P(EO-co-LLA) functionalized Fe3O4@mSiO2 nanocomposites for thermo/pH responsive drug controlled release and hyperthermia

Article abstract of DOI:10.1039/c4dt02441a

The Fe₃O₄@mSiO₂ nanocarrier that consisted of a magnetic Fe₃O₄ nanoparticle core and a mesoporous silica (mSiO₂) shell was synthesized. It shows a uniform sphere morphology about 65 nm in diameter. Considering the magnetic hyperthermia of Fe₃O₄ under an alternating magnetic field (AMF), a thermo-sensitive polymer, poly[(ethylene glycol)-co-(l-lactide)] (P(EO-co-LLA)), was used as gatekeeper coating outside Fe₃O₄@mSiO₂ to regulate the drug release behavior. The design of the nanocarrier was expected to block off the pores at low temperature and to reopen them at high temperature reversibly. The obtained hybrid nanocomposites were capable of loading the anti-cancer drug doxorubicin (DOX) and controlled drug release behavior trigged by the hyperthermia of Fe₃O₄ under AMF. Besides, the nanocarriers also show pH-sensitive drug release based on the slight differences between the tumor (weakly acid) and the normal tissue (weakly alkaline). What's more, the chemotherapy of DOX combined with magnetic hyperthermia can improve the cytotoxicity obviously. On the basis of the high stability and excellent controlled release performance, the multifunctional nanocarriers exhibit potential applications in targeted-control drug release and hyperthermia for cancer treatment.

Full text of DOI:10.1039/c4dt02441a

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P(EO-co-LLA) functionalized Fe3O4@mSiO2
nanocomposites for thermo/pH responsive drug
controlled release and hyperthermia†
Cite this: Dalton Trans., 2014, 43,
18056
Wei Guo,a Chunyu Yang,a Huiming Lin*a,b and Fengyu Qu*a
The Fe3O4@mSiO2 nanocarrier that consisted of a magnetic Fe3O4 nanoparticle core and a mesoporous
silica (mSiO2) shell was synthesized. It shows a uniform sphere morphology about 65 nm in diameter.
Considering the magnetic hyperthermia of Fe3O4 under an alternating magnetic field (AMF), a thermo-
sensitive polymer, poly[(ethylene glycol)-co-(L-lactide)] (P(EO-co-LLA)), was used as “gatekeeper” coating
outside Fe3O4@mSiO2 to regulate the drug release behavior. The design of the nanocarrier was expected
to block off the pores at low temperature and to reopen them at high temperature reversibly. The
obtained hybrid nanocomposites were capable of loading the anti-cancer drug doxorubicin (DOX) and
controlled drug release behavior trigged by the hyperthermia of Fe3O4 under AMF. Besides, the nano-
carriers also show pH-sensitive drug release based on the slight differences between the tumor (weakly
acid) and the normal tissue (weakly alkaline). What’s more, the chemotherapy of DOX combined with
magnetic hyperthermia can improve the cytotoxicity obviously. On the basis of the high stability and
excellent controlled release performance, the multifunctional nanocarriers exhibit potential applications
in targeted-control drug release and hyperthermia for cancer treatment.
Received 11th August 2014,
Accepted 4th October 2014
DOI: 10.1039/c4dt02441a
www.rsc.org/dalton
However, without the specific discrimination between
normal cells and cancer cells, chemotherapy always induces a
Introduction
Cancer, which has been regarded as a major cause of mortality huge side effect besides the efficacy. To obtain a smart drug
worldwide, is a large group of diseases produced by rapid release behavior and beneficial therapy effects, much attention
unregulated cell growth. Chemotherapy remains one of the has been focused on the mesoporous silica nanocontainers
most commonly used methods of many cancer treatments. To equipped with “gatekeepers” as the delivery vehicles, in which
improve the therapy effect, nanomedicine has been employed a regulated drug release at specific time intervals and targeted
as the platform, showing a good performance. To date, multi- sites can be achieved by taking advantage of various
functional nanoparticles, including polymers, micelles, lipo- “gatekeepers”.17–19 Currently, nanoparticles, organic molecules
somes, dendrimers, ceramics, and even virus capsids, have and supramolecular nanovalves have been employed as “gate-
been used in medical diagnostics, drug delivery, therapy, keepers” for MSNs, and the controlled release process can be
etc.1–11 Among these, mesoporous silica nanoparticles (MSNs) regulated by many special stimuli, such as thermal/electro-
have been considered to be the most promising inorganic statics/magnetic actuation, light, photoirradiation, pH and
hosts to store and release drug molecules due to their excellent enzymes.20–26 For example, using DNA block copolymers as
properties, such as uniform and tunable morphology and capping agents, Herrmann et al. realized light-triggered tar-
