Synthesis of Size-Tunable Hollow Polypyrrole Nanostructures and Their Assembly into Folate-Targeting and pH-Responsive Anticancer Drug-Delivery Agents

Article abstract of DOI:10.1002/chem.201702945

Chemotherapeutic drugs currently used in clinical settings have high toxicity, low specificity, and short half-lives. Herein, polypyrrole-based anticancer drug nanocapsules were prepared by tailoring the size of the nanoparticles with a template method, controlling drug release by means of an aromatic imine, increasing nanoparticle stability through PEGylation, and improving tumor-cell selectivity by folate mediation. The nanoparticles were characterized by TEM and dynamic light scattering. α-Folate receptor expression levelsof tumor cells and normal cells were investigated by western blot and quantitative polymerase chain reaction analyses. Flow cytometry and fluorescence imaging were used to verify the cell uptake of the different-sized nanoparticles. From the different-sized polypyrrole nanoparticles, the optimally functionalized nanoparticles of 180 nm hydrodynamic diameter were chosen and further usedfor in vitroandin vivotests. The nanoparticles showed excellent biocompatibility and the drug-loaded nanoparticles exhibited effective inhibition of tumor cell growth in vitro. Moreover, the drug-loaded nanoparticles showed substantially enhanced accumulation in tumor regions and effectively inhibitedin vivotumor growth. Furthermore, the nanoparticles showed reduced doxorubicin-induced toxicity andno significant side effects in normal organs of tumor-bearing mice, as measured by body-weight shifts and evaluationof drug distribution. Overall, the functionalized nanoparticles are promising nanocarriers for tumor-targeting drug delivery.

Full text of DOI:10.1002/chem.201702945

A Journal of
Accepted Article
Title: Synthesis of Size-tunable Hollow Polypyrrole Nanostructures and
Their Assembly into Folate Targeting and pH-responsive Anti-
cancer Drug Delivery
Authors: Jian Chen, Xiufang Li, Yanhua Sun, Yuwei Hu, Yulong Peng,
Yimin Li, Gang Yin, Hui Liu, Jiangfeng Xu, and Shian Zhong
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201702945
Link to VoR: http://dx.doi.org/10.1002/chem.201702945
Supported by

10.1002/chem.201702945
Chemistry - A European Journal
Synthesis of Size-tunable Hollow Polypyrrole Nanostructures and Their
Assembly into Folate Targeting and pH-responsive Anti-cancer Drug Delivery
Jian Chen1, Xiufang Li1, Yanhua Sun1, Yuwei Hu1, Yulong Peng2, Yimin Li2, Gang
Yin2,** ,Hui Liu1, Jiangfeng Xu1, Shian Zhong1,*
1College of Chemistry and Chemical Engineering, Central South University,
Changsha Hunan, 410083, P.R. China
2School of Basic Medical Science, Central South University, Changsha, Hunan,
410083, P.R. China
*Corresponding author. College of Chemistry and Chemical Engineering, Central
South University, Changsha Hunan, 410083, P.R. China
E-mail: zhongshian@aliyun.com
**Co-corresponding author. School of Basic Medical Science, Central South
University, Changsha, Hunan, 410083, P.R. China
E-mail: gang.yin@csu.edu.cn
ABSTRACT
Chemotherapeutic drugs currently used in clinical settings have a high toxicity, low
specificity, and short half-life. Herein, polypyrrole (PPy)-based anticancer drug
nanocapsules were prepared by tailoring the nanoparticles size with a template
method, controlling drug release by aromatic imine, increasing nanoparticles stability
through PEGylation, and improving tumor cell selectivity via folate (FA) mediation.
