Redox-responsive polymer-drug conjugates based on doxorubicin and chitosan oligosaccharide- g -stearic acid for cancer therapy

Article abstract of DOI:10.1021/mp500710x

Here, a biodegradable polymer-drug conjugate of doxorubicin (DOX) conjugated with a stearic acid-grafted chitosan oligosaccharide (CSO-SA) was synthesized via disulfide linkers. The obtained polymer-drug conjugate DOX-SS-CSO-SA could self-assemble into nanosized micelles in aqueous medium with a low critical micelle concentration. The size of the micelles was 62.8 nm with a narrow size distribution. In reducing environments, the DOX-SS-CSO-SA could rapidly disassemble result from the cleavage of the disulfide linkers and release the DOX. DOX-SS-CSO-SA had high efficiency for cellular uptake and rapidly released DOX in reductive intracellular environments. In vitro antitumor activity tests showed that the DOX-SS-CSO-SA had higher cytotoxicity against DOX-resistant cells than free DOX, with reversal ability up to 34.8-fold. DOX-SS-CSO-SA altered the drug distribution in vivo, which showed selectively accumulation in tumor and reduced nonspecific accumulation in hearts. In vivo antitumor studies demonstrated that DOX-SS-CSO-SA showed efficient suppression on tumor growth and relieved the DOX-induced cardiac injury. Therefore, DOX-SS-CSO-SA is a potential drug delivery system for safe and effective cancer therapy.

Full text of DOI:10.1021/mp500710x

Article
pubs.acs.org/molecularpharmaceutics
Redox-Responsive Polymer−Drug Conjugates Based on Doxorubicin
and Chitosan Oligosaccharide‑g‑stearic Acid for Cancer Therapy
Yigang Su,†,§ Yingwen Hu,†,§ Yongzhong Du,† Xuan Huang,‡ Jiabei He,† Jian You,† Hong Yuan,†
and Fuqiang Hu*,†
†College of Pharmaceutical Sciences, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, People’s Republic of China
‡Department of Pharmacy, School of Medicine Science, Jiaxing University, Jiaxing, Zhejiang 314001, People’s Republic of China
S
* Supporting Information
ABSTRACT: Here, a biodegradable polymer−drug conjugate
of doxorubicin (DOX) conjugated with a stearic acid-grafted
chitosan oligosaccharide (CSO-SA) was synthesized via
disulfide linkers. The obtained polymer−drug conjugate
DOX-SS-CSO-SA could self-assemble into nanosized micelles
in aqueous medium with a low critical micelle concentration.
The size of the micelles was 62.8 nm with a narrow size
distribution. In reducing environments, the DOX-SS-CSO-SA
could rapidly disassemble result from the cleavage of the
disulfide linkers and release the DOX. DOX-SS-CSO-SA had
high efficiency for cellular uptake and rapidly released DOX in
reductive intracellular environments. In vitro antitumor activity tests showed that the DOX-SS-CSO-SA had higher cytotoxicity
against DOX-resistant cells than free DOX, with reversal ability up to 34.8-fold. DOX-SS-CSO-SA altered the drug distribution in
vivo, which showed selectively accumulation in tumor and reduced nonspecific accumulation in hearts. In vivo antitumor studies
demonstrated that DOX-SS-CSO-SA showed efficient suppression on tumor growth and relieved the DOX-induced cardiac
injury. Therefore, DOX-SS-CSO-SA is a potential drug delivery system for safe and effective cancer therapy.
KEYWORDS: redox-responsive, polymer−drug conjugates, controlled release, chitosan, drug resistance
Doxorubicin (DOX) is widely used for the treatment of
various tumors, including hematological malignancies, soft
tissue sarcomas, and many others. The exact action mechanism
of DOX remains unclear, but generally accepted that it interacts
with DNA through intercalation and inhibits topoisomerase II.
Despite its high antitumor activity, DOX is limited in the
clinical application due to severe side effects and multiple drug
resistance (MDR). DOX induces cytotoxicity in normal tissue
and cardiotoxicity with a lack of specific biodistribution.15 In
MDR carcinoma cells, overexpression of P-glycoprotein (P-gp)
can lead to efflux of the drug from cells.16
In previous studies, we have synthesized glycolipid-like
copolymer based on chitosan oligosaccharide (CSO) and
stearic acid (SA).17 The obtained chitosan oligosaccharide
copolymer (CSO-SA) has a stable physicochemical property,
which is widely developed for drug delivery, including
chemicals and genetic drugs.18−21 Moreover, CSO-SA can be
internalized into cells excellently attributed to the hydrophobic
microdomains near the surface of the micelles.18 However,
along with many copolymers, the drug release rate of CSO-SA
1. INTRODUCTION
Over the past 20 years, polymer−drug conjugates have
attracted abundant attention and several of them are currently
under clinical investigation as controlled drug delivery systems
for anticancer therapy.1,2 Usually, a polymer−drug conjugate
consists of a hydrophilic polymer and a hydrophobic drug,
which are joined via chemical linkers. Chemically conjugating
drugs onto polymer not only benefits the polymer micelles but
also effectively prevents drug leaking into the circulation.3
Smart polymer−drug conjugate is a prodrug, which remains
inactive in systemic circulation and releases activated drugs in
response to specific stimuli at the target sites, such as
temperature, pH, redox potential, light, and enzymes.4−9
Among the stimuli, redox potential is often used to trigger
the intracellular drug delivery.4,10,11 In the human body, the
intracellular concentration of reduced substances is on average
1−11 mM, but only 1−10 μM in fluids outside cells, such as
plasma. In addition, the intracellular thiol concentration is
much higher in cancer cells than corresponding normal
tissues.12,13 Disulfide bonds (−S−S−) have great biological
value, which can be cleaved into thiols via abundant reducing
substances, including glutathione (GSH).14 This phenomenon
provides an opportunity for triggered drug to release from
disulfide-linked polymer−drug conjugates within the tumor
cells.
