Water-soluble dendritic-linear triblock copolymer-modified magnetic nanoparticles: Preparation, characterization and drug release properties

Article abstract of DOI:10.1039/c1jm11613d

One route has been employed to prepare dendritic-linear block copolymer modified superparamagnetic iron oxide nanoparticles (SPIONs), which consist of a Fe₃O₄ magnetic nanoparticle core and a dendritic-linear block copolymer, the focal point polyamidoamine-type dendron-b-poly(2- dimethylaminoethyl methacrylate)-b-poly(N-isopropylacrylamide) (PAMAM-b-PDMAEMA-b-PNIPAM) shell by two-step atom transfer radical polymerization (ATRP). Firstly, Fe₃O₄ nanoparticles were prepared by a high-temperature solution phase reaction in the presence of iron(iii) acetylacetonate [Fe(acac)₃], oleic acid and oleylamine. Then propargyl focal point PAMAM-type dendron (generation 2.0, denoted as propargyl-D_2.0) with four carboxyl acid end groups as a cap displaced the oleic acid and oleylamine on the surfaces. Subsequently, an initiator for ATRP was introduced onto the propargyl-D_2.0-modified Fe ₃O₄ nanoparticle surfaces via click chemistry with 2′-azidoethyl-2-bromoisobutylate (AEBIB). PDMAEMA and PNIPAM were grown gradually from nanoparticle surfaces using two-step copper-mediated ATRP. Finally, a crosslinking reaction between PDMAEMA block with 1,2-bis(2- iodoethoxy)ethane (BIEE) was used to stabilize the nanoparticles and reverse aggregation. The modified nanoparticles were subjected to detailed characterization using FT-IR, DLS, XRD and TGA. Magnetization measurements confirmed the characteristic superparamagnetic behavior of all magnetic nanoparticles under room temperature. In addition, doxorubicin (DOX) as an anticancer drug model was loaded into the dendritic-linear block copolymer shell of the modified nanoparticles, and subsequently the drug release was performed in phosphoric acid buffer solution (pH 7.4) at 25 °C or 37 °C. The results verify that dendritic-linear block copolymer-modified nanoparticles as a drug carrier possess thermosensitive drug release behaviors. Furthermore, a methyl tetrazolium (MTT) assay of DOX-loaded dendritic-linear block copolymer-modified nanoparticles against Hela cells was evaluated. The results show that the modified nanoparticles can be used for drug delivery.

Full text of DOI:10.1039/c1jm11613d

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Materials Chemistry
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PAPER
Water-soluble dendritic-linear triblock copolymer-modified magnetic
nanoparticles: preparation, characterization and drug release properties†
a
Xiaomeng Wu, Xiaohua He,
ab
a
b
c
c
Liang Zhong, Shaoliang Lin, Dali Wang, Xinyuan Zhu and Deyue Yanac
*
*
Received 15th April 2011, Accepted 16th June 2011
DOI: 10.1039/c1jm11613d
One route has been employed to prepare dendritic-linear block copolymer modified superparamagnetic
iron oxide nanoparticles (SPIONs), which consist of a Fe3O4 magnetic nanoparticle core and
a dendritic-linear block copolymer, the focal point polyamidoamine-type dendron-b-poly(2-
dimethylaminoethyl methacrylate)-b-poly(N-isopropylacrylamide) (PAMAM-b-PDMAEMA-b-
PNIPAM) shell by two-step atom transfer radical polymerization (ATRP). Firstly, Fe3O4
nanoparticles were prepared by a high-temperature solution phase reaction in the presence of iron(III)
acetylacetonate [Fe(acac)3], oleic acid and oleylamine. Then propargyl focal point PAMAM-type
dendron (generation 2.0, denoted as propargyl-D2.0) with four carboxyl acid end groups as a cap
displaced the oleic acid and oleylamine on the surfaces. Subsequently, an initiator for ATRP was
introduced onto the propargyl-D2.0-modified Fe3O4 nanoparticle surfaces via click chemistry with 20-
azidoethyl-2-bromoisobutylate (AEBIB). PDMAEMA and PNIPAM were grown gradually from
nanoparticle surfaces using two-step copper-mediated ATRP. Finally, a crosslinking reaction between
PDMAEMA block with 1,2-bis(2-iodoethoxy)ethane (BIEE) was used to stabilize the nanoparticles
and reverse aggregation. The modified nanoparticles were subjected to detailed characterization using
FT-IR, DLS, XRD and TGA. Magnetization measurements confirmed the characteristic
superparamagnetic behavior of all magnetic nanoparticles under room temperature. In addition,
doxorubicin (DOX) as an anticancer drug model was loaded into the dendritic-linear block copolymer
shell of the modified nanoparticles, and subsequently the drug release was performed in phosphoric
acid buffer solution (pH 7.4) at 25 ꢀC or 37 ꢀC. The results verify that dendritic-linear block copolymer-
modified nanoparticles as a drug carrier possess thermosensitive drug release behaviors. Furthermore,
a methyl tetrazolium (MTT) assay of DOX-loaded dendritic-linear block copolymer-modified
nanoparticles against Hela cells was evaluated. The results show that the modified nanoparticles can be
used for drug delivery.
Introduction
Over the past decade, magnetic nanoparticles (MNPs) have been
intensively pursued not only for their fundamental scientific
interest but also for rich technological applications.1–8 Among
them, biological applications have developed dramatically for
magnetic resonance imaging (MRI),7–12 bacterial detection,9
hyperthermia,6,13 protein purification,9 cell separation6 and drug
delivery6,9,14 etc. Among the wide variety of magnetic nano-
particles, iron oxide nanoparticles (usually Fe3O4 and Fe2O3)
have certainly been and still are the most intensively investigated.