Nanoparticles were characterized by transmission electron microscopy (TEM) and
dynamic light scattering (DLS). α-folate receptor (α-FR) expression level between
tumor cells and normal cells was investigated by western blot and quantitative
polymerase chain reaction (qPCR) analyses. Flow cytometry and fluorescence
imaging were used to verify the cell uptake of the different size nanoparticles. In
terms of different size polypyrrole nanoparticles, the optimal functionalized
nanoparticles of 180 nm hydrodynamic size were chosen and further used in vitro and
in vivo tests. The nanoparticles showed excellent biocompatibility and the
drug-loaded nanoparticles exhibited effective tumor cell growth inhibition in vitro.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
Moreover, the drug-loaded nanoparticles showed a substantially enhanced
accumulation in tumor regions and effectively inhibited in vivo tumor growth.
Furthermore, the nanoparticles showed reduced doxorubicin (Dox) -induced toxicities
and led to no significant side effects in normal organs of tumor bearing mice as
measured by body weight shifts and drug distribution evaluation. Overall, the
functionalized nanoparticles are a promising nanocarrier candidate for tumor targeting
drug delivery.
Keywords: polypyrrole, nanotube, drug carrier, tumor, folate
INTRODUCTION
With recent developments in nanobiotechnology, novel nanoparticles have gained
increasing attention as nanodevices for application in cancer therapy[1]. Drug
encapsulation within nanoparticles offers various advantages, including efficiently
controllable and targeted drug delivery as well as a reduction in adverse effects. It is
well-known that nanoparticles size and surface properties play a key role in
determining their in vivo fate[2]. Defective tumor tissue, which contains gap ranging
from 100 to 1000 nm, favors nanoparticles permeation. However, healthy tissues are
regular and have tight intercellular junctions of less than 10 nm[3]. Therefore, certain
size drug delivery nanocarriers are able to selectively leak into tumor tissue but
cannot be transported into healthy tissue. Therefore, polymeric nanoparticles of less
than 200 nm can easily escape the reticuloendothelial system (RES), thus be able to
passively accumulate in tumor tissues due to the enhanced permeation and retention
(EPR) effect[4]. Furthermore, nanoparticles smaller than 200 nm can prolong the in
vivo circulation time and enhance drug bioavailability[5]. Besides, actively targeted
drug delivery has emerged as a significant method to transport antitumor drugs to
specific tumor sites and decrease the internalization of drugs within normal tissues,
thus reducing side effects and improving the clinical efficiency[6]. In order to further
improve the accumulation of nanocarriers at the tumor sites, their surface is
commonly functionalized with targeting ligands, such as antibodies[7], polypeptides[8],
and FA[9], which have been proved to selectively bind to the overexpressed receptors
on the surface of tumor cells[10]. Normal cells usually also express such receptors but
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
just at a lower level. Analogously, FA (vitamin B9) is involved in 1-carbon transfer
reactions essential for RNA and DNA synthesis. Moreover, the FR is a
glycosylphosphatidylinositol-anchored membrane protein that transports FA into the
cell. FR is differentially expressed in normal and tumor tissues. α-FR, the most
extensively studied family member, is generally expressed in urogenital organs, the
female genital tract, and the placenta. However, high α-FR level have been detected in
different carcinomas, including non-mucinous ovarian carcinoma, endometrial
carcinoma, and cervix carcinoma[11], as well as in non-genital tumors, and are related
to elevated tumor cell cycle progression and proliferation. Therefore, the high
expression of α-FR in cancer cells and the high affinity of FR for FA have led to
extensive researches, which concern its potential role as a target for chemotherapy and
as a diagnostic marker in tumor imaging[12]
.
PPy is an excellent electroactive polymer, which is commonly used for nerve
regeneration[13], drug delivery[14], and other biological applications[15], given its
excellent biocompatibility[16]. PPy materials applied in cancer therapy are in its
primary stage and lead to a breast of research activities. Tang et al. prepared
spindle-like polypyrrole hollow Nanocapsules for chemo–photothermal combination
therapy of cancer cells in Vivo[17]. Wang et al. synthesized near-infrared light and
pH-responsive polypyrrole@polyacrylic acid/fluorescent mesoporous silica
nanoparticles for imaging and chemo-photothermal cancer therapy[18]. Zare et al.