Received: October 23, 2014
Revised: January 27, 2015
Accepted: March 9, 2015
Published: March 9, 2015
© 2015 American Chemical Society
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was relatively slow according to the in vitro drug release assay,
resulting from the slow degradation of the amide linkages
between the hydrophilic shell and the hydrophobic core. Thus,
polymer−drug conjugates with DOX attached to CSO-SA via a
disulfide linkage responding to intracellular high concentrations
of GSH might be a feasible approach for triggered drug release.
In this study, we present a polymer−drug conjugate system
for anticancer drug delivery and redox-triggered drug release.
When polymer−drug conjugate passively targeted the solid
tumor tissue and was internalized by tumor cells, DOX was
released from the micelles due to cleavage of disulfide linkers in
response to the reductive intracellular microenvironment and
trans-located into nuclei to take effect. Doxorubicin base
(DOX), as a model antitumor drug, was covalently conjugated
to the backbone of the CSO-SA micelles via disulfide bonds to
form redox-responsive doxorubicin−S−S−chitosan oligosac-
charide−g-stearic acid conjugate micelles. The characteristics, in
vitro drug release behavior in a reductive environment and the
cytotoxicity were then investigated. In vivo experiments were
conducted to confirm the passive targeting ability and the
antitumor activities of DOX-SS-CSO-SA.
unreacted reagent. Finally, the product was redispersed in
deionized water and lyophilized, and the CSO-SA were
received.
2.3. Synthesis of DOX-Conjugated CSO-SA (DOX-SS-
CSO-SA). Doxorubicin hydrochloride (DOX·HCl) was reacted
with triethylamine in dimethyl sulfoxide overnight to produce
the DOX.23 DOX was conjugated to CSO-SA micelles via
disulfide links to dithiobis (succinimidyl propionate) (DSP) in
two reaction steps. All reactions were under dark conditions.
DOX and DSP were dispersed in DMSO. DOX/DMSO
solution (20 mg/mL) was added dropwise to 20 mg/mL DSP/
DMSO solution (DOX/DSP = 1:1, molar ratio). A catalytic
amount of triethylamine (0.5 μL) was added into the reaction
mixture under stirring for 2 h at room temperature. The
reactant mixture was dialyzed (MWCO 3000 Da) against DI
water for 24 h followed by freeze-drying to obtain the DOX-
DSP intermediates.
Then a certain weight of CSO-SA was dissolved in DI with
ultrasonic dispersion uniformity to form a micelle solution. The
pH of the solution was adjusted to 7.4. DOX-DSP was
dissolved in DMSO to a concentration of 10 mg/mL and added
to the CSO-SA micelle solution in drops (DOX/CSO-SA =
1:10, w/w). The solution was stirred for 12 h and then dialyzed
against DI water (MWCO 7000 Da) for 48 h followed by
freeze-drying. Afterward, the lyophilized product was washed
thrice with DMSO to remove unreacted DOX-DSP molecules.
Then the solution was lyophilized and the DOX-SS-CSO-SA
was obtained.
2.4. Physicochemical of CSO-SA and DOX-SS-CSO-SA.
2.4.1. 1H NMR Analysis. The 1H NMR spectra of the chemicals
were used to verify the chemical structures. The chemicals were
dissolved in 20.0 mg/mL of dimethyl sulfoxide-d6 or D2O.
2.4.2. LC/MS Analysis. The reaction mixture was transferred
into a sample vial and analyzed by LC/MS.
LC/MS analysis: Agilent 1200 Series UPLC and 6460 Series
mass spectrometers were used (Agilent Technologies, Palo
Alto, CA, USA). Mobile phase: methanol/0.01 M ammonium
acetate/acetic acid = 68:30:3 (v/v/v).
2. MATERIALS AND METHODS
2.1. Materials and Animals. Chitosan oligosaccharide (Mw
=17.5 kDa, 95% deacetylated degree) was obtained by
enzymatic degradation from chitosan (Mw = 450.0 kDa),
which was supplied by Yuhuan Marine Biochemistry Co., Ltd.
(Zhejiang, China).22 Stearic acid (SA) was purchased from
Chemical Reagent Co., Ltd. (Shanghai, China). 1-Ethyl-3-(3-
(dimethylamino)propyl) carbodiimide (EDC) was purchased
from Shanghai Medpep Co., Ltd. (Shanghai, China).
Doxorubicin hydrochlorate (DOX·HCl) was a gift from
Hisun Pharm Co., Ltd., China. Fluorescein isothiocyanate
(FITC), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5 -diphenyl-tetra-
zolium bromide (MTT) were purchased from Sigma (St. Louis,
MO, USA). Dithiobis (succinimidyl propionate) (DSP) was
obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo,
Japan). Pyrene was purchased from Aldrich Chemical Co.
(USA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocya-
nine iodide (DiR) was a product of Molecular Probes, Inc.
