A facile preparation of targetable pH-sensitive polymeric nanocarriers with encapsulated magnetic nanoparticles for controlled drug release

Article abstract of DOI:10.1039/c2jm34817a

Novel multifunctional nanocomposites were successfully prepared for the controlled release of anti-cancer drug and magnetic resonance imaging (MRI) via a simple self-assembly process. In this strategy, superparamagnetic iron oxide nanoparticles (SPIONPs)

Full text of DOI:10.1039/c2jm34817a

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PAPER
A facile preparation of targetable pH-sensitive polymeric nanocarriers with
encapsulated magnetic nanoparticles for controlled drug release†
*
Shun Yang, Dongyun Chen, Najun Li, Xiao Mei, Xiuxiu Qi, Hua Li, Qingfeng Xu and Jianmei Lu
*
Received 21st July 2012, Accepted 10th October 2012
DOI: 10.1039/c2jm34817a
Novel multifunctional nanocomposites were successfully prepared for the controlled release of anti-
cancer drug and magnetic resonance imaging (MRI) via a simple self-assembly process. In this strategy,
superparamagnetic iron oxide nanoparticles (SPIONPs) were ‘‘fixed’’ between the hydrophobic
segment of the pH-sensitive amphiphilic polymer (HAMAFA-b-DBAM) and the surface of hollow
mesoporous silica nanoparticles (HMS) which were modified by the long-chain hydrocarbon
octadecyltrimethoxysilane (C18). Since the amphiphilic polymer was conjugated with a folic acid (FA)
group, the nanocomposites could target the folic acid receptor (FR) of over-expressed tumor cells
efficiently. Moreover, high drug loading content was obtained simultaneously due to the hollow core of
HMS. The loaded drug could release from the HMS core triggered by the mildly acidic pH environment
in the cancer cells due to the hydrolysis of the pH-sensitive polymer shell. The targeting process of the
nanocomposites could be easily tracked by MRI due to the magnetism of the SPIONPs. Therefore, a
nanocarrier with high drug-loading capacity and controlled drug release property for tumor diagnosis
and therapy was obtained via the self-assembly of HMS core, magnetic Fe3O4 nanoparticles and
targetable pH-sensitive polymer shell.
nanocomposites are able to achieve controlled release when acting
as drug carriers despite their complicated preparation process.
Recently, stimuli-responsive (e.g. pH changes, temperature,
enzyme, redox or light) block copolymer micelles are particularly
attractive for controlled drug release.13–21 The promising method
is the modification of inorganic nanoparticles with stimuli-
responsive copolymers to achieve controlled release for drug
delivery applications.22,23 In our previous works, pH-responsive
amphiphilic polymers were employed to encapsulate HMS for
targeted anti-cancer drug release. High drug loading capacity was
obtained in this approach and they could release incorporated
drugs selectively in mildly acidic tumor sites.24 Thus, we envi-
sioned that if we introduce the hydrophobic SPIONPs into this
self-assembly process, the pH-responsive amphiphilic polymers
would encapsulate SPIONPs and HMS together and the obtained
nanocomposites could act as both a contrast agent for MRI and
highly efficient nanocarriers for cancer therapy.
Introduction
Inorganic nanoparticles such as gold nanoparticles,1 mesoporous
silica nanoparticles,2,3 Fe3O4 nanoparticles4 have been increas-
ingly used to prepare nanocomposites for biomedical applica-
tions. Among them, superparamagnetic iron oxide nanoparticles
(SPIONPs) and hollow mesoporous silica nanoparticles (HMS)
have attracted great attention in the past few years. As a
magnetic resonance imaging (MRI) contrast agent, SPIONPs
have been widely used for cell imaging due to their super-
paramagnetism, narrow size distributions and imaging sensi-
tivity.5,6 HMS are also currently used in the field of drug delivery
due to their unique biocompatibility and chemical stability.
Notably, the hollow core and silica shell endow the HMS with a
high drug loading capacity and a modifiable outer surface.7–10
In view of the above, many researchers fabricated various
nanocomposites containing both Fe3O4 and silica. For instance,
Gai and co-workers reported a facile method in which the
hydrophobic SPIONPs were absorbed on the cetyl-
trimethylammonium bromide (CTAB) template, and then silicate
molecules were assembled on the surface to form a silica shell for
Herein, we report a facile strategy to prepare a multifunctional
nanocomposite for controlled anti-cancer drug delivery and
MRI imaging via a simple self-assembly process. SPIONPs
stabilized by oleic acid are ‘‘fixed’’ between the hydrophobic part
of the polymer and the surface of HMS which were modified with
the long-chain hydrocarbon octadecyltrimethoxysilane (C18)
(Scheme 1). The resulting nanocomposites have several advan-
tages as follows. First, a high drug loading content could be
achieved due to the hollow core of the HMS; the long alkyl
chains grown on the shell are able to delay the drug release.
