can conjugate DOX molecules
via a pH-sensitive hydrazone linkage. The PDMAPMA blocks
have been shown to efficiently condense nucleic acids at phys-
iological pH.32
The mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA copolymer is
amphiphilic and can self-assemble in aqueous solutions into
cationic nanoparticles which could condense siRNA to form
nanocomplexes (Fig. 1B). The nanocomplexes were expected to
stably preserve payloads under physiological conditions (pH
7.4), be selectively taken up by MCF-7 breast cancer cells via
folate receptor-mediated endocytosis, and rapidly release
payloads in the intracellular reductive acidic microenviron-
ment, thereby improving the antitumor efficacy of DOX.
2 ꢂ Af
Mn ¼ 2170 + 219 ꢂ m
Here, 2170 and 219 are the molecular weights of PEG blocks and
BLG units, respectively. 46 is the number of repeating units in
PEG. A represents the integral peak value. Subscripts b and f
correspond to the shis of the methylene protons of PEG blocks
and the methylene protons adjacent to the benzene rings of
PBLG blocks, respectively.
In comparison with the spectrum in Fig. 2B, some new
chemical shis at 3.2, 2.3–2.4, 2.0 and 1.0 ppm were observed
in the 1H NMR spectrum in Fig. 2C and attributed to the
protons of DMAPMA.35 At the same time, the molecular weight
(12 910) of the mPEG-b-PBLG-b-PDMAPMA triblock copolymer
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Fig. 2 1H NMR spectra of (A) FA-PEG-Cys in DMSO-d6, (B) mPEG-b-PBLG diblock copolymer in DMSO-d6, (C) mPEG-b-PBLG-b-PDMAPMA
triblock copolymer in CDCl3 and (D) mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA triblock copolymer in DMSO-d6.
and mPEG-b-PBLG-b-PDMAPMA triblock copolymers (Fig. 3B
and C), a decrease in the molecular weight provided an exper-
imental proof of the hydrazinolysis reaction. Moreover, the
chemical shis at 0.82, 1.13–1.47, 4.93 and 7.20–7.92 ppm
attributed to DOX molecules were also observed in Fig. 2D,
which meant that DOX molecules were chemically conjugated
onto the P(LG-Hyd) blocks via hydrazone bonds.36 This result
was further veried by comparing the FTIR spectra of the
Fig. 3 GPC traces of (A) mPEG-b-PBLG diblock copolymer (Mn,GPC
8770 g molꢁ1, PDI ¼ 1.07), (B) mPEG-b-PBLG-b-PDMAPMA (Mn,GPC
¼
¼
12 910 g molꢁ1, PDI ¼ 1.06) and (C) PEG-b-P(LG-Hyd)-b-PDMAPMA
triblock copolymers (Mn,GPC ¼ 10 250 g molꢁ1, PDI ¼ 1.14).
(Fig. 3B) was much higher than that of the mPEG-b-PBLG
diblock copolymer. These results suggested the successful
synthesis of the mPEG-b-PBLG-b-PDMAPMA triblock copoly-
mer. As shown in Fig. 2D, the absence of the chemical shis
at 7.3 and 5.1 ppm due to the protons of benzyl groups indi-
cated the complete debenzylation of PBLG blocks. By
comparing the GPC traces of mPEG-b-P(LG-Hyd)-b-PDMAPMA
Fig. 4 FTIR spectra of (A) mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA
and (B) mPEG-b-P(LG-Hyd)-b-PDMAPMA triblock copolymers.
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mPEG-b-P(LG-Hyd)-b-PDMAPMA copolymer before and aer following dilution in a large volume of body uid aer intra-
conjugation with DOX (Fig. 4). In the FTIR spectrum of the venous injection, which is desirable for the systemic delivery of
mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA triblock copolymer in drugs.
