Journal of Colloid and Interface Science 469 (2016) 8692

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Journal of Colloid and Interface Science

journal homepage: www.elsevier.com/locate/jcis

Dual-targeting superparamagnetic iron oxide nanoprobes with high and

low target density for brain glioma imaging
Juan Zhang a,1, Ning Chen b,1, Hao Wang c, Wei Gu a, Kang Liu a, Penghui Ai d, Changxiang Yan d,,
Ling Ye a,
a

School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, PR China
Department of Anatomy, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China
d
Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing 100093, PR China
b
c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The FA-c(RGDyK) dual-targeting,

Cy5.5 labeled Fe3O4 nanoprobes were

prepared.
 The dual-targeting nanoprobes were
applied for MR/NIR imaging of
gliomas.
 The synergistic targeting ability of
dual-targeting nanoprobes was
demonstrated.
 The density of dual-target plays an
important role in targeting specificity.

a r t i c l e

i n f o

Article history:
Received 16 December 2015
Revised 30 January 2016
Accepted 2 February 2016
Available online 2 February 2016
Keywords:
FA-c(RGDyK) dual-target
Iron oxide nanoparticles
MR/NIR imaging
Brain gliomas

a b s t r a c t
A major limit of superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic resonance (MR)
imaging nanoprobe in clinical applications is that the SPIONs are unable to reach sufficient concentrations at the tumor site by passive targeting to produce an obvious contrast effect for tumor imaging.
Single-targeting SPIONs systems have been applied to improve the contrast effect. However, they still
suffer from a lack of efficiency and specificity of the SPIONs to tumors. Herein, we developed folic acid
(FA) and cyclic Arg-Gly-Asp-D-Tyr-Lys (c(RGDyK)) dual-targeting nanoprobes based on Cy5.5 labeled
Fe3O4 nanoparticles (NPs). The synergistic targeting ability of the dual-targeting Fe3O4 NPs and the effect
of the dual-target density on targeting specificity were investigated in brain glioma-bearing mice. In vivo
T2-weighted MR imaging of brain glioma-bearing mice and ex vivo near-infrared imaging of brains
harboring gliomas suggested that the combination of dual-target increased the uptake of NPs by glioma,
consequently, enhanced the contrast effect. Moreover, it was revealed that the density of dual-target
plays an important role in targeting specificity.
2016 Published by Elsevier Inc.

1. Introduction
Corresponding authors.
1

E-mail addresses: yancx65828@163.com (C. Yan), lingye@ccmu.edu.cn (L. Ye).

These authors contribute equally to this work.

http://dx.doi.org/10.1016/j.jcis.2016.02.004
0021-9797/ 2016 Published by Elsevier Inc.