Second, the drug delivery process could be easily tracked by MRI
further
modification.11,12
However,
these
inorganic
Key Laboratory of Organic Synthesis of Jiangsu Province, College of
Chemistry, Chemical Engineering and Materials Science, Soochow
University, Suzhou, China, 215123. E-mail: lujm@suda.edu.cn; linajun@
suda.edu.cn; Fax: +86 (0) 512-6588 0367; Tel: +86 (0)512-6588 0368
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c2jm34817a
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the PS latex templates, 0.5 g AETAC (80 wt% in H2O) was dis-
solved in 180 g water in a 500 ml round-bottom flask. Then 20.0 g
styrene was added slowly and kept stirring for 30 min. The
mixture was purged with nitrogen for 20 min and then heated to
90 ꢀC. Afterwards, 5 ml of an aqueous solution containing 0.5 g
V-50 was added. The emulsion was kept at 90 ꢀC for 24 h under
nitrogen to complete the polymerization. The polystyrene latex
was collected by centrifugation and washed with ethanol several
times. To prepare the HMS, 0.8 g CTAB was dissolved in a
mixture of 80.0 g water, 60.0 g ethanol and 1.5 ml ammonium
hydroxide solution. 0.93 g of PS powders was dispersed in 10.0 g
water by sonication and then added dropwise to the above CTAB
solution at room temperature with vigorous stirring, followed by
sonication for 10 min. The derived milky mixture was then stirred
for 30 min before adding 4.0 g TEOS. The mixture was kept
stirring at room temperature for 48 h. The precipitate was
collected by centrifugation and washed with ethanol, then dried
at room temperature. Finally the material was calcined in air at
Scheme 1 Schematic depiction of the structure of HAMAFA-b-DBAM-
coated DOX@HMS@C18@SPIONPs and controlled release by degra-
dation under weakly acidic conditions.
due to the SPIONPs encapsulated in the nanocomposites.
Moreover, compared to other amphiphilic polymers, our pH-
responsive polymer conjugated with folic acid (FA) moiety is
more suitable for targeted anti-cancer drug delivery. When
recognized by the FA receptor of positive tumor cells, the
nanocarriers could be internalized efficiently with subsequent
controlled release of the loaded drug. Most importantly, in this
facile strategy, an amphiphilic polymer was introduced to
encapsulate hydrophobic SPIONPs and HMS in one nanosystem
and this could also be used to combine another kinds of small size
inorganic nanoparticles with HMS to form multifunctional
nanocomposites for further applications.
ꢀ
600 C for 8 h to remove any organic matter.
Modification of the external surface of HMS with
octadecyltrimethoxysilane (C18)
100 mg HMS was first dispersed in 20 ml anhydrous acetonitrile,
then 5 ml C18 was added, and the obtained suspension was
stirred for 24 h and collected by centrifugation, washed with
acetonitrile and ethanol several times, and dried under vacuum.
Experimental section
Preparation of superparamagnetic iron oxide nanoparticles
(SPIONPs)
Materials
(2-(Acryloyloxy)ethyl)trimethylammonium chloride (AETAC,
80 wt% in water), 2,20-azobis(2-methylpropionamidine) dihy-
drochloride (V-50, >97.0%), hexadecyltrimethylammonium
bromide (CTAB, >99.0%) and octadecyltrimethoxysilane (C18)
were purchased from Aldrich. Tetraethoxysilane (TEOS) was
purchased from Alfa without any further purification. Styrene
(St, >99.0%) was washed through an inhibitor remover column
for removal of tert-butyl catechol and then distilled under
reduced pressure prior to use. Ferric trichloride hexahydrate
(FeCl3$6H2O), sodium oleate, sodium methoxide, potassium
ferricyanide, p-hydroxybenzaldehyde, benzyl chloride, 1-bro-
mododecane, p-toluene sulfonic acid (PTSA) and glycerol were
all purchased from Shanghai Chemical Reagent Co. Ltd as
analytical regents and used without further purification. 4,4-
Azobis(4-cyanovaleric acid) (V-501) and azobisisobutyronitrile
(AIBN) were purchased from Aldrich. N-Hydroxysuccinimide
(NHS, 99%), N-(3-aminopropyl)methacrylamide hydrochloride
(APMA), dicyclohexacarbodiimide (DCC, 99%) and 4-dime-
thylaminopyridine (DMAP, 99%) were purchased from Alfa
Aesar. Methacryloyl chloride was produced from Haimen Best
Fine Chemical Industry Co. Ltd and used after distillation.