This is due to their remarkable magnetic properties and low
toxicity. To date, several synthetic methods have been applied to
prepare magnetic iron oxide nanoparticles, including co-precip-
itation of iron(II) and iron(III) ions in basic solution,15–18 an
emulsion method using pre-synthesized magnetite stabilized with
oleic acid,19 organic solution-phase decomposition of the iron
precursor at high temperature10,20–23 etc. In these methods, the
aDepartment of Chemistry, East China Normal University, 3663
Zhongshan North Road, Shanghai, 200062, China. E-mail: xhhe@chem.
ecnu.edu.cn
bThe Key Laboratory of Advanced Polymer Materials of Shanghai, School
of Materials Science and Engineering, East China University of Science
and Technology, Shanghai, 200237, China
cSchool of Chemistry & Chemical Engineering, Shanghai Jiao Tong
University, 800 Dongchuan Road, Shanghai, 200240, China. E-mail:
xyzhu@sjtu.edu.cn
† Electronic supplementary information (ESI) available: Experimental
procedures for the synthesis and characterization of 20-azidoethyl
2-bromoisobutylate (AEBIB) and propargyl focal point PAMAM-type
dendrons with four carboxyl acid end groups (generation 2.0, denoted
as propargyl-D2.0–COOH), the procedure for obtaining functional
block copolymers from the modified magnetic nanoparticles, the graft
density of the modified Fe3O4 nanoparticles and the stability of Fe3O4
nanoparticles. See DOI: 10.1039/c1jm11613d
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organic solution-phase decomposition of the iron precursor at
high temperature has been proved to be effective in the prepa-
ration of magnetic nanoparticles with good crystallinity, and
a relatively monodisperse and controlled size distribution.
In practical biological applications, magnetic iron oxide
nanoparticles are required to either disperse in water easily or be
superparamagnetic,1 which can be helpful to improve the
stability in the internal environment of the human body.
Generally, there are two kinds of strategies to enhance the
stability and biocompatibility of the magnetic iron oxide nano-
particles. The first strategy is to control the particle size to obtain
the superparamagnetism by using suitable synthetic methods,
such as organic solution-phase decomposition of the iron
precursor at high temperature.22 Superparamagnetic iron oxide
nanoparticles (SPIONs) do not retain any magnetism after
removal of the magnetic field and can prevent magnetic aggre-
gation after exposure to a magnetic field.24 The second one is to
modify the particle surface to enhance the biocompatibility and
stability. Up to now, some surface-modified methods have been
achieved with the help of monomeric stabilizers,25,26 inorganic
stabilizers27–29 or polymer stabilizers.10,11,15–18,20,21,30–39 It must be
pointed out that the latter not only provides stability to the
corresponding magnetic nanoparticles but also allows further
biological derivatization. Usually, two different approaches can
be adopted to modify nanoparticles with polymers: ‘‘grafting
onto’’ and ‘‘grafting from’’ the nanoparticle surface. The
‘‘grafting onto’’ method involves the as-synthesized polymers
being grafted onto the nanoparticle surface by electrostatic,
hydrophobic interactions or through the affinity of certain
chemical groups.17,33,38,40 However, grafting the as-synthesized
polymer chains onto a single small particle surface may be more
difficult, since polymer chains can bind more than a particle at
the same time, and then forming polymer-coated nanoparticle
clusters.33 On the contrary, the ‘‘grafting from’’ method,
a surface-initiated polymerization method, can be more suitable
to modify the small particle surfaces and prevent the occurrence
of nanoparticle clusters because the coating polymers are grown
directly from the nanoparticle surface. At present, many poly-
mer-coated magnetic iron oxide nanoparticles have been ach-
ieved by the ‘‘grafting from’’ method using different
polymerization processes, such as atom transfer radical poly-
merization (ATRP),6,20,19,31,34 ring-opening polymerization
(ROP),20,41 etc. Especially mentioned, ATRP is a controlled/
living polymerization process which can produce polymer chains
with controllable lengths and for which a broad variety of
monomers can be used.42 So, either the thickness of the polymer
shell or the properties of polymer-coated nanoparticles can be
easily tuned. Up to now, ATRP has been widely used for
nanoparticle surface-initiated modification.43
initiator was introduced onto the propargyl group by a click
reaction. Then, using two-step surface-initiated ATRP, poly(2-
dimethylaminoethyl methacrylate) (PDMAEMA) chains and
poly(N-isopropylacrylamide) (PNIPAM) chains were sequen-
tially introduced onto the magnetic nanoparticle surfaces by
means of a ‘‘grafting from’’ approach. PAMAM-b-PDMAEMA-
b-PNIPAM block copolymer-modified magnetic iron oxide
nanoparticles were successfully achieved. Moreover, cross-link-
ing reactions within the PDMAEMA chains were carried out to
further stabilize the magnetic iron oxide nanoparticles. Our
synthesis protocol possesses the following merits: (1) further
enhancing the stability of SPIONs. Dendritic polymers are
relatively novel polymers with many functional groups on the
surface, perfect chemical structures, internal cavities and nano-
size etc, which provide dendritic polymers with many potential
biomedical applications.44,45 The as-synthesized focal point
PAMAM-typed dendron bearing four carboxyl acid end groups
was chelated to SPION surfaces like a cap, which was beneficial
to further stabilize SPIONs. On the other hand, the stability of
polymer-modified nanoparticles can be also increased by cross-
linking.46 Cross-linking reactions within PDMAEMA chains can
be helpful to stabilize magnetic iron oxide nanoparticles. (2)
Offering more internal cavities due to PAMAM-type dendron
coating can improve the encapsulation efficiency of functional
molecules (such as drugs) into the polymer-modified shell layer
of SPIONs. (3) PNIPAM possesses temperature sensitivity and
biocompatibility, which has been widely applied in the biomed-
ical area.47 SPIONs coated with PNIPAM chains can not only
prevent the agglomeration but also increase biocompatibility. To
expand the biomedical applications of SPIONs, doxorubicin
(DOX), as an anticancer drug model, can be loaded into the as-
synthesized nanoparticle system and the drug release behaviors
at different temperatures were investigated. At the same time, the
cytotoxicity of the DOX-loaded nanoparticle system was also
evaluated. To the best of our knowledge, there have been few
reports concerning the synthesis of dendritic-linear block
copolymer-modified magnetic nanoparticles. The synthesis and
surface modification of SPIONs is illustrated schematically in
Scheme 1.
