An integrated nanoplatform for theranostics via multifunctional core-shell ferrite nanocubes

Article abstract of DOI:10.1039/c5tb01910a

Magnetic core-shell ferrite nanocubes (MNCs) were prepared by a two-step pyrolysis. The MNCs not only exhibit an excellent magnetothermal effect, but also can be used as T₂ magnetic resonance (MR) imaging agents. To obtain their good biocompati

Full text of DOI:10.1039/c5tb01910a

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ARTICLE
An Integrated Nanoplatform for Theranostics via
Multifunctional Core/Shell Ferrite Nanocubes
Cite this: DOI: 10.1039/x0xx00000x
Li Wanga, Yuping Yan a, Min Wang a, Hong Yang*a, Zhiguo Zhoua, Chen Peng*b
and Shiping Yang*a
Magnetic core/shell ferrite nanocubes (MNCs) were prepared by a twoꢀstep pyrolysis. MNCs
not only exhibit an excellent magnetothermal effect, but also can be used as T2 magnetic
resonance (MR) imaging agents. To perform their good biocompatibility and targeting ability,
MNCs were modified with poly(ethylene glycol) (PEG) and hyaluronic acid (HA). To further
construct an integrated nanoplatform for theranostics, doxorubicin (DOX) was loaded onto the
surface of MNCs with the pH and heatꢀresponsible chemical bond. Notably, MNCs revealed
the great stability and magnetothermal effect. Moreover, they showed the negligible toxicity
and synergistic therapy in vitro. Meanwhile, the MR imaging in vivo was further verified. The
novel integrated nanoplatform facilitates the excellent targeting MR imaging guided
synergistic therapy of the magnetothermal and chemoꢀtherapy. The multifunctional nanocubes
will be capable of playing a vital role in future cancer therapy.
Received 00th Autumn 2015,
Accepted 00th Autumn 2015
DOI: 10.1039/x0xx00000x
www.rsc.org/
well known, hyaluronic acid (HA) is a targeting moiety to various
cancer cells due to the specific interaction with overexpressed cluster
determinant 44 (CD44) 35ꢀ38. Considering the targeting property of
Introduction
Cancer has been one of the main causes of death throughout the
world for centuries. Though tremendous cancer therapies, such as
surgery, chemotherapy, and radiotherapy, have been developed
there are still the great risk for side effects and recurrence
HA to CD44 overꢀexpressing cancer cells, HA modified MNCs were
1, 2
prepared for the targeted antiꢀcancer drug delivery 38ꢀ40
.
,
.
3
Various novel drug delivery strategies have been explored to
improve the therapeutic effect 41ꢀ44. Doxorubicin (DOX) is a classical
anticancer drug and has been applied in patient treatment early. It
has been reported that a drug delivery system by tethering DOX onto
the surface of NPs via an acidꢀlabile chemical bond can significantly
inhibit the growth of cancer cells for the high efficiency of cellular
Therefore, it is necessary to search for the stateꢀofꢀtheꢀart therapeutic
strategy to solve this problem. Recently, photothermal therapy (PTT)
10ꢀ13
4ꢀ6, photodynamic therapy (PDT) 7ꢀ9, magnetothermal therapy
and chemotherapy
,
14ꢀ17 18, 19
have been developed. Among these
novel developed cancer therapies, magnetothermal therapy, which is
powerful, nonꢀinvasive, harmless and has no problem of tissue
10, 20,
uptake by endocytosis and subsequent acid responsive release in
penetration, has been the promising tumor treatment approach
45,
46. In recent work, hyperthermic effect, magnetic
21. Magnetic nanoparticles (NPs) induce hyperthermia can generate
efficient heat when they are subjected to an alternating magnetic
field (AMF), which has been demonstrated in numerous cancer
cancer cells
hyperthermia/photothermal effect can increase the release kinetics of
DOX 47ꢀ49
.
therapy 10, 12, 13, 21ꢀ23. Currently, they have been used in biomedicine
fields mainly including Fe2O3 24, 25, Fe3O4
and MFe2O4 (M =
26, 27
Inspired by the above mentioned reports, we developed an integrated
nanoplatform by modifying PEG, HA and DOX onto the surface of
MNCs via the chemical reaction, which can significantly improve
cancer therapy efficiency by taking advantage of the combined
benefits from the functional units (Scheme. 1). Furthermore, the
therapy effect of a novel integrated nanoplatform in vitro has been
demonstrated.