Superparamagnetic iron oxide nanoparticles (SPIONs), such as

magnetite (Fe3O4) nanoparticles (NPs), are the first T2 magnetic

J. Zhang et al. / Journal of Colloid and Interface Science 469 (2016) 8692

resonance (MR) nanoprobe used for clinical diagnosis of solid

tumors [14]. Nevertheless, SPIONs have limited extravasation
ability and therefore are subject to uptake by the reticuloendothelial system (RES) [5], which makes SPIONs less efficient for MR
imaging of tumors. Various targeting molecules have been
attached to the surface of SPIONs to enhance the specific targeting
and increase the uptake of SPIONs in tumor by taking advantage of
their high affinity to cell surface receptors that are overexpressed
in tumor cells [610]. For instances, monoclonal antibodies,
transferrin (Tf) protein, cyclic arginine-glycine-aspartic acid
(cRGD), and folic acid (FA) are commonly used for the design of
tumor-targeted MR nanoprobes [11]. However, single-targeting
SPIONs still suffer from a lack of efficiency and specificity to
tumors and exhibit insufficient MR imaging contrast effects due
to receptor saturation [12].
Considering the fact that multiple types of receptors are typically overexpressed on the surface of tumor cells [1315], several
dual-targeting systems have been developed to enhance the
targeting specificity and increase the uptake of NPs in tumors
[1620]. Improved efficacy in cancer therapy and imaging has been
demonstrated with combinations of folic acid (FA) and mAb225
monoclonal antibody [21], angiopep-2 peptides (ANG) and cyclic
arginine-glycine-aspartic acid (cRGD) [22], transferrin (Tf) and
wheat germ agglutinin (WGA) [23], and Tf and RGD [24]. It has
been demonstrated that in single targeting systems, the targeting
efficiency not only depends on the target specificity but also on
the target density [25]. Understanding the effect of dual-target
density on targeting specificity is therefore of great importance
but rarely studied.
In this study, we developed FA and cyclic Arg-Gly-Asp-D-TyrLys (c(RGDyK)) dual-targeting Fe3O4 nanoprobes to enhance the
targeting specificity toward brain glioma. FA and c(RGDyK) were
adopted because c(RGDyK) is able to target avb3 integrin receptors
overexpressed in endothelial cells in brain tumors [26,27], whereas
FA targets the folate receptor (FR) that is overexpressed in both
brain capillary endothelial cells and brain glioma cells [28].
Moreover, in comparison with antibody targets, FA and c(RGDyK)
could decrease the steric hindrance of nanoprobes across BBB
and increase the binding efficiency of the targeting molecules to
the target sites [29]. Therefore, the FA and c(RGDyK) dual-target
could not only enable Fe3O4 nanoprobes to cross the BBB but also
increase their uptake by glioma cells [30], which would consequently enhance the MR contrast effect between glioma and normal tissue. Meanwhile, the incorporation of near infrared (NIR)
fluorescent dye Cy5.5 into the nanoprobes provides additional
NIR imaging mode. The synergistic targeting ability of dual-target
and the effect of dual-target density on targeting specificity toward
gliomas were investigated in brain glioma-bearing mice by
T2-weighted MR and NIR imaging.

87

2.2. Synthesis of oleate-capped Fe3O4 (Fe3O4-OA) NPs

The Fe3O4-OA NPs were synthesized according to the reported
method [31]. Briefly, 2 mmol of Fe (acac)3, 10 mmol of 1,2hexadecanediol, 6 mmol of oleic acid, 6 mmol of oleylamine, and
20 mL of benzyl ether were mixed and heated at 100 C for 1 h
under vacuum, 200 C for 2 h and 300 C for 1 h under a nitrogen
flow. After cooling to room temperature, the mixture was precipitated with ethanol, separated by centrifugation. The precipitate
was dissolved in hexane containing small amount of oleic acid
and oleylamine and the undissolved impurity was removed by centrifugation. The Fe3O4-OA NPs were obtained by the precipitation
with ethanol and then centrifugation and dispersed in hexane for
further use.
2.3. Synthesis of TETT-modified Fe3O4 (Fe3O4-TETT) NPs
The synthesis of TETT-modified Fe3O4 was performed according
to the reported method with minor modification [32]. Typically,
100 mg of Fe3O4-OA NPs, 60 mL of anhydrous toluene, and 60 lL
of acetic acid were mixed and sonicated for 15 min. Subsequently,
3 mL of TETT was added and the mixture was stirred at 70 C for
48 h. Then, the precipitate was collected by centrifugation, washed
with toluene and ethanol, and dialyzed against deionized water,
finally lyophilized to obtain the powder product.
2.4. Synthesis of dual-targeting, Cy5.5-labeled Fe3O4 NPs
Prior to synthesize the dual-targeting nanoprobes, the Cy5.5
was labeled to Fe3O4 NPs. In brief, 1.11 mg of Cy5.5-NHS and
6.99 mg of H2N-PEG2000-NH2 at molar ratio of 1:4 (pH = 8) were
mixed at room temperature to produce Cy5.5-PEG2000-NH2. Then,
100 mg of Fe3O4-TETT NPs was activated by adding 0.25 mg of
EDC and 0.38 mg of NHS in 10 mL of PBS (0.1 M, pH = 6.0), followed
by reacting with Cy5.5-PEG2000-NH2 (pH = 8) at room temperature
for 12 h. The resulting product was purified using a centrifugal filter (MWCO = 5000) and lyophilized to yield Fe3O4-TETT-Cy5.5 NPs.
To synthesis dual-targeting nanoprobes, 100 mg of Fe3O4-TETTCy5.5 NPs was activated by EDC and NHS, followed by adding
53 mg of H2N-PEG3500-RGD and 45.2 mg of H2N-PEG3500-FA. The
reaction was proceeded at room temperature for 12 h (pH = 8) to
obtain the high density dual-targeting nanoprobes (Fe3O4-PEGRGD-FAh). Meanwhile, the reaction of activated Fe3O4-TETT-Cy5.5
NPs with 13.3 mg of H2N-PEG3500-RGD, 11.3 mg of H2N-PEG3500-FA,
and 67.7 mg of H2N-PEG3500-COOH produced the low density
dual-targeting nanoprobes (Fe3O4-PEG-RGD-FAl). For comparison
purpose, single-targeting (Fe3O4-PEG-RGD) and non-targeting
(Fe3O4-PEG) nanoprobes were prepared using the same procedure.
Specifically, 53 mg of H2N-PEG3500-RGD and 45.2 mg of H2NPEG3500-COOH were used to yield the single-targeting nanoprobes
while 90.3 mg of H2N-PEG3500-COOH was added to obtain the nontargeting nanoprobes.