pore size, high pore volume and surface area, easily geted cargo release.27 In our previous study, the Konjac oligo-
modified surface properties, good biocompatibility and saccharide (KOGC) was adopted as the “gatekeeper”. Owing to
biodegradation.12–16
the degradation of the “gate” (KOGC), the cargos can be
released triggered by β-mannanase, a normal enzyme in colon
of the human body.28
aLaboratory for Photon and Electronic Bandgap Materials, Ministry of Education,
College of Chemistry and Chemical Engineering, Harbin Normal University, Harbin
150025, China. E-mail: qufengyu2013@gmail.com, linhuiming@hrbnu.edu.cn
Magnetic (Fe3O4 or γ-Fe2O3) nanoparticles with strong mag-
netic properties and low toxicity have been widely used as tar-
geted and magnetic resonance imaging agents.29–31,33 For
example, Wang and coworkers synthesized a bicontrollable
drug release system with PAH/PSS multilayers on Fe3O4/mSiO2,
showing magnetic-targeted and pH-controllable release behav-
bInstitute of Functional Material Chemistry, Faculty of Chemistry, Northeast Normal
University, Changchun, 130024, P. R. China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4dt02441a
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ior.32 Ultra-small γ-Fe2O3 nanoparticles have also been adopted as the model to investigate the drug release kinetics
described as the potential specific positive contrast agents for of the system. Besides the thermo-sensitive control release be-
magnetic resonance molecular imaging.33–35 Moreover, mag- havior triggered by the hyperthermia of Fe3O4, the nano-
netic nanoparticles can produce localized hyperthermia by carriers also show pH-sensitive control release based on the
hysteresis heating upon exposure to an alternating magnetic slight acidity difference between the tumor and the normal
field (AMF).36 When the temperature is raised to 42–45 °C, tissue. The magnetic hyperthermia assisted thermo/pH drug
tumor/cancer cells are damaged or killed due to overheating, release can improve the therapeutic effect on tumor treatment.
while most of the normal cells far away from the magnetic
nanoparticles survive.37,38 At present, there are few reports on
the combination of hyperthermia with thermo-sensitive con-
Experimental section
trolled release.39,40 Vallet-Regí et al. synthesized mesoporous
Materials
silica nanoparticles with iron oxide nanocrystals encapsulated
Unless specified, all the chemicals were of analytical grade
and were used without further purification. Cetyltrimethyl-
ammonium bromide (CTAB), tin(II) 2-ethylhexanoate, 3-amino-
propyltriethoxysilane (APTES), tetraethyl orthosilicate (TEOS),
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
inside the silica matrix and decorated on the surface with a
thermoresponsive copolymer of poly(ethyleneimine)-b-poly-
(N-isopropylacrylamide) (PEI/NIPAM).41 Lin and coworkers pre-
pared multifunctional nanocarriers based on the up-conver-
sion luminescent nanoparticles and the thermo/pH-coupling
sensitive polymer poly[(N-isopropylacrylamide)-co-(methacrylic
acid)].42
Herein, the core–shell nanomaterial (Fe3O4@mSiO2), with
magnetic Fe3O4 nanoparticles as the core and mesoporous
silica as the shell, was synthesized as the drug loading host.
Poly[(ethylene glycol)-co-(L-lactide)] (P(EO-co-LLA)) was chosen
to graft outside Fe3O4@mSiO2 as blocking caps. It is known
that P(EO-co-LLA) is a typical thermo-sensitive polymer, which
exhibits reversible phase transitions between sol and gel in
aqueous solution based on the change in temperature.43 The
hyperthermia of Fe3O4 can serve as the “trigger” to control the
“on–off” of the thermo-sensitive polymer which is capable of
(MTT),
2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-
1H-benzimidazole, trihydrochloride (Hoechst 33342), and
monomethoxy poly(ethylene oxide) (PEO, Mn ∼ 5000) were pur-
chased from Sigma-Aldrich. L-Lactide (LLA) was purchased
from Changchun SinoBiomaterials Co. Ltd. Doxorubicin
hydrochloride (DOX), sodium oleate and oleic acid were
obtained from Aladdin, China. Dimethyl sulfoxide, ferric
trichloride hexahydrate (FeCl3·6H2O), ethanol, n-hexane and
triethylamine were purchased from Tianjin Chemical Corp. of
China.
Synthesis of Fe3O4 nanoparticles
regulating the drug release. From Scheme 1, firstly, In a typical synthesis of the iron-oleate complex, 10.8 g of iron
Fe3O4@mSiO2 nanomaterials were synthesized by the typical chloride (FeCl3·6H2O, 40 mmol) and 36.5 g of sodium oleate
Stöber method. After the modification of Fe3O4@mSiO2 with (120 mmol, 95%) were dissolved in a mixture solvent com-
–COOH, P(EO-co-LLA) can be grafted onto Fe3O4@mSiO2 as posed of 80 mL ethanol, 60 mL distilled water and 140 mL
the “gatekeeper”. Doxorubicin hydrochloride (DOX) was hexane. The resulting solution was heated to 70 °C and kept at
Scheme 1 Illustration of the preparation and controlled release process of Fe3O4@mSiO2-P(EO-co-LLA).
