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Tumor-penetrating peptide functionalization enhances the anti-glioblastoma effect of

doxorubicin liposomes

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2013 Nanotechnology 24 405101

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IOP PUBLISHING NANOTECHNOLOGY
Nanotechnology 24 (2013) 405101 (8pp) doi:10.1088/0957-4484/24/40/405101

Tumor-penetrating peptide
functionalization enhances the
anti-glioblastoma effect of
doxorubicin liposomes
Yiyi Yang1,2,5 , Zhiqiang Yan2,5 , Daixu Wei2 , Jian Zhong2 , Lu Liu2 ,
Lin Zhang3 , Fei Wang4 , Xiaoli Wei4 , Cao Xie4 , Weiyue Lu4 and
Dannong He1,2
1
School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240,
People’s Republic of China
2
National Engineering Research Center for Nanotechnology, Shanghai 200241,
People’s Republic of China
3
Department of Pharmacy, Shaoxing People’s Hospital, Shaoxing Hospital of ZheJiang University,
Shaoxing 312000, People’s Republic of China
4
Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, Department of
Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China

E-mail: yanzhiqiang2009@gmail.com, wylu@shmu.edu.cn and hdnbill@sh163.net

Received 20 June 2013, in final form 5 August 2013

Published 12 September 2013
Online at stacks.iop.org/Nano/24/405101
Abstract
The targeted therapeutic effect of nano drug delivery system for glioblastoma has been hampered by the
weak enhanced permeability and retention (EPR) effect of glioblastoma and the low delivering efficiency
of NDDS in glioblastoma tissue. In this study, a tumor-penetrating peptide (RGERPPR), the specific
ligand of neuropilin-1 overexpressed on glioblastoma and endothelial cells, was used as a targeting
moiety to enhance the anti-glioblastoma effect of doxorubicin liposomes. Firstly, RGERPPR-PEG-DSPE
was synthesized and used to prepare the RGERPPR peptide-functionalized liposomes (RGE-LS), which
showed vesicle sizes of around 90 nm and narrow size distributions. The cellular uptake and in vivo
near-infrared fluorescence imaging test displayed that RGE-LS exhibited increased uptake by
glioblastoma cells and intracranial glioblastoma tissues. The cytotoxicity assay and anti-glioblastoma
study proved that RGERPPR functionalization significantly enhanced the in vitro inhibitory effect of
doxorubicin liposomes on glioblastoma cells and prolonged the median survival time of nude mice
bearing intracranial glioblastoma. Finally, the immunofluorescence analysis evidenced that RGE-LS
were able to penetrate through tumor vessels and stroma and deep into the whole tumor tissue. The
results indicated that tumor-penetrating peptide functionalization is an effective strategy for enhancing
the anti-glioblastoma effect of doxorubicin liposomes.
(Some figures may appear in colour only in the online journal)

1. Introduction regions for brain function makes it highly risky to be treated

Glioblastoma is the most aggressive and frequent primary
malignant brain tumor in humans, and the life times of patients
are quite short [1]. The proximity of glioblastoma to critical
5 These authors contributed equally to this work.
by surgery and radiotherapy [2]. Conventional chemotherapy
often leads to serious systemic toxicity [3]. Thus, it is urgent
to develop an efficient treatment for glioblastoma.
The blood–brain barrier (BBB) precludes access for
most compounds to the brain tissue [4]. However, when
glioblastoma is clinically diagnosed, the BBB has usually

0957-4484/13/405101+08$33.00 1 c 2013 IOP Publishing Ltd Printed in the UK & the USA


