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Self-Assembled Autophagy-Inducing Polymeric
Nanoparticles for Breast Cancer Interference In-Vivo
Yi Wang, Yao-Xin Lin, Zeng-Ying Qiao, Hong-Wei An, Sheng-Lin Qiao, Lei Wang,
R P Yeshan J Rajapaksha, and Hao Wang*
Autophagy is a lysosome-based evolutionarily conserved pro-
cess that plays an important role in cellular degradation for the
clearance of damaged or superfluous proteins and organelles.[1]
Abnormalities in autophagy may directly correlate to various
pathologic diseases, including neurodegenerative disease,[2]
cardiac disease,[3] and cancer.[4] Recently, researchers have
attempted to regulate autophagy and prevent further tumor pro-
gress.[5,6] Compared to present cancer therapy strategies, such
as chemo-,[7] immuno-,[8] gene,[9] radiation,[10] photothermal,[11]
and photodynamic[12] therapies, the autophagy-based concep-
tual pilot studies for cancer therapy are of particular – and
growing – interest in autophagy-deficient cancers, for example,
breast, ovarian, and prostate cancers.[6]
Beclin-1 (Bec1) is the peptide encoded by the human gene
BECN1, a mammalian homolog of the yeast autophagy-
related gene (Atg) 6, and participates in the regulation of
autophagy through binding to phosphatidylinositol-4,5-bispho-
sphate 3-kinases (PI3Ks, signal transducer enzymes), which
are required for initiation of autophagosome formation in
autophagy.[13] BECN1 has been identified as a haploinsufficient
tumor suppressor, and is commonly deleted in human breast,
ovarian, and prostate tumors.[14] Furthermore, upregulation of
Bec1 enhances Ras-induced cell death in MCF-7 breast cancer
cells.[15] Therefore, it has been suggested that overexpression of
Bec1 could inhibit tumor development.[6,16] However, the thera-
peutic Bec1 peptide has two major drawbacks that hamper their
in vivo application, namely poor chemical stability in vivo and
non-specific bio-distribution in tissues. Chemical modification
and/or self-assembly are two useful approaches for overcoming
these limitations.[17] pH-sensitive polymers have been devel-
oped widely to realize the controlled disassembly and release of
payloads in biological conditions.[18] Our previous studies dem-
onstrated that a pH-sensitive poly(β-amino ester) copolymer
self-assembled into a micelle-like nanoparticle by means of a
Y. Wang, Y.-X. Lin, Dr. Z.-Y. Qiao, H.-W. An, S.-L. Qiao,
Prof. L. Wang, R. P. Y. J. Rajapaksha, Prof. H. Wang
CAS Key Laboratory for Biological Effects of
Nanomaterials and Nanosafety
National Center for Nanoscience
and Technology (NCNST)
No. 11 Beiyitiao, Zhongguancun 100190
Beijing, (P. R. China)
E-mail: wanghao@nanoctr.cn
Y. Wang, Y.-X. Lin, H.-W. An, S.-L. Qiao
University of Chinese Academy of Science (UCAS)
No.19A Yuquan Road, 100049 Beijing, (P. R. China)
DOI: 10.1002/adma.201405926
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COMMUNICATION
one-pot synthesis method and effectively delivered drugs.[19,20]
Although numerous nanomaterials influence the autophagy
process,[21] few examples, to the best of our knowledge, have
been reported to effectively control autophagic flux and ulti-
mately achieve therapeutic potential.
Herein, we describe the development of autophagy-inducing
peptides engineered into polymeric nanoparticles for highly
efficient induction of autophagy and interference of breast
cancer in vitro and in vivo. Bec1, as an autophagy-inducing pep-
tide, covalently grafts onto pH-sensitive polymers (Ps), which
self-assemble into micelle-like nanoparticles (P-Bec1) with
poly(ethylene glycol) (PEG) as hydrophilic shells (Scheme 1).
The P-Bec1 nanoparticles improved the stability and enhanced
the cellular uptake of Bec1 in vitro. P-Bec1 can dissociate in the
weakly acidic lysosomal environment (pH 4–5), which resulted
in the alkalization and impairment of lysosomes.[22] The disso-
ciated P-Bec1 can escape from lysosomes and effectively induce
autophagy, leading to autophagic cell death. Moreover, P-Bec1
is also able to effectively inhibit the growth of tumors and slow
down tumor development in vivo.