(Eugene, OR, USA). Dulbecco’s modified Eagle’s medium
(DMEM) and trypsin-EDTA were purchased from Gibco-
BRLC (MD, USA). Fetal bovine serum (FBS) was purchased
from Sijiqing Biologic Co., Ltd. (Zhejiang, China). Commercial
doxorubicin hydrochloride injection was obtained from Pfizer
(USA). All other chemicals were of analytical or chromato-
graphic grade.
HPLC analysis was performed at a flow rate of 0.8 mL/min.
Mass spectra were acquired in positive ion modes.
2.4.3. SA Content in CSO-SA. SA content in CSO-SA was
determined by the 2,4,6-trinitrobenzenesulfonic acid (TNBS)
method. 0.3 mL of CSO-SA solution at 1 mg/mL was
incubated with 2.0 mL NaHCO3 (4.0%) and 2.0 mL of TNBS
(0.1%) at 37 °C for 2 h. Then 2.0 mL of HCl (2 mol/L) was
added and the results were measured at 344 nm by an
ultraviolet spectrophotometer. The degree of SA in CSO-SA is
calculated by the following formula:
Male BALB/C nude mice (6−8 weeks old) were purchased
from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai,
China). All animal studies were performed under Institutional
Animal Care and Use Committee-approved protocols.
ACSO
ACSO−SA
MCSO−SA
MCSO
nNH2
nNH2 − mNH2
=
×
2.2. Synthesis of CSO-SA. As previously reported, CSO-
SA was synthesized by a one-step reaction. We used the
carboxyl group of SA reacting with the free amino groups of
CSO by an amine-reactive coupling, which was catalyzed by
EDC.17,21 Briefly, 1.0 g of CSO (Mw = 17.5 kDa) was dissolved
in 60 mL of DI water. A total of 1.65 g of SA and 11.0 g of EDC
was dissolved in ethanol and activated at 60 °C for 30 min.
Then the ethanol solution was added dropwise into the
chitosan solution with stirring at 80 °C for 4 h. The reactant
mixtures were dialyzed with a dialysis membrane (MWCO
7000 Da) against DI water for 2 days followed by freeze-drying.
The lyophilized product was washed with ethanol to remove
where ACSO is the UV absorbance of CSO, ACSO‑SA is the UV
absorbance of CSO-SA, MCSO‑SA is the molecular weight of
CSO-SA, MCSO is the molecular weight of CSO, nNH2 is the
total number of moles of −NH2 in the CSO chain, and mNH2 is
the content of SA on CSO.
2.4.4. DOX Content in DOX-SS-CSO-SA. We used an
ultraviolet spectrophotometer (DU640, Beckman-Counter,
USA) to measure the DOX content in DOX-SS-CSO-SA.24
DOX and DOX-SS-CSO-SA were dispersed in a mixture of
DMSO and H2O (5:95, v/v), and the DOX content was
calculated compared to a standard curve obtained using DOX.
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2.4.5. Critical Micelle Concentration. To measure the
critical micelle concentration (CMC) of DOX-SS-CSO-SA,
pyrene was used as a probe and detected by the fluorescence
spectroscopy.25 To determine the intensity ratio (I1/I3) of the
first peak (I1, 374 nm) to the third peak (I3, 385 nm) in the
pyrene emission spectra, sample solutions of different
concentrations containing pyrene (5.93 × 10−7 mol/L) were
excited at 337 nm, and the emission spectrum of pyrene was
obtained in the range of 360−450 nm with a fluorometer (F-
2500, Hitachi Co., Japan). The slit openings for excitation and
emission were at 10.0 and 2.5 nm, respectively.
2.4.6. Particle Size and Zeta Potential. The sizes of the
micelles with CSO-SA were detected by dynamic light
scattering (DLS) using a Zetasizer (Malvern Instrument
Ltd.). The zeta potential was also measured by the Zetasizer.
2.4.7. Morphology of Particles. The morphology examina-
tions were performed by TEM (JEOL JEM-1230, Japan). The
samples were placed on carbon-coated copper grids and stained
with 2% (w/v) phosphotungstic acid.
2.5. Redox-Responsive Behaviors and in Vitro DOX
Release. To demonstrate that the disulfide bonds are cleavable
and DOX was quickly released from micelles in the reductive
environment, DOX-SS-CSO-SA micelles were dissolved in PBS
(pH, 7.4) containing 10 mM or 10 μM dithiothreitol (DTT) at
a drug concentration of 5 μg/mL. The fluorescence intensity
was scanned at different time points (0−50 min) using a
fluorescence spectrometer with an excitation wavelength of 505
nm. Additionally, the fluorescence intensity of DOX at the
same concentration was detected under the same conditions as
the control.
The in vitro release profile of DOX from DOX-SS-CSO-SA
micelles was investigated by dialysis in PBS (pH 7.4) medium
containing 10 mM or 10 μM DTT. The DOX-SS-CSO-SA
containing 100 μg of DOX was dialyzed against 20 mL of buffer
(MWCO 7000 Da). At the designated time points, the media
was collected and replaced with fresh medium. Drug
concentrations were measured by fluorometer (Em = 505
nm; Ex = 565 nm) and calculated in comparison with a
standard curve. Free DOX was performed under the same
conditions as a control. All release tests were performed in
triplicate.
2.6. Cell Culture. Human breast cancer MCF-7 cells and
multidrug resistant variant MCF-7/ADR cells were gifts from
the first Affiliated Hospital, College of Medicine, Zhejiang
University. Human liver tumor BEL-7402 cells were obtained
from the Institute of Biochemistry and Cell Biology. Cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum (FBS) in a
humidified atmosphere containing 5% CO2 at 37 °C. Cells were
regularly subcultured using trypsin/ethylenediaminetetraacetic
acid (EDTA).
