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Engineering magnetic-molecular sequential targeting
nanoparticles for anti-cancer therapy†
Qi Zhang,
a
Jundong Zhu,
a
Lichao Song,
ab
Ju Zhang,
c
Deling Kong,
c
Yanjun Zhao*
a
and Zheng Wang*
a
Nanoparticle drug delivery to tumors via the enhanced permeability and retention (EPR) effect is usually
limited by the step of blood circulation and extravasation. Only less than 10% of the administered dose
would eventually reach the tumor tissue. To enhance the drug delivery efficiency, we report the
approach of magnetic plus molecular dual targeting nanoparticles to combine tumor targeting, drug
delivery, and in situ imaging together. The surface of superparamagnetic iron oxide nanoparticles
(SPIONs) was coated with biocompatible poly(ethylene glycol)–poly(lactic acid) and then anchored with
folic acid (FA). Despite the presence of FA, the hydrodynamic size of SPIONs was less than 100 nm.
Increasing the surface FA density sacrificed the aqueous stability of SPIONs, but 20% FA did not induce
noticeable particle aggregation. The existence of 20% FA maintained the superparamagnetic property
of SPIONs with a saturation magnetization level at ca. 30 emu g
1
. The drug release profile was not
significantly different between SPIONs with (20%) and without FA. However, the presence of FA
dramatically increased the intracellular uptake of SPIONs when using the MCF-7 breast cancer cell line.
These results highlighted the role of surface ligand optimization in the design of desired magnetic-
molecular dual tumor-targeting nanoparticles.
Introduction
Cancer remains one of the major threats to human health and
the leading cause of death worldwide. Chemotherapy together
with radiotherapy and surgery is currently the main choice of
tumor medication. Chemotherapy refers to the treatment of
tumors with one or more cytotoxic active agents, but it oen
suffers from off-target toxicity, drug resistance and clinical
relapse.
1
Nanotechnology has been popularly employed to
address these above issues via a diverse range of approaches.
First, the solubility and stability of the “difficult-to-deliver”
chemotherapeutics with extreme physicochemical properties
can be enhanced via nanoparticle encapsulation and/or conju-
gation.
2
Additionally, synergistic effects are likely via co-loading
multiple active agents with different mechanisms of action in a
single nanoparticle platform.
3
Moreover, drug delivery and
tumor imaging can be integrated in precisely engineered
multifunctional nanoparticles for improved clinical efficacy.
4
The pharmacokinetics and tissue distribution of the active drug
can also be modied by nanoparticles with preferred homing to
the tumor, whilst the accumulation in normal tissues and cells
is reduced to minimize toxicity.
5
Currently, the “passive targeting”of nanoparticles to tumors
is based on the “enhanced permeability and retention (EPR)
effect”, one common feature that distinguishes tumors from
healthy tissues.
6
The EPR effect is a consequence of the pres-
ence of leaky vasculature and poor lymphatic drainage in tumor
tissues. The surface modication of nanoparticles via hydro-
philic poly(ethylene glycol) coating (i.e. PEGylation) reduces the
uptake of nanoparticles by the reticuloendothelial system,
extends their systemic circulation and facilitates their parti-
tioning into tumors via the EPR effect. With the rapid progress
of particle engineering technology, “active targeting”mediated
by the interaction between the overexpressed receptors on the
tumor cell surface and the ligands attached to the exterior of
nanoparticles complements the EPR effect.
7
Commonly
employed targeting ligands include folic acid (FA), transferrin,
epidermal growth factor and various antibodies. Many types of
nanoparticle chemotherapy utilize a dual mode (active plus
passive) targeting strategy for improved therapeutic outcome.
8
Despite the large number of publications in this eld, to
date, the targeting efficiency of anti-cancer nanoparticles via
passive and/or active targeting has not been as good as expected
a
Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency, School of
Pharmaceutical Science & Technology, Tianjin University, 92 Weijin Road, Tianjin
300072, China. E-mail: zhaoyj@tju.edu.cn; wangzheng2006@tju.edu.cn; Fax: +86-
22-27404018; Tel: +86-22-27404018
b
Department of Pharmacy, Breast Cancer Prevention Key Laboratory of Ministry of
Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin
300060, China
c
Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering,
Chinese Academy of Medical Science, Tianjin 300192, China
†Electronic supplementary information (ESI) available: The synthetic routes of
MPEG-PLA-NHS, FITC-PEG-NHS, and Fe
3
O
4
-APS, and the aqueous dispersibility
of Fe
3
O
4
-Polym and Fe
3
O
4
-Polym-FA
20
. See DOI: 10.1039/c3tb20715c
Cite this: J. Mater. Chem. B, 2013, 1,
6402
Received 20th May 2013
Accepted 30th September 2013
DOI: 10.1039/c3tb20715c
www.rsc.org/MaterialsB
6402 |J. Mater. Chem. B, 2013, 1, 6402–6410 This journal is ªThe Royal Society of Chemistry 2013
Journal of
Materials Chemistry B
PAPER
and the majority (more than 90%) of the administered dose
ends up in healthy organs and tissues, causing signicant
toxicity.