Experimental section
Materials
Iron(III) acetylacetonate (Fe(acac)3, 98%), oleic acid (OA, 90%)
and oleylamine (OAm, 80%) were purchased from Aladdin-
Reagent (Shanghai) Ltd. 2-Dimethylaminoethyl methacrylate
(DMAEMA, 99%), 1,1,4,7,7-pentamethyldiethylenetriamine
(PMDETA, 99%), 2,20-bipyridine (bpy, 99%), N-iso-
propylacrylamide (NIPAM, 99.8%) and 3-(4,5-dimethyl-thiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
from Aldrich. CuBr and CuCl (Shanghai Chemical Reagent Co.,
A.R. grade) were purified by stirring in glacial acetic acid over-
night, filtered, washed with ethanol and then dried in a vacuum
oven at 60 ꢀC overnight. Phenyl ether (99.9%), N,N-dime-
thylformamide (DMF, A.R. grade) and dimethyl sulfoxide
(DMSO) were purchased from Shanghai Chemical Reagent
Company and dried over calcium hydride (CaH2) and distilled
under reduced pressure. 1,2-bis(2-iodoethoxy)ethane (BIEE) was
In this report, dendritic-linear block copolymer-modified
magnetic iron oxide nanoparticles were designed and prepared
by the combination of the ‘‘grafting to’’ and ‘‘grafting from’’
approach. Firstly, magnetic iron oxide nanoparticles were
prepared by the method of the organic solution-phase decom-
position of the iron precursor at high temperature. Second, the
propargyl focal point polyamidoamine (PAMAM)-type den-
dron, bearing four carboxyl acid end groups, can cap on
magnetic iron oxide nanoparticle surfaces to stabilize the nano-
particles by means of a ‘‘grafting onto’’ approach. The surface-
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Scheme 1 The synthesis and surface modification of superparamagnetic Fe3O4 nanoparticles.
synthesized according to the published procedure.48 20-Azidoethyl
2-bromoisobutylate (AEBIB) and propargyl focal point
PAMAM-type dendrons with four carboxyl acid end groups
(generation 2.0, denoted as propargyl-D2.0–COOH) were synthe-
sized according to the ESI†. Tris(2-(dimethylamino)ethyl)amine
(Me6TREN) was synthesized according to the literature proce-
dures.49 Doxorubicin hydrochloride (DOX-HCl) was purchased
from Zhejiang Hisun Pharmaceutical Co., Ltd. Other chemical
reagents were purchased from Shanghai Chemical Reagent
Company and purified by conventional procedures if needed.
dichlorobenzene, DMF and methanol (volume ratio 2 : 2 : 1),
and sonicated for 10 min, to which the solution of 1.22 g of
propargyl-D2.0-COOH dissolved in dried methanol (10 mL) was
added. The mixture was then mechanically stirred at 65 ꢀC under
nitrogen for 48 h. After cooling to room temperature, an excess
of methanol was added to the mixture, and the black particles
were magnetically precipitated and subsequently separated by
centrifugation. The nanoparticles were washed with methanol to
remove propargyl-D2.0-COOH residues and dried in a vacuum
oven at room temperature.
Synthesis of oleic acid-coated magnetic iron oxide nanoparticles
(Fe3O4-ol)
Synthesis of magnetic nanoparticle macroinitiators
(Fe3O4-D2.0-Br)
Magnetic nanoparticles were prepared according to the similar
procedure reported by Lattuada et al.20 The procedure was as
follows: Fe(acac)3 (5.7 g, 16 mmol), OA (13.6 g, 48 mmol), OAm
(12.8 g, 48 mmol) and phenyl ether (120 mL) were mixed in a four-
neckedroundbottomflaskandstirred vigorouslyunder a constant
flow of nitrogen. The mixture was heated to 100 ꢀC for 40 min and
Fe3O4-D2.0-propargyl (0.15 g) was dispersed in DMF (8 mL), to
which AEBIB (0.4 g, 1.7 mmol) was added. The mixture was
purged with pure nitrogen for 30 min, CuBr (26.5 mg, 0.18
mmol) and PMDETA (38.0 mg, 0.18 mmol) were then added.
Under the protection of nitrogen, the mixture was mechanically
stirred for 24 h at 45 ꢀC. After cooling to room temperature, 30
mL of diethyl ether was added to the mixture, and the black
particles were magnetically precipitated and subsequently sepa-
rated by centrifugation. The nanoparticles were washed with
methanol to remove any residues and dried in a vacuum oven at
room temperature.
ꢀ
then heated to 200 C for 2 h under nitrogen. Sequentially, the
mixture was heated to reflux (ꢁ265 ꢀC) for another 1 h under
a blanket of nitrogen. The dark-brown mixture was cooled to
room temperature under nitrogen. Ethanol (ꢁ200 mL) was added
to the mixture and the black precipitates were collected via
centrifugation. The black precipitates were dissolved in hexane
and purified by centrifugation for removal of any residue. The
black products were dried in a vacuum oven at room temperature.