Mn, Zn, Co etc.) 10, 13. The heat generation under an AMF is resulted
13
from Brownian and Néel relaxations of magnetic moments of NPs
.
The chemical component, size, morphology, and crystal form of NPs
have a crucial impart on the heat generation under an AMF.
Furthermore, exchangeꢀcoupled magnetic NPs could result in the
significant enhancement of the magnetothermal effect. Therefore, we
prepared the exchangeꢀcoupled nanocubes (Zn0.4Co0.6)Fe2O4
(Zn0.4Mn0.6)Fe2O4 (denoted as MNCs) with the excellent
magnetothermal effect 13
@
.
In order to improve the water solubility and targeting property of
NPs, poly(ethylene glycol) (PEG) has been widely used for the
modification because of its excellent performance in vivo 28ꢀ34. It is
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Synthesis of poly(ethylene glycol) PEG modified MNCs (MNCs-
PEG). For the further functionalization of the obtained MNCs, the
prepared 3ꢀ(3,4ꢀdihydroxyphenyl)propanehydrazide ((DPPH) was
synthesized according to the previous report 51, which was verified
by 1HꢀNMR (Fig. S1). DPPH conjugated methoxypoly(ethylene
glycol) carboxyl acid (DPPHꢀPEG) was synthesized as follows.
Methoxypoly(ethylene glycol) carboxyl acid (PEGꢀCOOH, 20 mg),
Nꢀhydroxysuccinimide
(NHS,
30
mg)
and
3ꢀ(3ꢀ
dimethylaminopropyl)ꢀ1ꢀethylcarbodiimide (EDC, 25 mg) were
dissolved in pyridine (20 mL). After 2 h, DPPH (60 mg) was added
to the above solution. The solution was reacted for 8 h, and 20 mg of
the prepared MNCs was added, then the mixture was shaken for
overnight. The obtained PEGylated MNCs (MNCsꢀPEG) were
centrifuged and washed with ethanol and H2O. The obtained MNCsꢀ
PEG were dispersed in water (10 mL) for further modification.
Synthesis of HA modified MNCs-PEG (MNCs-PEG/HA)52. HA
o
(20 mg) was dissolved in 10 mL water, and heated to 70 C. Then
EDC (50 mg) and NHS (100 mg) were added to the HA solution for
the reaction of 2 h. The mixture was dropped into the above aqueous
suspension of MNCsꢀPEG (20 mg, 10 mL) under ultrasound. After
for 24 h in oscillators, the obtained HA modified MNCsꢀPEG
(MNCsꢀPEG/HA) were subjected to multiple cycles of
centrifugation/redispersion in water to remove the impurities.
Synthesis of doxorubicin (DOX) modified MNCs-PEG/HA
(MNCs-PEG/HA-DOX). DOX (20 mg) was added to the aqueous
suspension of 20 mL MNCsꢀPEG/HA (1 mg/mL). The mixed
o
solution was reacted at 50 C in oscillators for 48 h. The prepared
DOX modified MNCsꢀPEG/HA were centrifuged/ redispersed for 3
times and kept in water for further use.