2. Experimental
2.5. Characterization
2.1. Materials
Iron(III)
acetylacetonate
(Fe(acac)3),
benzyl
ether,
1,2-hexadecanediol, and oleylamine were purchased from
SigmaAldrich (USA). Oleic acid (OA, >90%) was obtained from Alfa
Aesar (Johnson Matthey, UK). N-(Trimethoxysilylpropyl) ethylene
diamine triacetic acid, trisodium salt (TETT, 45% in water) was
provided by Gelest Inc (USA). Bi-functional polyethylene glycol
(H2N-PEG3500-COOH), folic acid conjugated poly(ethylene glycol)
(FA-PEG3500-NH2) and c(RGDyK) were received from JenKem
Technology Co. Ltd. (Beijing, China). Other chemicals were of
analytical grade and used as received.

Transmission electron microscopy (TEM) images were acquired

on a JEM-2100F (JEOL, Tokyo, Japan) microscope at an operating
voltage of 120 kV. X-ray diffraction (XRD) patterns were obtained
on a PANalytical Xpert Pro MPD diffractometer (PANalytical,
Holland) using Cu Ka radiation (k = 1.54056 , 40 kV, 40 mA) with
2h scanning mode. The magnetic properties of NPs were
determined on a SQUID MPMSXL-7 (Quantum Design, USA). The
UVvis absorption spectra of NPs with Fe concentration of
160 lM were recorded on a UV-2600 spectrophotometer
(Shimadzu, Japan). The fluorescence emission spectra of NPs with
Fe concentration of 800 lM was collected on an F-2500

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J. Zhang et al. / Journal of Colloid and Interface Science 469 (2016) 8692

fluorescence spectrophotometer (Hitachi, Japan). The content of Fe

was determined on an inductively coupled plasma optical emission
spectrometry (ICP-OES, Varian 710-ES, USA).

intensities within the tumor and a normal brain region respectively and rn is the standard deviation of noise measured from
the background noise in the same slice. The DCNR was calculated
from |CNRpost CNRpre|/CNRpre [34].

2.6. Relaxivity measurements

2.9. NIR and confocal imaging
Relaxivity measurements were conducted on a 7T MR scanner
(Bruker Pharmascan, Germany) using the RARE-T1 + T2-map
sequence. The measurement parameters were set as follows: repetition times (TR) = 3000 ms, multiple echo time (TE) = 11 ms,
33 ms, 55 ms, 77 ms, 99 ms, matrix size = 256 mm  256 mm, field
of view (FOV) = 4.0  4.0 cm2, flip angle (FA) = 180 and slice thickness = 1 mm. The T2 relaxation times and corresponding MR T2
mappings of NPs at various concentrations were acquired. The
relaxivity value of r2 was calculated from the slope of the linear
plot of 1/T2 versus the Fe concentration.
2.7. Brain glioma model
All animal experiments were performed according to the guidelines of Capital Medical University animal committee. Mice C6
brain glioma model was established according to the method
reported in literature [33]. Briefly, ICR mice were fixed in a stereotactic frame after anesthetized with 6% chloral hydrate
(0.10 mL/20 g). 5 lL of suspension containing 5  105 C6 glioma
cells were implanted into the burr hole on the skull. The injection
was done slowly over 5 min, stay 10 min and the needle was withdrawn after another 10 min. The burrhole was filled with bone wax
and the skin was closed with nonmagnetic structures.
2.8. In vivo MR imaging
MR T2-weighted images of the mice brain bearing glioma before
and after the intravenous injection of 0.2 mL of nanoprobes at a
dosage of 10 mg Fe kg 1 body were acquired on a 7T MR scanner
(Bruker Pharmascan, Germany) using the RARE sequence: TR/
TE = 3000/45 ms, matrix size = 256  256, field of view = 2.5 
2.5 cm2, flip angle = 180, slice thickness = 1 mm and number of
averages = 4. The contrast to noise ratio (CNR) was calculated as
follows: CNR = (St Sb)/rn, where St and Sb are the averaged signal