studied polypyrrole nanoparticles for tunable, pH-sensitive and sustained drug
release[19]. The reported PPY-based nanoparticles focus on chemo/photothermal
synergistic therapy combining pH-/photoresponsive drug release, which is very
helpful for the development of efficient anticancer strategy. Given their large inner
volume, nanotubes offer advantages over sheet, rod, and spherical nanoparticles with
regard to the transport of drug molecules[20]. Meanwhile, a number of methods have
been demonstrated to generate hollow PPy nanostructures, while the most versatile
one is based on template-directed synthesis[21]. As well as determining the
morphology of the prepared nanotubes, the template approach offers control of the
dimensions of nanotubes fabrication[22]. The surface of a template, such as a porous
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
membrane, can be directly coated with a layer of oxidants to initiate polymerization.
The main advantages of the template approach are its adaptability to large-scale
fabrication and the tailorability of the diameter of the PPy nanotubes through the use
of templates with various diameters. Meanwhile, given the ease of preparation of PPy
and the flexibility of its surface characteristics, targeting ligands attach onto suitably
sized nanoparticles to achieve both active targeting via the conjugation of receptors
and passive targeting via enhanced permeation and retention effect[23]. In addition,
nanoparticles may be tailored through the use of physical or chemical stimuli
responsiveness, including pH[24], light[25], redox[4], and enzymes[26], to exploit a series
of cellular drug release mechanisms. Among the various stimuli, pH responsivity is
frequently utilized since pH values vary in different tissues and cellular compartments.
The tumor extracellular environment is more acidic (pH 6.2–6.9) than blood and
normal tissues (pH 7.3–7.4), and even more so in cellular endo/lysosomes (pH
4.0–6.0)[24b, c]. The aromatic imine bond, easily formed by amidogens and aromatic
aldehydes[24a], is stable under neutral and alkaline conditions but becomes labile with
an acidic trigger. Therefore aromatic imine bond is an ideal pH-sensitive bond for
tumor environment.
The main objective of the present study was to investigate the effects of particle
size of polymeric nanoparticles functionalized through surface modification on
cellular uptake. Tubular PPy with tunable 60-200 nm diameters were successfully
synthesized using a TiO2 nanotubes array template in the presence of FeCl3 and
pyrrole monomer. Aromatic imine bonds were introduced into the PPy nanoparticles
through reaction with tetra-arm aldehyde (TAA) to prepare a novel pH-triggered
cleavable backbone. A hydrophilic or amphiphilic surface coating, such as
polyethylene glycol (PEG), was added to solubilize the carrier, thus further
prolonging the nanoparticles suspension time in vivo and reducing systemic clearance.
One end of PEG was functionalized with FA as the targeting ligand to bind with α-FR
on the tumor cell surface, whereas the other end was chemically conjugated to the
surface of PPy nanoparticles via a cleavable aromatic imine bond to control release of
drug. Flow cytometry and fluorescence microscopy were used with commercially
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
available fluorescent tagged PPy nanoparticles in the evaluation of particle size effects
on cell endocytosis in vitro. Guided by the optimal size of PPy nanoparticles, the
therapeutic efficacy of Dox-loaded PPy nanoparticles was demonstrated both in vitro
and in vivo. The overall procedure for size-tunable PPy nanotubes synthesis
combining with pH-triggered drug release and α-FR targeting was designed for
targeted drug delivery to tumor tissues (Scheme 1).
Scheme 1. Schematic illustration of PPy-based drug delivery system responding to
mildly acidic tumor tissues.