Scheme 1. The fabrication process of an integrated nanoplatform and
the subsequent integrated tumor therapy
Magnetothermal experiments in the aqueous solution. 2 mL of
aqueous solution of MNCsꢀPEG/HAꢀDOX with different
concentrations (50, 100, 200, and 500 ꢀg/mL, respectively) was
placed into a 4 mL sample cell under an AMF (60.6 kA/m, 200 kHz)
Experimental
Materials
Oleylamine, oleic acid, and 3ꢀ(3,4ꢀdihydroxyphenyl)propanoic
acid were purchased from Shanghai Sinopharm Chemical
reagent Co., Ltd. 1,2ꢀhexadecanediol and phenyl ether were
purchased from TCI chemical Industry Co., Ltd. Hyaluronic
acid (HA) and doxorubicin (DOX) were purchased from Alfa
Aesar Chemical Co., Ltd. Fe(acac)3, Zn(acac)2, Co(acac)2,
Mn(acac)2 were purchased from Sigma Adrich company. Water
used in all experiments was purified using a MilliꢀQ Plus 185
water purification system (Millipore, Bedford, MA) with the
resistivity higher than 18 Mꢁ·cmꢀ1.
Synthesis of Zn- and Co-Doped iron oxide NPs [(Zn0.4Co0.6)Fe2O4
NPs]50. Fe(acac)3 (2 mmol), Zn(acac)2 (0.4 mmol), Co(acac)2 (0.6
mmol), 1,2ꢀhexadecanediol (10 mmol), oleylamine (6 mmol), oleic
acid (6 mmol), and phenyl ether (20 mL) were mixed and
magnetically stirred under the flow of nitrogen. The reaction mixture
for 20 min. The temperature was recorded using
a digital
thermocouple device with an accuracy of ± 0.1°C. The specific loss
power (SLP) was calculated using an equation (1). Where dT/dt is
the initial slope of the heating curve, C is the volumetric specific
heat capacity of the solution, Vs is the volume of the sample, and m
is the mass of magnetic nanoparticles in the sample.
CVS dT
SLP =
(1)
m
dt
MR imaging in solution. The longitudinal relaxation time (T1)
and transverse relaxation time (T2) were measured (298 K) and T2ꢀ
weighted MR images were performed with a 0.5 T system (Shanghai
Niumag Electronic Technology Co., Ltd, NM120ꢀAnalyst). T1ꢀ
weighted MR images were acquired using a conventional spinꢀecho
sequence under the following parameters: TE = 12 ms, TR = 100 ms
，150 ms，200 ms, 300 ms, 500 ms, 700 ms, 1000 ms, 1500 ms,
2500 ms, 220×320 matrices, 82×120 mm field of view, 140 Hz/Px of
bandwidth, a slice thickness of 3 mm. T2ꢀweighted MR images using
a fast spinꢀecho sequence was used to reduce acquisition time under
the following parameters: TR = 1000 ms，TE = 13.8 ms， 27.6 ms
，41.4 ms，55.2 ms，69 ms，82.8 ms, 220×320 matrices, 82×120
mm field of view, 220 Hz/Px of bandwidth, and a slice thickness of 3
mm. Longitudinal relaxation rate (r1) and transverse relaxation rate
(r2) were calculated from the fitting of 1/T1, 1/T2 and metal ion
concentration, respectively.
Drug release in solution. 3.0 mL of PBS buffer solution (pH
7.4 and 5.2, respectively) containing MNCsꢀPEG/HAꢀDOX (5
mg) was sealed in dialysis membranes (Mw cutoff = 3500). The
dialysis bags were transferred to a cube containing PBS buffer
solution (17 mL) of the same pH value at 37 °C under the
gentle shake (150 rpm min−1). The amount of released DOX
was recorded at the given time interval by a UV–vis spectrum
ο
was heated to 200 C for 2 h, then the temperature was increased to
280 οC for another 1 h. The blackꢀbrown mixture was cooled to
room temperature, then the black NPs were precipitated by ethanol,
purified by the centrifugation and sonication, and dried overnight
under the vacuum in sequence.
Synthesis of Zn- and Mn-Doped iron oxide coated Zn- and Co-
Doped Iron Oxide NPs [(Zn0.4Co0.6)Fe2O4@(Zn0.4Mn0.6)Fe2O4)
NPs, MNCs]. The synthesis of Znꢀ and Mnꢀdoped iron oxide coated
(Zn0.4Co0.6)Fe2O4 NPs was similar to that of (Zn0.4Co0.6)Fe2O4 NPs.