Hydroxyethyꢀlacrylate (HEA) was distilled under vacuum and
stored at 15 C under an inert gas atmosphere. Other reagents
were commercially available and used as received.
SPIONPs were synthesized according to the literature with some
modifications.26 2.7 g (10 mmol) FeCl3$6H2O and 9.1 g (30
mmol) sodium oleate were dissolved in a mixed solvent
composed of 20 ml ethanol, 15 ml water and 35 ml hexane. After
heating at 70 ꢀC for 4 h, the organic layer was washed and
evaporated to obtain an iron–oleate complex. Then 9.0 g (10
mmol) of the iron–oleate complex and 1.4 g oleic acid (5 mmol)
were dissolved in 50 g trioctylamine at room temperature. The
obtained nanoparticles were washed several times with ethanol
ꢀ
and dried at 60 C for 24 h.
Synthesis of RAFT agent 4-cyanopentanoic acid dithiobenzoate
(CAD)
CAD was synthesized according to the literature with some
modifications.27 Briefly, 12.8 g benzyl chloride was added drop-
wise to a sodium methoxide methanol solution (172 g, 12.6 wt%)
containing 12.8 g elemental sulfur. Subsequently, the obtained
mixture was refluxed for 10 h under an inert atmosphere. The
crude sodium dithiobenzoate solution was extracted by diethyl
ether, 1.0 M hydrochloric acid and sodium hydroxide aqueous
solution and finally yielded a solution of sodium dithiobenzoate.
Potassium ferricyanide solution (500 ml, 6.5 wt%) was added
dropwise to the sodium dithiobenzoate over a period of 1 h with
vigorous stirring. The obtained red precipitate was filtered,
washed with deionized water, and dried under vacuum at room
temperature overnight. Dithiobenzoyl disulfide (8.50 g, 0.28 mol)
was added slowly to the distilled ethyl acetate (150.0 ml) solution
containing 11.68 g V-501 (0.42 mol). After refluxing for 18 h, the
Synthesis of hollow mesoporous spherical (HMS) particles
The hollow mesoporous spherical particles were synthesized
according to the literature with little modification.25 To prepare
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reaction solution was concentrated under vacuum and purified
by column chromatography (ethyl acetate–hexane, 2 : 3). 1H
NMR (400 MHz, CDCl3), d (ppm): 7.91 (d, J ¼ 7.59 Hz, 2H,
C6H4), 7.58 (t, J ¼ 7.47 Hz, 1H, C6H4), 7.41 (t, J ¼ 7.79 Hz, 2H,
C6H4), 2.75 (m, 2H, CCH2), 2.45 (m, 2H, CH2COOH), 1.95 (s,
3H, CH3).
was added to the aqueous solution containing 2.5 mg V-501,
1.22 g HEA (10.5 mmol) and 268 mg APMA (1.5 mmol). Then
the tube was sealed after three cycles betweeꢀn vacuum and
nitrogen. After 5 h reaction in an oil bath at 70 C, the mixture
was concentrated and washed with a large amount of acetone.
The obtained copolymer was dried under vacuum and stored in
desiccators for further polymerization.
The amphiphilic diblock polymer HAMA-b-DBAM was
synthesized using HAMA macro chain transfer agent. In a
typical polymerization procedure, 1.2 g HAMA was added to a
dimethylsulfoxide solution containing 3 mg AIBN and 2ꢀ00 mg
(0.31 mmol) DBAM and then placed in an oil bath at 70 C for
5 h. The mixture was concentrated and washed with a large
amount of ethyl ether. After washing, the obtained diblock
polymer was dried under vacuum overnight and stored in
desiccators.