The amphiphilic poly(β-amino ester) copolymer composed
of the hydrophobic monomer 1,6-hexanediol diacrylate (HDDA,
1.2 mM), the pH-sensitive monomer 3-(dibutylamino)-1-pro-
pylamine (DBPA, 0.7 mM), and hydrophilic amino-terminated
PEG (PEG-NH2, MW = 2 kDa, 0.3 mM) was synthesized by
Michael addition.[19,20] The structure of the polymer was charac-
terized by 1H NMR (Figure S4, in the Supporting Information,
SI). The degree of polymerization (DP) and molecular weight
(MW) of copolymers were deduced to be 27 and ca. 16 kDa,
respectively.
Furthermore, Bec1 was engineered to have a thiol group
at the N terminal to conjugate with the poly(β-amino ester)
copolymer (Figures S2–S4, SI). The 1H NMR spectrum of
P-Bec1 showed the disappearance of the acrylate double bonds
of the polymer P at 5.8–6.3 ppm and the appearance of Bec1
peaks at 6.7–8.5 ppm (Figure 1a), indicating that the acrylate
groups on both ends of the polymer had completely reacted
with Bec1. Circular dichroism (CD) spectra of Bec1 and P-Bec1
were analyzed using standard CONTINLL algorithms,[23] which
revealed the secondary structure of Bec1 to be 33.8% α-helix,
and P-Bec1, 18.8% α-helix, proving that 56% of the secondary
structure of Bec1 was not destroyed during the process of copol-
ymer synthesis (Figure 1c). The amphiphilic P-Bec1 polymers
simultaneously self-assemble into micelle-like nanoparticles
during dialysis from dimethyl sulfoxide (DMSO) into water
(Scheme 1). The morphologies and hydrodynamic sizes of P
and P-Bec1 were observed by transmission electron microscopy
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Scheme 1. Schematic illustration of P-Bec1 nanoparticles for a highly efficient autophagy-inducing process.
(TEM) and dynamic lighting scattering (DLS), respectively.
Average hydrodynamic sizes of 73.7 ± 26.3 nm and 32.4 ±
11.3 nm were obtained for P and P-Bec1, respectively, which
were in accordance with the diameters obtained from TEM
(Figure 1b). Moreover, cryo-TEM and scanning electron micros-
copy (SEM) measurements further validated the self-assembled
spherical morphology of P-Bec1 (Figures S6 and S7, SI). The
zeta potential of the P-Bec1 nanoparticles was about 24.1 ± 4.0
mV, implying favorable interaction with negatively charged cells
(Figure S8, SI). In addition, the diameter of P-Bec1 changed
from 32.4 ± 11.3 nm at pH 7.4 to 275.2 ± 20.2 nm at pH 5.0
(Figure 1b), implying that P-Bec1 disassociates and reorganizes
into larger nanoparticles under weakly acidic conditions.
In order to investigate the biological effects of P-Bec1 nano-
particles, we first evaluated the cytotoxicity of P-Bec1 nano-
particles on MCF-7 cells. Bec1 itself was barely taken up by cells
and therefore exhibited little cytotoxicity even at a high concen-
tration (ca. 50 µM). A high dose of P (ca. 284 µg mL−1) showed
a little cytotoxicity, which could be explained by the pH-sensi-
tive nanoparticles inducing autophagic cell death, as we showed
previously[24] (Figure S9, SI). However, the P-Bec1 nanoparticles
(half maximal inhibitory concentration IC50 = 186.7 µg mL−1)
exhibited much higher cytotoxicity than the polymer P (IC50 =
269.7 µg mL−1), which may be reasonably explained with the
conjecture that P effectively carried autophagy-inducing peptide
Bec1 into the cells. To prove that cytotoxicity is from induction
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of autophagy we then measured the expression of the protein
light chain 3-II (LC3-II), as the conversion of LC3-I to LC3-II is
widely used to monitor autophagosome formation.[25,26] Using
the MCF-7 cell line, which stably expresses green fluorescent
protein (GFP)-tagged LC3 (GFP-LC3),[26,27] we found that both
P and P-Bec1 indeed induced accumulation of GFP-LC3–posi-
tive puncta. The quantitative analysis of induce GFP-LC3 dots
of MCF-7 cells showed that P-Bec1 treatment gave better elimi-
nation than Bec1 and P treatment (p = 0.008) (Figure 2a), which
indicated that P-Bec1 induced autophagosome accumulation
effectively.
Acridine Orange (AO) is a pH-sensitive dye that can stain
acidic vesicular organelles and is used for autophagy detection.