Fe(acac)3 (2 mmol), Zn(acac)2 (0.4 mmol), Mn(acac)2 (0.6 mmol),
1,2ꢀhexadecanediol (10 mmol), oleylamine (2 mmol), oleic acid (2
mmol), the prepared NPs (84 mg) and phenyl ether (20 mL) were
mixed and magnetically stirred under the flow of nitrogen. The
ο
reaction mixture was heated to 200 C for 1 h, then the temperature
increased to 280 οC for another 0.5 h. The other procedures were the
same to the above step.
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at 490 nm. Each experiment was repeated three times. The diameter and surface charge of NPs were obtained by dynamic light
upper part (containing the sample in the dialysis cassette) was scattering (DLS) with a Malvern Zetasizer Nano ZS model
exposed to an AMF for 20 min before each measurement.
ZEN3600. The sample absorbance was recorded on a Beckman
Flow cytometry. HeLa and Uꢀ87MG cells were seeded in 6ꢀwell Coulter DU 730 spectrophotometer. The hysteresis loops of NPs
culture plate with the density of 2×105 cells per well in 2 mL of were measured on a Quantum Design SQUID MPM S XLꢀ7.
RPMI 1640 medium and incubated at 37 oC and 5 % CO2. After
the incubation overnight, the medium was replaced with 2 mL
fresh medium containing PBS buffer solution (control) or
Results and discussion
MNCsꢀPEG/HAꢀDOX. After 0, 5, 10, 15, 20, 30, 60, 120, and Synthesis and characterization of MNCs-PEG/HA-DOX
240 min, respectively, the cells were washed with PBS, and
In this present work, (Zn0.4Co0.6)Fe2O4 NPs were prepared
according to the method reported 50, which were monodispersed
with an average diameter of 7.6 ± 1.4 nm (Fig. 1a). The similar
method was used to coat (Zn0.4Mn0.6)Fe2O4 NPs to form
core/shell NPs. TEM images of the coreꢀshell structured
then dealt with trypsinꢀEDTA solution (0.25%). Thereafter, the
cells were separated by centrifugation and washed with PBS.
The collected cells were reꢀsuspended in 1 mL PBS and
determined by flow cytometer (Cell Lab QuantaTM SC,
Beckman Coulter). Flow Jo software 7.6.5 was used to analyze
the data.
(Zn0.4Co0.6)Fe2O4@(Zn0.4Mn0.6)Fe2O4 nanocubes
(MNCs)
showed that the mean diameter was 11.2 ± 1.4 nm (Fig. 1b).
The increase of the diameter demonstrated that the successful
construction of core/shell structure of MNCs. The
crystallography of NPs was verified by powder Xꢀray
diffraction (XRD) (Fig. S2). It is clear that all the diffraction
peaks of the asꢀprepared (Zn0.4Co0.6)Fe2O4 and MNCs can be
indexed as an inverse spinel Fe3O4 (JCPDS card No. 19ꢀ0629)
phase. High resolution TEM image (Figure 1b inset) revealed
the high crystalline nature of MNCs. The lattice spacing
between two adjacent planes is 0.33 nm, which corresponds to
Laser scanning confocal microscopy. The cellular uptake process
of MNCsꢀPEG/HAꢀDOX was also confirmed on laser scanning
confocal microscopy (LSCM, Leica TCS SP5ꢀII, Germany).
After HeLa cells were incubated with MNCsꢀPEG/HAꢀDOX
(100 ꢀg/mL) for 0.5, 1, 2, and 4 h, respectively, excess MNCsꢀ
PEG/HAꢀDOX were removed by PBS. Then the cells were
fixed with paraformaldehyde (2.5%) for 15 min, and stained
with DAPI (100 ng/mL) for 15 min using a standard procedure.
The cells on the coverslips were imaged using a 63 × oilꢀ
immersion objective lens. DOX and DAPI were excited by the
488 nm and 405 nm lasers, respectively.
the
d spacing for the (220) lattice plane very well.