Fig. 4A, the peaks at 3435 and 1075 cmꢁ1 were the characteristic
absorption peaks of DOX.37,38 To reveal the form of DOX existing
3.3 Formation and characterization of nanocomplexes
in the DOX-NPs, HPLC was employed to separate and determine
The cationic DOX-NPs had a high positive zeta potential of
31.5 ꢃ 7.0 mV when dispersed in PBS (pH 7.4), conrming their
suitability to condense siRNA via an electrostatic interaction to
form nanocomplexes. To determine the optimal N/P ratio for
P-gp siRNA complexation, the complex stability of a series of
nanocomplexes at different N/P ratios was investigated by
agarose gel electrophoresis, using the complexes of siRNA and
the mPEG-b-P(LG-Hyd)-b-PDMAPMA triblock copolymer as a
control. The retardation of siRNA mobility in gel electrophoresis
was used to characterize the ability of the cationic NPs to
the amount of DOX unconjugated to the DOX-NPs. The results
of HPLC analysis (Fig. S1†) showed that the mPEG-b-P(LG-Hyd-
DOX)-b-PDMAPMA copolymer eluted at 5 min and the uncon-
jugated DOX eluted at 10 min. The content of free DOX absor-
bed in the DOX-NPs was less than 3%. These results conrmed
the successful chemical conjugation of DOX onto the mPEG-b-
P(LG-Hyd)-b-PDMAPMA triblock copolymer.
3.2 Preparation and characterization of DOX-NPs
The mPEG-b-P(LG-Hyd-DOX)-b-PDMAPMA triblock copolymer complex siRNA. As illustrated in Fig. 6, the cationic DOX-NPs
was amphiphilic and could self-assemble into cationic DOX- and the triblock copolymer could completely retard siRNA
NPs in aqueous solutions. The DLC and DLE of DOX deter- migration at N/P ratios greater than 1 and 4, respectively,
mined using uorescence measurements were 14.1% and indicating the complexation of full negatively charged siRNA
65.5%, respectively. To reveal the properties of the DOX-NPs, chains.
the particle size, zeta potential and the CMC value of mPEG-b-
It is well known that the N/P ratio has a signicant effect on
P(LG-Hyd-DOX)-b-PDMAPMA triblock copolymer were charac- the size and zeta potential of carrier–siRNA complexes, which
terized. In this study, an ultrasonic method was selected to are important parameters of carriers for drug and gene deliv-
prepare the DOX-NPs. DLS and TEM were used to characterize eries.40 The average size and zeta potential of the nano-
the size and morphology of the DOX-NPs, respectively, and the complexes at different N/P ratios are shown in Fig. 6C, and the
results are shown in Fig. 5. The DOX-NPs had an average corresponding size distributions are presented in Fig. S3.† It
hydrodynamic diameter of 211.9 ꢃ 3.3 nm and a zeta potential was found that the zeta potential of the nanocomplexes
of 31.5 ꢃ 7.0 mV (Fig. 5A). The TEM image indicated that the signicantly increased from 8.6 to 30.2 mV as the N/P ratio
DOX-NPs were well dispersed with a spherical morphology and increased from 4 to 32, and the average size increased from
their average size was about 150 nm. The difference in the 153.1 to 197.2 nm. The reason why the average size increased
average size of the DOX-NPs between the DLS and TEM results with increasing N/P ratio is that a higher N/P ratio will lead to a
was caused by the shrinkage of the DOX-NPs during the TEM poorer complexing effect. The polydispersity indices (PDIs) of
sample preparation. The CMC value of the mPEG-b-P(LG-Hyd- the nanocomplexes at N/P ratios from 4 to 32 were 0.194, 0.226,
DOX)-b-PDMAPMA triblock copolymer was determined from 0.214, 0.184 and 0.248 respectively. It has been reported that
the plot of uorescence intensity ratio of the peaks at 373 and 200 nm is a rough upper size limit for cellular uptake by
384 nm (I373/I384) in the pyrene uorescence spectra versus its phagocytosis,41 and the nanoparticles bigger than this size
concentration. As shown in Fig. S2,† the CMC value of the might be excluded from cellular internalization altogether,
copolymer was about 5.79 ꢂ 10ꢁ3 mg mLꢁ1. Such a CMC value resulting in the inability to mediate gene knockdown. There-
is signicantly lower than those of PEG-based amphiphilic fore, an N/P ratio of 16 was used for in vitro cellular uptake and
linear polymers reported in the literature.39 In general, a low cytotoxicity studies in the present study. The nanocomplexes
CMC value means that the nanoparticles can keep their stability obtained at an N/P ratio of 16 exhibited a uniformly spherical
Fig. 5 (A) DLS result and (B) TEM image of DOX-NPs (sample concentration: 1 mg mLꢁ1). Scale bar: 500 nm.