RESULTS AND DISCUSSION
Synthesis and Characterization of PPy@TAA@PEG@FA. The detailed
synthesis protocol of PPy@TAA@PEG@FA is shown in Scheme 1 and Supporting
Information (Figure S1-S5). PPy nanotubes were initially prepared via chemical
polymerization of 1-(2-aminoethyl)pyrrole (Supporting Information, Figure S 1)
through a template method. Figure 1 (a) shows the TEM images of the synthesized
PPy nanotubes within the pores of the TiO2 template membrane. The dimensions of
the PPy nanotubes were tailored in accordance with the diameter of the TiO2 template
used. PPy nanotubes of 60, 98, 120, 170, and 200 nm in diameter were obtained. The
synthesis and characterization of the TiO2 template were detailed in Figure 1 (a) and
Supporting Information (Table S 1)[27]
.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
PPy/PEG nanoconstructs were engineered by conjugation of the intermediate TAA
through a aromatic imine bond. Aromatic imine bond formation was difficult in the
acidic environment, which introduced a dynamically pH-sensitive manner. TAA,
formed through the reaction of pentaerythrityl tetrabromide(Supporting Information,
Figure S 2)[28] with p-hydroxy benzaldehyde[29], is a four-arm aldehyde (Supporting
Information, Figure S 3). TAA plays an essential role in the controlled release of
drugs and in the formation of a hydrophobic layer. One arm of TAA was attached onto
the PPy nanotubes backbone, while the remaining three arms were present at the end
of the PEG or FA@PEG (Supporting Information, Figure S 4) chains. The aldehyde
group of TAA reacted with the amino group of PPy surface to form aromatic imine
bond. Consequently, the TAA was coupled with PPy, as shown in enlargement of TAA
in Scheme 1.The star-branched linker (TAA) was designed to increase the proportion
of PEG on the PPy nanotubes surface. The overall functionalization of the
nanoparticles was considered to act as visualized microcapsules for drug
encapsulation and additionally as drug delivery.
The surface functionalization of PPy was characterized by Fourier Transform
Infrared Spectroscopy (FT-IR, Figure 1 (b)) and Thermaogravimetric analysis (TGA,
Figure 1 (c)). At the beginning of polymerization, the pyrrole monomer was modified
with an amino group, namely 1-(2-aminoethyl)pyrrole[30]. The FT-IR spectrum of PPy
showed a C-H stretching vibration at 2920 cm–1and a N-H stretching vibrations at
3452 cm–1, thus confirming that the amino ethyl group was retained on the PPy
surface. Peaks at 1639 and 1480 cm–1 were associated with the C-C and C-N
stretching vibrations of the PPy ring. The surface of PPy was first functionalized with
TAA to obtain PPy@TAA. The presence of the aromatic aldehyde group on the PPy
surface was confirmed by two symmetrical peaks, one at 2827 cm–1 and another at
2728 cm–1, representing the C-H stretching vibration of CHO. The peak at 1688 cm–1
was assigned to the C=N stretching vibration of the aromatic imine bond. Peaks at
1604, 1570 and 1507 cm–1could be ascribed to benzene C=C vibration of TAA and the
peak of 3071 cm–1 could be assigned to the benzene C-H vibration of TAA. A
characteristic peak at 2920 cm–1 was due to an overlap of the alkane segment of PPy
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
and TAA. For PPy@TAA@PEG, the C-O ether absorption bands at 1267, 1183, 1135,
and 1068 cm–1 confirmed the existence of PEG. In comparison, the spectra of
PPy@TAA@PEG@FA showed a significant increase in adsorption at 3076 and 3119
cm–1, which could be attributable to the absorption O-H of COOH group and N-H of
NH2 group of FA as a result of the conjugation of FA on PEG. The characteristic
peaks at 1742, 1508 and 1441 cm–1 corresponded to the C=N, C=O and C-N
stretching bands of FA. Furthermore, a slight shift in peak positions and the additional
number of peaks also suggested the presence of FA.