It is membrane permeable so that DNA and cytoplasm can be
stained green and acid vesicular organelles red; the ratio of red
to green can be used to evaluate the effect of autophagy.[26,28]
The confocal laser scanning microscopy (CLSM) images indi-
cated that there were more red spots in P-Bec1–treated cells,
which indicated high accumulation of autophagosomes. The
CLSM images also revealed the occurrence of cellular mor-
phologic changes from spreading to rounded states after
P-Bec1 treatment for 24 h (Figure 2c). In addition, flow cytom-
etry analysis showed that the ratio of red to green fluorescent
intensity increased almost six-fold compared to the control
group when treated with P-Bec1 (Figure S10, SI). These results
demonstrated that P-Bec1 nanoparticles strongly induced
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Figure 1. Characterizations of P-Bec1 nanoparticles. a) 1H NMR spectra of Bec1, P, and P-Bec1 in d6-DMSO. Peaks corresponding to the double
bonds of the acrylate end group are labeled *; the partial Bec1 peptide characteristic peaks are enclosed in dashed boxes. b) TEM image of P-Bec1 in
pH 7.4 aqueous solution (scale bar, 100 nm), which shows spherical nanoparticles. Inset: Hydrodynamic size of P-Bec1 in pH 7.4 and pH 5.0 aqueous
solutions by DLS. c) Circular dichroism (CD) spectra of Bec1, P, and P-Bec1 at the same mass concentration (1 mg mL−1).

autophagy and potentially led to autophagic cell death. Western
blot also confirmed that P-Bec1 treatment increased conver-
sion of LC3-I to LC3-II significantly, whereas Bec1 treatment
induced practically no accumulation of LC3-II. Moreover, we
found that P-Bec1 treatment did not cause p62 (a substrate that
can be degraded by autophagy) degradation but instead caused
accumulation of p62, which indicated possible impairment of
autophagic degradation capacity (Figure 2d and Figure S11, SI).
The lysosome is closely related to the autophagic cellular
process and is believed to be the key organelle involved in
autophagic cell death.[29] Mounting evidence strongly implies
that the nanoparticles influence lysosomes remarkably, owing
to their endocytosis cell uptake pathway and high accumula-
tion in the lysosome compartment.[30] P-Bec1 nanoparticles
are taken up through the endocytosis process, as we proposed
(Figure S12, SI). To determine whether P-Bec1 could inter-
fere with lysosome function and induce lysosome-related
autophagic cell death, we first labeled P-Bec1 nanoparticles with
the cyanine dye Cy5 (P-Bec1-Cy5) and measured the subcel-
lular localization by CLSM. The MCF-7 cells were treated with
P-Bec1-Cy5 for 2 h and then stained with LysoTracker Green
DND-26. CLSM images exhibited that the P-Bec1-Cy5 signal
was co-localized with endosomes/lysosomes (Figure 3a). This
result proved that P-Bec1 nanoparticles entered the cell effec-
tively by endocytosis. Second, the MCF-7 cells, which stably
express GFP-LC3, were treated with P-Bec1 for 24 h and then
incubated with LysoTracker Red DND-99. CLSM images dis-
playing both wavelengths of fluorescence were also obtained,

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Figure 2. P-Bec1 induces autophagic cell death. a,b) Representative images (a) and the corresponding quantitative analysis (b) of GFP-LC3–positive
puncta of non-treated and treated MCF-7/GFP-LC3 cells (15 µM, 24 h). c) AO staining of MCF-7 cells after the same treatment as in (a). d) Western
blot (and its analysis) of LC3-II/LC3-I and the protein p62 after the same treatment as in (a). All treatment concentrations are referred to Bec1 and an
equal amount of copolymer in P-Bec1; ** p < 0.01, *** p < 0.001; one-way analysis of variance (ANOVA) for indicated comparison.

displaying in large areas of yellow signals where the images
overlapped (Figure 3c), which means that P-Bec1–induced
GFP-LC3 puncta almost overlapped with the lysosome signal
(LysoTracker Red DND-99). These results confirmed that most
of the autophagosomes induced by P-Bec1 could be trapped in
the lysosomes but not degraded. Therefore, we can conclude
that P-Bec1 might impair lysosomal degradation capacity.
As we reported, pH-sensitive nanoparticles can damage lys-
osomes by increasing lysosomal pH.[24] To test whether P-Bec1
can affect lysosomal pH, the MCF-7 cells were labeled with
LysoSensor Green DND-189 dye to monitor lysosome acidity.