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Fig. 6 Electrophoretic mobility of free siRNA and siRNA condensed with (A) mPEG-b-P(LG-Hyd)-b-PDMAPMA triblock copolymer and (B)
cationic DOX-NPs at different N/P ratios. (C) Size and zeta potential of nanocomplexes at various N/P ratios. (D) TEM image and (E) DLS result of
nanocomplexes at an N/P ratio of 16 (sample concentration: 1 mg mLꢁ1). The scale bar denotes 500 nm.
morphology and had a diameter in the range of 150–250 nm were released from the nanocomplexes in a pH and reduction
(Fig. 6D), and the corresponding average hydrodynamic size was dual-dependent fashion. The release rates of DOX and P-gp
196.8 ꢃ 3.5 nm (Fig. 6E). Compared with the DOX-NPs, the siRNA increased with decreasing pH no matter whether GSH
nanocomplexes had a slightly bigger size because of the loading was present or not. The reason for this result should be due to
of siRNA.
the acidic hydrolysis of the hydrazone linkage between DOX and
the triblock copolymer and the protonation of the triblock
copolymer, respectively. In the reducing acidic environment
(pH 5.0, 10 mM GSH), about 52.6% of DOX and 36.5% of FAM-
siRNA were released in the initial burst phase over 12 h. Aer
144 h, the cumulative amounts of released DOX and P-gp siRNA
were approximately 90.1% and 74.4%, respectively. In contrast,
only about 19.8% of DOX and 18.8% of FAM-siRNA were
released at pH 7.4 aer 144 h. On the other hand, DOX and
siRNA were released more quickly in the presence of GSH than
in the absence of GSH when other conditions were the same.
These results veried that the release of DOX and P-gp from the
nanocomplexes could be triggered by the levels of intracellular
pH and GSH. The stimuli-responsive release behaviors should
be ascribed to both the acid-induced cleavage of the hydrazone
linkage and the GSH-triggered PEG detachment.
3.4 Release proles of DOX and P-gp siRNA from
nanocomplexes
To date, disulde and hydrazone bonds have extensively been
used in the design and synthesis of stimuli-triggered vehicles
for the delivery and controlled release of payloads because they
can sense the reductive acidic environments inside cancer
cells.42,43 To reveal the stimuli-triggered release of DOX and P-gp
siRNA from the nanocomplexes, their in vitro release kinetics
were investigated in PBS solutions (pH 5.0 and 7.4) with or
without GSH (10 mM). The pH and GSH levels were chosen to
simulate those in the endosomal and lysosomal vesicles inside
the cancer cells, whose pH values and GSH concentrations are
4–6 and 2–10 mM, respectively. Fig. 7A shows the release
proles of DOX and P-gp siRNA from the nanocomplexes under
different conditions by the dialysis method as well as the
diffusion proles of free DOX from a dialysis bag. It is seen from
3.5 Cellular uptake studies using ow cytometry
Fig. 7A that the diffusion proles of free DOX from the dialysis Two-color ow cytometry was used to assess the ability of the
bags at pH 5.0 and 7.4 were nearly the same and the diffusion nanocomplexes to simultaneously deliver DOX and P-gp siRNA
was essentially accomplished within 7 h. This result indicated to MCF-7 cells. Fig. 8 shows the light-scatter proles of the MCF-
that there was a negligible difference in the diffusion rates of 7 cells treated with the different formulations by the two-color
free DOX out of the dialysis bags at the studied pH values. The ow cytometry. It could be seen that the MCF-7 cells incubated
release proles of DOX and P-gp siRNA from the nano- with the DOX-NPs and FAM-siRNA/triblock copolymer
complexes (Fig. 7B and C) revealed that DOX and P-gp siRNA complexes displayed red and green uorescence, respectively. A
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Fig. 7 (A) Diffusion profiles of free DOX from a dialysis bag at pH 5.0 and 7.4. Release profiles of DOX and P-gp siRNA from the nanocomplexes in
the absence and presence of 10 mM GSH at (B) pH 7.4 and (C) 5.0.