The successful functionalization of the nanoparticles was further confirmed by
TGA (Figure 1 (c)). The weight losses of PPy, PPy@TAA, PPy@TAA@PEG and
PPy@TAA@PEG@FA from 100 to 900°C were 53.5%, 60.4%, 70.4% and 71.3%,
respectively. For PPy, there was no significant mass loss from 100 to 300°C . Major
weight loss began at 400°C , possibly due to the decomposition of PPy[31]. The turning
point at 600°C was possibly ascribed to the breakdown of aminoethyl group grafted
on the PPy surface. For PPy@TAA, the weight loss percentage was 6.9% larger
compared to PPy, indicating that TAA was successfully immobilized onto the surface
of the nanotubes. For PPy@TAA@PEG and PPy@TAA@PEG@FA, a faster
decomposition was observed compared to PPy and PPy@TAA due to PEG
degradation occurring at 300°C [32]. Given the similarities between PEG and PEG@FA,
no significant mass loss differences were observed between PPy@TAA@PEG and
PPy@TAA@PEG@FA.
The component analysis of the sample was carried out using X-ray photoelectron
spectroscopy (XPS, Figure 1 (d)). The coexistence of C,N and O elements was
evidenced by the main peaks at 284, 400 and 532 eV. The O1S spectra of PPy might be
derived from surface adsorbed water. The relatively atomic ratio of C:N:O, which
could express the material composition from PPy to PPy@TAA, PPy@TAA@PEG
and PPy@TAA@PEF@FA. The data analysis was carried out using XPS PEAK41
software by least-sqares fitting of Gaussian-Lorentzian lineshapes to the
photoelectron peaks after subtracting a Shirley background, as shown in Figure S 6
[33].The C1S spectra of PPy could be divided into two peaks: C-N of PPy at 285.43 eV
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
and C=C of PPy at 284.53 eV. The C1S spectrum of PPy@TAA was fitted into three
contributions, which could be assigned to C-O of TAA and C=C of PPy@TAA at
284.5 eV, C-N of PPy and C=N of PPy@TAA at 286.02 eV and C=O of TAA at 287.1
eV. The C1S spectrum of PPy@TAA@PEG exhibited two resolved doublets, resulting
from C-C and C-O-C of PEG located at 283.8 eV and 285.3 eV and the C1S spectra of
PPy and TAA could still be fitted[34]. The shift in peak pattern of PPy@TAA@PEG
when comparing the C1S spectrum of PPy@TAA provided evidence of the
composition. The C1S spectra of PPy@TAA@PEG@FA could be separated into four
peaks. The two main peaks, present at 283.7 and 285.01 eV, were characteristic peaks
of PEG. The other two peaks with a lower intensity, located at 284.5 and 286.01 eV,
were due to C element of PPy and FA. It was worth noting that one characteristic peak
belonged to several species, whose chemical environment were too close. The binding
energy N1S peak of PPy at 399.52 and 400.97 eV were due to NH2 and C-N-C of PPy.
Comparing to N1S peak of PPy, the N1S peaks of PPy@TAA had a C=N (402.01 eV)
signal and exhibited a large distribution. There was NH2 signal in N1S spectra of
PPy@TAA that was related to unreacted NH2 group with aromatic aldehyde group.
The similar trend could be seen for N1S spectra of PPy@TAA@PEG and
PPy@TAA@PEG@FA. The increase of the signal at 402.09 eV for N1S spectra of
PPy @TAA@PEG@FA could attribute to FA. The O1S fitting of PPy@TAA was
obtained due to C=O of TAA at 530.98 eV and C-O-C of TAA at 532.01 eV. The bing
energy at 531.94 eV of PPy@TAA@PEG when comparing PPy@TAA provided
evidence of C-O-C signal of PEG. O1S peak of PPy@TAA@PEG@FA became much
broadened and could be overlapping at 531.5 eV assigned to C=O of TAA and COO-
of FA, and at 532.98 eV assigned to C-O-C of PEG and OH of FA.