Synthesis of PDMAEMA-modified magnetic iron oxide
nanoparticles (Fe3O4-D2.0-b-PDMAEMA-Br)
Fe3O4-D2.0-Br (0.15 g), methanol (4 mL) and DMAEMA (3.0 g,
19.1 mmol) were charged into a reaction tube. After purging with
pure nitrogen for 30 min, CuBr (89.0 mg, 0.6 mmol) and bpy (100
mg, 0.6 mmol) were added into the mixture under the protection
of nitrogen. The mixture was stirred for 24 h at room
Synthesis of propargyl-D2.0-COOH-coated magnetic iron oxide
nanoparticles (Fe3O4-D2.0-propargyl)
Fe3O4-ol (0.8 g) prepared as described above was dispersed in 40
mL of
a mixture of anhydrous solvent containing 1,2-
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temperature under nitrogen atmosphere. Then, an excess of
diethyl ether was added to the mixture, and the black particles
were magnetically precipitated and subsequently separated by
centrifugation. The nanoparticles were purified from methanol
to diethyl ether three times and dried in a vacuum oven at room
temperature.
selected time intervals (5 h or 24 h) in the dark, the nanoparticles
were recovered by magnetic separation and centrifugation. The
supernatant was further diluted with methanol to constant
volume and then monitored by a UV-vis spectrometer at 485 nm
to determine the rate of drug release. The DOX concentration
was determined according to the standard curves of DOX solu-
tion. Cumulative release was expressed as the total percentage of
drug release and calculated from the following relationship:
Synthesis of PDMAEMA-b-PNIPAM block copolymer-modified
magnetic iron oxide nanoparticles (Fe3O4-D2.0-b-PDMAEMA-
b-PNIPAM)
Cumulative release (%) ¼ 100 ꢃ Wt/W
where Wt was the weight of drug released from block copolymer-
modified nanoparticles at time t and W was the total weight of
drug loaded into the block copolymer-modified nanoparticles.
Fe3O4-D2.0-b-PDMAEMA-Br (0.15 g), NIPAM (0.7 g, 6.2
mmol), Me6TREN (23 mg, 0.1 mmol) and 2-propanol (4 mL)
were charged into a reaction tube. After purging with pure
nitrogen for 30 min, CuCl (10 mg, 0.1 mmol) was introduced into
the mixture under the protection of nitrogen flow. After poly-
merization at room temperature under nitrogen for 24 h, the
mixture was exposed to air and 30 mL of diethyl ether was added.
The black particles were magnetically precipitated and subse-
quently separated by centrifugation. The nanoparticles were
purified from 2-propanol to diethyl ether three times and dried in
a vacuum oven at room temperature.
Cell culture and cell viability assay
Hela cells (human cervix carcinoma cell line) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplied with
10% FBS (fetal bovine serum) and antibiotics (50 units mLꢂ1
penicillin and 50 units mLꢂ1 streptomycin) at 37 ꢀC in a humid-
ified atmosphere containing 5% CO2 atmosphere.
Hela cells were chosen to assess the anticancer activity of the
released DOX from DOX-loaded iron oxide magnetic nano-
particles as described above. Briefly, a series of solutions of
DOX-loaded iron oxide magnetic nanoparticles were prepared in
DMEM media, in which the final concentration of DOX ranged
from 0.1 to 2 mg mLꢂ1. Hela cells with approximately 8000 cells
wellꢂ1 were seeded in 96-well plates in 200 mL medium for incu-
bating for 24 h at 37 ꢀC. Then, the culture medium was removed
and replaced with 200 mL of fresh medium containing serial
concentrations of DOX-loaded iron oxide magnetic nanopartices
to incubate the cells. After 24 h of incubation at 37 ꢀC, 20 mL of 5
mg mLꢂ1 MTT assays stock solution in PBS was added to each
well and the cells were incubated for another 4 h at 37 ꢀC. Then,
the medium containing unreacted dye was removed carefully and
200 mL DMSO per well was added to dissolve the obtained blue
formazan crystals. The absorbance was measured in a BioTek
Elx800 at a wavelength of 490 nm. To compare with the anti-
cancer activity of the released DOX from DOX-loaded iron
oxide magnetic nanoparticles, the same concentration of the free
DOX was also assayed.
Cross-linking of Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM
nanoparticles
Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM (0.15 g) prepared as
described above was dissolved in ethanol (4 mL) and BIEE (0.2 g,
0.54 mmol) was then introduced into the mixture. The [BIEE]/
[DMAEMA] molar ratio was fixed at 1 : 2, targeting a 100%
degree of cross-linking. After stirring at room temperature under
nitrogen for 48 h, the black particles were magnetically precipi-
tated and subsequently separated by centrifugation. The black
nanoparticles were further washed using diethyl ether for removal
of any residues and dried in a vacuum oven at room temperature.
Loading block copolymer-modified nanoparticles with DOX and
in vitro drug release
DOX-HCl (3.0 mg) was dissolved in methanol (6 mL), and trie-
thylamine (0.05 mL) was then added into the solution to remove
hydrochloride. The DOX solution was added dropwise with stir-
ring to 3 mL of Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM nano-
particles (or Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM cross-linked
nanoparticles) in methanol (concentration of 2.5 mg mLꢂ1). The
mixture was shaken for 24 h in the dark at room temperature to
allow the drug partition into the polymer shell. The black particles
were magnetically precipitated and subsequently separated by
centrifugation. The supernatant was also collected. The drug-
loaded nanoparticles collected were further washed three times
using methanol to remove completely the free DOX. All super-
natant were gathered together to obtain the amount of free DOX.