considerable part of the cells treated with the nanocomplexes nanocomplexes were studied by CLSM, and the results are
showed both green and red uorescence, suggesting that the shown in Fig. 9A and B. Aer 1 h of incubation, both the red
nanocomplexes could simultaneously transport siRNA and DOX uorescence of DOX and the green uorescence of FAM-siRNA
into the same MCF-7 cell. It was also found that the percentage were observed in the MCF-7 cells, though the uorescence
(59.5%) of the MCF-7 cells treated with the FA-nanocomplexes signals were weak. This result indicated that all of the nano-
exhibiting both red and green uorescence was signicantly complexes could be internalized by the MCF-7 cells irrespective
higher than that (42%) of the MCF-7 cells treated with the of the attachment of targeting moieties. Aer 4 h of incubation,
nanocomplexes. This result indicated that the FA decoration the red uorescence started to separate from the green one and
facilitated the internalization of the nanocomplexes by MCF-7 to aggregate in the nuclei, indicating that DOX had been
cells, which meant that folate-receptors were overexpressed on partially released from the nanocomplexes. Aer 24 h of incu-
the surface of the MCF-7 cells.44
bation, the red and green uorescent intensities became
stronger. By comparing Fig. 9A with 9B, it was obvious that both
the red and green uorescent intensities in the MCF-7 cells
treated with the FA-nanocomplexes were stronger than those in
the MCF-7 cells treated with the nanocomplexes, though the
concentrations of the two kinds of nanoparticles in the culture
media were the same. This result was consistent with that from
3.6 Microscopic studies on cellular uptake
Aer MCF-7 cells were incubated with the nanocomplexes
decorated and undecorated with FA for 1, 4 and 24 h,
the cellular uptake and intracellular distribution of the
Fig. 8 Cellular uptake of different formations in MCF-7 cells after 4 h of incubation. MCF-7 cells were treated with (A) pristine triblock copolymer
(0.5 mg mLꢁ1), (B) free DOX, (C) DOX-NPs, (D) triblock copolymer/siRNA complexes, (E) nanocomplexes and (F) FA-nanocomplexes (dose:
6 mg mLꢁ1 DOX and 200 nM siRNA-FAM).
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Fig. 9 CLSM images of MCF-7 cells after incubation with (A) FA-nanocomplexes, (B) nanocomplexes, and (C) FA-nanocomplexes in
the presence of 1 mg mLꢁ1 of free FA for 1, 4 and 24 h. Cell nuclei were stained blue with DAPI. The concentrations of DOX and FAM-siRNA were
6 mg mLꢁ1 and 200 nM, respectively. Scale bars represent 30 mm.
the two-color ow cytometry analysis (Fig. 8). It should be suggested that excess free FA molecules inhibited the cellular
closely related to the FA receptor-mediated endocytosis. uptake of the nanocomplexes by competitive binding to the
Importantly, the strong DOX uorescence was observed in the folate-receptors on the surface of MCF-7 cells.