The hydrodynamic diameter of PPy nanoparticles following hydrophilic PEG
coating in an aqueous medium was assessed. The hydrodynamic particle size and the
zeta potential of PPy (I, II, III, IV, and V), PPy@TAA (I, II, III, IV, and V),
PPy@TAA@PEG (I, II, III, IV, and V), and PPy@TAA@PEG@FA (I, II, III, IV, and
V) were measured by DLS (the results are presented in Supporting Information Figure
S 7 (a) and Table S 2). The PPy and PPy@TAA nanoparticles were highly
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
agglomerated prior to PEG or PEG@FA functionalization (Figure S 7 (b)). Following
surface modification by PEG or PEG@FA, the obtained nanoparticles were
homogeneously suspended in a PBS solution (Figure S 7 (b)), with the hydrodynamic
size of the nanoparticles being reduced universally from the micro- to the nano-scale
(Figure S 7 (a)). As shown in Table S 2, the reversed change in zeta potential of
PPy@TAA was relative to the incorporation of TAA in comparison with pure PPy.
The zeta potential of PPy@TAA@PEG and PPy@TAA@PEG@FA fell within -20 to
-40 mV, indicating the successful introduction of PEG or PEG@FA groups.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
Figure 1 (a) TEM images (top) of PPy nanotubes (I, II, III, IV, and V) with the
corresponding scanning electron microscopy (SEM) images (bottom) of different
TiO2 nanotubes template dimensions as well as the schematic of template method
(middle). (b) FT-IR spectra of PPy, PPy@TAA, PPy@TAA@PEG, and
PPy@TAA@PEG@FA. (c) TGA curves of PPy, PPy@TAA, PPy@TAA@PEG, and
PPy@TAA@PEG@FA. (d) XPS spectra of PPy, PPy@TAA, PPy@TAA@PEG, and
PPy@TAA@PEG@FA.
α-FR Expression Level Evaluation between Tumor Cells and Normal Cells. In
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
order to verify the FA targeting capability of the prepared nanoparticles, cell surface
α-FR expression was first assessed. The expression of α-FR protein on the tumor cells
was investigated with western blotting[35] using a highly specific polyclonal antibody.
Two clear α-FR protein bands were observed for tumor cells (human ovarian
carcinoma (Skov3) cells) but was negligible for normal cells (human embryonic
kidney (HEK 293T) cells) (Figure 2). To gain further insight on α-FR expression, we
analyzed the effect in microRNA (miRNA) level. qPCR is a sensitive technique to
estimate the expression level of circulating miRNAs[36], with reference genes being
used as an internal control (β -actin) for reliable normalization of target miRNA data.
qPCR analysis showed a significantly higher α-FR mRNA (FOLR1)[37] level in Skov3
compared with HEK 293T, a result consistent with the western blot analysis (Figure
2). Therefore, the high levels of FOLR1 gene expression led to high α-FR protein
levels.
Figure 2. Assessment of α-FR protein expression in Skov3 and HEK 293T cells using
western blotting (molecular weight markers were noted and the bands were indicated
by dashed boxes) and relative expression levels of FOLR1 gene determined by qPCR.
P values of less than 0.05 were considered significant (*P < 0.005). β -actin is used
to normalize protein expression due to its high conservation as an endogenous
housekeeping gene.
In Vitro Cell Uptake of Different Sized Nanoparticles. In order to verify the
tumor-targeted uptake capacity of different sized nanoparticles in tumor cells, flow
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
cytometry analysis was carried out to quantitatively evaluate the cellular uptake
behavior of different sized coumarin-6-labeled PPy@TAA@PEG@FA nanoparticles
according to mean fluorescence intensity (MFI) in the two cell lines (Skov3 and HEK
293T) (Figure 3). In order to assess cell uptake of nanoparticles according to particle
size, the incubation time and coumarin-6 concentration were fixed. For Skov3 cells,
PPy@TAA@PEG@FA (Ⅰ) had a MFI value of 3834, approximately 1.44-fold higher
than that of PPy@TAA@PEG@FA (Ⅴ), having efficient interfacial interaction with
the cell membrane and suggesting prominent cellular uptake of the smaller sized
nanoparticles. The Skov3 cellular uptake of PPy@TAA@PEG nanoparticles was at a
low MFI, indicating that nanoparticles unconjugated FA could not interact with α-FR
of Skov 3 cells. Regardless of particle size, no obvious fluorescence was observed
from HEK 293T incubated with PPy@TAA@PEG and PPy@TAA@PEG@FA
nanoparticles. Certainly, the two cell lines decelerated the internalization of
PPy@TAA@PEG nanoparticles attributed to non-specific endocytosis. Therefore, the
selectivity of PPy@TAA@PEG@FA for tumor cells over normal cells was high, in
particular for smaller sized nanoparticles, due to the overexpression of α-FR on the
surface of Skov3.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
Figure 3. Flow cytometry analysis of Skov3 (a) and HEK 293T (b) cells treated with
different sized coumarin-6-labelled nanoparticles for 12 h. Each bar represents the
mean ± SD (n = 3).