The drug loading efficiency was calculated as follows:
Characterization
Fourier transform infrared (FT-IR) spectra were recorded on
a Perkin-Elmer Spectrum One spectrometer at frequencies
ranging from 400 to 4000 cmꢂ1. Samples were thoroughly mixed
with KBr and pressed into pellet form.
Transmission electron microscopy (TEM) analysis of iron
oxide magnetic nanoparticles was performed on a JEOL 2100CX
(200 kV) microscope. Drops of the solution of iron oxide
magnetic nanoparticles were deposited and dried at room
temperature on a carbon-coated copper grid.
Dynamic light scattering (DLS) measurements were per-
formed with a Malvern Instruments particle sizer (HPPS-ET
5002, Malvern Instruments, UK) equipped with a He-Ne laser
(l ¼ 632.8 nm). Scattered light was collected at a fixed angle of
173ꢀ for the duration of 5 min. All data were averaged over three
measurements.
Loading efficiency (%) ¼ 100 ꢃ ((initial amount of DOX –
amount of free DOX)/initial amount of DOX)
The drug-loaded nanoparticles quantified were immersed into
the quantitative volume of phosphate-buffered solution (PBS)
(0.01 M) with pH ¼ 7.4 at 25 ꢀC or 37 ꢀC. After shaking for the
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Wide-angle X-ray diffraction (XRD) patterns of powder
samples were obtained at room temperature by a Cu Ka radia-
ꢀ
tion (l ¼ 1.788965 A) and using a D8 Advance X-ray diffrac-
tometer (Bruker, Germany). The supplied voltage and current
were set to 40 kV and 120 mA. Samples were exposed at a scan-
ning rate of 2q ¼ 4ꢀ minꢂ1 with 2q values from 1.5ꢀ to 60ꢀ.
Thermogravimetric analysis (TGA) measurements were per-
formed on a TGA/SDTA851e/SF/100 instrument. All samples
were dried in a vacuum oven at 60 ꢀC prior to each TGA
measurement to remove most of the water or volatile solvent.
Samples weighing between 5 and 10 mg were heated from 20 to
850 ꢀC at a heating rate of 10 C minꢂ1 in nitrogen.
ꢀ
Magnetic studies were carried out with a MMVFTB Quantum
Design SQUID magnetometer (Petersen Instruments Corpora-
tion) with fields up to 10 KOe at room temperature.
The UV-vis spectra and transmittance were recorded on an
ultraviolet–visible spectrometer (UV/vis, Unico UV2102). The
transmittance of the solution was measured at a wavelength of
485 nm using a thermostatically controlled cuvette.
Results and discussion
Preparation of polymer-modified superparamagnetic iron oxide
nanoparticles and their characterization
Magnetic nanoparticles prepared by different synthetic methods
may possess different properties, such as particle size, shape,
magnetic properties, and so on. Sun et al.50 proposed a high-
temperature organic phase decomposition of an iron precursor
method to synthesize iron oxide magnetic nanoparticles, which
possess good crystallinity, and a relatively monodisperse and
size-controlled distribution. Lattuada et al.20 exploited the
synthetic method to prepare OA/OAm-coated iron oxide
magnetic nanoparticles using 1,2-tetradecane diol instead of the
much more expensive 1,2-hexadecane diol. In the procedure, the
heating stage was adjusted to control the average size and size
distribution of nanoparticles. In our work, a similar procedure
was adopted to prepare iron oxide magnetic nanoparticles, in the
absence of expensive 1,2-hexadecane diol and 1,2-tetradecane
diol, and the reaction mixture was first heated from room
Fig. 1 TEM image (A) and high resolution TEM image (B) of Fe3O4-ol
nanoparticles.
and polydispersity indexes (PDI) of 0.116 (Fig. 3a). The differ-
ence from the particle sizes confirmed by TEM and DLS
measurements was owing to the thicknesses of the OA/OAm
layers on the nanoparticles surfaces.20 The OA/OAm layers on
the Fe3O4 nanoparticles can be proved by FT-IR measurement.
The characteristic absorption bands at 2927 and 2856 cmꢂ1 for
methine groups originating from OA/OAm molecules, and the
symmetric COOꢂ stretch peak at 1406 cmꢂ1 can be observed in
the FT-IR spectrum (Fig. 4a) of the synthetic Fe3O4 nano-
particle.21 On the other hand, the C]O absorption peak at 1700
cmꢂ1 of the oleic acid cannot be observed in the FT-IR spectrum
of the freshly prepared Fe3O4 nanoparticles. The bonding of the
temperature to 100 ꢀC at the slow heating rate of 2 ꢀC minꢂ1
.
TEM and X-ray diffraction measurements were employed to
characterize the structures of the synthetic iron oxide nano-
particles. TEM images of these nanoparticles are shown in Fig. 1.
It can be seen from Fig. 1A that nanoparticles are roughly
spherical or ellipsoidal shapes with a particle size of about 10 nm.
Fig. 1B is the high-resolution TEM image, in which the lattice
corresponds to a group of atomic plane fringes, indicating that
the synthetic nanoparticles possess a good crystalline structure.
Fig. 2A shows the XRD pattern of the synthetic iron oxide
nanoparticles, in which six characteristic diffraction peaks for
Fe3O4 (2q ¼ 30.2ꢀ, 35.5ꢀ, 43.6ꢀ, 53.6ꢀ, 57.3ꢀ, 63.1ꢀ),17,50 marked
by their indices ((220), (311), (400), (422), (511), (440)) can be
observed. These results reveal that Fe3O4 nanoparticles can be
prepared using the modified synthetic method. The sizes and size
distributions of the freshly prepared Fe3O4 nanoparticles, Fe3O4-
ol, can be also obtained by DLS measurement. DLS measure-
ment of the freshly prepared Fe3O4 nanoparticles diluted in
hexane provided an average hydrodynamic diameter of ꢁ17 nm
Fig.