Nanotechnology 24 (2013) 405101
been damaged by the invasion of tumor cells, and
endothelial gaps have formed on the microvessels of brain
tumors [5]. Then the enhanced permeability and retention
(EPR) effect comes into action, leading to the tendency of
nanoscale particles (for example, nanoparticles, liposomes
and macromolecular drugs) to accumulate in tumor tissue
much more than they do in normal tissues [6]. However,
the EPR effect of intracranial tumors is relatively weak
compared with that of peripheral tumors, as the critical pore
size for solute passage of intracranial tumors is smaller than
peripheral tumors [7, 6]. For example, the critical pore size
for intracranial human primary glioblastoma cell line U87MG
is as low as 7–100 nm [7]. The weak EPR effect decreases
the extravasation of nano drug delivery system (NDDS) from
blood vessel to the glioblastoma tissue, thereby resulting in
the low delivering efficiency to the targets [6].
Another problem affecting the efficient delivery of NDDS
in glioblastoma is the difficulty of permeation from the tumor
vessel vicinity deep into tumor tissue. This results from
several delivering barriers, including the rapidly increased
IFP in the periphery of tumor tissue, dense stromal cells
and ECM between tumor vessels and cancer cells [8]. These
barriers prevent the transport of NDDS sufficiently deep to
reach as many glioblastoma cells as possible. Several reports
have proved that the NDDS can penetrate only a few cell
diameters into the extravascular tumor tissue, leaving most
cancer cells in deep tumor tissue unable to be exposed to
antitumor drugs [9–11]. This problem has resulted in the
incomplete inhibition of tumor growth, arousing extensive
concern among researchers [12, 11].
Neuropilin-1 (NRP-1) is a receptor overexpressed on
both glioblastoma cells and tumor endothelium [13]. As
the co-receptor of VEGF165, NRP-1 plays an essential
role in tumor angiogenesis, metastasis and regulation of
vascular permeability [13, 14]. The expressing level of NRP-1
increases with the increase in the degree of glioblastoma
malignancy [15]. RGERPPR peptide, derived from phage
display screens, is the specific ligand of NRP-1 [16, 17]. It
was proved to be a ‘tumor-penetrating peptide’ (TPP), able
to penetrate through tumor vessels and tumor stroma [18, 16].
Several reports demonstrated that TPP functionalization could
promote the transport of NDDS through blood vessel to
tumor tissue and the penetration through tumor stroma
and deep into the whole tumor tissue, which had been
evidenced in pancreatic, breast, prostatic and liver cancer
[18, 16, 19]. Therefore, TPP makes it possible to solve the
two above-mentioned problems of NDDS in the targeting
therapy of glioblastoma. On the one hand, the tumor
vessel-penetrating ability of TPP-functionalized NDDS may
compensate for the weak EPR effect of glioblastoma; on the
other, the tumor stroma-penetrating ability may compensate
for the low delivering efficiency of NDDS in glioblastoma.
In this study, we prepared RGERPPR-functionalized
liposomes (RGE-LS) to enable them to penetrate through
tumor vessel and tumor stroma, thereby enhancing the
inhibitory effect to glioblastoma. Firstly, we conjugated
RGERPPR to the surface of liposomes and tested their
targetability to U87MG glioblastoma cells in vitro and
2
Y Yang et al
to glioblastoma in vivo. Then the growth inhibitory
effect of RGERPPR-functionalized doxorubicin liposomes
(RGE-LS/DOX) to glioblastoma was investigated. Finally,
we studied the tumor-penetrating ability of RGE-LS by
immunofluorescence analysis.
2. Materials and methods
2.1. Materials
Mal-PEG-DSPE was obtained from Laysan Bio Co. (USA).
Fetal bovine serum (FBS) and DMEM medium were from
Gibco Co. (USA). PEG2000-DSPE and HSPC were supplied
by Shanghai AVT Pharmaceutical (China). DiR was from
Invitrogen (USA). Cholesterol was supplied by Sinopharm
Chemical Reagent Co. (China). Sephadex G50 was from GE
Healthcare (UK). Rat anti-mouse CD31 was from Abcam
(UK). Goat anti-rat IgG-R was from SantaCruz (USA). DAPI
was purchased from Molecular Probes (USA). All chemicals
were analytic reagent grade. U87MG cell line was provided
by Shanghai Institute of Cell Biology, which was cultured
according to Zhan et al [20]. Male Balb/c Nu/Nu mice
(four weeks old) of 20–25 g body weight were supplied
by Shanghai SLAC laboratory animal company (China) and
maintained under SPF conditions. All manipulations were
performed in accordance with procedures approved by the
ethics committee (Shanghai Jiao Tong University).
2.2. Synthesis of RGERPPR-PEG-DSPE
Firstly, the thiolated RGERPPR (C-RGERPPR) with was
synthesized via Boc-protected solid-phase peptide synthesis
strategy, followed by reaction with Mal-PEG-DSPE as
described previously [21]. The reaction solution was purified
by dialysis (MWCO 3.5 kDa cut off, Millipore) against
distilled water, followed by lyophilization. The purity was
verified by 1 H-NMR and FTIR.
2.3. Preparation of liposomes
The thin-film hydration and extrusion method was used
to prepare the liposomes loaded with DOX, FAM or DiR
as previously [22, 23]. HSPC, cholesterol, mPEG-DSPE
and RGERPPR-PEG-DSPE were used with the molar ratio
of 55:40:5:1 (for RGERPAR-modified liposomes, including
RGE-LS/FAM, RGE-LS/DiR and RGE-LS/DOX) or 0 (for
unmodified liposomes, including LS/FAM, LS/DiR and
LS/DOX). DOX was loaded into liposomes by the ammonium
sulfate gradient method according to the procedure described
previously [24].
2.4. Characterization of liposomes
The vesicle size of liposomes was measured by dynamic
light scattering method (Zetasizer Nano-ZS, Malvern Instru-
ments, Westborough, MA). Transmission electron microscopy
(TEM) (JEM-2010FEF, JEOL, Japan) was used to evaluate the
morphology of liposomes.
Nanotechnology 24 (2013) 405101 Y Yang et al