2.7. Internalization and Intracellular Trafficking of
DOX-SS-CSO-SA Micelles. DOX-SS-CSO-SA micelles were
labeled with fluorescein isothiocyanate (FITC) via the amino
group of chitosan and the isothiocyanate group of FITC. Cells
were transferred and cultured on 20 mm cover glass in a 24-
well plate at 1.0 × 105 mL−1 cells/well and allowed to grow for
24 h. DOX·HCl and FITC-labeled DOX-SS-CSO-SA micelles
of a certain concentration (DOX content was 5 μg/mL) were
added. After different incubation periods, cell nuclei were
stained with Hoechst 33342. Cell monolayers on cover glasses
were repeatedly rinsed with PBS and mounted for microscopic
examination. A confocal laser scanning microscope (Olympus,
Japan) was used to image the intracellular fluorescence. For a
quantitative study, cells were harvested and resuspended in
PBS. The intensity of cellular fluorescence was determined by
flow cytometer (FC500MCL, Beckman Coulter).
2.8. In Vitro Antitumor Activity. A cytotoxicity compar-
ison was performed using BEL-7402, MCF-7, and MCF-7/
ADR cells with in vitro proliferation using the MTT method.
Briefly, cells were seeded at 1.0 × 104 mL−1 cells/well in a 96-
well plate (Nalge Nunc International, Naperville, IL, USA) and
allowed to grow for 24 h. After treating DOX·HCl, DOX, CSO-
SA, and DOX-SS-CSO-SA micelles with a series of
concentrations, the cells were further cultured for 48 h. Then,
20 μL of MTT solution (5 mg/mL) was added. After
incubating for an additional 4 h at 37 °C, the medium was
removed, and 200 μL of DMSO was added into each well for
dissolution of the MTT formazan crystals. Finally, the
absorbance at 570 nm was measured by a microplate reader
(Bio-Rad, Model 680, USA). All experiments were performed
in triplicate. The overcome ratio of drug resistance was
calculated from formula 1:20,26
overcome power = (Rf /Rm)/(Sf /Sm)
(1)
where Rf was the IC50 value of the drug against the drug-
resistant cells; Rm was the IC50 value of DOX-SS-CSO-SA
micelles against the drug resistant cells; Sf was the IC50 value of
the drug against drug-sensitive cells; and Sm was the IC50 value
of the DOX-SS-CSO-SA micelles against the drug-sensitive
cells.
2.9. In Vivo Distribution. To prepare the tumor-bearing
mice models, a tumor cell suspension of approximately 1.0 ×
107 BEL-7402 cells was inoculated subcutaneously in the
BALB/C nude mice (6−8 weeks). To investigate the in vivo
distribution, DOX-SS-CSO-SA micelles was labeled with the
near-infrared dye DiR. DiR/DMSO stock solution was slowly
added dropwise into DOX-SS-CSO-SA micelle solution. After
stirring for 1 h, the mixture was ultrasonic dispersion. Finally,
the mixture was subjected to dialysis and centrifugation to
remove the free DiR. There was almost no DiR released from
the micelles during the experiment, suggesting that DiR could
be used to trace the micelles.
The tumor-bearing mice were observed with an in vivo
imaging system (CRI Inc., Woburn, MA, USA) at a preset time
point after intravenous injection with DOX-SS-CSO-SA
micelles containing DiR. At the end of the experiment, the
mice were sacrificed and various tissues and tumors were
collected, weighed, and observed by an in vivo imaging system.
To calculate the accumulation of DOX-SS-CSO-SA micelles in
various tissues as %ID/g (the percentage of the injected dose
per gram of tissue), the fluorescent intensity was also read by
the imaging system.
2.10. In Vivo Antitumor Efficacy. When the tumor
volume was approximately 150 mm3, the nude mice were
randomly divided into different groups and treated with various
formulations by i.v. injection.
Group 1, 0.9% saline; Group 2, commercial doxorubicin
hydrochloride injection (2.0 mg/kg); Group 3, DOX-SS-CSO-
SA (2 mg/kg). The injections were continued for 7 days after
the first injection. The tumor volume and mice weight were
measured every 3 days thereafter. On the 24th day, the mice
were sacrificed and tumors were removed and weighed. In
addition, the hearts were collected and fixed with formalin for
48 h. After paraffin sectioning, tissue sections were stained with
hematoxylin/eosin (H&E) and observed by optical microscopy.
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Figure 1. Synthetic route of the DOX-DSP and DOX-SS-CSO-SA (A). 1H NMR spectra of DOX, DSP, DOX-DSP, CSO-SA, and DOX-SS-CSO-SA
(B).
2.11. Statistical Analysis. All data represent the mean
anthracene proton peaks of DOX at approximately 8.2 ppm
were found. These results confirmed that DOX-SS-CSO-SA
was successfully synthesized. The DOX content in DOX-SS-
CSO-SA was measured as 5.5% (weight ratio) by UV
spectrophotometer.24 The weak peaks of DOX in the 1H
NMR spectrum from DOX-SS-CSO-SA may result from the
low DOX content and poor solubility of DOX in D2O.