Flow cytometry analysis showed that the LysoTracker Green
DND-189 fluorescent intensity of cells that had been treated
with P-Bec1 was decreased, which determined the alkaliza-
tion of the lysosomes and corresponding lysosome dysfunction
(Figure 3b). These results revealed that P-Bec1 induced lyso-
somal dysfunction through lysosome alkalization.
That P-Bec1 induces autophagic cell death significantly in
vitro led us to consider that P-Bec1 may serve as a novel can-
didate for breast cancer therapy. We used P-Bec1-Cy5, injected
intravenously, to monitor the bio-distribution of P-Bec1 in
MCF-7 tumor-bearing nude mice. Mice injected with phos-
phate-buffered saline (PBS) through the tail vein were used as
controls. Strong fluorescence illuminations around the tumor
were observed using a multispectral imaging system 4 h after
injection of P-Bec1-Cy5, indicating the specific tumor targeting
of P-Bec1 by the enhanced permeability and retention (EPR)
effect. In addition, we used ex vivo experiments to further
evaluate the bio-distribution of P-Bec1. The tumor exhibited a
strong signal, and the ratio of tumor signal to liver and kidney
signals was 1:1.5 and 1:1.9, respectively (Figure 4a,b). Some
other harvested organs exhibited fluorescence signals likewise,
especially the liver and kidneys, which are the two main meta-
bolic organs of mammals. However, fluorescence signals from
the liver and kidneys became weaker at 24 h post-injection,
unlike signals from tumor tissue: at the tumor site stronger
fluorescence was observed at 24 h than at 4 h post-injection
(Figure S13, SI), suggesting that metabolism of P-Bec1 is rapid
in the liver and kidneys but that it could accumulate in tumor
tissue. The ex vivo results at 24 h suggest to us that P-Bec1 may
have little toxicity to normal tissue but will effectively inhibit a
tumor.
In order to further confirm the bio-distribution of P-Bec1
nanoparticles, besides the fluorescence method, we further
encapsulated gold nanoparticles into the nanoparticles and uti-
lized inductively coupled plasma mass spectrometry (ICP-MS)
to monitor the bio-distribution of P-Bec1 nanoparticles (Figure
S14, SI). According to the data of ICP-MS, we calculated that
the P-Bec1 accumulated in the tumor had increased by 7.8%
compared with initially injected amount of P-Bec1. To further
confirm the systemic toxicity of P-Bec1 nanoparticles, hematox-
ylin-eosin (H&E) staining was applied to liver, kidney, spleen,
heart, and lung tissues. We observed that there was no lesion in
these major organs, which was probably attributable to the fact

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Figure 3. P-Bec1 destroys lysosomal degradation capacity through alkalization of lysosomes. a) Co-localization of P-Bec1 micelles (red) and lysosomes
(green); cells treated with P-Bec1-Cy5 for 2 h and stained with LysoTracker Green DND-26, BF (bright field). b) Flow cytometry analysis of cells with
control, Bec1, P, or P-Bec1 treatment and staining with LysoSensor Green-189 (15 µM, 2 4h). c) Co-localization of GFP-LC3–positive puncta and lys-
osomes. MCF-7/GFP-LC3 cells treated with P-Bec1 for 24 h and stained with LysoTracker Red DND-99.

that the P-Bec1 was rapidly cleared from these major organs
(Figure S15, SI). The high accumulation of P-Bec1 nanoparticles
at the tumor site indicated the potential anticancer effect in vivo
as a result of its efficient autophagy-inducing effect.
To evaluate the effect of P-Bec1 on tumor suppression in
vivo, we used the MCF-7 cancer cells xenografted tumor nude
mice model. We compared the tumor size, body weight, and
pathological morphology of tumor tissues treated with Bec1,
P, or P-Bec1. The process of tumor growth was observed
and recorded for 13 days. As shown in Figure 4c, the tumor
size of MCF-7 tumor-bearing nude mice treated with P-Bec1
nanoparticles increased significantly more slowly than that of

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the control group. The tumor volume ratio

(V/Vinitial) of the control group increased
more than two times more than that of the
P-Bec1–treated group; the tumor inhibitory
rate was calculated to be 51.6%. It is possible
that nanometer-scale P-Bec1 nanoparticles
can slow down the tumor growth by the
effect ascribed to Bec1, which was protected
and accumulated in the tumor site by the
EPR effect (p = 0.004). The Bec1-treated
group showed almost no difference to the
control group, so it can be deduced that the
free peptide Bec1 was degraded and removed
quickly in vivo. However, the nanometer-
scale polymer P can accumulate in the
tumor site by the EPR effect and exhibited
a minute effect on tumor growth, which can
be explained by the pH-sensitive polymers
with autophagy-inducing function. Further-
more, P-Bec1 nanoparticles slowed down the
tumor growth significantly compared to P
nanoparticles and Bec1, demonstrating that
P-Bec1 can kill tumor cells synergistically
and effectively.