2 XRD patterns of (A) Fe3O4-ol and (B) Fe3O4-D2.0-b-
PDMAEMA-b-PNIPAM cross-linked.
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Fig. 3 DLS curves of (a) Fe3O4-ol in hexane, (b) Fe3O4-D2.0-b-
PDMAEMA-Br in water and (c) cross-linking Fe3O4-D2.0-b-
PDMAEMA-b-PNIPAM in water.
Fig. 5 TGA curves of (a) Fe3O4-ol, (b) Fe3O4-D2.0-propargyl, (c) Fe3O4-
D2.0-b-PDMAEMA-Br, (d) Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM and
(e) Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM cross-linked.
1622 and 1550 cmꢂ1 attributed to the acid amides stretch (d(N–H)
)
of propargyl-D2.0 appear in the FT-IR spectrum of propargyl-
2.0-modified Fe3O4 nanoparticles, Fe3O4-D2.0-propargyl, as
D
shown in Fig. 4b. The TGA profile of Fe3O4-D2.0-propargyl
shows a weight loss of about 10% over the entire temperature
range and a slow rate of weight loss (Fig. 5b) between 300 ꢀC and
650 ꢀC compared with that of Fe3O4-ol. Moreover, no weight
losses are observed above 650 ꢀC, which is obviously different
from the TGA profile of Fe3O4-ol.
On the other hand, the functional propargyl groups were also
introduced onto the nanoparticle surfaces because of coating
propargyl-D2.0–COOH dendrons, which was helpful to further
modify the nanoparticles. To date, different controlled radical
polymerization techniques have been utilized to modify the
nanoparticle surfaces. Among these techniques, atom transfer
radical polymerization (ATRP) has been most extensively used
to modify the nanoparticles.17,39,43,51–53 Herein, the surface-initi-
ated ATRP macroinitiator was prepared by Fe3O4-D2.0-prop-
argyl reacted with AEBIB by click chemisty. The sequential two
steps of copper-mediated ATRP were used to polymerize
monomers DMAEMA and NIPAM to the nanoparticle surfaces
(see Scheme 1). After the first step of copper-mediated ATRP,
Fig. 4 FT-IR spectra of (a) Fe3O4-ol, (b) Fe3O4-D2.0-propargyl, (c)
Fe3O4-D2.0-b-PDMAEMA-Br and (d) Fe3O4-D2.0-b-PDMAEMA-b-
PNIPAM.
OA/OAm to Fe3O4 nanoparticle surfaces can certainly be
claimed. Themogravimetry (TG) measurements were also carried
out to verify the bonding of the OA/OAm to Fe3O4 nanoparticle
surfaces. As shown in Fig. 5a, the absolute mass loss of freshly
prepared Fe3O4 nanoparticles, Fe3O4-ol, is about 21% over the
D
2.0-b-PDMAEMA-modified Fe3O4 nanoparticles (Fe3O4-D2.0-
b-PDMAEMA-Br) were obtained. From the FT-IR spectrum
(Fig. 4c) of the purified Fe3O4-D2.0-b-PDMAEMA-Br, an
intense adsorption peak at 1728 cmꢂ1 corresponding to the ester
group stretch is clearly observed. The absorption band provides
strong evidence that PDMAEMA has been grown from the
nanoparticle surfaces via ATRP. At the same time, it demon-
strated that the macroinitiator was successfully prepared by click
chemistry. Compared with that of Fe3O4-D2.0-propargyl
(Fig. 5a), the TGA curve of PDMAEMA-modified nanoparticles
(Fig. 5c) shows obviously a two-stage weight loss upon the
heating process in nitrogen. A weight loss of about 14% is
completed at 350 ꢀC at the first stage, which is close to the weight
ꢀ
temperature range from room temperature to 850 C.
The freshly prepared Fe3O4 nanoparticles can stably disperse
in organic solvents such as toluene and hexane (see ESI,
Figure S1:A†).20,50 To obtain water-soluble magnetic nano-
particles and improve their stability, hydrophilic polymers are
introduced onto the magnetic nanoparticle surfaces by ‘‘grafting
onto’’ and ‘‘grafting from’’ methods.1,3 In this contribution, the
propargyl-D2.0–COOH dendron carrying four carboxyl groups
was firstly chosen as a ligand to displace oleic acid from the
Fe3O4 nanoparticle surfaces by the ‘‘grafting onto’’ method. It
should be noted that the molecules bearing multiple carboxyl
groups possess stronger binding capabilities and can be adsorbed
more efficiently onto the iron oxide nanoparticle surfaces.20 After
modifying the OA/OAm-coated nanoparticles with propargyl-
loss of Fe3O4-D2.0-propargyl and may be assigned to the D2.0
-
propargyl dendron degradation. The second stage accounts for
about another 22% of the weight loss at the range of 380–850 ꢀC,
indicating the contribution of PDMAEMA. The DLS measure-
ment of freshly PDMAEMA-modified nanoparticles diluted in
D
2.0–COOH dendron, the characteristic absorption bands at
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water provided an average hydrodynamic diameter of particles in
solution equal to ꢁ23 nm (Fig. 3b). The number-average
molecular weight (Mn) and polydispersity index (PDI) of the
grafted copolymers, D2.0-b-PDMAEMA-Br, obtained from
Fe3O4-D2.0-b-PDMAEMA-Br nanoparticles through the
removal of Fe3O4 cores, were 4800 g molꢂ1 and 1.23 (see ESI,
Figure S2†), respectively. The relatively narrow PDI indicated
that the molecular weight of the grafted PDMAEMA on the
surfaces of magnetic nanoparticles can be controlled by the
linking reaction, a weight loss of about 52% can be observed in
the TGA curve over the entire temperature range (Fig. 5e) and
the behavior of thermal degradation is obviously different from
that of Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM. After the cross-
linking reaction, the magnetic nanoparticles can be stabilized in
water, forming a stable brown solution and no precipitation
occurs for 4 months (see ESI, Figure S1:C†). However, Fe3O4-
D
2.0-b-PDMAEMA-b-PNIPAM nanoparticles can be only
stabilized in water for 2 months (see ESI, Figure S1:B†). It is also
verified that the cross-linking reaction can be helpful to stabilize
magnetic iron oxide nanoparticles.46 DLS measurements of
freshly cross-linked block copolymer-modified nanoparticles
diluted in water provided an average hydrodynamic diameter of
particles in solution equal to ꢁ32 nm (Fig. 3c). Its XRD pattern
is shown in Fig. 2b. Compared with that of the OA/OAm-coated
nanoparticles, the relative intensity and positions of the peaks
were consistent. This revealed that the modification process did
not result in the phase change of Fe3O4.17
ATRP approach. The chemical structures of
PDMAEMA-Br were further characterized by 1H NMR (see
ESI†). The characteristic peaks of 2.0-b-PDMAEMA-Br
D2.0-b-
D
macromolecules such as those of methylene (–CH2–) originating
from PDMAEMA block and methylene (–CH2–) originating
from D2.0 block clearly appeared in the spectrum (see ESI,
Figure S3†) at 4.35 and 3.55–3.49 ppm, respectively.