Magnetothermal therapy and chemotherapy in vitro. HeLa
cells were continuously cultured and seeded into 96ꢀwell cell
culture plate at a density of 5 × 104 cells/well in RPMIꢀ1640
medium supplemented with 10% FBS and 1% penicillinꢀ
streptomycin at 37°C and 5% CO2 for 24 h. After the incubation
for 12 h, the medium was replaced by the different
concentrations of MNCsꢀPEG/HA, MNCsꢀPEG/HAꢀDOX (0,
10, 20, 50, 100, and 200 ꢂg/mL in RPMIꢀ1640, respectively,
DOX content in the samples is 0.625, 1.25, 2.5, 5, and 10
µg/mL, respectively), and DOX (0.625, 1.25, 2.5, 5, and 10
µg/mL) for 24 h at 37°C under 5% CO2. Meanwhile, the upper
part incubated with MNCsꢀPEG/HAꢀDOX was exposed to an
AMF for 20 min before a measurement. After removal of the
medium, the purple formazan product was dissolved with
DMSO for 15 min. Finally, the optical absorption of formazan
at 490 nm was measured by an enzymeꢀlinked immunosorbent
assay reader (Multiskan MK3, USA.), and the background
subtraction at 690 nm was applied.
MR imaging in vivo. The MR imaging in vivo was performed
on HeLa tumorꢀbearing mice using
a 3.0 T system
(MAGNETOM VERIO, Siemens) before and after the tail
injection of MNCsꢀPEG/HAꢀDOX of 0.5, 1, 2, 3, and 4 h,
respectively. All animals were lawfully acquired and their
retention and use were in every case in compliance with federal,
state and local laws and regulations, and in accordance with the
Institutional Animal Care and Use Committee of SLAC
(IACUC) Guide for Care and Use of Laboratory Animals. T2ꢀ
weighted MR images were acquired using a conventional spinꢀ
echo sequence with the following parameters: TR/TE =
7500/77, 256 × 256 matrices, 90 × 90 mm field of view, 140
Hz/Px of bandwidth, a slice thickness of 0.9 mm.
Fig. 1. TEM images of Zn0.4Co0.6)Fe2O4 NPs (a), MNCs (b),
MNCsꢀPEG (c), and MNCsꢀPEG/HAꢀDOX (d). The bottom
right inset in (a) and (b) is the size distribution of NPs. The top
right inset in (b) is the HRTEM imaging of MNCs. The bottom
right inset in (d) were the photographs of MNCs (in hexane),
MNCsꢀPEG (in water), and MNCsꢀPEG/HAꢀDOX (in water),
respectively. The scale bar is 100 nm and the scale bar of inset
HRTEM is 5 nm.
Characterization.
It is well known that PEGylation is a successful strategy to
endow NPs with the water solubility and biocompatibility. The
amine moiety of 3ꢀ(3,4ꢀdihydroxyphenyl)propanehydrazide
(DPPH) on the surface of MNCs should be introduced for
further graft of HA and DOX. In the first step, DPPHꢀPEG and
The morphology of materials was characterized using JEOLJEMꢀ
2010 transmission electron microscope. Xꢀray diffraction (XRD)
was performed using a Rigaku DMAX 2000 diffractometer equipped
with Cu/Kα radiation in the 2θ range from 20 to 80° (λ = 0.15405
nm, 40 kV, 40 mA) at a scanning rate of 4°/min. The hydrodynamic
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DPPH were coated on the surface of MNCs by the ligand
exchange method. Then HA was reacted with a portion of the
primary amine via an amide linkage to form MNCsꢀPEG/HA.
MNCsꢀPEG/HAꢀDOX was obtained by conjugating DOX to
MNCsꢀPEG/HA by a hydrazide linker.