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that temperature for four hours. When the reaction was com- under nitrogen for
4
h
to obtain amine-functionalized
plete, the upper organic layer containing the iron-oleate Fe3O4@mSiO2 (Fe3O4@mSiO2-NH2). Later on, 200 mg of
complex was washed three times with 30 mL distilled water in Fe3O4@mSiO2-NH2 was added into 10 mL DMSO solution con-
a separatory funnel. After washing, hexane was evaporated, taining succinic anhydride (60 mg) and triethylamine (60 mg)
resulting in an iron-oleate complex in a waxy solid form.
and stirred at 40 °C for 48 h to produce the carboxyl-functiona-
Following a literature procedure, Fe3O4 nanoparticles were lized Fe3O4@mSiO2 (Fe3O4@mSiO2-COOH). 330 mg of
prepared.44 36 g (40 mmol) of the iron-oleate and 5.7 g of oleic Fe3O4@mSiO2-COOH was added into 20 mL methylene chlor-
acid (20 mmol, 90%) were dissolved in 200 g of 1-octadecene ide solution containing 350 mg of dichlorosulfoxide and
(90%) at room temperature. The reaction mixture was heated stirred at 50 °C for 4 h, and then 4.0 g of P(EO-co-LLA) (Mn
=
to 320 °C at a constant heating rate of 3.3 °C min−1, and then 7250) was added and stirred for another 4 h to obtain
kept at that temperature for 30 min. When the reaction temp- Fe3O4@mSiO2-P7250. Using P(EO-co-LLA)s with other mole-
erature reached 320 °C, a severe reaction occurred and the cular weights (8750 and 10 000), the samples were named
initial transparent solution became turbid and brownish Fe3O4@mSiO2-P8750 and Fe3O4@mSiO2-P10000.
black. The resulting solution containing the nanocrystals was
then cooled to room temperature, and 500 mL of ethanol was
added to the solution to precipitate the nanocrystals, which
Preparation of FITC labeled Fe3O4@mSiO2-P(EO-co-LLA)
In a typical synthesis of FITC-APTES, 15.6 mg of FITC and
100 μL of (3-aminopropyl)triethoxysilane (APTES) were dis-
solved in 5 mL DI water. The resulting solution was stirred at
room temperature and kept in the dark for 24 hours. FIT-
C-APTES is attached to Fe3O4@mSiO2-P(EO-co-LLA) via the
reaction among silica coupling groups of FITC-APTES and the
residual Si–OH on the silica spheres. In a typical process,
60.0 mg of Fe3O4@mSiO2-P(EO-co-LLA) was dispersed in 3 mL
of DI water, and then 2 mL of the obtained FITC-APTES solu-
tion was added. The reaction was performed in the dark at
ambient temperature overnight. The solid was collected by
centrifugation, followed by washing 6 times with DI water and
freeze drying.
were further collected by centrifugation and then dispersed in
chloroform.
Synthesis of Fe3O4@mSiO2 nanoparticles
In a typical procedure, 0.5 mL of the Fe3O4 nanocrystals in
chloroform (10 mg mL−1) was poured into 8 mL of a 0.2 M
aqueous CTAB solution and the resulting solution was stirred
vigorously for 30 min. The formation of an oil-in-water microe-
mulsion resulted in a turbid brown solution. Then, the
mixture was held at 60 °C for 30 min to evaporate the chloro-
form, resulting in a transparent black Fe3O4/CTAB solution.
Then, 20 mL of distilled water was added to the obtained black
solution and the pH value of the mixture was adjusted to 8 to
9 using 0.1 M NaOH. After that, 100 μL of 20% TEOS in
ethanol was injected six times in 30 min intervals. The reac-
tion mixture was reacted for 24 h under vigorous stirring. The
as-synthesized Fe3O4@mSiO2 NPs were washed three times
with ethanol to remove the unreacted species.
Drug loading and release
Fe3O4@mSiO2-P(EO-co-LLA) (60 mg) was added to the DOX
solution (3 mL, 1 mg mL−1) and stirred at 45 °C for 12 h.
Then, the solid was shock cooled, centrifuged, and washed
several times with distilled water. The loading amount of DOX
was determined by the UV/vis spectroscope at 480 nm, and the
drug loading sample was named DOX-Fe3O4@mSiO2-P(EO-co-
LLA). The loading efficiency (LE%) of DOX can be calculated
using formula (1). The experiment was repeated three times.