Following quantitative measurement of the cellular uptake of the different sized
nanoparticles, qualitative analysis demonstrated the cellular association of
coumarin-6-labelled nanoparticles in a time-dependent manner under a fluorescent
microscope (Figure 4 and Supporting Information Figure S 8). The smaller the
PPy@TAA@PEG@FA nanoparticles size, the faster the cellular uptake by Skov3
cells, especially for nanoparticles I and II (Figure 4 (a, b)), while far less
PPy@TAA@PEG nanoparticles were internalized in Skov3 cells (Figure S 8 (a, b)).
These results also indicate that the presence of the FA motif improved the
nanoparticles’ ability to bind onto the tumor cells, leading to enhanced cellular uptake.
As expected, a rather feeble fluorescence in HEK 293T cells was observed as a
function of time regardless of treatment with PPy@TAA@PEG@FA or
PPy@TAA@PEG nanoparticles. This was attributed to the rare internalization and not
easily hydrolyzing aromatic imine bond of these nanoparticles in HEK 293T cells.
Thus, the smaller nanoparticles exhibited a noticeable enhancement in tumor cell
uptake , which is consistent with the results of flow cytometry. The still weak
fluorescence was observed after 24 h incubation in Supporting Information Figure S 8
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
(a, d) , which could be the ascribed to cells desquamation and fluorescence
quenching of coumarin-6. Nevertheless, the nanoparticles I system exhibited the
lowest drug loading (only 6.15 wt%, Table S 2) and was therefore not used for the
follow-up experiments. Instead, the nanoparticles II system (180 nm hydrodynamic
size) were chosen, which had a reasonable size, suitable drug loading, and the
efficient cellular uptake.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
Figure 4. fluorescence images of Skov3 cells incubated with coumarin-6-loaded
PPy@TAA@PEG@FA under different indicated times. Coumarin-6-loaded
nanoparticles appeared in green and the nuclei stained with DAPI in blue, Scale bars:
20 m.
In Vitro Release of Dox from Nanoparticles. The release behavior of Dox-loaded
nanoparticles was discussed in medium of different pH values (pH 2.5, 4.5, 6.5, 7.4,
8.0, and 9.1). As shown in Figure 5 (a), the release behavior of nanoparticles showed a
clear pH-dependent feature: the lower the pH, the faster the drug release. The
accumulative Dox release was approximately 80% at pH 2.5 after 15 h, with similar
release profiles at pH 4.5 and 6.5 (simulating the pH of endosome and lysosome,
respectively). At these pH values, the assembled nanostructure would dissociate due
to cleavage of the aromatic imine bond. To be more specific, the hydrophobic TAA
shell attached onto the PPy nanotubes surface would maintain Dox within a
hydrophobic chamber, and be hydrolyzed in an acidic aqueous environment gradually.
Interestingly, no significant burst release was observed at pH 7.4 (simulating the pH
of blood), 8.0, and 9.1, and only less than 10% of Dox leaked out throughout the 40-h
period. Yet, after acidification of the medium, the drug was released (Figure 5 (b)),
indicating the relatively robust host-guest interactions of the nanocarrier. Therefore,
drug encapsulation at physiological conditions (pH 7.4) would minimize premature
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
drug leakage in the blood circulation before reaching tumor tissues, resulting not only
in reduced systemic toxicity but also in enhanced drug bioavailability at the target
sites.