To demonstrate the successful modification of PEG, HA and
DOX, the thermogravimetric analysis (TGA) has been recorded
firstly. The percentage contents of surfactants, PEG, HA and
DOX were determined to be 8.4 %, 2.5%, 26.4 % and 3.9 %,
respectively (Fig. S3). TEM images (Fig. 1c and 1d) showed
that functionalized MNCs attained the similar morphology to
those without PEG, HA and DOX modification, which were
water dispersible (Fig. 1d inset). Zeta potential measurements
showed that the surface potential increased from ꢀ30.4±1.4 mV
of MNCsꢀPEG to ꢀ45.7±0.9 mV of MNCsꢀPEG/HA, which can
be explained by the surface modification with negatively
charged HA. MNCsꢀPEG/HA grafted with DOX on the surface
of MNCsꢀPEG/HA gave rise to an increase of the surface
potential, which resulted from the neutralization of the negative
surface charge. It is clear that the characteristic band at 490 nm
in the UVꢀvis spectroscopy (Fig. 2b) corresponded to the
absorption of DOX, which verified the graft of DOX. The drug
Fig. 3. The r1 and r2 values of MNCsꢀPEG/HAꢀDOX (a) and T2ꢀ
weighted MR imaging (b).
loading capacity was calculated to be
∼ 50 ꢀg/mg.
Magnetothermal and drug release property of MNCs-
PEG/HA-DOX in solution
Magnetic hyperthermia therapy highly depends on the
magnetothermal effect of nanoparticles. Therefore, the SLP value of
MNCsꢀPEG/HAꢀDOX was determined firstly. As seen from Fig. 4a,
the temperature was elevated with the increase of the concentration
of MNCsꢀPEG/HAꢀDOX. The SLP value of MNCsꢀPEG/HAꢀ
DOX was calculated to be
∼1343.0 W/g by an equation (1).
Fig. 2. The zeta potential (a), and UVꢀvis spectra (b) of MNCsꢀ
PEG, MNCsꢀPEG/HA, and MNCsꢀPEG/HAꢀDOX, respectively.
Under our alterative magnetic field condition, it is an excellent
performance and laid the foundation for magnetic hyperthermia
therapy. The acidꢀsensitive hydrazone linker can be cleaved
effectively at low pH value. Therefore, the degradation of
hydrazone bond of MNCsꢀPEG/HAꢀDOX will lead to the
release of DOX. 45, 53 To determine the drug release content, the
absorbance at 490 nm was measured. As shown in Fig. 4b, the
drug release profiles of DOX from MNCsꢀPEG/HAꢀDOX in
Magnetic and relativity property
As the magnetic property of nanomaterials is crucial for their
biomedical application, the hysteresis loops of MNCs, MNCsꢀ
PEG/HA and MNCsꢀPEG/HAꢀDOX were recorded firstly (Fig.
S4). The saturation magnetization of MNCs was ∼ 69 emu gꢀ1 and
decreased with the more ligand conjugated to MNCs. The hysteresis
loop of MNCsꢀPEG/HAꢀDOX showed the remarkable superꢀ
paramagnetic property. To validate the MR contrast ability, the
longitudinal relaxation time (T1) and transverse relaxation time (T2)
of MNCsꢀPEG/HAꢀDOX were examined in a 0.5 T MR system. The
r1, r2 and T2ꢀweighted MR imaging were presented in Fig. 3a and 3b.
The r1 and r2 value of MNCsꢀPEG/HAꢀDOX was 10.9 and 124.1
mMꢀ1sꢀ1, respectively. It can be deduced that MNCsꢀPEG/HAꢀDOX
should be a good candidate as T2 MR imaging agents. The T2ꢀ
weighed MR imaging of MNCsꢀPEG/HAꢀDOX (Fig. 3b) indicated
the apparent T2ꢀweighed MR imaging effect.
o
PBS buffer solution (pH 7.4 and 5.3, respectively) at 37 C
were recorded, respectively. The result revealed that the release
amount of DOX was up to
∼ 26.8% after 48 h in PBS at pH 7.4.