Syntheses of diblock copolymers (P(EO-co-LLA)) with different
molecular weights
P(EO-co-LLA)-7250 was synthesized following a literature pro-
cedure.45 PEO (8 g, 1.60 mmol) was added to 80 mL of dried
toluene and the residual water in the solution was removed by
azeotropic distillation to a final volume of 30 mL. L-Lactide
(3.6 g, 25.00 mmol) and stannous octoate (8.7 mg,
21.47 mmol) were added to this PEO/toluene solution and
refluxed under a dry nitrogen atmosphere for 24 h. The solu-
m
ðoriginal DOXÞ ꢀ m
ðresidual DOXÞ
LE% ¼
ꢁ 100% ð1Þ
m
þ m
ꢀ m
ðmSiO2Þ
ðoriginal DOXÞ
ðresidual DOXÞ
The gating protocol was investigated by studying the release
tion was precipitated in diethyl ether and filtered, and the profiles of DOX from the DOX-Fe3O4@mSiO2-P(EO-co-LLA) at
residual solvent was eliminated by vacuum drying. Diblock 25/45 °C in certain media solutions (pH 5.8 or 7.4, phosphate
copolymers with various molecular weights P(EO-co-LLA)-8750 buffer solution). Briefly, 10 mg of DOX loaded Fe3O4@mSiO2-
and P(EO-co-LLA)-10 000 were synthesized by a similar method P(EO-co-LLA) (DOX-Fe3O4@mSiO2-P(EO-co-LLA)) was dispersed
with 6 g and 8 g of L-lactide. The NMR spectrum of as- in 5 mL of media solution and sealed in a dialysis bag (mole-
synthesized P(EO-co-LLA) is shown in Fig. S1† that testifies the cular weight cutoff 8000), which was submerged in 20 mL of
polymerization between PEO and L-lactide.
media solution. At selected intervals, the solution was taken
out to determine the release amount by UV. The drug release
of DOX from the DOX-Fe3O4@mSiO2-P(EO-co-LLA) in AMF
Synthesis of Fe3O4@mSiO2-P(EO-co-LLA)
In a typical procedure, as-synthesized Fe3O4@mSiO2 nano- experiments was performed with a similar method, but the
particles (300 mg) were suspended in 20 mL of dry toluene difference is that the drug loading system was put in the AC
containing 100 μL of APTES. The solution was stirred at 50 °C magnetic field (250 kHz).
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Cell culture
netic field was generated by the alternating magnetic field
(Generator-SP04C).
HeLa cells (cervical cancer cell line) were grown in a monolayer
of Dulbecco’s modified Eagle’s medium (DMEM, Gibco)
supplemented with 10% (v/v) fetal bovine serum (FBS,
Tianhang Bioreagent Co., Zhejiang) and penicillin/strepto-
mycin (100 U mL−1 and 100 μg mL−1, respectively, Gibco)
under a humidified 5% CO2 atmosphere at 37 °C.
Results and discussion
Morphology, structure and phase
TEM was used to display the core–shell structure of the
samples (Fig. 1). Fig. 1A shows the TEM image of Fe3O4 nano-
particles that reveals a uniform and well dispersed spherical
morphology with the average diameter of 20 nm in size.
Confocal laser scanning microscopy (CLSM)
To determine the cellular uptake, HeLa cells were cultured in a
12-well chamber slide with one piece of coverglass at the
bottom of each chamber in the incubation medium (DMEM)
for 24 h. The cell nucleus was labeled with Hoechst 33342.
Fe3O4@mSiO2-P(EO-co-LLA) was added to the incubation
medium at a concentration of 100 μg mL−1 for 6 h of incu-
bation in 5% CO2 at 37 °C. After the medium was removed, the
cells were washed twice with PBS (pH 7.4) and the coverglass
was visualized under a laser scanning confocal microscope
(FluoView FV1000, Olympus).
Cell viability
The viability of cells in the presence of nanoparticles was
investigated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTT, Sigma) assay. The assay was per-
formed in triplicate in the following manner. For the MTT
assay, HeLa cells were seeded into 96-well plates at a density of
1 × 104 per well in 100 μL of the medium and grown overnight.
The cells were then incubated with various concentrations of
the need tested samples for 48 h. Afterwards, cells were incu-
bated in media containing 0.5 mg mL−1 of MTT for 4 h. The
precipitated formazan violet crystals were dissolved in 100 μL
of 10% SDS in 10 mmol HCl solution at 37 °C overnight. The
absorbance was measured at 570 nm with a multidetection
microplate reader (Synergy™ HT, BioTek Instruments Inc.,
USA).
Characterization
Powder X-ray patterns (XRD) were recorded on a Siemens D
5005 X-ray diffractometer with Cu Kα radiation (40 kV, 30 mA).
The nitrogen adsorption/desorption, surface areas, and
median pore diameters were measured using a Micromeritics
ASAP 2010 M sorptometer. The surface area was calculated
according to the conventional BET method and the adsorption
branches of the isotherms were used for the calculation of the
pore parameters using the BJH method. Fourier transform
infrared (FTIR) spectra were recorded on a PerkinElmer 580B
infrared spectrophotometer using the KBr pellet technique.