3.2. Characteristics of DOX-SS-CSO-SA. Synthesized
DOX-SS-CSO-SA easily self-assembled into micelles in
aqueous solution due to the conjugation of the hydrophilic
chain (CSO) and hydrophobic segment (SA and DOX).17
Table 1 shows the characteristics of DOX-SS-CSO-SA. The
values
standard deviation of the independent experiments.
Differences between groups were performed using Student’s t-
test (two-tailed), and p < 0.05 was considered statistically
significant for all cases. Statistically significant differences
between pairs of mean values were determined with ANOVA
followed by Tukey−Kramer tests. Mean differences with p-
values < 0.05 were considered statistically significant.
3. RESULTS
3.1. Synthesis of CSO-SA and DOX-SS-CSO-SA. CSO-
SA was synthesized by amide reaction of the amino groups of
CSO and the carboxyl group of SA catalyzed by EDC. The
content of SA on CSO was determined to be 10.03% (molar
ratio). DOX-SS-CSO-SA was then synthesized using DSP as
the linker. Figure 1A shows the synthetic route for DSP-DOX
and DOX-SS-CSO-SA. The structures of products were
confirmed by 1H NMR spectra (Figure 1B) and LC/MS
spectra (Figure S1). The identity of DSP-DOX could be
deduced by the molecular ions: [M + H]+ at m/z 819 in the
positive mode. The obtained molecular mass of DSP-DOX
product matched with the theoretical value. The area
normalization method was used as quantitative analysis of the
purity of DSP-DOX, which was 75.8%. After the purification by
LC, no molecular ions matching the molecular mass of DSP-
DOX-DSP were found in the product. In the 1H NMR
spectrum of DSP-DOX, peaks at approximately 8.1 and 2.8
ppm were attributed to the anthracene protons of DOX and the
NHS group protons of DSP, respectively.27 In the spectrum of
CSO-SA, peaks at approximately 0.9 and 1.1 ppm belonged to
−CH3 and −CH2− of SA. The spectrum of DOX-SS-CSO-SA
was similar to the spectrum of CSO-SA but the small
Table 1. Characteristics of CSO-SA and DOX-SS-CSO-SA
zeta potential
(mV)
CMC
a
material
CSO-SA
size (nm)
PI
(μg/mL)
40.5 4.8
62.8 5.4
0.401 0.044
0.266 0.025
32.7 1.1
21.6 1.2
59.8
49.1
DOX-SS-
CSO-SA
a
PI presents the polydispersity index of the micelle size. The data
represent the mean standard deviation (n = 3).
CMC confirmed the self-aggregation ability of CSO-SA and
DOX-SS-CSO-SA.18,22 As shown in Figure 2A, at low
concentration, the fluorescence intensity ratio of the first
peak to the third peak (I1/I3) in the emission spectra of pyrene
remained constant at approximately 1.7. When the concen-
tration increased to form micelles, the I1/I3 value sharply
decreased as a result of pyrene incorporation into the micelles.
The CMC values for DOX-SS-CSO-SA and CSO-SA in DI
water was approximately 49.1 and 59.8 μg/mL, respectively,
which indicated that the CSO-SA after DOX conjugation had
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Figure 2. Characteristics of CSO-SA and DOX-SS-CSO-SA. Variations in the fluorescence intensity ratio I1/I3 with logarithm concentrations of
⧫
●
CSO-SA ( ) and DOX-SS-CSO-SA ( ) (A). TEM image (B), size distribution (C), and zeta potential (D) of CSO-SA micelles. TEM image (E),
size distribution (F), and zeta potential (G) of DOX-SS-CSO-SA micelles.
Figure 3. Redox-responsive behavior and the in vitro controlled DOX release of DOX-SS-CSO-SA micelles. Fluorescence emission spectra of DOX-
SS-CSO-SA micelles after incubation in PBS (pH, 7.4) containing 10 mM DTT (A) or 10 μM DTT (B) at different times. Fluorescence intensity
■
(C) or in vitro DOX release profile (D) of DOX-SS-CSO-SA micelles. DOX-SS-CSO-SA micelles in PBS (pH, 7.4) containing 10 mM DTT ( ),
⧫
▲
DOX-SS-CSO-SA micelles in PBS (pH, 7.4) containing 10 μM DTT ( ), and DOX in PBS (pH, 7.4) containing 10 mM DTT ( ). *p < 0.05, **p
< 0.01, compared with 10 μM DTT group. The error is the standard deviation from the mean (n = 3).
excellent dispersity and self-assembly ability in aqueous
environments.
3.3. Redox-Responsive Behaviors and in Vitro DOX
Release. To demonstrate that the disulfide bonds in DOX-SS-
CSO-SA micelles are responsive to the reductive environment,
the fluorescence emission of DOX-SS-CSO-SA micelles
incubated with DTT was measured at different time points.
As previously reported, the fluorescence emission of DOX
quenches when DOX is covalently conjugated with a polymer,
and it recovers when it is separated from polymers.7,8 The DTT
concentrations were set at 10 mM and 10 μM to simulate the
intracellular thiol concentration and extracellular level,
respectively. As shown in Figure 3A, the fluorescence emission
of the DOX-SS-CSO-SA micelle solution increased to a plateau
that was close to the fluorescence intensity of free DOX within
TEM images, size distributions, and zeta potentials of CSO-
SA and DOX-SS-CSO-SA micelles are presented in Figure 2B−
G. We found that DOX-SS-CSO-SA micelles were larger in size
(62.8 5.4 nm) than CSO-SA micelles (40.5 5.8 nm) as
determined by DLS, which was in accordance with the results
shown in TEM images. It was obvious that the DOX-SS-CSO-
SA micelles had a lower positive zeta potential (21.6 1.2 mV)
compared with the CSO-SA micelles (32.7 1.1 mV) due to
the decreased amine groups on the micelle surface and
enhanced micelle size after DOX conjugation.