The in vitro stability of liposomes loaded with DiR or

FAM was investigated according to the procedure reported
previously [25]. A FL Spectrophotometer (F-1000, Hitachi,
Japan) was used to determine the concentrations of DiR or
FAM at Ex/Em 741/776 nm and 493/520 nm, respectively.

2.5. Cellular uptake and flow cytometry tests

The cellular uptake test was performed according to previous
reports with minor modification [26, 27]. The U87MG cells
with 80% confluence were treated with RGE-LS/FAM for 2 h
in DMEM supplemented with 10% FBS, and LS/FAM and
FAM were as control. At the end of incubation, the cells were
washed thrice with PBS, fixed in 0.5% paraformaldehyde
for 15 min, treated by DAPI for 10 min and imaged
using a laser scanning confocal microscope (DMI4000 B,
Leica, Germany). For the flow cytometry test, following the
incubation, the U87MG cells were trypsinized, centrifuged,
resuspended in PBS and analyzed by flow cytometer equipped
with a 488 nm Ar ion laser (FACSAria, BD, USA).

2.6. In vivo fluorescence imaging study

The targeting ability of RGE-LS to glioblastoma was
investigated by using near-infrared in vivo imaging study.
The nude mice model of glioblastoma was first established
according to a previously reported procedure with slight
modifications [28]. The U87MG cells (5 × 105 ) were
inoculated into the right striatum by using a stereotactic
fixation device with mouse adaptor. The coordinates were
determined based on the Paxinos mouse brain atlas as
follows: 0.6 mm anterior, 1.8 mm lateral to the bregma and
3 mm deep [29, 30]. On the 24th day post-inoculation, the
intracranial glioblastoma-bearing mice were i.v. injected with Figure 1. The molecular structure (A), NMR spectra (B) and FTIR
spectra (C) of Mal-PEG-DSPE and RGERPPR-PEG-DSPE; the
100 µl of RGE-LS/DiR via the tail vein, and LS/DiR were as characteristic peak of maleimide group can be found in the NMR
the control. After 2, 4, 8 and 24 h, the mice were anesthetized, spectrum of Mal-PEG-DSPE, but disappears in that of
and the fluorescent images were detected using an in vivo RGERPPR-PEG-DSPE; the intensity of both N–H and C=O band
imaging system (CRi, MA, USA) equipped with a DiR filter significantly enhanced in the FTIR spectrum of
sets (excitation/emission, 730/790 nm). RGERPPR-PEG-DSPE compared with that of Mal-PEG-DSPE.

2.7. Tumor-penetrating ability study

Immunofluorescence method was used to investigate the
tumor-penetrating ability of RGE-LS. RGE-LS/FAM were
i.v. injected to glioblastoma-bearing nude mice 24 days
after implantation. The mice were sacrificed 4 h later,
and glioblastoma-bearing brains were collected, fixed,
dehydrated, and sectioned. The sections (10 µm) were stained
with rat anti-mouse CD31 (1:10, Abcam) followed rhodamine
labeled goat anti-rat IgG (1:100, SantaCruz) and DAPI.
2.9. Anti-glioblastoma study of RGE-LS/DOX
The nude mice models of glioblastoma were established
as mentioned in section 2.6. Four groups (n = 7) of mice
were i.v. injected of normal saline (NS), DOX, LS/DOX and
RGE-LS/DOX at the 20th, 23th and 26th day post-inoculation,
respectively. The total doxorubicin dose was 22.5 mg kg−1 .
The survival times of mice in each group were recorded.