The ex vivo images of the brains harboring gliomas were captured on an optical imaging system (NightOWL II LB983, Germany)
with a 630 nm excitation filter and a 680 nm emission band-pass
filter set. Afterwards, the brains were fixed in 4% paraformaldehyde, dehydrated with 30% sucrose solution, sliced in 20 lm
thickness, stained with DAPI (100 ng/ml), and imaged on a LEICA
TCSSP5 confocal microscope.
2.10. Prussian blue staining
Fixed brain slices were stained with a 1:1 mixture of 2% potassium ferrocyanide (II) trihydrate solution and 2% HCl for 15 min,
washed in distilled water, counterstained with nuclear fast red
for 10 min, and examined under an optical microscope.

3. Results and discussion

The Fe3O4-OA NPs were synthesized by thermal decomposition
of Fe(acac)3 precursors to obtain high crystallinity, purity, and
reproducibility. As water-dispersible and colloidal stability are
the perquisites for bio-imaging applications of NPs, carboxylic
silane TETT were used to replace OA on the surface of Fe3O4 NPs.
Next, the Fe3O4-TETT NPs were labeled with Cy5.5 to ensure
equivalent fluorescence intensity. Finally, PEG, c(RGDyK), and
FA-c(RGDyK) were conjugated onto Fe3O4-TETT-Cy5.5 to obtain
non-targeting (Fe3O4-PEG), single-targeting (Fe3O4-PEG-RGD),
low density dual-targeting (Fe3O4-PEG-RGD-FAl) and high density
dual-targeting (Fe3O4-PEG-RGD-FAh) nanoprobes, respectively
(Scheme 1). The use of PEG not only confers the nanoprobes
additional solubility and stability in aqueous solutions but also
prolongs the blood circulation time, which, to some extent,
enhances the chance for the nanoprobes to cross the BBB.

Scheme 1. Schematic illustration of the non-targeting, single-targeting, high density dual-targeting, and low density dual-targeting nanoprobes.

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89

Fig. 1. TEM images of Fe3O4-OA (A), Fe3O4-TETT (B), Fe3O4-PEG (C), Fe3O4-PEG-RGD (D), Fe3O4-PEG-RGD-FAl (E) and Fe3O4-PEG-RGD-FAh (F). Scale bar = 50 nm. The insets are
the photographs of corresponding NPs dispersed in hexane (A) or distilled water (BF).

Fig. 2. XRD patterns of Fe3O4-OA and Fe3O4-TETT NPs (A), the fielddependent magnetization curve (MH curve) (B), the inset displays the temperature-dependence of ZFC
and FC magnetization at a magnetic field of 100 Oe, and UVvis absorbance (C) and fluorescence emission (D) spectra of various NPs.

Fig. 1 shows the TEM images of Fe3O4-OA, Fe3O4-TETT,

Fe3O4-PEG, Fe3O4-PEG-RGD, Fe3O4-PEG-RGD-FAl and Fe3O4-PEGRGD-FAh NPs, respectively. The NPs were nearly spherical and
monodispersed with an average diameter of 8 nm as calculated
from 150 individual NPs. All of the nanoprobes exhibit high
colloidal stability after being dispersed in water for at least one

month (Fig. 1BF, insets). The color of the dispersions changed

from yellow (Fig. 1B, inset) to2 green (Fig. 1CE, inset), implying
the successful conjugation of Cy5.5.
2
For interpretation of color in Fig. 1, the reader is referred to the web version of
this article.