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Figure 4. Confocal laser scanning micrographs of MCF-7 cells after incubation with DOX-SS-CSO-SA for 1, 4, 8, and 12 h: DOX (red), FITC-
labeled micelles (green), and nuclei stained with Hoechst 33342 (blue).
Table 2. Cytotoxicity of CSO-SA, DOX·HCl, DOX, and DOX-SS-CSO-SA micelles against BEL-7402, MCF-7, and MCF-7/Adr
a
Cells
IC50
material
BEL-7402
MCF-7
MCF-7/Adr
reversal power
CSO-SA (μg/mL)
DOX·HCl (μM)
DOX (μM)
347 32
307 26
488 66
0.94 0.01
14.88 1.64
12.45 1.10*
0.57 0.05
8.10 0.70
3.59 0.39*
67.63 11.64
DOX-SS-CSO-SA (μM)
12.28 1.42**
34.7
a
*p < 0.05, **p < 0.01, compared with DOX·HCl group. The error is the standard deviation from the mean (n = 3).
50 min after incubation in PBS (pH, 7.4) containing 10 mM
DTT, which was the result of the cleavage of disulfide linkers
and separation of DOX from the polymer. In contrast, no
significant increase in fluorescence intensity was observed in
PBS (pH, 7.4) containing 10 μM DTT (Figure 3B). In Figure
3C, it is quantitatively shown that the disulfide bonds were
rapidly cleaved within 20 min in a reductive environment and
that they were stable in a low reductive environment within 50
min (p < 0.05).
In vitro drug release of DOX-SS-CSO-SA in different
mediums is shown in Figure 3D. As a control, free DOX was
completely released within 48 h. Approximately 38.3% DOX
was released from the DOX-SS-CSO-SA micelles within 48 h in
media simulating the extracellular thiol concentration (10 μM).
However, approximately 81.4% DOX was released within 48 h
when the polymer−drug conjugate was incubated in PBS (pH,
7.4), simulating the intracellular thiol concentration (10 mM).
The drug release rate of DOX-SS-CSO-SA micelles with 10
mM DTT was significantly faster compared with that of 10 μM
of DTT (p < 0.05). These results indicate that DOX-SS-CSO-
SA micelles are relatively stable in nonreducing environments,
and they are sensitive to intracellular reductive environments,
which result in the fast release of DOX.
3.4. Internalization and Intracellular Trafficking.
Confocal laser scanning microscopy (CLSM) analysis was
performed with MCF-7 cells to investigate the internalization
and intracellular trafficking behavior of micelles. In Figure 4, the
yellow color (merged, the yellow arrow mark) is an indication
for DOX-SS-CSO-SA micelles in which DOX (red fluores-
cence) did not release from the FITC-conjugated micelles
(green fluorescence). When MCF-7 cells were treated with
DOX-SS-CSO-SA micelles (DOX content was 5 μg/mL) for 1
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Figure 5. Cytotoxicity of DOX-SS-CSO-SA ( ), DOX·HCl ( ), and DOX ( ) against MCF-7 (A), MCF-7/Adr (B), and BEL-7402 (C) cells. Data
represent the mean standard deviation (n = 3). Fluorescence images of MCF-7 and MCF-7/Adr cells after incubation with DOX·HCl and DOX-
SS-CSO-SA micelles (DOX content was 5 μg/mL) for 4 and 8 h, respectively (D). Quantitative cell uptake as analyzed by a flow cytometer into
MCF-7/Adr cells incubated with DOX·HCl (red line) and DOX-SS-CSO-SA micelles (blue line) (DOX content was 5 μg/mL) for 4 and 8 h. MCF-
7/Adr cells without treatment (black line) are shown as a control (E).
Figure 6. In vivo fluorescence images of a tumor-bearing nude mouse at different times after the i.v. injection of DOX-SS-CSO-SA micelles
encapsulating DiR (A). Fluorescence imaging of various tissues of the mouse at 48 h after i.v. injection (B). The accumulation of DOX-SS-CSO-SA
micelles in various tissues was calculated as %ID/g (the percentage of the injected dose per gram of tissue). The fluorescent intensity, responding to
the amount of the micelles, was read by the imaging system (C).
h, the yellow fluorescence was evident in cells, indicating that
the micelles had good internalization capacity. At 4 and 8 h, in
merged images, there was green fluorescence from responding
micelles without drug but not at 1 h. DOX effectively migrated
into the nucleus where it presumably intercalated into DNA,
inducing cell death. This process was observed when DOX
fluorescence colocalized with the nucleus (labeled with Hoechst
33342, the white arrow marked), while the micelle fluorescence
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Figure 7. In vivo antitumor effect and systemic toxicity of DOX-SS-CSO-SA micelles. Body weight changes (n = 4) (A) and tumor volume changes
⧫
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▲
(n = 4) (B) of BEL-7402 cell tumor-bearing mice treated with saline ( ), adriamycin ( ), and DOX-SS-CSO-SA micelles ( ). *p < 0.05, **p <
0.01, compared with saline group. #p < 0.05, ##p < 0.01, compared with adriamycin group. The error is the standard deviation from the mean (n = 4).