9
The reason behind this unsatisfactory truth is that
targeted tumor drug delivery via the EPR effect is limited by the
process of blood circulation and extravasation of nanoparticles.
In fact, liver, spleen and bone marrow with a leaky endothelial
wall oen contribute to the substantial uptake of nanoparticles
during the course of blood circulation, resulting in compro-
mised tumor targeting.
5
Furthermore, for the active targeting,
no matter how affinitive the nanoparticle surface ligand is to the
tumor biomarker, the ligand–receptor interaction takes place
only upon the nanoparticles reaching the tumor sites.
10
As a
result, the passive and active dual targeting approach still
cannot fundamentally circumvent the pitfalls of the EPR effect,
which necessitates other efficient tumor targeting strategies.
Biodegradable iron oxide (Fe
3
O
4
) nanoparticles provide an
alternative versatile platform for tumor targeting.
11
Compared
to passive targeting, magnetic targeting utilizes external
magnetic eld to achieve guided drug delivery to the tumor,
making possible the improved clinical efficacy and reduced side
effects.
12
In addition, a targeting ligand can be anchored to the
surface of Fe
3
O
4
nanoparticles to combine the magnetic and
active targeting together.
13
It was reported that magnetic tar-
geting coupled with active targeting could deliver ca. 60% of
applied dose to the tumor sites, which brought about a dramatic
increase of therapeutic efficacy and reduction of harm to the
healthy tissues.
14
The core of such particles can be designed to
generate superparamagnetic iron oxide nanoparticles (SPIONs),
which enables the non-invasive real-time magnetic resonance
imaging (MRI) with SPIONs as the contrast agents.
Efficient tumor targeting and drug delivery via SPIONs
involves highly complex biological, mechanical, chemical and
transport phenomena, of which the characteristics differed
spatiotemporally.
9,15
This requires tailored design and surface
manipulation of SPIONs. For example, the iron core should be
small enough (ca. <25 nm), so it is feasible to overcome
the energy barrier of magnetic ipping that is proportional to
the particle volume.
16
Typically the SPIONs were generated by
two means. The rst method involves loading the iron core
within a polymer/lipid nanocarrier (i.e. hybrid nanoparticles)
that is oen surface-tailored with a targeting ligand.
17
The
second approach requires the surface engineering of the iron
core.
18
For the latter, the surface coating of SPIONs should be
created to prolong the half-life in blood circulation, facilitate
the conjugation of targeting ligands, and enable sufficient drug
loading.
12,13
The size of SPIONs should be precisely tuned to
avoid the rapid elimination by the clearance organs, aid tumor
accumulation and uptake, and achieve applicable magnetiza-
tion properties.
5,13
It is challenging to engineer desired SPIONs
with integrated capability of efficient dual tumor-targeting,
drug delivery and in situ imaging due to the interplay of these
design parameters and the complexity of tumors. Particularly,
the effect of the presence and density of surface targeting
ligands on the drug delivery performance of SPIONs was not
fully elucidated. The aims of this study were to generate
magnetic-molecular dual targeting surface-engineered SPIONs
and investigate the inuence of surface ligands on the
pharmaceutical properties of SPIONs in vitro. Amphiphilic
and biocompatible poly(ethylene glycol)–poly(lactic acid), i.e.
PEG–PLA was employed for surface coating of the Fe
3
O
4
core
and folic acid (FA) was used as the active targeting molecule
with Paclitaxel (PTX) as the model anti-tumor drug being
physically loaded.
Materials and methods
Materials
Methoxypoly(ethylene glycol) (i.e. MPEG, M
w
: 2000) and uo-
rescein isothiocyanate (FITC) were obtained from Sigma-Aldrich
(Beijing, China). D,L-Lactide was purchased from Yuan Shen-
grong (Beijing, China). Ferric chloride (FeCl
3
$6H
2
O), ferrous
chloride (FeCl
2
$4H
2
O), folic acid, oleic acid (OA), and succinic
anhydride were sourced from Guangfu Fine Chemical Research
Institute (Tianjin, China). Dicyclohexylcarbodiimide (DCC),
di-tert-butyl dicarbonate (Boc
2
O), N-hydroxysuccinimide (NHS),
stannous octanoate [Sn(Oct)
2
], triethylamine (TEA), triuoro-
acetic acid (TFA), 3-aminopropyltrimethoxysilane (APS), and
4-dimethylamino pyridine (DMAP) were provided by Aladdin
Reagent (Shanghai, China). Dulbecco's modied Eagle's medium
(DMEM), fetal bovine serum, and penicillin–streptomycin were
from HyClone Inc. (Logan City, Utah, USA). The breast cancer
cell line (MCF-7) was provided by Prof. Deling Kong from Nankai
University.