Synthesis of 4-n-dodecyloxybenzalacetal monomer (DBAM)
4-n-Dodecyloxybenzaldehyde (DBD) was synthesized according
to the literature with some modification.28 1-Bromododecane
(29.9 g, 120 mmol) was added dropwise to the mixture of
p-hydroxybenzaldehyde (12.2 g, 10 mmol) and anhydrous
potassium carbonate (20.7 g, 150 mmol) in 150 ml acetone. After
heating under reflux with stirring for 14 h, the mixture was
filtered off and acetone was removed by a rotary evaporator. The
crude product was purified by column chromatography with a
mixture of ethyl acetate–petroleum ether (boiling range 60–90
1
ꢀC) (1/10, v/v). H NMR (400 MHz, CDCl3), d (ppm): 9.88 (s,
Synthesis of folate conjugated diblock polymer HAMAFA-b-
DBAM
1H, CHO), 7.83 (d, J ¼ 8.72 Hz, 2H, C6H4), 6.99 (d, J ¼ 8.69 Hz,
2H, C6H4), 4.04 (t, J ¼ 6.56 Hz, 2H, CH2O), 1.86–1.76 (m, 2H,
CH2CH2O), 1.46 (m, 2H, CH2CH2CH2O), 1.26 (m, 16H, CH2),
0.88 (t, J ¼ 6.79 Hz, 3H, CH3).
The conjugation procedure was carried out according to the
literature reported elsewhere.29–31 First, the thiocarbonylthio end
group of HAMA-b-DBAM was removed according to the liter-
ature with some modifications. Briefly, 500 mg HAMA-b-
DBAM was dissolved in 10 ml N,N-dimethylformamide (DMF)
containing 30 mg AIBN. The DMF solution was sealed after
cycling between vacuum and nitrogen three times. After stirring
at 70 ꢀC for 5 h, the mixture was precipitated in anhydrous ether.
The obtained diblock polymer was dried under vacuum and
stored in desiccators for further use. Second, folate NHS ester
was prepared by the following procedure: 0.62 g DCC and 0.51 g
NHS were added to a dry dimethylformamide (50 ml) solution
containing 2.0 g folic acid and then the solution was stirred for
12 h at room temperature in the dark. The solution was filtered
off and precipitated in diethyl ether. The obtained yellow powder
was washed several times with anhydrous ether and used
immediately for the next step. Then, FA–NHS (30 mg) was
added to dry pyridine containing 100 mg of the above polymer.
The solution was shaken for 12 h at room temperature and the
mixture was precipitated in 40% acetone in diethyl ether. The
filtrate was concentrated and stored in the dark at 4 ꢀC. 1H NMR
(400 MHz, DMSO-d6), d (ppm): 8.63–6.62 (folic acid and
CH2NH2), 4.21–3.76 (COOCH2CH2 and CH2OH), 2.85
(NHCH2 and CH2NH2), 1.21 (CH3).
After that, DBD (8.7 g, 30 mmol) was reacted with glycerol
(2.76 g, 30 mmol) in 50 ml toluene using PTSA (0.5 g) as the
catalyst. The solution was refluxed for 14 h and the water formed
by dehydrogenation reaction was removed by the oil–water
separator. Then, the mixture was concentrated and washed with
potassium carbonate solution (1%, 80 ml) to remove the acid
catalyst and any residual glycerol. After that, the precipitate was
collected and purified by column chromatography with a mixture
of ethyl acetate–petroleum ether (1/2, v/v) to obtain 4-n-dode-
cyloxybenzalacetal (DBA). 1H NMR (400 MHz, CDCl3), d
(ppm): 7.41 (d, J ¼ 8.51 Hz, 2H, C6H4), 6.90 (d, J ¼ 8.67 Hz, 2H,
C6H4), 5.51 (s, 1H, C6H4CH), 3.57–4.38 (m, 8H, C6H4OCH2,
CHCH2O, CHOH, OH), 1.86–1.76 (m, 2H, CH2CH2O), 1.50–
1.39 (m, 2H, CH2CH2CH2O), 1.26 (m, 16H, CH2), 0.88 (t, J ¼
6.78 Hz, 3H, CH3).
Methacryloyl chloride (2.3 g, 22 mmol) was added slowly to
the anhydrous tetrahydrofuran (20 ml) solution containing tri-
ethylamine (4.5 g, 44 mmol) and DBA (4.0 g, 11 mmol) and
ꢀ
cooled to 0 C in a water–ice bath. After constantly stirring for
another 12 h at room temperature, the mixture was filtered off to
remove the byproduct. The obtained filtrate was concentrated
and purified by column chromatography with a mixture of ethyl
acetate–petroleum ether (1/8, v/v). 1H NMR (400 MHz, CDCl3),
d (ppm): 7.41 (d, J ¼ 8.39 Hz, 2H, C6H4), 6.89 (d, J ¼ 8.41 Hz,
2H, C6H4), 6.30 (s, 1H, CCH2), 5.65 (s, 1H, CCH2), 5.52 (s, 1H,
C6H4CH), 4.75 (s, 1H, CHO), 4.31 (d, J ¼ 12.88 Hz, 2H,
CHCH2O), 4.18 (d, J ¼ 12.92 Hz, 2H, CHCH2O), 3.95 (t, J ¼
6.59 Hz, 2H, C6H4OCH2), 2.01 (s, 3H, CH3), 1.81–1.72 (m, 2H,
CH2CH2O), 1.49–1.38 (m, 2H, CH2CH2CH2O), 1.26 (m, 16H,
CH2), 0.88 (t, J ¼ 6.68 Hz, 3H, CH2CH3).