However, when the pH of the solution was decreased to 5.3,
57.6% of DOX was released after 48 h in PBS at 5.3, which
was attributed to the acceleration of cleavage of hydrazine bond
at low pH value. Additionally, the heat of magnetothermal
effect also accelerated pHꢀresponsive release of DOX from
MNCsꢀPEG/HAꢀDOX. The cumulative content of DOX
released from MNCsꢀPEG/HAꢀDOX under an AMF for 20 min
increased to 38.8 and 87.4% at pH 7.4 and 5.3, respectively. It
can be explained that an AMF induced the heat, which
accelerated the cleavage of hydrazine bond and increased the
the efficiency of the drug release. Therefore, the
magnetothermal effect could be utilized as an external stimulus
to effectively promote the drug release from MNCsꢀPEG/HAꢀ
DOX.
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Fig. 4. (a) Heating curves of water and aqueous solution of MNCsꢀ
PEG/HAꢀDOX with different concentrations (50, 100, 200, and 500
ꢂg/mL, respectively) under an AMF (200 kHz, 60.6 kA/m). (b) The
quantitative analysis of the released content of DOX from MNCsꢀ
PEG/HAꢀDOX with and without an AMF at pH 7.4 and 5.3,
respectively.
Fig. 5. (a) The mean fluorescence intensity of HeLa cells and Uꢀ
87MG cells as a function of time. (b) The relative fluorescence
intensity of HeLa cells and Uꢀ87MG cells were incubated with
o
MNCsꢀPEG/HAꢀDOX (100 ꢀg/mL) for 1 h at 37 oC and 4 C,
respectively. The block experiment: HA preꢀincubated (30 ꢂg/mL)
Uꢀ87MG cells were then incubated with MNCsꢀPEG/ꢀHAꢀDOX
(100 ꢀg/mL) for 1 h at 37 oC.
The property of MNCs-PEG/HA-DOX in vitro
The synergetic therapy in vitro
To confirm the targeted ability of the MNCsꢀPEG/HAꢀDOX to
cancer cells, both HeLa cells with CD44 receptor overꢀexpression
and Uꢀ87MG cells with CD44 receptor lowꢀexpression were
According to the excellent magnetothermal effect and the high
release efficiency of DOX of MNCsꢀPEG/HAꢀDOX in solution, the
combined therapy for the enhanced effect for cancer cells was
further verified. In our study, HeLa cells were incubated with free
DOX, MNCsꢀPEG/HA and MNCsꢀPEG/HAꢀDOX with the same
DOX concentration (0.625, 1.25, 2.5, 5, and 10 µg/mL, respectively)
(Fig. 6).When HeLa cells were only exposed under an AMF or
incubated with MNCsꢀPEG/HA only, the killing effect on HeLa cells
was negligible. The cell viability of HeLa cells incubated with free
DOX or MNCsꢀPEG/HA, then exposed to an AMF showed the
slight drop because of their chemotherapy effect of DOX and the
incubated with MNCsꢀPEG/HAꢀDOX (100
ꢀg/mL) for 0, 5, 10,
15, 20, 30, 60, 120, and 240 min, respectively. The amount of
MNCsꢀPEG/HAꢀDOX uptaken by cancer cells was evaluated
by the fluorescence intensity of DOX on the MNCs, which was
determined by the flow cytometric analysis. By plotting the mean
fluorescence intensity of HeLa cells and Uꢀ87MG cells as a function
of time (Fig. 5a), we can see that HeLa cells showed remarkably
higher fluorescence intensity than that of Uꢀ87MG cells, which
indicated that the higher uptaken content of MNCsꢀPEG/HAꢀDOX
to HeLa cells than that of Uꢀ87MG cells. To further confirm the
receptorꢀmediated process of MNCsꢀPEG/HAꢀDOX in HeLa cells,
the negative, temperature, and block control experiments were
conducted (Fig. 5b). The incubation time of 1 h was selected for
further investigation according to the relationship between the
uptaken content of MNCs and incubation time. For the negative
control experiment, Uꢀ87MG cells were chosen. After the incubation
magnetothermal
effect
of
MNCsꢀPEG/HA,
respectively.