Synthesis and activation of MPEG-PLA
MPEG-PLA was obtained by the ring-opening polymerization of
D,L-lactide. The carboxyl-terminated copolymer (MPEG-PLA-
COOH) was prepared by reacting MPEG-PLA with succinic
anhydride in the presence of a DMAP catalyst (Scheme S1,
ESI†).
19
The reaction was carried out at ambient temperature
with the molar ratio of MPEG-PLA : succinic anhy-
dride : DMAP : TEA at 2 : 3 : 3 : 3. The product was precipitated
with cold diethyl ether and dried under vacuum. MPEG-PLA was
activated by the reaction between MPEG-PLA-COOH and NHS,
DCC, DMAP (2 : 3 : 3 : 3) in dichloromethane (DCM) for 24 h at
ambient temperature. The nal product (MPEG-PLA-NHS) was
puried by ltration, precipitation (with diethyl ether), and
then vacuum-dried.
Synthesis and activation of FA-PEG-PLA
The synthesis of FA-PEG-PLA involved ve major steps (Scheme
1). First, Boc
2
O (1.0 mmol) dissolved in dioxane was slowly
added to 10 mL ice-cold sodium bicarbonate aqueous solution
(0.2 M) containing NH
2
-PEG-OH (1.0 mmol). The reaction was
maintained at ambient temperature for 20 h followed by
adjusting the pH to 7.0 and solvent evaporation. Then the
residue was re-dissolved in DCM and the insoluble precipitate
was removed by ltration. Thereaer Boc-NH-PEG-OH was
obtained by precipitation using cold diethyl ether followed by
vacuum-drying. Second, Boc-NH-PEG-OH (1.0 mmol) as well as
D,L-lactide (1.5 mmol) was melted at 125 C under a nitrogen
atmosphere. The polymerization was maintained for 24 h at
125 C since the addition of Sn(Oct)
2
(1.5 mmol). Thereaer,
This journal is ªThe Royal Society of Chemistry 2013 J. Mater. Chem. B, 2013, 1, 6402–6410 | 6403
Paper Journal of Materials Chemistry B
Boc-PEG-PLA-OH was puried using a similar approach
described above. Third, Boc-NH-PEG-PLA-OH (1.0 mmol) and
TFA (2 mL) were dissolved in DCM (20 mL). The reaction lasted
for 1 h at ambient temperature followed by solvent removal by
evaporation; the residue was re-dissolved in DCM and extracted
with 0.05% (w/v) sodium bicarbonate aqueous solution. The
water in the organic phase was cleared by anhydrous sodium
sulphate; the precipitate was isolated by ltration and the
product (NH
2
-PEG-PLA-OH) was obtained by a repeated
precipitation procedure described above. Fourth, FA was acti-
vated to generate FA-NHS using a previously reported method.
Equal molar NH
2
-PEG-PLA-OH and FA-NHS were employed to
generate FA-PEG-PLA-OH in the presence of TEA. The reaction
was maintained in dimethyl sulfoxide (DMSO) for 36 h. The
crude product was dialyzed against excess water using the
regenerated cellulose membrane (molecular weight cut off/
MWCO: 2000 Da) for 24 h followed by ltration through a
0.45 mm membrane and lypophilization. Finally, FA-PEG-PLA-
OH was converted to FA-PEG-PLA-COOH and then activated to
FA-PEG-PLA-NHS using a similar method of MPEG-PLA activa-
tion described earlier.
Synthesis of iron oxide nanoparticles
Iron oxide (Fe
3
O
4
) nanoparticles were produced using a simple
co-precipitation method with slight modication (Scheme S2,
ESI†).
20
In brief, FeCl
3
$6H
2
O (0.500 g) and FeCl
2
$4H
2
O (0.184 g)
were mixed in 20 mL deionized water followed by rapid addition
of 5.0 mL 25% (w/w) ammonia solution at ambient temperature.
Under vigorous stirring, 0.125 mL OA was dropped into the
dispersion gradually; the temperature was raised to 80 C and
maintained for 1 h under a nitrogen atmosphere. Then 50 mL
toluene was added into the above OA-stabilized Fe
3
O
4
(Fe
3
O
4
-
OA) aqueous dispersion followed by reuxing for 15 min at 120
C. Aerwards, the mixture was cooled down and Fe
3
O
4
-OA
transformed into the toluene phase was separated by using a
separatory funnel; additional toluene was added to achieve the
nal toluene dispersion with Fe
3
O
4
content at ca. 2mgmL
1
.
Subsequently, APS was used to introduce amino groups to the
Fe
3
O
4
surface based on a ligand-exchange process. APS (0.5 mL)
together with TEA (0.4 mL) was added into the 50 mL OA-Fe
3
O
4
toluene dispersion, stirred for 12 h, and magnetically separated
to get the APS-modied Fe
3
O
4
(Fe
3
O
4
-APS) that was further
cleaned by toluene and DCM under sonication.