Synthesis of HAMAFA-b-DBAM-coated
HMS@C18@SPIONPs
Oleic acid-stabilized SIONPs (5 mg) and HMS (10 mg) were
dispersed in 750 ml tetrahydrofuran by sonication for 5 min to get
solution A, then HAMAFA-b-DBAM (20 mg) was dissolved in
tetrahydrofuran (750 ml) to get solution B. Solution B was
combined with solution A in an ultrasonic bath at room
temperature for 5 min. Then, 5 ml distilled water was added to
the above solution with vigorous shaking and the resulting
colloid was stirred vigorously for 24 h to evaporate the
tetrahydrofuran.
Synthesis of HAMA-b-DBAM
HAMA was prepared by RAFT polymerization of HEA and
APMA using CAD as a transfer agent according to the literature
with some modification.23 Typically, 5.3 mg (0.02 mmol) CAD
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In vitro experiments
was used as model anticancer agent owing to its fluorescence that
is convenient to be observed by confocal laser fluorescence
microscopy. KB cells were seeded in 96-well plates (1.3 ꢂ 104
In vitro drug-release. To evaluate the drug loading capacity
and release properties, doxorubicin (DOX) was used as the
model drug according to the literature with some modifica-
tions.9,32 DOX was extracted from doxorubicin hydrochloride
(DOX$HCl) according to the procedure reported previously.33
HMS@C18 (4, 2 and 0.4 mg mlꢁ1) were dispersed into 1 ml SBF
buffer solution, then DOX solution (5 mg mlꢁ1, 40 mL) was
added. After dispersion and stirring under light-sealed conditions
for 24 h, DOX@HMS@C18 were obtained by centrifugꢀation
and washed with 20 ml of PBS (pH 7.4), then dried at 60 C in
vacuum. HAMAFA-b-DBAM-coated DOX@HMS@C18@
SPIONPs was prepared via self-assembly as discussed before.
Then, 5 mg of sampleꢀwas immersed in simulated body fluid
(SBF) incubated at 37 C to maintain a constant temperature.
The obtained colloid was divided into two equivalent parts and
adjusted to different pH values by acetate buffer.
ꢀ
cells per well) and incubated overnight at 37 C in a humidified
incubator. The sample of HAMAFA-b-DBAM-coated
DOX@HMS@C18@SPIONPs was dispersed in RPMI-1640
medium. Cells were cultured with the HAMAFA-b-DBAM-
coated DOX@HMS@C18@SPIONPs solution for a certain
time and observed using confocal laser fluorescence microscopy
(Olympus, FV 1000) after washing three times with PBS.
MRI experiments
MRI experiments were carried out with a 0.5 tesla (T) super-
conducting unit (GE Vectra, International General Electric,
Slough, UK). HAMAFA-b-DBAM-coated HMS@C18@
SPIONPs was loaded into Eppendorf tubes for imaging. Multi-
section T2-weighted fast spin-echo sequences were performed in
at least two orthogonal planes to obtain MR phantom images.
MRI signal intensity was calculated using the in-built software.
Here, the DOX concentration was determined using a fluo-
rescence spectrophotometer at lex ¼ 480 nm and lem ¼ 627 nm. A
standard plot was prepared under identical conditions to confirm
the amount of drug loaded by HMS@C18. Drug loading content
and drug loading efficiency can be calculated as follows:
Characterization
FT-IR measurements were performed as KBr pellets on a Nicolet
4700 spectrometer (Thermo Fisher Scientific) in the range of 400–
4000 cmꢁ1. 1H NMR spectra were measured by an INOVA 400
MHz NMR instrument. TEM images were obtained using a
TecnaiG220 electron microscope at an acceleration voltage of
200 kV. Energy dispersive spectrometry (EDS) was taken on a
Hitachi S-4700 equipped with an energy-dispersive X-ray spec-
trometer. Brunauer–Emmett–Teller (BET) and Barrett–Joyner–
Halenda (BJH) analyses were used to determine the surface area,
pore size and pore volume and measurements were obtained with
a Quantachrome Autosorb 1C apparatus at ꢁ196 ꢀC under
continuous adsorption conditions. Confocal laser scanning
microscopy (CLSM) images were observed by a confocal laser
scanning microscope (Olympus, FV 1000).