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J. Zhang et al. / Journal of Colloid and Interface Science 469 (2016) 8692

Fig. 3. The linear plotting of relaxation rate R2 versus Fe concentration (A) and
corresponding MR T2 mappings (B).

The crystal structure of Fe3O4-OA NPs was characterized by XRD

(Fig. 2A). For Fe3O4-OA, six characteristic diffraction peaks at 30.1,
35.4, 43.1, 56.9, 62.5 and 74.0 can be indexed as a cubic
spinel-structured Fe3O4-OA (JCPDS card No. 19-0629). These characteristic diffraction peaks also observed in the XRD pattern of
Fe3O4-TETT NPs, indicating that the TETT replaced the OA on the
surface of Fe3O4-OA NPs without affecting the crystalline structure.
The additional broad diffraction peak observed between 22 and
27 was assigned to the amorphous TETT. Furthermore, the magnetic properties of Fe3O4-OA were studied using a SQUID. As shown
in Fig. 2B, the fielddependent magnetization curve (MH curve)
revealed no coercivity and remanence at 300 K, suggesting the
superparamagnetic behavior of Fe3O4-OA NPs. The temperaturedependent magnetization under zero-field cooling (ZFC) and field
cooling (FC) measurements (Fig. 2B, inset) gave the estimated
blocking temperature of about 50 K for Fe3O4-OA NPs. The MH
curve indicated that the saturation magnetization (Ms) of
Fe3O4-OA NPs was approximately 85.5 emu g 1 at 300 K.
The conjugation of FA-c(RGDyK) dual-target onto NPs was verified by UVvis spectra (Fig. S1). As can be seen, FA possesses a
characteristic absorbance peak at 280 nm while c(RGDyK) presents
a peak at 260 nm. These two peaks were simultaneously presented
in the UVvis spectra of both Fe3O4-PEG-RGD-FAl and Fe3O4-PEGRGD-FAh dual-targeting nanoprobes, confirming the successful
conjugation of FA and c(RGDyK). Conjugation of Cy5.5 was also
confirmed by the absorption peak at 675 nm in the UVvis spectra
and the emission peak at 700 nm in the fluorescence spectra of NPs
(Fig. 2C).
Next, the relaxivity property of nanoprobes was evaluated by
acquiring the T2 relaxation time and the corresponding MR T2 mapping on a 7T MR scanner. The relaxivity r2 was obtained from the

Fig. 4. MR T2-weighted and pseudo-colored MR T2-weighted images of mouse

brains bearing gliomas before and after i.v. injection of nanoprobes (A), and
corresponding quantification of the signals on the MR images of brain gliomas by
DCNR (B). DCNR = |CNRpost CNRpre|/CNRpre. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

slope of linear fitting the inverse relaxation time (1/T2) versus

the Fe concentration (measured by ICP-OES) (Fig. 3A). It was found
that the r2 value of Fe3O4-PEG NPs was 232.6 mM 1 s 1. Upon conjugating the dual-target, the r2 decreased slightly (in the range
between 199.6 and 214.6 mM 1 s 1), probably due to the shield
effect of FA and RGD. Nevertheless, these r2 values are comparable
to the reported value [35] and are suitable for in vivo MR T2 imaging. Consistently, MR T2 mapping of nanoprobes dispersed in water
became progressively darker with the increase of Fe concentration
(Fig. 3B).
The targeting specificity of the dual-targeting Fe3O4 nanoprobes
with a low and high FA-c(RGDyK) density (Fe3O4-PEG-RGD-FAl and
Fe3O4-PEG-RGD-FAh) was first evaluated by in vivo MR imaging of