Surface engineering of iron oxide nanoparticles
Concurrent coating of the surface of iron oxide nanoparticles
with amphiphilic polymers (MPEG-PLA and FA-PEG-PLA) was
achieved by the introduction of amide bonds. MPEG-PLA-NHS
(0.2 g) alone or its mixture with FA-PEG-PLA-NHS at different
molar ratios was added into 50 mL Fe
3
O
4
-APS dispersion in
DCM and stirred for 24 h at ambient temperature. The polymer-
coated iron oxide nanoparticles were separated by a permanent
magnet and dispersed in double-distilled water. The aqueous
mixture was then dialyzed against water with a regenerated
cellulose membrane (MWCO: 7000 Da) for 24 h to remove the
remaining polymers. The nal polymer-coated Fe
3
O
4
aqueous
solution was ltered through a 0.45 mmlter membrane to
remove aggregates and freeze-dried ready for use.
Characterization of iron oxide nanoparticles
The successful synthesis of polymers was veried by proton
nuclear magnetic resonance (
1
H NMR). The
1
H NMR spectra of
the polymers were recorded using a Bruker AVANCE III 400 MHz
NMR instrument with tetramethylsilane as an internal standard
for chemical shis and deuterated chloroform or DMSO (d
6
)as
the solvent. Number average molecular weights (M
n
) of the
synthesized polymers were calculated based on the
1
H NMR
analysis. Fourier-transform infrared (FTIR, Bruker Tensor 27)
analyses were carried out from the wavelength of 450 cm
1
to
4000 cm
1
. The crystal structures of the Fe
3
O
4
with differing
coatings were characterized by powder X-ray diffraction (XRD,
Rigaku D/MAX-2500) at 40 kV and 25 mA with a Cu Ka(l¼
0.154 nm) radiation source. The elemental analysis on the
surface of particles was carried out by X-ray photoelectron
spectroscopy (XPS, PE PHI1600). The morphology and size of
the particles were investigated by transmission electron
microscopy (TEM, JEOL JEM-100CX II). The thermogravimetric
analysis (TGA) was carried out using a STD Q600 thermogravi-
metric analyser (TA instruments) at a heating rate of 10 C
min
1
and a nitrogen ow of 20 mL min
1
. The magnetization
measurements of samples were performed by using a vibrating
Scheme 1 The synthesis route of FA-PEG-PLA-NHS.
6404 |J. Mater. Chem. B, 2013, 1, 6402–6410 This journal is ªThe Royal Society of Chemistry 2013
Journal of Materials Chemistry B Paper
sample magnetometer (VSM, LDJ9600-1) with a eld from 0 to
10 000 Oe at 300 K. The hydrodynamic diameter and size
distribution of magnetic particles were measured with a Zeta-
Plus (Brookhaven Nicolet 6700). The contents of Fe and Si in the
SPIONs were analysed with inductively coupled plasma atomic
emission spectroscopy (ICP-AES, Varian VISTA-MPX).
Hemolytic analysis of iron oxide nanoparticles
Hemolytic tests were performed using a human erythrocyte
suspension in vitro.
2
The human blood provided by Huang-Hua
Orthopaedic Hospital (China) was centrifuged at 800 gfor
10 min and the supernatant was removed. The red blood cells
(RBC) at the bottom of the centrifuge tube were washed three
times with normal saline (0.9%, w/v) to obtain a clear, colour-
less supernatant, and then re-suspended in normal saline at a
concentration of 2% (w/v) for further hemolytic investigations.
The RBC suspension (2 mL) was mixed with either controls or
magnetic samples (2 mL) followed by incubation at 37 C for
4 h. Normal saline and double distilled water were used as
negative (0% hemolysis) and positive (100% hemolysis) control,
respectively. Hemolytic activity of polymer-coated SPIONs with
or without terminal FA was evaluated at a concentration up to
500 mgmL
1
in normal saline. Aer incubation, the samples
were centrifuged at 800 gfor 15 min and the supernatants
were analysed spectrophotometrically (UV-2450, Shimadzu,
Japan) at 545 nm. The degree of hemolysis was calculated by the
following equation:
Hemolysis ð%Þ¼ AsA0
A100 A0
100
where A
S
,A
0
, and A
100
are the absorbance of SPION sample,
negative and positive control, respectively.
Drug loading and release
The model drug, Paclitaxel (PTX), was loaded into the magnetic
nanoparticles via physical encapsulation. Briey, a certain
amount of magnetic power was added into 5 mL DCM, followed
by addition of excess PTX. The mixture was stirred for 2 h and
the organic solvent was evaporated. Then 5 mL distilled water
was added to dissolve the dried power. The solution was then
centrifuged for 5 min at 1000 g; the supernatant was ltered
through a 0.45 mmlter membrane to remove free PTX and
magnetic aggregates followed by lyophilisation. The loading
capacity was determined by extracting PTX from drug-loaded
particles with methanol at ambient temperature for 1 h with
sonication. Aer centrifugation, the PTX in the supernatant was
assayed by high performance liquid chromatography (Dionex
ULTIMATE 3000 HPLC). The separation was achieved by using a
reverse-phase Kromasil C
18
column (250 mm 4.6 mm, 5 mm).