A UV-vis spectrum was used to describe the amount of drug
released (Shimadzu UV2550 spectrophotometer). Transmission
electron microscopy (TEM) images were recorded on a TECNAI
F20. Zeta potential and dynamic light scattering (DLS) were
carried out with a ZetaPALS zeta potential analyzer. The mag-
netic properties of samples were characterized with a vibrating
sample magnetometer (Lake Shore 7410). The alternating mag-
Fig. 1 TEM images of (A) Fe3O4, (B) Fe3O4@mSiO2, (C) Fe3O4@mSiO2-
P7250, (D) Fe3O4@mSiO2-P8750, (E) Fe3O4@mSiO2-P10000 and the
hydrodynamic size distribution of (F) Fe3O4@mSiO2, (G) Fe3O4@mSiO2-
P7250, (H) Fe3O4@mSiO2-P8750, and (I) Fe3O4@mSiO2-P10000.
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Fe3O4@mSiO2 shows the obvious core–shell structure with an
Fe3O4 core and a mesoporous silica shell about 20 nm in thick-
ness (Fig. 1B). Through esterification, P(EO-co-LLA) can be
gated onto the surface of Fe3O4@mSiO2. The polymer
layers result in a rough surface and less dispersion of
all Fe3O4@mSiO2-P(EO-co-LLA) nanoparticles (Fig. 1C–E).
Additionally, the hydrodynamic diameters of Fe3O4@mSiO2
and Fe3O4@mSiO2-P(EO-co-LLA)s were measured using a zeta
potential analyzer. As illustrated in Fig. 1F, the diameter of
Fe3O4@mSiO2 centers at 82 nm is larger than that observed
from TEM because of the hydrate layer in the aqueous environ-
ment. As depicted in Fig. 1G–I, the diameter of Fe3O4@mSiO2-
P(EO-co-LLA)s increases from 103 nm to 121 and 135 nm with
the polymer molecular weight increasing from 7250 to 8750
and 10 000, respectively. The zeta-potential test was further
used to monitor the difference between Fe3O4@mSiO2 and
Fe3O4@mSiO2-P(EO-co-LLA)s (Fig. 2). The zeta-potential value
increased from −16.02 2.17 mV for Fe3O4@mSiO2 to −2.2
4.57 mV for Fe3O4@mSiO2-P7250. With abundant surface
Si–OH, silica always shows the negative charge (−16.02
2.17 mV). After grafting with P(EO-co-LLA), the decrease of
surface Si–OH induces an increase of zeta-potential
for Fe3O4@mSiO2-P(EO-co-LLA). Fe3O4@mSiO2-P8750 and
Fe3O4@mSiO2-P10000 show
a zeta-potential of −3.08
4.17 mV and −4.83 5.86 mV, respectively (Fig. 2). Further-
more, with the same addition amount of P(EO-co-LLA), P(EO-
co-LLA)-7250 possesses the most molecules to interact with
surface Si–OH and to make the highest zeta-potential of all.
Eager to further verify the successful grafting of P(EO-co-
LLA) on Fe3O4@mSiO2, FT-IR spectroscopy was monitored to
study the organic and inorganic components of the samples.
The corresponding FT-IR spectra of PEO, L-lactide, P(EO-
co-LLA), Fe3O4@mSiO2 and Fe3O4@mSiO2-P(EO-co-LLA) are
shown in Fig. 3. As depicted in Fig. 3A, the absorption bands
at 2948 and 2887 cm−1 are assigned to the C–H stretching
vibrations, and the absorption bands at 1110 and 962 cm−1
can be assigned to the C–O–C stretching vibrations and C–H
rocking vibrations of PEO. The obvious absorption band at
Fig. 3 FTIR spectra of (A) PEO, L-lactide and P(EO-co-LLA); (B)
Fe3O4@mSiO2 and Fe3O4@mSiO2-P(EO-co-LLA).
1762 cm−1 testifies the CvO stretching vibrations of the ester
group in L-lactide. In addition, L-lactide also gives the charac-
teristic absorption bands at 935 and 656 cm−1 due to its six-
membered ring structure. After the polymerization, P(EO-co-
LLA) also shows the stretching vibration adsorption peaks of
CvO (1758 cm−1) and C–O–C (1110 cm−1), but the six-mem-
bered rings’ characteristic absorption bands (935 and
656 cm−1) disappear, testifying the open-ring polymerization
of L-lactide. In Fig. 3B, the obvious absorption band at
1086 cm−1 testifies the Si–O–Si framework of Fe3O4@mSiO2.