nuclei, indicating that the acidic environment of the endo-
TEM was also used to further reveal the cellular uptake of
somes and lysosomes triggered the release of DOX from the nanovehicles by MCF-7 cells. It is seen from Fig. 10 that MCF-7
nanovehicles. It is well known that the accumulation of free cells could take up the two kinds of nanovehicles aer 4 h of
DOX in the nuclei is essential for its antiproliferative activity on incubation. Compared with the nanocomplexes, more FA-
cancer cells. To further evaluate the role of FA in the cellular nanocomplexes were taken up by the MCF-7 cells. The reason
uptake of the nanocomplexes, MCF-7 cells were incubated with for this is that the FA-nanocomplexes could actively recognize
the FA-nanocomplexes in a culture medium containing 1 and bind to the FA receptors overexpressed in the MCF-7 cells,
mg mLꢁ1 of free FA,45 and the corresponding CLSM images are which facilitated the endocytosis of the FA-nanocomplexes into
shown in Fig. 9C. In comparison with Fig. 9A, the intensities of MCF-7 cells. The TEM results supported those from the above
both red and green uorescence were much weaker. This result CLSM studies.
3.7 In vitro cytotoxicity
To evaluate the synergistic effect of DOX and P-gp siRNA and the
active targeting ability of the FA-decorated nanocomplexes, the
in vitro cytotoxicities of the triblock copolymer, free DOX, DOX-
NPs, nanocomplexes and FA-nanocomplexes were evaluated in
MCF-7 cells by the MTT assay aer incubation for 24 and 48 h,
and the results are presented in Fig. 11. The N/P ratio was
xed at 16 and the DOX loading content varied from 0.03 to
6.4 mg mLꢁ1. As shown in Fig. 11A, no signicant cytotoxicity
Fig. 10 TEM images of MCF-7 cells treated with (A) FA-nano-
against MCF-7 cells was observed for the mPEG-b-P(LG-Hyd)-
b-PDMAPMA triblock copolymer at concentrations up to
complexes and (B) nanocomplexes for 4 h. The nanoparticles are
indicated by the white arrows. Scale bars: 2 mm.
Fig. 11 Cell viabilities of MCF-7 cells treated with different formulations with different concentrations determined by the MTT assay. MCF-7 cells
were treated with (A) the triblock copolymer for 24 and 48 h and payload-contained formulations for (B) 24 and (C) 48 h. *p < 0.05.
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100 mg mLꢁ1, indicating that the copolymer showed good cells being positive for Annexin V-FITC and negative for PI were
biocompatibility. The reason for this is that the existence of the classied as being apoptotic. The cells being positive for PI were
stealthy PEG blocks signicantly reduced the cytotoxicity of the classied as being necrotic. As expected, the nanocomplexes
copolymer.46 All the formulations containing DOX exhibited a group showed a higher apoptotic percentage (37.4%) than the
dose-dependent cytotoxic effect in the MCF-7 cells (Fig. 11B and DOX-NPs group (28.0%) and the free DOX group (26.9%). This
C). On the basis of the IC50 values (concentrations that kill 50% should be attributed to the fact that the suppression of P-gp
of MCF-7 cells) of the formulations, their killing efficiencies efflux pumps in the MCF-7 cells by P-gp siRNA will lead to the
were ranked as follows: FA-nanocomplexes > nanocomplexes > increase in the intracellular drug concentration and induce cell
free DOX > DOX-NPs. The IC50 values of the three nanovehicles apoptosis.48 It was also found that the DOX-NPs group had a
were 1.99 ꢃ 0.25, 2.77 ꢃ 0.31, 3.44 ꢃ 0.29 and 5.09 ꢃ 0.79 mg lower cytotoxicity on MCF-7 cells than the free DOX group. The
mLꢁ1 for 24 h and 1.08 ꢃ 0.09, 2.49 ꢃ 0.18, 2.87 ꢃ 0.31 and 3.12 FA-nanocomplexes group exhibited the highest apoptotic
ꢃ 0.33 mg mLꢁ1 for 48 h, respectively. It was found that the IC50 percentage of 77.2%, and the result was consistent with that
value of the FA-nanocomplexes was over 2.5 times lower than from the MTT assay. These results further conrmed that the
that of the DOX-NPs. The results showed that the co-delivery of nanocomplexes could co-deliver DOX and P-gp siRNA into the
DOX and P-gp siRNA into the MCF-7 cells could enhance the MCF-7 cells and increase anti-tumor efficacy.