Drug loading content (wt%) ¼ (weight of loaded drug/weight of
nanocomposites) ꢂ 100%;
Drug loading efficiency (%) ¼ (weight of loaded drug/weight of
drug in feed) ꢂ 100%.
Cell culture and preparation. A549 human alveolar adenocar-
cinoma (FRꢁ) and human KB (FR+) cell lines (purchased from
Shanghai Cell Institute Country Cell Bank, China) were cultured
as monolayers in RPMI-1640 medium supplemented with 10%
heat-inactivated fetal bovine serum at 37 ꢀC in a humidified
incubator (5% CO2 in air, v/v).
In vitro cytotoxicity. The aminoxanthene dye, sulforhodamine
B (SRB), was used as an assay for assessing the effects of drug
carriers in various concentrations.34 In brief, well-growing KB
cells were placed in 96-well plates (1.3 ꢂ 104 cells per well) and
four duplicate wells were set up in the sample. The culture
medium was replaced with a medium containing different
concentrations of drug carriers and cultured at 37 ꢀC in a
humidified incubator (5% CO2 in air, v/v) with the cells anchored
to the plates. After being cultured for 24 h, the medium was
poured away and 10% (w/v) trichloroacetic acid in Hank’s
balanced salt solution (100 ml) was added and stored at 4 ꢀC for
1 h. Then the stationary liquid was discarded, the cells were
washed with deionized water five times before air drying and
stained with 0.4% (w/v) SRB solution (100 ml per well) for 30 min
at room temperature. After removing the SRB, the cells were
washed with 0.1% acetic acid solution five times. Bound SRB dye
was solubilized with 10 mmol lꢁ1 Tris-base solution (150 ml, pH
¼ 10.5). The optical density (OD) value of each individual well
was calculated using a spectrophotometer at 531 nm absorbance.
Results and discussion
Synthesis of HAMAFA-b-DBAM and DOX@HMS@C18
The amphiphilic copolymer used here was synthesized as in a
1
previous report,23 which is shown in Scheme 2. The H NMR
spectrum of HAMAFA-b-DBAM (not listed here, but the
characteristic data are listed above in the experimental section)
showed the successful conjugation of FA. When specifically
recognized and internalized by tumor cells, the pH-responsive
polymer would hydrolyze into a biocompatible polymer due to
cleavage of acetal moieties in the weakly acidic endosomal/
lysosomal compartments, the hydrophobic segments hydrolyzed
into hydrophilic segments, resulting in encapsulated drug release
from unblocked pores of HMS.
The surface of HMS has many Si–OH groups,2,3,35 which could
be simply modified with C18. As can be seen, there was no
change in the diameter of particles between HMS and
HMS@C18. N2 adsorption–desorption isotherms and the cor-
responding pore size distributions of HMS, HMS@C18 are
shown in Fig. S1.†
Cellular
uptake
of
HAMAFA-b-DBAM-coated
DOX@HMS@C18@SPIONPs. Herein, doxorubicin (DOX)
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Scheme 2 Schematic illustration of the synthesis of HAMAFA-b-
DBAM.
The FT-IR spectra of HMS, HMS@C18 and
DOX@HMS@C18 are shown in Fig. 1. The Si–OH bond (950
cmꢁ1) in the spectrum of HMS suggested that a large number of
silanol groups exist on the surface of HMS for coupling alkyl
chains. As shown in the spectrum of HMS@C18, a strong bond
of C–Hx (2890 cmꢁ1) revealed the successful growth of alkyl
chains on the silica. For the DOX@HMS@C18, a new bond of
C]O (1740 cmꢁ1) appeared according to the spectrum of
HMS@C18, implying that the DOX molecules were loaded into
the nanoparticles successfully.
Assembly of HAMAFA-b-DBAM-coated
HMS@C18@SPIONPs
Fig. 2 TEM images of HMS (a), HMS@C18 (b), oleic acid-capped
SPIONPs (c), HAMAFA-b-DBAM-coated HMS@C18@SPIONPs
(d and e), EDS spectrum of HAMAFA-b-DBAM-coated HMS@
C18@SPIONPs (f) and DLS of HMS and HAMAFA-b-DBAM-coated
HMS@C18@SPIONPs (g).
The surface of the HMS shell was first modified with C18 to
change the wettability of the particle, and TEM images of HMS
(Fig. 2a) and HMS@C18 (Fig. 2b) did not show any difference.