After P(EO-co-LLA) was grafted, two peaks assigned to C–H
stretching vibrations at 2982 and 2938 cm−1 appear. The
absorption peaks at 1758, 1693 and 1639 cm−1 assigned to the
CvO stretching vibrations of P(EO-co-LLA) can also be found
in Fe3O4@mSiO2-P(EO-co-LLA), confirming that P(EO-co-LLA)
has been successfully grafted on Fe3O4@mSiO2.
The X-ray diffraction patterns (XRD) were collected from
Fe3O4@mSiO2 and Fe3O4@mSiO2-P(EO-co-LLA)s. In Fig. S2,†
all samples show only one diffraction peak at about 2θ = 2.26°,
Fig. 2 Zeta-potential
Fe3O4@mSiO2-P8750 and Fe3O4@mSiO2-P10000.
of
Fe3O4@mSiO2,
Fe3O4@mSiO2-P7250,
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Table 1 Pore parameters and loading efficiency of the samples
BET
Vp
Pore size
(nm)
LE
(%)
Samples
(m2 g−1
)
(cm3 g−1
)
Fe3O4@mSiO2
326
152
100
68.0
0.285
0.156
0.133
0.095
2.42
2.31
2.30
2.30
—
DOX-Fe3O4@mSiO2-P7250
DOX-Fe3O4@mSiO2-P8750
DOX-Fe3O4@mSiO2-P10000
5.9 0.4
6.2 0.6
6.8 0.5
suggesting that they possess the ordered mesoporous struc-
ture. It is clearly observed that the relative intensities of the
peaks of the pattern collected from Fe3O4@mSiO2-P(EO-co-
LLA)s was reduced obviously as compared to that of
Fe3O4@mSiO2 without P(EO-co-LLA) grafted. Moreover, the
larger the amount of P(EO-co-LLA) grafted onto Fe3O4@mSiO2,
the lower the diffraction intensity of Fe3O4@mSiO2-P(EO-co-
LLA)s, which is consistent with the previous report.28 The pore
structure and related textural properties of Fe3O4@mSiO2 and
Fe3O4@mSiO2-P(EO-co-LLA)s were followed using nitrogen
adsorption–desorption measurements. As can be seen in
Fig. S3A,† Fe3O4@mSiO2 displays the typical IV adsorption iso-
therm and a steep capillary condensation step at P/P0
=
0.2–0.4. The typical H4 hysteresis loop further testifies the
mesoporous structure of Fe3O4@mSiO2.
As depicted in Fig. S3A,† there is a much smaller uptake of
nitrogen for Fe3O4@mSiO2-P(EO-co-LLA) taking its counterpart
(Fe3O4@mSiO2) as a comparison. Additionally, the surface
area (SBET) and pore volume are reduced from 326 m2 g−1 and
0.285 cm3 g−1 for Fe3O4@mSiO2 to 152 m2 g−1 and 0.156 cm3
g−1 for Fe3O4@mSiO2-P7250, 100 m2 g−1 and 0.133 cm3 g−1 for
Fe3O4@mSiO2-P8750, 68.0 m2 g−1 and 0.095 cm3 g−1 for
Fe3O4@mSiO2-P10000 (Table 1).
The magnetization characterization of the samples was also
studied. Fig. 4 presents the magnetic hysteresis loop of Fe3O4
and Fe3O4@mSiO2-P(EO-co-LLA) at room temperature. In
Fig. 4, Fe3O4 nanoparticles possess magnetism with the satur-
ation magnetizations (Ms) about 80.5 emu g−1. Fe3O4@mSiO2-
P(EO-co-LLA)s has super-paramagnetism with the reduced Ms
about 59.5, 55.6, and 48.7 emu g−1, which is ascribed to the
nonmagnetic mSiO2 and P(EO-co-LLA).
Fig. 4 Representative hysteresis-loop measurements of the obtained a:
Fe3O4@mSiO2, b: Fe3O4@mSiO2-P7250, c: Fe3O4@mSiO2-P8750 and d:
Fe3O4@mSiO2-P10000.
at a low temperature (25 °C). However, the release became
more easy when the temperature was increased to 45 °C. In
Fig. 5A(a–c), the cumulative release amount of DOX reaches
24.7, 28.6 and 38.0% for DOX-Fe3O4@mSiO2-P7250, DOX-
Fe3O4@mSiO2-P8750 and DOX-Fe3O4@mSiO2-P10000 within
24 h, respectively. With a higher molecular weight of the
polymer, DOX-Fe3O4@mSiO2-P10000 shows a higher release
amount (38.0%).
Considering the pH-sensitivity of P(EO-co-LLA), the release
profile of DOX-Fe3O4@mSiO2@P10000 was also studied under
different pH conditions (pH 7.4, 5.8 and 4.0). In Fig. 5B, the
fast burst release can be found at pH 4. It just takes 24 h to
reach maximum release (92.7%). However, when the pH value
increases to 5.8, the release rate decreases as well as the
release amount. Fig. 5B shows about 89.4% release amount
until 24 h at pH 5.8. Going on to increase the pH value to 7.4,
the release decreases more obviously (38.0%). From the above,
the acidic condition is beneficial for the drug release. Compar-
ing the release profiles in Fig. 5B and C, it can be also found
that the release at 45 °C is freer than that at 25 °C.