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91

Fig. 5. (A) Ex vivo fluorescence images of the excised brain harboring gliomas after injection of (a) Fe3O4-PEG, (b) Fe3O4-PEG-RGD, (c) Fe3O4-PEG-RGD-FAl, and (d) Fe3O4-PEGRGD-FAh nanoprobes at an equivalent dosage. (B) Prussian blue staining of mouse brain sections harboring gliomas upon injection of (a) Fe3O4-PEG, (b) Fe3O4-PEG-RGD, (c)
Fe3O4-PEG-RGD-FAl, and (d) Fe3O4-PEG-RGD-FAh nanoprobes. Scale bar = 100 lm. (C) CLSM images of the brain slices bearing C6 glioma after i.v. administration of different
nanoprobes. Blue: cell nuclei, Red: Cy5.5 labeled NPs. White arrow points to the glioma region. Scale bar = 250 lm. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

brain glioma-bearing mice on a 7T MR scanner. The acquired

T2-weighted MR images of glioma-bearing brains before and after
injection of nanoprobes are presented in Fig. 4A. Notably, both
dual-targeting nanoprobes led to a darkened contrast effect surrounding the tumors. On the contrary, less contrast enhancement
effect was detected after injection of either the single-targeting
or non-targeting nanoprobes at the equivalent dosage. To better
visualize the negative enhancement effect, pseudo-colored
T2-weighted MR images are provided. The color of the MR images
of brain glioma treated with Fe3O4-PEG-RGD-FAh appeared bluer
than those of images of brain glioma treated with Fe3O4-PEGRGD-FAl, signifying the synergistic targeting ability toward glioma.
The corresponding quantitative analysis of MR signal enhancement, which was performed and compared via DCNR [34], is
shown in Fig. 5B. It was found that there were approximately
54% and 31% increases in DCNR for Fe3O4-PEG-RGD-FAh and
Fe3O4-PEG-RGD-FAl, respectively, suggesting that a greater dualtarget density results in a higher targeting specificity. However,
due to the fact that an increased number of target moieties would
lead to greater steric hindrance that impedes the uptake of the
nanoprobes by tumor, a detailed study to fully clarification the
relationship between dual-target density and the targeting specificity is required.
Additionally, ex vivo NIR fluorescence imaging of brain harboring
gliomas was conducted. As can be seen from Fig. 5A, the order of
fluorescence intensity from weak to strong is non-targeting,
single-targeting, low- and high-density dual-targeting NPs, suggesting that the high density dual-targeting nanoprobes have the
highest accumulation in gliomas due to the high specificity toward
glioma. This was further supported by Prussian blue staining of
brain tissue sections (Fig. 5B). In addition, the synergistic targeting
ability of dual-targeting nanoprobes toward glioma was investi-

gated by CLSM. As shown in Fig. 5C, only weak red fluorescence originating from Cy5.5 was observed around the glioma treated with
non-targeting nanoprobes. Although single-targeting (Fe3O4-PEGRGD) nanoprobes resulted in slightly higher fluorescence intensity,
the much stronger fluorescence signals were observed from brain
slices treated with either Fe3O4-PEG-RGD-FAl or Fe3O4-PEG-RGDFAh, which further proves the enhanced uptake of dual-targeting
nanoprobes by glioma due to an improved targeting specificity.
Moreover, it was noted that a higher dual-target density induced a
greater accumulation of nanoprobes in the glioma region (Fig. S2).
4. Conclusions
In sum, the dual-targeting iron oxide nanoprobes with low and
high FA-c(RGDyK) dual-target density were prepared and characterized. The in vivo MR imaging of brain glioma-bearing mice
demonstrated that both dual-targeting nanoprobes exhibited an
improved targeting specificity toward glioma due to the synergistic
targeting ability. This consequently led to an enhanced MR negative contrast. More importantly, it was disclosed that the density
of dual-target plays an important role in targeting specificity. On
the one hand, the targeting specificity elevates with the increasing
of dual-target density. On the other hand, an increased dual-target
density might cause a steric hindrance that impedes the uptake of
nanoprobes by glioma. Therefore, optimization the density of dualtarget is needed to further improve the targeting specificity of
dual-targeting nanoprobes toward gliomas.
Acknowledgements
The authors gratefully acknowledge the financial supports from
National Natural Science Foundation of China (81271639),

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J. Zhang et al. / Journal of Colloid and Interface Science 469 (2016) 8692

National Key Technology Research and Development Program of

the Ministry of Science and Technology of China (2014BAI04B01),
and the Basic-clinical Key Research Grant (13JL02, 15JL07) from
Capital Medical University.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jcis.2016.02.004.
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