Then, the nanocomposite was prepared through a self-assembly
process. First, SPIONPs and HMS@C18 were dispersed in
tetrahydrofuran by sonication to get solution A, the oleic acid-
capped SPIONPs could be absorbed on the surface of
HMS@C18 via hydrophobic interactions. HAMAFA-b-DBAM
was dissolved in tetrahydrofuran (solution B) and the solution
was immersed into solution A slowly. Then, 5 ml of distilled
water was added to the above solution, and THF was evaporated
under continued shaking, the amphiphilic polymer would form
micelles to encapsulate the hydrophobic guest molecules. The
obtained HAMAFA-b-DBAM-coated HMS@C18@SPIONPs
remained hydrophilic because of the hydrophilic segment of
HAMAFA-b-DBAM. The detailed morphological and struc-
tural features of the prepared nanoparticles were examined by
TEM. Comparing with HMS@C18 and SPIONPs (Fig. 2c), we
can clearly see that SPIONPs stabilized by oleic acid were fixed
between the hydrophobic part of the polymer and the outer
surface of HMS@C18. The prepared HAMAFA-b-DBAM-
coated HMS@C18@SPIONPs (Fig. 2d and e) have an average
diameter of about 200 nm and maintained good monodipersity
and the DLS shows nearly the same result (Fig. 2g). The EDS
spectrum (Fig. 2f) of HAMAFA-b-DBAM-coated HMS@
C18@SPIONPs was used to further confirm the presence of
silicon (HMS) and iron (SPIONPs) (10%). Thus, the
Fig. 1 FT-IR spectra of HMS, HMS@C18 and DOX@HMS@C18.
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nanoparticles formed by HMS, SPIONPs and HAMAFA-b-
DBAM were successfully synthesized.
Drug loading and release
To discuss the drug loading and release behaviours of the
HAMAFA-b-DBAM-coated HMS@C18@SPIONPs, DOX was
used as the model anticancer agent. The drug loading content
was determined by measuring drug concentrations before and
after being loaded by HMS@C18. The theoretical drug loading
contents were set at 5%, 10% and 50%. DOX loading efficiency
was measured at 82, 81 and 77% (Table 1). Then HAMAFA-b-
DBAM-coated DOX@HMS@C18@SPIONPs were prepared
through a self-assembly process at a low drug loading content
(ca. 5 wt%),36 and by evaluating the DOX concentration after the
centrifugation of nanocomposites, 5.6% of the DOX loaded in
the HMS was lost in this process. However, the drug loading
content presented that HMS was much more suitable for drug
delivery than common mesoporous silica owing to its hollow
core. Furthermore, the hydrophobic surface of HMS modified by
long alkyl chains made the insoluble drug molecules more easily
loaded.
Fig. 3 Release of DOX in vitro from drug-loaded HAMAFA-b-DBAM-
coated DOX@HMS@C18@SPIONPs at different pH at 37 ꢀC.
These characteristics are necessary for further biomedical
application.
Since DOX is a fluorophore, the fluorescence intensity could
be used to evaluate the intracellular activity of the obtained
nanocomposites in folate receptor positive (KB cells) and nega-
tive (A549) cancer cell lines. Herein, in vitro experiments based
on these two cells were used to demonstrate the selective release
and targeting properties of the nanocomposites. KB and A549
cells’ uptake and the intracellular distribution of the nano-
composites with FA groups were studied by CLSM. And after
being incubated with nanocomposites for 0.5 h and 2 h, the DOX
distributions are shown in Fig. 5.
Herein, we used SBF with different pH values (pH ¼ 5.0 and
7.4) at 37 ꢀC to simulate the tumor and normal cells and the
in vitro drug release behaviour of HAMAFA-b-DBAM-coated
DOX@HMS@C18@SPIONPs is shown in Fig. 3. About 70% of
DOX was released from the HMS at pH 5.0 while only less than
5% of DOX was released in a neutral environment after 150 h.
This phenomenon strongly demonstrated that the polymer could
degrade in an acidic environment due to its pH-sensitive groups
and then turned hydrophilic to allow drug release from the
unblocked pores of HMS.37 However, the SPIONPs attached to
silica did not affect the DOX release a lot. Hence, the highly
pH-sensitive polymer used here was able to prevent the drug
release in a normal physiological environment, which was
desirable for selective drug release. Besides, the long alkyl chains
could delay the drug release to about 150 h due to the hydro-
phobic interaction between drug and long alkyl chains, so that a
long-term drug release was obtained, which is important for
further cancer treatment.