The prepared Fe3O4-D2.0-b-PDMAEMA-Br was used as the
macroinitiator to initiate monomer NIPAM polymerization for
the preparation of D2.0-b-PDMAEMA-b-PNIPAM-modified
nanoparticles, Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM, in the
second step of copper-mediated ATRP. The peak at 1728 cmꢂ1
(Fig. 4d) corresponding to the ester group stretch becomes weak,
while the peaks at 1627 and 1556 cmꢂ1 attributed to the amido
bands of PNIPAM become stronger. As shown in Fig. 5d,
a three-stage weight loss upon heating process can be clearly
observed in the TGA curve of Fe3O4-D2.0-b-PDMAEMA-b-
PNIPAM. The weight losses of about 17%, 10%ꢀand 20%, which
The magnetization curves measured at room temperature for
the OA/OAm-coated Fe3O4 nanoparticles and the polymer-
modified Fe3O4 nanoparticles are shown in Fig. 6. For the OA/
OAm-coated Fe3O4 nanoparticles, after subtracting the organic
layer weights based on the TGA result, the normalized saturation
magnetization was 61.1 emu gꢂ1, which was lower than the bulk
magnetic value of 90 emu gꢂ1 54
. This reduction in magnetization
may be the result of noncollinear spins at the nanoparticle
surface. At the same time, the saturation magnetizations for
Fe3O4-D2.0-b-PDMAEMA, Fe3O4-D2.0-b-PDMAEMA-b-PNI-
PAM, and cross-linking Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM
nanoparticles were normalized to 61.0, 41.5 and 17.7 emu gꢂ1
based on subtracting the organic layer weights from the TGA
results, respectively. The saturation magnetizations decreased
with the increase of the organic layer thicknesses. On the other
hand, it should be noted that no hysteresis curves can be
observed in all samples, indicating the characteristic super-
paramagnetic behavior of all magnetic nanoparticles under room
temperature (Fig. 6). These magnetic properties of polymer-
modified nanoparticles also indicate that they are capable of
ꢀ
ꢀ
are sequentially completed at 235 C, 235–340 C, 340–850 C,
can be clearly observed in the first, second and third stage,
respectively. At the same time, it is estimated that the main
contribution to the weight losses in the first, second and third
stage should be originated from the degradation of the coated
D2.0 dendron, PDMAEMA and PNIPAM, respectively. After
removal of Fe3O4 cores, Mn and PDI of the grafted copolymers
D
2.0-b-PDMAEMA-b-PNIPAM were 7400 g molꢂ1 and 1.17 (see
ESI, Figure S2†), respectively. The increasing molecular weight
and relatively narrow PDI indicated that the grafting PNIPAM
process can be controlled by the ATRP approach. 1H NMR was
further used to characterize the chemical structures of D2.0-b-
PDMAEMA-b-PNIPAM (see ESI†). The inherent peaks of D2.0
-
b-PDMAEMA-b-PNIPAM macromolecules such as those of
methylene (–CH2–) assigned to PDMAEMA block, methylene
(–CH2–) originating from D2.0 block and methyl (CH3–) assigned
to PNIPAM block clearly appeared in the spectrum (see ESI,
Figure S3†) at 4.35, 3.55–3.49 and 1.05 ppm, respectively. The
obtained results also indicated that D2.0-b-PDMAEMA-b-PNI-
PAM-modified nanoparticles were successfully prepared. On the
other hand, according to the density of Fe3O4, the diameter of
the magnetic nanoparticles, and the weight losses and molecular
weight obtained from the Fe3O4-D2.0-b-PDMAEMA-Br and
Fe3O4-D2.0-b-PDMAEMA-b-PNIPAM nanoparticles, the graft
density can be approximately 0.56 and 0.50 chains nmꢂ2
,
respectively (see ESI†). After surface-grafting with D2.0-b-
PDMAEMA-b-PNIPAM, the magnetic nanoparticles can be
dispersed in many organic solvents as well as water, such as
ethanol, methanol, DMF and DMSO etc.