The mobile phase was a mixture of methanol, acetonitrile and
water (35 : 40 : 25, v/v); the UV wavenumber was 229 nm and the
ow rate was set at 1 mL min
1
with a 20 mL injection volume.
The kinetics of PTX release from magnetic nanoparticles was
investigated at 37 C by dialysis. In detail, 5 mL of PTX-loaded
nanoparticle dispersion (2 mg mL
1
) was dialyzed (MWCO:
2000 Da) against 95 mL phosphate buffer solutions (PBS) at pH
7.4. At xed time intervals, PTX concentration in the release
medium was quantied by the HPLC method described above.
Intracellular uptake of iron oxide nanoparticles
To investigate the active targeting capability of FA-containing
SPIONs, uorescein isothiocyanate (FITC) was further cova-
lently linked to the particle surface, which would enable in situ
analysis of the intracellular uptake of SPIONs. FITC-conjugated
SPIONs without FA were also generated as the control. Prior to
the labelling process, FITC-PEG-NHS was rstly synthesised by
three major steps (Scheme S3, ESI†). In detail, equal molar FITC
(0.117 g) and NH
2
-PEG-OH (0.6 g) were mixed in 30 mL dime-
thylformamide and maintained for 24 h under nitrogen
protection at ambient temperature. The crude product was
puried by dialysis against deionized water (MWCO: 1000 Da)
for 24 h followed by ltration through a 0.45 mm membrane.
The remaining ltrate was freeze-dried to obtain the interme-
diate FITC-PEG-OH. Then FITC-PEG-OH (0.328 g), succinic
anhydride (0.021 g) together with DMAP (0.026 g) were put into
20 mL DCM followed by adding 30 mL TEA; the reaction was
kept at ambient temperature in the dark for 24 h. The product
FITC-PEG-COOH was obtained by repeated precipitation in
ice-cold diethyl ether and then vacuum-drying. Aerwards,
FITC-PEG-COOH (0.25 g), NHS (0.018 g), and DCC (0.035 g) were
stirred together in 20 mL DCM at ambient temperature in
the dark for 24 h. FITC-PEG-NHS was puried using the same
approach that was employed to rene FITC-PEG-COOH.
Subsequently the conjugation of a uorescent moiety to the
particle surface was simply achieved by replacing 5% (molar
ratio) MPEG-PLA-NHS with FITC-PEG-NHS during the process
of SPION (with or without FA) surface engineering as described
previously.
The MCF-7 cells were seeded in 35 mm culture plates at a
density of 2.5 10
4
cells and maintained in a 5% CO
2
incubator
at 37 C. The culture medium was DMEM containing 10% fetal
bovine serum and 100 kU L
1
penicillin–streptomycin. Aer
24 h, the cells were washed with PBS and then 200 mL FITC-
labelled SPION suspension (in DMEM, 500 mgmL
1
) was added.
Aer another 6 hours of incubation under the same conditions,
the culture medium was removed followed by washing the cells
with PBS in triplicate and then the addition of fresh medium.
Aerwards, the cells were incubated with the dye 40,6-dia-
midino-2-phenylindole (DAPI, 2 mg) for 5 min. The unbound
DAPI was removed by washing with PBS. The uorescence of
FITC and DAPI was assessed by using a confocal laser scanning
microscope (CLSM) (LSM 710, Zeiss, Germany) with the excita-
tion wavelength at 488 nm and 358 nm, respectively.
Results and discussion
Synthesis and surface engineering of SPIONs
The successful synthesis of activated MPEG-PLA was veried by
1
H NMR analysis (Fig. 1). Compared to the strong characteristic
peaks of MPEG (3.66 ppm) and PLA (1.58 ppm), the methylene
peak at 2.73 ppm from the NHS moiety was evident. The pres-
ence of multiple repeating units in the copolymer creates a large
This journal is ªThe Royal Society of Chemistry 2013 J. Mater. Chem. B, 2013, 1, 6402–6410 | 6405
Paper Journal of Materials Chemistry B
number of protons that absorb radiation and thus produce the
intense peaks. The results agreed well with previous investiga-
tions.
21
The molecular weight (M
n
) of MPEG-PLA-NHS was
calculated to be 3300 Da by
1
H NMR. The synthesis of FA-PEG-
PLA was associated with multiple steps and several interme-
diate polymers. The structure of activated FA-PEG-PLA-NHS was
conrmed by
1
H NMR analysis (Fig. 2), where the characteristic
proton signals from FA, PEG, PLA, and NHS were clear.