Excised BEL-7402 solid tumors from different treatment groups on the 24th day (n = 4) (C). Histologic evaluation of the cardiac tissue
corresponding to the saline treated group (D), the DOX-SS-CSO-SA micelle treated group (E), and the adriamycin treated group (F).
did not. Dramatic changes in cell and nuclear morphology were
observed within 12 h because of tumor cell death. In summary,
DOX is rapidly released from redox-sensitive polymer−drug
conjugates into cancer cells. DOX then accumulates in target
subcellular organelles and induces the death of tumor cells.
3.5. In Vitro Cytotoxicity. The in vitro cytotoxicity of
DOX-SS-CSO-SA was evaluated against MCF-7, MCF-7/Adr,
and BEL-7402 cells. DOX base and DOX·HCl served as
controls. Previous reports demonstrated that attachment of
DOX via a low molecular weight amine group did not reduce
the potency of the drug.28,29 Fifty percent cell growth inhibition
(IC50) of BEL-7402, MCF-7, and MCF-7/Adr cells was
determined by MTT assay (Table 2). As shown in Figure
5A−C, the IC50 values of CSO-SA micelles for BEL-7402,
MCF-7, and MCF-7/Adr cells were approximately 347, 307,
and 488 μg/mL, respectively, indicating that the micelles have
low cytotoxicity. From Table 2 and Figure 5A,C, the IC50 value
of DOX-SS-CSO-SA micelles is lower than that of DOX (p <
0.05) in BEL-7402 and MCF-7 cells, and the cell viability
percentage for both cell lines is reduced with increasing DOX-
SS-CSO-SA concentration in the micelles. In MCF-7/Adr cells,
the IC50 value of DOX·HCl was 118.6-fold higher than that of
the MCF-7 cells, which confirmed the DOX resistance of MCF-
7/Adr cells. However, the IC50 value was only 3.4-fold between
the MCF-7/Adr and MCF-7 cells by DOX-SS-CSO-SA. The
overcoming ability of drug resistance for DOX-SS-CSO-SA was
34.6. These results indicated that DOX-SS-CSO-SA could
overcome the drug resistance of MCF-7/Adr cells.
micelles, the intracellular DOX concentration was similar in
MCF-7 and MCF-7/Adr cells. In addition, in drug resistant
cells, the drug concentration of DOX-SS-CSO-SA micelles was
significantly higher than that of those in the DOX·HCl group,
which was further confirmed by analysis with flow cytometry
(Figure 5E).
3.6. In Vivo Distribution. Far-red and near-infrared light in
the spectral range of 650−900 nm, was a “clear” window for in
vivo optical imaging because it is separated from the major
absorption peaks of organisms.30 The in vivo distribution image
of DiR-marked DOX-SS-CSO-SA micelles is shown in Figure
6A. There was an increased accumulation of DOX-SS-CSO-SA
micelles in BEL-7402 tumors from 1 to 36 h, and the
accumulated amount of micelles in tumors peaked at 36 h. It
was indicated that DOX-SS-CSO-SA micelles could passively
target tumors, which was attributed to the EPR effect of the
nanoscale micelles. To further investigate the distribution of
DOX-SS-CSO-SA micelles, organs and tumors were collected,
weighed, and observed at 48 h after injection. In Figure 6B,C,
we could find that DOX-SS-CSO-SA micelles accumulated in
the liver (41.4%, ID/g), spleen (35.5%, ID/g), tumors (14.7%,
ID/g), and lungs (18.1%, ID/g). Strikingly, there was nearly no
accumulation of DOX-SS-CSO-SA micelles in the heart (2.0%,
ID/g).
3.7. In Vivo Antitumor Efficacy. The tumor growth
inhibitions in vivo were performed on the BEL-7402 xenograft
tumor bearing nude mice. Commercial preparation adriamycin
was used as a positive control. Measurements of changes in
body weight (Figure 7A) demonstrated that DOX-SS-CSO-SA
micelles are safer than adriamycin. Figure 7B,C illustrates
changes in the tumor volume and excised BEL-7402 solid
tumors. Both adriamycin and DOX-SS-CSO-SA treatments
showed effectively inhibition of tumor growth. The tumor
inhibition rate (IR) was 82.7% for adriamycin treatment group
and 70.8% for DOX-SS-CSO-SA treatment group. These
To investigate the mechanism of how DOX-SS-CSO-SA
micelles overcome drug resistance, CLSM analysis with a flow-
cytometer was performed to estimate the intracellular DOX
concentration in MCF-7 and MCF-7/Adr cells. DOX·HCl
served as control. As shown in Figure 5D, after the cells were
incubated with 5 μg/mL DOX·HCl for 4 or 8 h, and the DOX
concentration in MCF-7/Adr cells was much lower than that in
MCF-7 drug-sensitive cells. In contrast, for DOX-SS-CSO-SA
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results confirmed the efficient suppression ability of tumor
growth in vivo of DOX-SS-CSO-SA.
CSO-SA entered resistance cells by endocytic pathways. The
internalization pathway of DOX-SS-CSO-SA substantially
increased the intracellular drug concentration of the drug
resistant cells and greatly avoided the efflux of the Pgp
transports on plasma membrane.
P-gp and MDR related proteins (MRP1 and MRP4) are
ATP-binding cassette transporters, which function as energy-
dependent efflux pumps to remove cytotoxic agents from drug
resistant cells.31 DOX-SS-CSO-SA micelles entered cells via
endocytosis, which was assumed to reduce the ATP production
in drug resistant cells. The decrease of ATP could also reduce
the effect of efflux pumps.