2.8. Cytotoxicity assay 2.10. Statistical analysis

The MTT assay was used to determine the cytotoxicity
of RGE-LS/DOX according to the procedure reported
previously [31]. U87MG cells were cultured in 96 well plate
for 24 h (2000/well), and treated by serial concentrations of
LS/DOX or RGE-LS/DOX for 72 h. The MTT assay was
performed and the percentage of cell viability was determined
by using an enzyme-labeling instrument (PowerWave XS,
Bio-TEK, USA). The IC 50 values were determined by curve
analysis software (GraphPad Prism 5.02).
Statistical differences were evaluated with two-tailed cor-
rected student’s t-test with p < 0.05 as significant.
3. Results
3.1. Characterization of RGERPPR-PEG-DSPE
Figure 1 shows the molecular structure, NMR and FTIR
spectra of Mal-PEG-DSPE and RGERPPR-PEG-DSPE. Both

3
Nanotechnology 24 (2013) 405101
Figure 2. LS, RGE-LS, LS/DOX and RGE-LS/DOX all exhibited uniform spherical morphology as displayed in the TEM images (scale
bar = 100 nm).
Table 1. The vesicle sizes and drug leakage rates of DOX, FAM or DiR loaded liposomes with or without RGERPAR modification.
Formulation Vesicle size (nm)
LS/DOX 88.5 ± 8.0
RGE-LS/DOX 87.7 ± 9.1
LS/FAM 93.1 ± 6.7
RGE-LS/FAM 92.3 ± 5.4
LS/DiR 86.6 ± 7.8
RGE-LS/DiR 88.1 ± 6.9
Data are represented with mean ± SD (n = 3); N.D. = not detectable.
NMR spectra contain the solvent peak of CDCl3 at 7.26 ppm,
the methylene protons peaks of DSPE at 1.26 ppm and the
repeat units of PEG at 3.7–3.8 ppm. The NMR spectrum
of Mal-PEG-DSPE indicates the presence of the maleimide
group by its characteristic peak at 6.7 ppm, which disappears
in that of RGERPPR-PEG-DSPE. The results suggested the
successful reaction of the thiol group of RGERPPR with
maleimide group.
In the FTIR spectrum of Mal-PEG-DSPE, a weak C=O
stretch band at 1666.8 cm−1 and a N–H stretch band at
3200–3600 cm−1 are found, which may be responsible for
the amide groups in Mal-PEG-DSPE. The intensity of the two
bands in the spectrum of RGERPPR-PEG-DSPE remarkably
enhance, which should be due to the increased number of
amide groups compared with Mal-PEG-DSPE. The results
suggested that the RGERPPR peptide had been conjugated to
Mal-PEG-DSPE.
3.2. Characterization of liposomes
As determined by dynamic light scattering method, the
liposomes loaded with DOX, FAM or DiR all exhibited
vesicle sizes of around 90 nm and narrow size distributions
(table 1). As displayed in TEM images (figure 2), LS,
RGE-LS, LS/DOX, and RGE-LS/DOX all exhibited uniform
spherical morphology. The modification of RGERPAR had
Y Yang et al
Polydispersity index Drug leakage (%)
0.022 ± 0.004 —
0.026 ± 0.007 —
0.029 ± 0.005 1.89 ± 0.32
0.030 ± 0.010 1.97 ± 0.55
0.024 ± 0.007 N.D.
0.025 ± 0.008 N.D.
no obvious influence on the vesicle size and morphology
of liposomes. The leakage of FAM from liposomes was
about 2% within 4 h and no leakage of DiR was
detected from liposomes within 72 h (table 1), suggesting
that the prepared fluorescein-loaded liposomes satisfied the
requirement of the cellular uptake, in vivo targeting ability and
immunofluorescence study [32].
3.3. Targeting property of RGE-LS
3.3.1. In vitro cellular uptake of RGE-LS. The images of
cellular uptake and flow cytometry for FAM, LS/FAM and
RGE-LS/FAM were shown in figure 3. The percentages of
FAM-positive cells for FAM, LS/FAM and RGE-LS/FAM
were 2.99%, 14.8% and 94.1%, and the mean fluorescent
intensities for them were 18.3, 18.7 and 361, respectively.
These data indicated that the cellular uptake of liposomes was
remarkably enhanced by RGERPPR modification.
3.3.2. In vivo targeting property of RGE-LS to glioblastoma.
RGE-LS/DiR were i.v. injected into glioblastoma-bearing
nude mice to investigate the in vivo targeting property
of RGE-LS to glioblastoma. As shown in figure 4, from
4 h post-injection, significantly more RGE-LS/DiR were
localized in glioblastoma than LS/DiR, indicating that
RGERPPR functionalization could increase the liposome
delivery to glioblastoma in vivo.
4
Nanotechnology 24 (2013) 405101 Y Yang et al