The Fe
3
O
4
core of the SPIONs was generated by the bottom-
up co-precipitation method, which enabled the rapid manu-
facture of adequate uniform Fe
3
O
4
in a reproducible manner.
The presence of OA is essential to prevent the potential oxida-
tion of Fe
3
O
4
nanoparticles and also maintain their physical
stability via the steric effect.
22
The OA coating at the Fe
3
O
4
surface was replaced by APS through a ligand exchange process;
this procedure introduced amino groups at the nanoparticle
surface. Such amino-terminated Fe
3
O
4
nanoparticles could
react with a number of ligands directly to get multifunctional
SPIONs.
12
In the current study, the activated polymers bearing the
succinimidyl ester (i.e. MPEG-PLA-NHS and FA-PEG-PLA-NHS)
acted as the reactive ligands. They reacted with the amino
groups forming the polymeric conjugates at the exterior of
SPIONs. Controlling the molar ratio between MPEG-PLA-NHS
and FA-PEG-PLA-NHS during the conjugation step could effec-
tively manipulate the surface density of the molecular targeting
moiety (i.e. FA), generating a series of dual-targeting SPIONs for
anti-cancer therapy. The biodegradability of amphiphilic PEG-
PLA coating would secure the biocompatibility of such nano-
particles. Depositing APS to the surface of the Fe
3
O
4
core was
the foundation of subsequent polymer coating, which was
conrmed by XPS assessment (Fig. 3). For the Fe
3
O
4
-APS
nanoparticles, the signals at 102 eV, 284 eV, and 399 eV were
attributed to Si2p, C1s and N1s, respectively.
23
The molar ratio
of the C : Si was calculated to be ca. 4, which was less than the
theoretical value of 6 for APS, indicating the formation of Fe
3
O
4
-
APS through APS hydrolysis and condensation.
The FTIR analysis veried the conjugation of both MPEG-
PLA and FA-PEG-PLA to the surface of SPIONs (Fig. 4). The
bands at 580 cm
1
were due to the stretching vibration of Fe–O
from the inner Fe
3
O
4
core. The broad N–Hstretch(ca.
3400 cm
1
)andtheC–Hstretch(ca. 2900 cm
1
)provedthe
attachment of APS to the Fe
3
O
4
surface, which was further
veried by the bands at 1122 cm
1
and 1031 cm
1
as the
Fig. 1 The
1
H NMR spectrum of MPEG-PLA-NHS in CDCl
3
with the specific
proton signals of MPEG, PLA and NHS being labeled.
Fig. 2 The
1
H NMR spectrum of FA-PEG-PLA-NHS in DMSO (d
6
) with enlarged
proton signals of FA and NHS.
Fig. 3 The XPS spectra of Fe
3
O
4
(bottom) and Fe
3
O
4
-APS (top) nanoparticles.
Fig. 4 The FTIR spectra of Fe
3
O
4
-APS, Fe
3
O
4
-Polym coated with MPEG-PLA, and
Fe
3
O
4
-Polym-FA
20
coated with mixed MPEG-PLA and FA-PEG-PLA (4 : 1, molar
ratio).
6406 |J. Mater. Chem. B, 2013, 1, 6402–6410 This journal is ªThe Royal Society of Chemistry 2013
Journal of Materials Chemistry B Paper
reection of Si–O asymmetric and symmetric stretching
vibration, respectively.
24
The spectrum of MPEG-PLA coated
Fe
3
O
4
, abbreviated as Fe
3
O
4
-Polym, exhibited the C]Oamide
stretch at 1645 cm
1
, which was a consequence of the forma-
tion of amide between Fe
3
O
4
-APS and MPEG-PLA. The band at
1753 cm
1
was assigned to the C]O ester stretch from the PLA
segment. Similarly, both the unique bands of the C]Oester
and C]O amide were still present in the spectrum of Fe
3
O
4
-
Polym-FA
20
coated with 80% MPEG-PLA and 20% FA-PEG-PLA.
The bands at 1604 cm
1
, 1560 cm
1
, 1510 cm
1
,and
1450 cm
1
were assigned to the aromatic C]C stretch from
the benzyl group, demonstrating the existence of FA at the
surface of Fe
3
O
4
-Polym-FA
20
.
Pharmaceutical evaluation of SPIONs
The aqueous stability of surface-engineered SPIONs is vital for
their in vivo application and function. To investigate the effect
of surface FA density on the physical stability of SPIONs, a series
of SPIONs with increasing FA molar percentage were generated,
including Fe
3
O
4
-Polym (0% FA), Fe
3
O
4
-Polym-FA
20
(20% FA),
Fe
3
O
4
-Polym-FA
40
(40% FA), and Fe
3
O
4
-Polym-FA
60
(60% FA).