Fig. 6 Field dependent magnetization at room temperature for (a)
Fe3O4-ol, (b) Fe3O4-D2.0-b-PDMAEMA-Br, (c) Fe3O4-D2.0-b-
PDMAEMA-b-PNIPAM and (d) Fe3O4-D2.0-b-PDMAEMA-b-PNI-
PAM cross-linked.
To further improve the stability of the block copolymer-
modified nanoparticles, the cross-linking reaction between
PDMAEMA chains and BIEE was carried out. Due to the cross-
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responding to an external magnetic field and will show high
trapping efficiency for magnetic targeting. To the best of our
knowledge, it is first report for the synthesis of dendritic-linear
block copolymer-modified magnetic nanoparticles.
DOX for cross-linking block copolymer-modified nanoparticles
only increased from 26.8% to 41.1%. This indicates that the
cross-linking structures retard drug release.
To assess the drug delivery potential of the DOX-loaded
nanoparticles and the anticancer activity of the released DOX, in
vitro cytotoxicity assays were performed by MTT assay against
a Hela cell line at different administered concentration of DOX
ranging from 0.1 to 2 mg mLꢂ1. To compare the cytotoxic activity
of the DOX-loaded nanoparticle with that of the free drug, the
free DOX was used as a control. The Hela cells proliferation
results are shown in Fig. 8. It is found that the free DOX exhibits
higher inhibition on Hela cells in 24 h compared with the loaded
DOX at the same DOX concentration when it is more than about
0.6 mg mLꢂ1. The doses required for 50% cellular growth inhi-
bition (IC50) of the DOX loaded in the modified nanoparticles
In vitro thermosensitive drug release behaviors and anticancer
activity assay
SPIONs have been utilized as a carrier for targeted drug delivery
through the EPR effect55 or by applying an external magnetic
field.56 Drug molecules were either entrapped in the SPION
surface polymer layer through physical interactions (electro-
static, hydrophobic interaction, etc) or covalently conjugated to
the functional groups on the SPION surface. DOX, as an anti-
cancer drug model, has been extensively explored in the SPIONs
system.2,57–61 In the contribution, to evaluate the block copoly-
mer-modified Fe3O4 nanoparticles potential as a drug carrier,
DOX was entrapped into D2.0-b-PDMAEMA-b-PNIPAM-
modified nanoparticles and cross-linking D2.0-b-PDMAEMA-b-
PNIPAM-modified nanoparticles, and the DOX release test in
pH 7.4 PBS buffer solutions was performed at 25 ꢀC and 37 ꢀC.
After removal of the free DOX, the drug loading efficiency of the
block copolymer-modified nanoparticle was 22.7%. The DOX
cumulative release results are shown in Fig. 7. In buffer solution
at pH 7.4 in 5 h, the cumulative release amounts of DOX from
uncross-linking block copolymer-modified nanoparticles were
28.8% and 15.5% at 25 ꢀC and 37 ꢀC, respectively, and those from
cross-linking block copolymer-modified nanoparticles were
26.8% and 13.7% at 25 ꢀC and 37 ꢀC, respectively. This indicates
that both uncross-linking and cross-linking polymer modified
nanoparticles possess thermosensitive drug release behaviors,
with uncross-linking and cross-linking are 1.0 and 1.49 mg mLꢂ1
,
respectively, which are higher than the IC50 of the free DOX
(ꢁ0.66 mg mLꢂ1). The results are most likely related to the slow
release of the drug from the drug-loaded nanoparticles in the
course of incubation. In addition, the same antiproliferation
effect on Hela cells in 24 h requires higher concentration of DOX
in the cross-linking block copolymer-modified nanoparticles
than in the uncross-linking ones. This proves that the cross-
linking structures can delay drug release. A possible reason is
that more compact structures result in a lower diffusion rate of
drug molecules after cross-linking. At the same time, the results
verify that the encapsulation of DOX in the block copolymer-
modified nanoparticles retards the toxic effect of DOX on the
cells.57 As a drug delivery system, the DOX-loaded nanoparticles
are beneficial to decrease the side effects of DOX on cells.
ꢀ
and the cumulative release amount was higher at 25 C than at
Conclusions
37ꢀ C. It can be explained that PNIPAM block chains are in the
collapsed and hydrophobic conformation at 37 ꢀC above the
LCST,47 which can retard drug release. This phenomenon can
also be observed after 24 h (Fig. 7). On the other hand, the
cumulative release amounts of DOX at 25 ꢀC for uncross-linking
block copolymer-modified nanoparticles increased from 28.8%
to 55.0% with the change in cumulative release times from 5 h to
24 h. At the same conditions, the cumulative release amounts of
The modified high-temperature solution phase reaction has been
successfully used to prepare relatively monodisperse, super-
paramagnetic magnetite nanoparticles with a diameter size of
about 10 nm. The propargyl focal point PAMAM-type dendrons
with four carboxyl acid end groups as a cap displace the oleic
acid and oleylamine on the magnetite nanoparticle surfaces by
the ‘‘grafted onto’’ method. An initiator for ATRP was
Fig. 7 Drug release profiles from DOX-loaded block copolymer-modi-
Fig.
8 Activity of DOX-loaded block copolymer-modified Fe3O4
fied Fe3O4 nanoparticles.
nanoparticles against Hela cells.
13618 | J. Mater. Chem., 2011, 21, 13611–13620
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Acknowledgements
This work was financially supported by Shanghai Natural
Scientific Foundation of China (10ZR1409500). Support from
the Scientific Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry, and the financial
support of the Shanghai Key Laboratory of Advanced Polymer
Materials (ZD20100101), and Open Foundation of East China
Normal University (ECNU2001-49) are also appreciated.
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