anti-tumor effect of DOX through their synergistic effect in
comparison to single DOX delivery. The FA-nanocomplexes
exhibited the lowest IC50 value, because they could be taken up
4. Conclusions
more efficiently by the MCF-7 cells through FA receptor-medi- In this study, novel multifunctional triblock copolymers, FA/m-
ated endocytosis than other formulations.47 These results PEG-b-P(LG-Hyd)-b-PDMAPMA, were successfully synthesized.
demonstrated that the co-delivery of DOX and P-gp siRNA using The triblock copolymers could conjugate DOX via a hydrazone
the multifunctional copolymers was a promising strategy for linkage and simultaneously complex P-gp siRNA via an elec-
breast cancer treatment.
trostatic interaction, resulting in the formation of spherical
nanocomplexes in aqueous solutions. The in vitro drug release
kinetics of DOX and P-gp siRNA from the nanocomplexes
exhibited a pH and reduction dual-dependent behavior. The co-
3.8 Annexin V-FITC apoptosis assay
To conrm the cytotoxicity of DOX in different transportation delivery of DOX and P-gp siRNA into MCF-7 breast cancer cells
modalities, we used Annexin V-FITC and PI to stain the MCF-7 enhanced the anticancer efficacy of DOX via their synergetic
cells treated with an equivalent DOX concentration (3 mg mLꢁ1
)
effects. Compared with FA-free nanocomplexes, FA-decorated
to determine their apoptotic efficiency, and the results are nanocomplexes were more efficiently internalized by MCF-7
shown in Fig. 12. The cells being negative for both Annexin cells via FA receptor-mediated endocytosis and showed higher
V-FITC and PI staining were classied as being alive, while the cytotoxicity. The present results demonstrate that the novel
Fig. 12 Apoptosis and cell death in the MCF-7 cells treated with different formulations. (A) Control group (untreated cells), (B) free DOX
group, (C) DOX-NPs group, (D) nanocomplexes group and (E) FA-nanocomplexes group. The concentrations of DOX and P-gp siRNA used were
3 mg mLꢁ1 and 100 nM, respectively. *p < 0.05.
J. Mater. Chem. B
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multifunctional triblock copolymers may be a promising 19 J. L. Zhu, H. Cheng, Y. Jin, S. X. Cheng, X. Z. Zhang and
vehicle for the co-delivery of DOX and siRNA to enhance the
anti-tumor effect of DOX.
R. X. Zhuo, J. Mater. Chem., 2008, 18, 4433–4441.
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21 N. Cao, D. Cheng, S. Y. Zou, H. Ai, J. M. Gao and X. T. Shuai,
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Acknowledgements
The authors gratefully acknowledge the nancial support from 22 Q. Yin, J. N. Shen, Z. W. Zhang, H. J. Yu and Y. P. Li, Adv. Drug
the National Natural Science Foundation of China (50603020 Delivery Rev., 2013, 65, 1699–1715.
and 50773062), the Project of Natural Science Foundation of 23 Y. L. Xiao, H. Hong, A. Javadi, J. W. Engle, W. J. Xu,
Shaanxi Province, China (2013K09-27) and the Fundamental
Research Funds for the Central Universities (XJJ2014124,
XJJ2013130 and XJJ2012146).
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