In addition, to validate the attenuated doxorubicin-induced
cardiac injury of DOX-SS-CSO-SA, the histology and pathology
of cardiac tissues from mice with different treatments were
observed by pathological sectioning (Figure 7D−F). In Figure
7F, cardiomyocytes from the adriamycin treatment group were
scattered and ruptured and had vacuolar degeneration (the
arrow mark). In contrast, after treatment with DOX-SS-CSO-
SA micelles, the cardiomyocytes (Figure 7E) were maintained
the same as the negative control group.
4. DISCUSSION
Early studies reported that proteins bearing O-linked single
or multiple N-acetylglucosamine (GIcNAc) moieties were
particularly abundant in the cytoplasmic and nucleoplasmic
(but not lumenal) sides of nuclear membranes.32,33 As the
monomer structure of chitosan is identical to the GIcNAc, we
assumed that CSO-SA may hold the potential to gather into the
cell nuclei. Since DOX interacts with DNA in the cell nuclei,
the delivery of DOX tends to enrich in the cell nuclei and could
substantially increase its efficacy. Thus, DOX-SS-CSO-SA
micelles have excellent properties for overcoming the drug-
resistance of MCF-7/Adr cells (Table 2 and Figure 5).
DOX-SS-CSO-SA was synthesized via amide reactions of the
NHS ester groups of DSP between DOX and CSO-SA micelles
at physiological pH and room temperature to form amide
bonds.3,4 Thus, DOX was covalently conjugated into CSO-SA
micelles via disulfide bonds. The obtained polymer−drug
conjugates maintained the properties of physicochemical
polymers and easily self-assembled into a core−shell structure
in an aqueous environment. Therefore, the polymer−drug
conjugates could improve the water solubility of the hydro-
phobic drug. The reaction between the DOX and CSO-SA was
carried out in the H2O/DMSO solution in which the amino
groups of CSO mainly appeared on the surface of the micelles.
On the basis of the collision theory, when DOX-DSP solution
was added dropwise in the CSO solution, succinyl groups of
DOX-DSP preferentially collided with the amino groups on the
surface of the micelles and reacted. So part of the hydrophobic
DOX may appear on the surface of the micelles. This result
could be demonstrated by the fact that the size of the DOX-SS-
CSO-SA was larger, and the zeta potential was lower compared
with the CSO-SA (Table 1). The structure of DOX-SS-CSO-
SA micelles made it possible for DOX to be rapidly released
from them in a reducing environment.
Disulfide bonds in the polymer−drug conjugates were
introduced as redox-sensitive linkers for intracellular release
of DOX,14 which were rapidly broken by the abundance of
intracellular thiols, resulting in DOX separation from micelles
and transportation into nuclei (Figures 3 and 4). In contrast, in
extracellular fluid or systemic circulation, which has low
concentration of thiols, nearly no DOX was leaked from the
DOX-SS-CSO-SA. Furthermore, DOX-SS-CSO-SA could
passively target to the solid tumor issue due to the EPR effect
and reduce nonspecific accumulation in biological systems
(particularly in the heart) (Figure 6). Thus, DOX-SS-CSO-SA
micelles have high antitumor effects and low toxicity, which
could attenuate doxorubicin-induced cardiac injury (Figure 7).
Multiple lines of evidence have demonstrated that the drug
resistance in tumor is caused by the overexpression of ATP-
binding cassette (ABC) transporters on the cell membrane. As
one of the ABC, P-gp is an energy-dependent efflux pump,
which could actively efflux cytotoxic agents from the cells.16 P-
gps are large, glycosylated membrane proteins that localize
predominantly to the plasma membrane. In the passive
diffusion process of internalization, free drug molecules could
be easily exported by the Pgp transports near plasma membrane
of the resistance cells. Therefore, the DOX concentration in
MCF-7/Adr cells was much lower than that in MCF-7 cells
(Figure 5D), and in the MCF-7/Adr cells, the viability rate
barely changed with an increase in drug concentration from 5
to 20 μg/mL (Figure 5B). Because of the membrane affinity of
cationic and special spatial structure of CSO-SA, DOX-SS-
5. CONCLUSIONS
We have developed a smart polymer−drug conjugate where
DOX is covalently bonded to CSO-SA via disulfide linkage for
redox triggered drug release. The polymer−drug conjugate
selectively accumulated at tumor sites and showed minor
nonspecific accumulation in the heart. After the polymer−drug
conjugate rapidly internalized into cancer cells, DOX was
rapidly released due to the cleavage of the disulfide bonds
mediated by an abundance of free intracellular thiols and then
trans-located into nuclei. Our data indicated that the polymer−
drug conjugate could overcome MDR in vitro by eluding the
drug efflux pumps. The polymer−drug conjugate showed a
good anticancer effect and low toxicity in vivo. All of these
results suggest that the polymer−drug conjugate holds certain
potentiality for safe and effective cancer therapy.
ASSOCIATED CONTENT
■
S
* Supporting Information
LC/MS analysis and quantitative analysis report. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: hufq@zju.edu.cn. Tel: +86-571-88208441. Fax: +86-
571-88208439.
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for financial support from National Natural
Science Foundation of China (81273442) and Zhejiang
Provincial Program for the Cultivation of High-level Innovative
Health Talents. We would also like to thank Mrs. Xiaodan Wu
from the Analysis Center of Agrobiology and Environmental
Science, Zhejiang University for the LC/MS analysis.
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