Figure 3. The representative images of cellular uptake and flow cytometry for FAM (A), LS/FAM (B) and RGE-LS/FAM (C). The cellular
uptake of liposomes by U87MG cells was remarkably enhanced by RGERPPR modification.

Figure 4. Representative in vivo fluorescent images of glioblastoma-bearing nude mice following i.v. administration of LS/DiR (A) and
RGE-LS/DiR (B). RGERPPR functionalization increased the liposome delivery to glioblastoma in vivo.

3.4. The tumor-penetrating ability of RGE-LS
The tumor-penetrating ability of RGE-LS in glioblastoma was
investigated by performing the immunofluorescence analysis
of frozen glioblastoma sections. The immunofluorescence
images showed that LS/FAM were mainly located adjacent
to blood vessels, whereas RGE-LS/FAM were located in
the whole tumor tissue (figure 5). The results suggested
that RGERPPR modification enabled liposomes to penetrate
through tumor vessels and tumor stroma and deep into the
whole tumor tissue.
3.5. Growth inhibitory effect of RGE-LS/DOX to
glioblastoma
3.5.1. Cytotoxicity of RGE-LS/DOX. The in vitro
cytotoxicity of RGE-LS/DOX was measured by MTT assay,
compared with that of LS/DOX. After incubation for 72 h,
the IC50 values were 1.445 µM for LS/DOX and 0.468 µM
for RGE-LS/DOX (figure 6), indicating that RGE-LS/DOX
exhibited significantly higher growth inhibitory effect to
U87MG cells than LS/DOX.
3.5.2. In vivo inhibitory effect of RGE-LS/DOX to
glioblastoma. The anti-glioblastoma effect of RGE-
LS/DOX was evaluated by the Kaplan–Meier survival curve
of intracranial U87MG glioblastoma-bearing nude mice. The
results (figure 7) showed that the median survival times of
mice injected with normal saline (NS), DOX, LS/DOX and
RGE-LS/DOX were 38, 39, 42 and 48 days, respectively. The
survival times of LS/DOX group (p < 0.05, log-rank analysis)
and RGE-LS/DOX group (p < 0.001) were significantly
longer than that of NS, but there was no significant difference
between free DOX group and the NS group (p > 0.05).
Such superiority of LS/DOX and RGE-LS/DOX over DOX