Remarkably, compared to the free DOX or MNCsꢀPEG/HA plus an
AMF group, MNCsꢀPEG/HAꢀDOX incubated HeLa cells under an
AMF showed the obviously low cell viability. Furthermore, with the
increase of the incubation of MNCsꢀPEG/HAꢀDOX under an AMF,
the cell viability decreased more and more. As we known, there are
three possible factors accounting for the enhanced therapeutic effect.
Firstly, the heat generated by MNCsꢀPEG/HAꢀDOX under an AMF
improved the release of DOX. Secondly, the chemotherapy of DOX
improved the sensitivity of cancer cells to heat. Lastly, the raised
temperature also improved the sensitivity of cancer cells to DOX.
Therefore, the combined therapy of chemotherapy and magnetic
hyperthermia showed a synergetic therapy in HeLa cells.
of
100 µg/mL MNCsꢀPEG/HAꢀDOX, Uꢀ87MG cells of
fluorescence signals decreased 54% relative to that of HeLa cells. As
the incubation temperature decreased to 4°C, the fluorescence
intensity of HeLa cells was also kept 55% compared with 37°C. For
the block experiment, free HA was preꢀincubated (30 ꢂg/mL) with
HeLa cells before the incubation with MNCsꢀPEG/HAꢀDOX, the
fluorescence intensity of HeLa cells was decreased to 66%
corresponding to the incubation with only MNCsꢀPEG/HAꢀDOX.
These results indicated that the binding of MNCsꢀPEG/HAꢀDOX
can be mediated by receptorꢀmediated endocytosis.
In order to figure out the DOX release process in HeLa cells, the
fluorescence of HeLa cells was recorded for the different incubation
of MNCsꢀPEG/HAꢀDOX by laser scanning confocal microscopy
(CLSM). The CLSM images of the intracellular distribution of DOX
(red color) and DAPI (blue color) were presented in Fig. S5.
Apparently, the fluorescence of the cytoplasm became stronger with
the increase of the incubation time. The overlay of red and blue color
in nucleus illustrated that DOX had slowly transferred to nucleus.
Subsequently, the DOX had an inhibiting effect on RNA and
resulted in the cancer therapeutic effect. Additionally, the observed
change of cellular morphology also indicated the therapeutic effect
of DOX. The fact indicated that MNCsꢀPEG/HAꢀDOX firstly were
taken up by cells via the specific targeted endocytosis, then DOX
was released under the acid intracellular condition, transferred to
nucleus, and played the therapeutic role.
Fig. 6. The synergetic therapy to HeLa cells. Error bars were based
on five parallel samples.
This journal is © The Royal Society of Chemistry 2014
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Journal of Materials Chemistry B
DOI: 10.1039/C5TBA01R9T10ICALE
The MR imaging in vivo was carried out on HeLa tumorꢀ
bearing mice. The T2ꢀenhanced MR imaging was conducted on
a 3 T MRI scanner before and after the tail vein injection of
MNCsꢀPEG/HAꢀDOX (Fe: 0.10 mmol kgꢀ1). The signal was
darken from 0.5 to 3 h gradually, and recovered at 4 h (Fig. 7a),
which indicated the targeted diagnostic property. To our pity,
the synergetic therapy in vivo hasn’t performed with the
limitation of our experiment condition.
*Eꢀmail: yanghong@shnu.edu.cn, shipingy@shnu.edu.cn; Fax: 86ꢀ21ꢀ
64322343.
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We constructed an integrated nanoplatform of MNCsꢀPEG/HAꢀ
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magnetothermal effect accelerated the pH release efficiency of DOX
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Acknowledgements
This work was partially supported by National Natural Science
Foundation of China (Nos.21271130, 21371122, and
81401458), Shanghai Science and Technology Development
Fund (No. 13520502800), Shanghai Municipal Education
Commission (Nos. 113ZZ110 and 14YZ073), and International
Joint Laboratory on Resource Chemistry (IJLRC).
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DOI: 10.1039/C5TB01910A
The novel integrated nanoplatform facilitates the excellent targeting MR imaging guided
synergistic therapy of the magnetothermal and chemo-therapy based on magnetic core/shell ferrite
nanocubes (MNCs).