Drug loading and release profiles
To investigate the sensitive and controlled release properties of
Fe3O4@mSiO2-P(EO-co-LLA)s, DOX was selected as the model
drug and the release performances were investigated in detail
(Table 1). The actual loading levels of DOX were calculated to
be 6.8 0.5, 6.2 0.6 and 5.9 0.4% with the increase of the
corresponding molecular weight of P(EO-co-LLA)s from 7250 to
8750 and 10 000, respectively. It is known that the drug
loading ability is related to the surface area of carriers. With a
large surface area, Fe3O4@mSiO2-P7250 (152 m2 g−1) possesses
a high drug loading amount (6.8 0.5%).
To further reveal the sensitive-release mechanism of the
system, the hydrodynamic sizes of all samples under different
temperatures/pH values/molecular amounts were investigated
and are summarized in Table 2. As can be seen in Table 2, as
the molecular amount of P(EO-co-LLA)s increases from 7250 to
8750 and 10 000, the hydrodynamic size of Fe3O4@mSiO2-
P(EO-co-LLA)s increases from 108 to 149 nm at pH 7.4 (25 °C).
The release profiles of DOX-Fe3O4@mSiO2-P(EO-co-LLA)s in
PBS buffer (pH 7.4) in response to the temperature (20 and
45 °C) are shown in Fig. 5A. In Fig. 5A(d and e), all samples
show low release cumulative amounts (below 10%) until 24 h
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Fig. 5 (A) Release profiles of DOX from Fe3O4@mSiO2-P10000 (a: 45 °C and f: 25 °C), Fe3O4@mSiO2-P8750 (b: 45 °C and e: 25 °C), Fe3O4@mSiO2-
P7250 (c: 45 °C and d: 25 °C) at pH 7.4. Release profiles of DOX from Fe3O4@mSiO2-P10000 under different pH conditions at 45 °C (B) and 25 °C
(C). (D) Release profiles of DOX from Fe3O4@mSiO2-P10000 (pH 5.8) a: with AMF from t = 0 h, b: with AMF from t = 2 h, and c: without AMF.
takes place not only at pH 7.4, but also at pH 5.8 and 4.0
(Table 2).
Table 2 The hydrodynamic size of the samples
Samples
T = 25 °C
T = 45 °C
As mentioned above, besides the temperature-triggered
release, DOX-Fe3O4@mSiO2-P10000 reveals also pH sensitive-
release behavior under different pH conditions. It is known
that the swelling of P(EO-co-LLA) in aqueous solution depends
on the hydrogen bond between P(EO-co-LLA) and H2O. When
decreasing the pH value of the solution, the hydrogen bond is
weakened, leading to the shrinking of polymer brushes. As
illustrated in Table 2, as the pH value decreases from 7.4 to 5.8
and 4.0, the hydrodynamic size of DOX-Fe3O4@mSiO2-P10000
DOX-Fe3O4@mSiO2-P7250 (pH 5.8)
DOX-Fe3O4@mSiO2-P7250 (pH 7.4)
DOX-Fe3O4@mSiO2-P8750 (pH 5.8)
DOX-Fe3O4@mSiO2-P8750 (pH 7.4)
DOX-Fe3O4@mSiO2-P10000 (pH 4.0)
DOX-Fe3O4@mSiO2-P10000 (pH 5.8)
DOX-Fe3O4@mSiO2-P10000 (pH 7.4)
97 nm
108 nm
115 nm
130 nm
128 nm
131 nm
149 nm
85 nm
102 nm
97 nm
122 nm
99 nm
105 nm
138 nm
With the high molecular amount of P(EO-co-LLA), the corres- decreases from 149 to 131 and 128 nm at 25 °C and from 138
ponding hydrodynamic size increases due to the long chain of to 105 and 99 nm at 45 °C. So, the shrinking of P(EO-co-LLA)
the polymer brushes that makes the release amount decrease makes the preponderant release in the acidic environment as
from 10.1% of DOX-Fe3O4@mSiO2-P7250 to 7.1% of DOX- depicted in Fig. 5B and C.
Fe3O4@mSiO2-P10000 (Fig. 5A).