5
Nanotechnology 24 (2013) 405101
Figure 5. Representative immunofluorescence images of frozen glioblastoma of nude mice following injection of LS/FAM and
RGE-LS/FAM. LS/FAM were mainly located adjacent to blood vessels (shown for CD31, red), whereas RGE-LS/FAM penetrated through
tumor vessels and tumor stroma and deep into the whole tumor tissue.
Figure 6. The cytotoxicity of LS/DOX and RGE-LS/DOX on
U87MG glioblastoma cells as measured by MTT assay.
RGE-LS/DOX showed significantly higher growth inhibitory effect
than LS/DOX.
should be attributed to the prolonged blood circulation
time and passive targeting delivery to tumor caused by
liposomes. In addition, the results showed that the survival
time of RGE-LS/DOX group was significantly longer than
LS/DOX group (p < 0.05), suggesting that the growth
inhibitory effect of DOX liposomes to glioblastoma could
be enhanced by RGERPPR modification. The enhancement
of anti-glioblastoma effect should be attributed to the
tumor-penetrating ability of RGE-LS as evidenced above.
4. Discussion and conclusion
In this work, RGERPPR was modified to the surface of
liposomes to improve the transport of liposomes through
the tumor vessel into glioblastoma tissue and further
penetration of liposomes from tumor vessel vicinity through
the tumor stroma into deep glioblastoma tissue. The results
indicated that RGE functionalization significantly increased
the uptake of liposome by glioblastoma and the inhibiting
6
Y Yang et al
Figure 7. The Kaplan–Meier survival curve of intracranial U87MG
glioblastoma-bearing nude mice. The median survival time of
RGE-LS/DOX group was significantly longer than that of LS/DOX
group (p < 0.05, log-rank analysis).
effect of DOX liposomes to glioblastoma, and that the
RGE-modified liposomes exhibited tumor-penetrating ability
in glioblastoma.
Peptide-modified NDDSs for tumor targeting have been
extensively studied due to their good therapeutic effect to
glioblastoma [6]. Many peptides, such as RGD peptides
[30, 33], NGR peptides [34] and Angiopep-2 [35], have
been widely used as the targeting moieties for glioblastoma.
However, the therapeutic effect of peptide-modified NDDS
for glioblastoma is limited by several factors, for instance,
the weak EPR effect of glioblastoma and the lack of
tumor-penetrating ability of delivery system. In fact, these
factors can further result in the incomplete inhibition, drug
resistance and recurrence of the tumor [36]. These problems
have seriously hindered the development of tumor-targeted
NDDS. The tumor-penetrating peptide modification of NDDS
provided an effective solution for the problem of delivery of
nanocarriers in tumor tissue. As evidenced by our results,
Nanotechnology 24 (2013) 405101
the tumor-penetrating peptide-modified liposomes penetrated
through tumor vessels and deep into the glioblastoma
tissues, enabling most tumor cells to be exposed to and be
effectively inhibited by drug-loaded liposomes. Furthermore,
it is possible to decrease the drug resistance and recurrence
of the tumor by using the tumor-penetrating peptide-modified
NDDS.
The NRP-1 is overexpressed on not only tumor cells
but also endothelial cells in tumor vessels. Accordingly,
the RGE-LS should possess targetability to tumor vessels
in addition to tumor cells, which has two advantages. On
the one hand, the targeting of tumor vessels can increase
the distribution of NDDS into tumor tissues. Generally, the
presence of tumor cell-targeted ligands on the nanocarrier
surface does not change the chances of nanocarriers to
enter tumor tissues, which is mainly determined by the EPR
effect [37]. In contrast, RGE peptide, a tumor vessel-targeted
ligand, can take more nanocarriers to the tumor vessels by
its specific binding with NRP-1 on tumor vessels, thereby
increasing the chances of nanocarriers to enter tumor tissues.
In addition, with the tumor-penetrating ability, RGE-LS
adjacent to the tumor vessels can penetrate through tumor
vessels and deep into the whole tumor tissue. On the other
hand, RGE-LS may be able to destroy the tumor vessels while
killing the tumor cells, which would significantly amplify the
antitumor effect of NDDS. Many reports have proved that
the elimination of endothelial cells can remarkably inhibit
the growth of tumor cells [38]. Besides, the destruction of
tumor vessels can improve the growth inhibitory effect on
both primary tumors and metastatic solid tumors [38].
In summary, RGE-LS exhibited an enhanced targeted
therapeutic effect on glioblastoma, based on their tumor
targetability and penetrating ability, indicating that tumor-
penetrating peptide functionalization is an effective strategy
of enhancing the anti-glioblastoma effect of doxorubicin
liposomes. This study may provide a solution for the current
dilemma of active targeted therapy of glioblastoma and other
tumors.
Acknowledgments
This work was supported by the National Basic Research
Program of China (2013CB932500), National Natural Sci-
ence Foundation of China (81202471, 51203024), Interna-
tional Cooperation Project from Science and Technology
Commission of Shanghai Municipality (No. 12520708000),
Nano Project from Science and Technology Commission
of Shanghai Municipality (11nm0505000), Shaoxing City
Public Welfare Technology Applied Research Projects
(2012B70050) and Open Project Program of Key Lab
of Smart Drug Delivery (Fudan University), Ministry of
Education & PLA, China (SDD2011-06).
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