Visual observation showed perceptible particle aggregation in
an aqueous medium when the FA content was above the
threshold of 20% (i.e. Fe
3
O
4
-Polym-FA
40
and Fe
3
O
4
-Polym-FA
60
)
(Fig. 5), while the physical stability was well maintained for
Fe
3
O
4
-Polym and Fe
3
O
4
-Polym-FA
20
. Interestingly, previous
studies reported that mixed micellar nanoparticles containing
surface FA ligands could only be prepared at a low level of FA
(0–20%).
25
These investigations concur surprisingly with the
above phenomena observed in the current study. The increasing
surface FA compactness would favour a diverse range of intra-
particle and inter-particle interactions, including hydrogen
bonding, hydrophobic interaction, and p–pstacking, hence
resulting in reduced surface hydrophilicity of SPIONs and
particle aggregation.
Based on the physical stability assessment of SPIONs, only
those with lower content of FA or those without FA were further
evaluated. Surface engineering of Fe
3
O
4
with either MPEG-PLA
only or blended copolymers (MPEG-PLA and FA-PEG-PLA) did
not change the face-cantered cubic structure of Fe
3
O
4
, which
was proven by XRD and electron diffraction analysis (Fig. 6).
Both Fe
3
O
4
-Polym and Fe
3
O
4
-Polym-FA
20
exhibited spherical
shape with the size in the range of 10–20 nm (Fig. 7). The
dynamic light scattering analysis revealed that the corre-
sponding mean hydrodynamic diameters were 70 nm (Fe
3
O
4
-
Polym) and 73 nm (Fe
3
O
4
-Polym-FA
20
). The latter exhibited a
wider size distribution with a polydisperse index (PI) of 0.37,
whereas the PI of the former was 0.23. However, both types of
SPIONs showed outstanding dispersibility in water (Fig. S1,
ESI†), which would benet their blood circulation. The sizes of
obtained SPIONs were not within the range of 100–200 nm that
was nearly the gold standard for designing tumor-targeting
nanoparticles based on the EPR effect. The reason for such a
design is that SPIONs are expected to target tumors mainly by
magnetic targeting other than the EPR effect. The relatively
smaller size of SPIONs in the current study would favour the
particle transport upon reaching the tumor tissue that was
characterized with the elevated interstitial uid pressure and
the abnormal extracellular matrix structure.
26
Preserving the magnetic property is an important parameter
for surface engineering of SPIONs. In the current study, both
types of SPIONs displayed the typical superparamagnetic
behaviour without magnetic hysteresis (Fig. 8). The value of
Fig. 5 The appearance of four types of SPIONs dispersed in deionized water pre
filtration (top) and post-filtration (bottom) through a 0.45 mm cellulose
membrane. (A and A0)Fe
3
O
4
-Polym, (B and B0)Fe
3
O
4
-Polym-FA
20
, (C and C0)
Fe
3
O
4
-Polym-FA
40
, and (D and D0)Fe
3
O
4
-Polym-FA
60
.
Fig. 6 The XRD patterns (left) of (A) Fe
3
O
4
standard (JCPDS no. 65-3107), (B)
Fe
3
O
4
-Polym, and (C) Fe
3
O
4
-Polym-FA
20
; the electron diffraction pattern of Fe
3
O
4
-
Polym-FA
20
was shown (right).
Fig. 7 The TEM images of (A) Fe
3
O
4
-Polym and (B) Fe
3
O
4
-Polym-FA
20
.
This journal is ªThe Royal Society of Chemistry 2013 J. Mater. Chem. B, 2013, 1, 6402–6410 | 6407
Paper Journal of Materials Chemistry B
saturation magnetization of Fe
3
O
4
as the control was calculated
to be 58.7 emu g
1
aer weight normalization (i.e. subtracting
the mass of oleic acid based on the TGA analysis). Compared to
the control, the level of saturation magnetization of surface-
engineered SPIONs reduced to 31.2 emu g
1
(Fe
3
O
4
-Polym) and
29.9 emu g
1
(Fe
3
O
4
-Polym-FA
20
) due to the polymer coating.
Regardless of the presence or absence of FA, the particle surface
engineering by the polymer did not change the magnetic
property of Fe
3
O
4
; the ICP-AES analysis showed that the Fe
3
O
4
core of weight-normalized SPIONs showed similar values of
saturation magnetization at 59.6 emu g
1
(Fe
3
O
4
-Polym) and
58.9 emu g
1
(Fe
3
O
4
-Polym-FA
20
), respectively, which are
consistent with the control. It has been suggested that the level
of saturation magnetization of SPIONs above ca. 10 emu g
1
is
sufficient for pharmaceutical and biomedical applications.
27
Therefore, the SPIONs obtained in the current work would show
superior magnetic stimulus-responsive properties.
The small size, unique physicochemical properties and
surface chemistry of SPIONs may trigger their interactions with
red blood cells. Hence, the assay of hemolysis (destruction of
red blood cells) is critical to evaluate their compatibility with
blood constituents at the early stage of pharmaceutical devel-
opment.