From the above investigation, the temperature-triggered
It is known that P(EO-co-LLA) is a typical temperature sensi- release behavior of the nanocomposites is very definite. Con-
tive polymer, which can change from sol to gel as the tempera- sidering the hyperthermia of the magnetic Fe3O4, AMF was
ture increases above its lower critical solution temperature also introduced to control the release process and the release
(LCST). As displayed in Table 2, when the temperature performances. As shown in Fig. 5D, under the stimulus of
increases to 45 °C, the hydrodynamic size of Fe3O4@mSiO2- AMF, the release is enhanced to 28.7% at 2 h and 89.4% at
P(EO-co-LLA)s deceases to 102, 122, and 138 nm (pH 7. 4) due 12 h. To further reveal the AMF enhanced release, the release
to the shrinking of the polymer brushes at high temperature. without AMF firstly and then with AMF was also carried out.
In short, when the temperature increases to 45 °C, all the The release is very slow (about 7.8%) at the first 2 h. Then,
releases were accelerated and the release amount increased AMF was introduced and the release increased to 41.3% at 4 h
obviously, while at 25 °C, the loose polymer brushes block the and even 86.8% at 12 h. In addition, the time-dependent temp-
pore and make the release below 10%. Furthermore, the erature of Fe3O4@mSiO2-P10000 under AMF treatment is
shrinking/loosening of polymer brushes based on temperature shown in Fig. S4.† After AMF treatment for 10 min, the temp-
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erature increases to 35 °C. It costs about 20 min to reach
To compare the anticancer efficacy of the DOX-loaded nano-
45 °C. As time goes on, the temperature can reach 47 °C composites and free DOX, HeLa cells were incubated in
under the AMF stimulus for 30 min. In Fig. 5D and S4,† AMF culture medium in the presence of free DOX, Fe3O4@mSiO2-
stimuli-release can be ascribed to the hyperthermia of the P10000 and DOX-Fe3O4@mSiO2-P10000 with various concen-
magnetic Fe3O4 that causes the temperature increase and drug trations for 48 h, and then the MTT assay was used for quanti-
release.
tative testing of the cell viability. As shown in Fig. 7A,
Fe3O4@mSiO2-P10000 shows a high cell viability (89.5%) even
after 48 h of incubation, with the sample concentration as
high as 480 μg mL−1, showing the neglectable toxicity and
good biocompatibility of the nanocomposites. However, the
cell viability decreases to 62.7% (480 μg mL−1) due to the
hyperthermia of the magnetic nanocomposites under AMF
treatment (Fig. 7A).
In vitro cytotoxic effect and cellular uptake
To investigate the cellular uptake of the sample, DOX-
Fe3O4@mSiO2-P10000 was incubated with HeLa cells at a
concentration of 100 μg mL−1 for 6 h. The cellular uptake and
subsequent localization of the sample is shown in Fig. 6. As can
be seen in Fig. 6, nanoparticles are localized in the cytoplasm
after 6 h of incubation, proving the fast cellular uptake of the
sample. In addition, DOX can be found in karyon that is
because the nanocomposites were taken into the cytoplasm and
the low-pH endosomal environment induced the drug release.15
In Fig. 7B, DOX-Fe3O4@mSiO2-P10000 exhibits 71.5% apop-
tosis at an equivalent DOX concentration of 50 μg mL−1
.
Assisted with AMF stimuli, the cellular apoptosis is enhanced
to 93.7%, revealing that the chemotherapy combined with
hyperthermia can improve the cellular cytotoxicity obviously.
Fig. 6 CLSM images of HeLa cells after incubation with 100 μg mL−1 of DOX-Fe3O4@mSiO2-P10000 for 6 h. (A) HeLa cells (bright), (B) DOX fluor-
escence in cells (red), (C) FITC labeled DOX-Fe3O4@mSiO2-P10000 (green), (D) Hoechst 33342 labeled cell nucleus (blue), and (E) merged.
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toxicity (93.7%) as an equivalent DOX (87.4%) concentration of
50 μg mL−1
.
Conclusion
In summary, Fe3O4@mSiO2 core–shell nanocomposites were
synthesized as the nanocarriers. Considering the magnetic
hyperthermia of Fe3O4 under AMF, a thermo-sensitive polymer
P(EO-co-LLA) was used as “gatekeeper” coating outside
Fe3O4@mSiO2 to regulate the drug release behavior. After the
loading of the anticancer drug DOX, the composites show con-
trolled drug release behavior trigged by the hyperthermia of
Fe3O4 under AMF. Besides this, the nanocarriers also demon-
strate the pH-sensitive control release based on the slight
acidity difference between the tumor and the normal tissue.
The chemotherapy of the anticancer drug combined with mag-
netic hyperthermia enhances the cytotoxicity obviously. There-
fore, with these unique properties, the nanocomposites have
potential applications in targeted-control drug release and
hyperthermia for cancer therapy.
Acknowledgements
Financial support for this study was provided by the National
Natural Science Foundation of China (21471041, 21171045,
21101046, 21441002), Natural Science Foundation of
Heilongjiang Province of China (ZD201214), Technology devel-
opment pre-project of Harbin Normal University (12XYG-11).
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Fig. 7 Cell viability of HeLa cells incubated with different amounts of
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