To quantify the cellular uptake of nanocarriers for the two
different cell lines, the mean fluorescence intensity of FR+ and
FRꢁ cells with HAMAFA-b-DBAM-coated DOX@HMS@
C18@SPIONPs in solution at different incubation times are
shown in Fig. S2.† No significant difference could be observed in
the fluorescence intensity between the two nanocomposites when
Evaluation of cytotoxicity and drug uptake
The sulforhodamine B (SRB) assay was used to assess the
cytotoxicity of the nanocomposite. After incubation in the
nanocomposite for 72 h, the cells displayed high cell viability
(>80%) as shown in Fig. 4. Even at a high concentration, the
cytotoxicity still remained at a low level. The result above
proved the polymer shell to be biocompatible and nontoxic.
Fig. 4 In vitro cell viability of DOX-free HAMAFA-b-DBAM-coated
HMS@C18@SPIONPs at different concentrations.
Table 1 Drug loading content and drug loading efficiency
Theoretical drug loading
content (wt%)
Drug loading efficiency
(%)
Drug loading contenta (wt%)
5
10
50
4.1
8.1
38.5
82
81
77
a
Drug loading content for DOX was determined by fluorescence measurements.
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J. Mater. Chem., 2012, 22, 25354–25361 | 25359

View Article Online
5.2 and 165 mMꢁ1 sꢁ1 for r1 and r2, respectively. In our study, the
relaxivity of r1/r2 is calculated to be 32, which is much larger than
that of commercial dextran-coated SPIONPs.43 To study the
intracellular accumulation of SPIONPs, KB cells were incubated
with nanocomposites for 0.5 h, and the Prussian Blue-staining
results are shown in Fig. S4.† Good magnetic responsivity and
effective imaging ability of HAMAFA-b-DBAM-coated
HMS@C18@SPIONPS revealed their potential applications for
targeting and separation as a drug carrier.
Conclusion
In summary, we have designed a multifunctional nanocomposite
for anti-cancer drug delivery via a simple self-assembly strategy.
In this strategy, the oleic acid-capped SPIONPs were used
without any modifications, the HMS were modified with long
alkyl chains and turned hydrophobic. And then a FA-conjugated
pH-sensitive amphiphilic polymer was introduced to cap the two
inorganic nanoparticles via a simple self-assembly process. The
as-prepared nanocomposite could not only act as an efficiency
drug carrier due to its high drug loading content, pH-dependent
degradation and cancer targeting group, but could also be used
to track the cancer targeting process due to its magnetism. Most
importantly, this reported facile strategy could be used to
combine other inorganic nanoparticles with silicon for further
applications.
Fig. 5 CLSM of FRꢁ and FR+ incubated with HAMAFA-b-DBAM-
coated DOX@HMS@C18@SPIONPs for 0.5 h and 2 h.
incubated for 0.5 h. But after 2 h, a significant increase in fluo-
rescence intensity can be seen in KB cells incubated with FA-
conjugated nanocomposites. This result demonstrated that in a
short time the nanocomposites were internalized by the KB and
A549 cells through a same endocytosis process. 2 h later, the
changes in fluorescence intensity demonstrated that the targeting
moiety offered by folic acid is efficient at enhancing the tumor
cell targeting in vitro because the FA-conjugated nanocomposite
was taken up by KB cells through a folate receptor-mediated
endocytosis.38–41
Acknowledgements
We acknowledge that funding for this work came from National
Natural Science Foundation of China (20902065, 21076134),
Supporting Project for Science and Technology of Jiangsu
Province (Industry) (BE2011074), Natural Science Foundation
of Jiangsu Province (BK2012625), a Project Funded by the
Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD) and Innovative Research Team
of Advanced Chemical and Biological Materials, Suzhou
University.
Magnetic properties
The as-fabricated nanocomposites have a strong magnetism due
to the Fe3O4 nanoparticles capped by the polymer. HAMAFA-b-
DBAM-coated HMS@C18@SPIONPs had homogeneous
dispersions in water and a serum-containing medium (Fig. S3†),
and showed a sensitive response to an external magnetic field: a
visible separation could be seen just about 5 min later (Fig. 6c).
Furthermore, to evaluate the detectability of the nanocomposite,
the magnetic resonance phantom imaging method was used. The
T2-weighted phantom images of the nanocomposites at different
concentrations are shown in Fig. 6a. Even at a low concentra-
tion, the prepared nanocarriers still have a strong signal inten-
sity.42 Fig. 6b shows the relaxation rate. The specific relaxivity is
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