28
The in vitro assessment results showed that both
Fe
3
O
4
-Polym and Fe
3
O
4
-Polym-FA
20
exhibited low hemolytic
potential; the extent of hemolysis of both types of SPIONs was
less than 3% at a concentration up to 500 mgmL
1
. It is usually
recognized that a hemolysis degree of less than 10% indicates
the low risk of hemolysis and thus good biocompatibility for
pharmaceutical excipients or carriers.
29
The superior hemo-
compatibility of SPIONs generated in the current study was
assumed due to the presence of PEG at the particle surface. A
previous investigation demonstrated the protective effect of
surface PEGylation against the hemolytic power of certain
nanoparticles via mechanical or biochemical action.
30
However,
the co-existence of low content of FA at the surface of SPIONs
did not boost the hemolysis. The paclitaxel loading in both
Fe
3
O
4
-Polym and Fe
3
O
4
-Polym-FA
20
was similar at ca. 2% (w/w).
In addition, they exhibited an analogous drug release prole in
PBS (pH ¼7.4) (Fig. 9). Both curves displayed a rapid drug
release prior to 6 h and then they started getting plateaued with
nearly 80% of the total drug being released at 24 h. The pacli-
taxel release was thought to be mainly controlled by diffusion.
As the surface compactness of FA is low, its presence did not
signicantly modify the drug release behaviour.
To track the SPIONs during the course of endocytosis and
evaluate the degree to which FA affects such a process, the
uorescent FITC moiety was conjugated to the particle surface,
as both the particles and the active agent show no uorescence.
The selection of covalent linking other than physical encapsu-
lation of FITC is because that the former means could exclude
the inuence of FITC release from the particles; thus the
localization of FITC could precisely indicate the position of
particles. DAPI has been shown to cross the intact cell
membrane and bind strongly to DNA; thus it was used to denote
the sites of nuclei. The localization of nanoparticles with or
without FA aer 6 hours of incubation in MCF-7 cells is shown
in Fig. 10 where the particles and nuclei are displayed in green
and blue, respectively. Irrespective of the presence of FA on the
nanoparticles surface, both types of FITC-labelled nano-
particles, i.e. Fe
3
O
4
-Polym and Fe
3
O
4
-Polym-FA
20
successfully
entered the cells. However, the higher intensity of green uo-
rescence observed in the upper row of Fig. 10 in contrast to that
in the lower row qualitatively conrmed that the FA indeed
enhanced the intracellular uptake of SPIONs via the FA
receptor-mediated endocytosis.
31
The enhanced molecular tar-
geting efficiency to MCF-cells was postulated to be the result of
multivalent interaction between FA on the particle exterior and
the FA-receptor on the cell surface. A previous study demon-
strated that FA-conjugated dendritic nanoparticles can
dramatically enhance the binding avidity between the particles
and the cells as a consequence of increased particle residence
on the cell.
32
In the current study, particles with more than 20%
FA on their surface were not investigated in terms of cellular
uptake. Firstly, further increasing the compactness of FA on
particle surfaces would weaken the particles' physical stability.
Moreover, increasing the FA content on the particle surface
would enhance the extent of particle–cell binding, leading to
the difficulty in disassociation of particles from the cell
membrane, which was evidenced in the previous work.
32
Fig. 8 The field-dependent magnetization curves of Fe
3
O
4
(stabilized with oleic
acid), Fe
3
O
4
-Polym, and Fe
3
O
4
-Polym-FA
20
.Fig. 9 The cumulative release profiles of paclitaxel from drug-loaded Fe
3
O
4
-
Polym and Fe
3
O
4
-Polym-FA
20
in buffer media (PBS, pH 7.4).
6408 |J. Mater. Chem. B, 2013, 1, 6402–6410 This journal is ªThe Royal Society of Chemistry 2013
Journal of Materials Chemistry B Paper
Conclusions
Magnetic-molecular dual tumor-targeting nanoparticles were
successfully generated with iron oxide, PLA-PEG, and FA as the
core, surface coating and targeting ligand, respectively. The
surface compactness of FA is critical in determining the phys-
ical stability of produced SPIONs; particle aggregation was
evident for particles containing a high content of FA. However, a
less compact FA at the surface of SPIONs (20%) did not
considerably change the pharmaceutical properties of SPIONs,
including particle size, hemocompatibility, drug loading capa-
bility, drug release behaviour, and magnetization performance.
In addition, the presence of a suitable amount of FA could
signicantly improve the particle avidity to the cell membrane
and hence enhance the cellular uptake of particles upon
reaching the cells. Such a magnetic-molecular sequential tar-
geting strategy holds the promise of enhanced tumor homing in
comparison to the mechanism of EPR effect.
Acknowledgements
This work was supported by the Tianjin Research Program of
Application Foundation and Advanced Technology
(11JCZDJC20600; 13JCQNJC13300), the National Natural
Science Foundation of China (21304068), and the Research
Fund for the Doctoral Program of Higher Education of China
(20110032120077).
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