None of the mice groups had significant
variations in body weight throughout the
treatment processes (ca. 16 g), so it can be
inferred that there was no extreme toxicity
with these treatments in vivo (Figure 4d). As
well as the biological effects tumor volume
and body weight, we further investigated the
pathological morphology of all the tumor
tissues. H&E staining showed that P-Bec1
reduced breast tumor growth and invasion in
a breast cancer nude mouse model in com-
parison with a control group (Figures 4e).
However, a Bec1-treated group and a P-treated
group showed that tumor cells were poorly
differentiated (Figure S16, SI). These results
indicate that P-Bec1 reduced breast tumor
growth and invasion.
To determine whether P-Bec1 could induce
autophagy in vivo, we used an immunohis-
tochemical method to detect the expression
of LC3-II in tumor tissues. The tumor tis-
sues were incubated with LC3-II antibody,
and the expression of LC3-II was detected by
a fluorescein isothiocyanate (FITC)-labeled
second antibody. Compared to the control
Figure 4. P-Bec1 effectively inhibits the growth of a tumor by inducing autophagy in vivo. a) Ex
vivo near-infrared (NIR) optical images of tumor tissue and major organs dissected from PBS-
group, there was stronger immunofluores-
and P-Bec1–injected mice with MCF-7 cells xenografted for 4 h. b) The corresponding average cence of LC3-II in the P-Bec1 treatment group
signal intensities. Each column represents the mean ± SD (standard deviation), number of (Figure 4f and Figure S17, SI). These results
samples n = 3. c,d) Tumor volume (c) and body weight (d) as a function of time for PBS, Bec1, suggested that P-Bec1 nanoparticles can effec-
P, P-Bec1 treatment groups after MCF-7 tumor xenograft for 7 days. The concentration of Bec1 tively induce autophagic cell death in vivo.
was 20 mg kg−1 in both the Bec1 and P-Bec1 groups. Each point represents a mean ± SD, n In conclusion, nanomaterials with diversi-
= 5, *p < 0.05, **p < 0.01,***p < 0.001; one-way ANOVA for indicated comparison. e) Patho-
fied physiochemical parameters have shown
logical features of tumor tissues in representative mice treated with PBS or P-Bec1. Tumor
issues were H&E stained. f) Immunohistochemical representative images of tumor tissues the possibility of autophagy regulation.[21]
for PBS or P-Bec1-treated mice. Stained with LC3-II; the entire nucleus was stained with DAPI The mechanism is not clear so far; it remains
(4’,6-diamidino-2-phenylindole). challenge for cancer therapy owing to the

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limited work on autophagic networks. We have demonstrated
a convenient approach to preparing self-assembled micelle-like
nanoparticles composed of pH-sensitive poly(β-amino ester)s
and an autophagy-inducing peptide (Bec1). The P-Bec1 nano-
particles displayed enhanced autophagy-inducing cytotoxicity to
MCF-7 cells that are Bec1 deficient, because P-Bec1 can con-
tribute to efficient internalization of the Bec1 peptide into cells
by the endocytosis pathway. Moreover, the P-Bec1 nanoparticles
effectively induced autophagy and inhibited tumor growth.
By modulating the Bec1 peptide autophagy-inducing effect
assisted by pH-sensitive polymers, we identified the autophagy
anticancer efficiency of P-Bec1. We believe that the P-Bec1 pep-
tide described here provides an important addition to existing
efforts in identifying new cancer therapies, and has a prom-
ising effect especially in autophagy-deficiency tumors.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the authors.
Acknowledgements
Y.W. and Y.-X.L. contributed equally to this work. This work was
supported by the National Basic Research Program of China (973
Program, 2013CB932701), the National Natural Science Foundation
of China (21374026, 21304023, 51102014, and 51303036), the Beijing
Natural Science Foundation (2132053), and the 100-Talent Program of
the Chinese Academy of Science (Y2462911ZX).
Received: December 30, 2014
Revised: February 5, 2015
Published online:
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DOI: 10.1002/adma.201405926