Figure 5. Release behavior of Dox-loaded nanoparticles in the buffer solution at
different pH values (a) and release behavior of Dox-loaded nanoparticles in the buffer
solution (pH 7.4) triggered by the addition of different acidic pH values buffer
solution (b). Data are presented as mean ± SD (n = 3).
In Vitro Cytotoxicity of Nanoparticles and Antitumor Effect of Dox Loaded
Nanoparticles. To determine whether the nanoparticles were a safe and efficient drug
vehicle, the in vitro cytotoxicity of nanoparticles was evaluated through cell viability
against the Skov3 and HEK 293T cells using a CCK-8 assay (Figure 6 (a, b)). Given
that the nanoparticles would dissociate during circulation, the two cell lines were
exposed to serial nanoparticles concentrations of the various functionalizations,
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
namely PPy, PPy@TAA, PPy@TAA@PEG, and PPy@TAA@PEG@FA, for 24 h to
evaluate their individual toxicity. The drug-free nanoparticles with different
functionalization showed no or low cytotoxicity, even up to the highest testing
concentration of 2000 g/mL (Figure 6 (a, b)), indicating their excellent
biocompatibility.
Following verification of the safety of the nanoparticles, The cell growth
inhibitory effect of Skov3 and HEK 293T cells treated with free Dox or an equivalent
dose of Dox-loaded onto either PPy@TAA@PEG or PPy@TAA@PEG@FA was
observed (Figure 6 (c, d)). The cytotoxic efficacy of Dox/PPy@TAA@PEG@FA
against Skov3 cells was higher than that of Dox/PPy@TAA@PEG, indicating that the
targeting of the nanoparticles to Skov3 cells through FR-mediated endocytosis
enhanced the internalization of nanoparticles and hence led to a significant percentage
of Dox release. In addition, the IC50 (half maximal inhibitory concentration) of Dox
against Skov3 was 1.126 g/mL, while that of Dox/PPy@TAA@PEG@FA was 6.594
g/mL. According to their IC50, the toxic effect of Dox/PPy@TAA@PEG@FA was
significantly lower than that of free Dox for the reason that Dox could easily cross the
tumor cell membrane via free diffusion, while Dox/PPy@TAA@PEG@FA required
endocytosis and traded with acidic endosomes/lysosomes to release the drug.
Furthermore, the release of Dox from PPy@TAA@PEG@FA was pH-sensitive as
indicated by the in vitro release studies, with less than 20% of the total Dox not being
released after 24 h (Figure 6 (b)). To further evaluate the selectivity of
Dox/PPy@TAA@PEG@FA against tumor cells over normal cells, the cell viability of
HEK 293T cells treated with free Dox, Dox/PPy@TAA@PEG, and
Dox/PPy@TAA@PEG@FA was assessed at different Dox doses. HEK 293T cells
were less sensitive to Dox/PPy@TAA@PEG@FA compared with Skov3 cells, while
both of them were equally sensitive to free Dox. The selective tumor cell growth
inhibition could be attributed to the cooperative effects of the right nanoparticles size,
FA-mediated endocytosis, and pH-triggered Dox release. In addition, the drug-free
nanoparticles tested at high concentrations caused no obvious cytotoxicity towards the
Skov3 cells, indicating that the cytotoxicity was only ascribed to the release of Dox
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
from nanoparticles rather than from the nanoparticles themselves.
Figure 6. Cell viability of Skov3 and HEK 293T cells following incubation with as
prepared (a, b) and Dox-loaded (c, d) nanoparticles at equivalent Dox concentrations
for 24 h. Data were presented as mean ± SD (n = 6).
In Vivo Biodistribution of Nanoparticles. To investigate the potential of
PPy@TAA@PEG@FA nanoparticles to specifically deliver drugs to tumor tissues,
the biodistribution of Dir-loaded nanoparticles (9.3 % Dir for PPy@TAA@PEG@FA
and 9.62 % Dir for PPy@TAA@PEG) in tumor xenograft mice was evaluated by
real-time fluorescence monitoring. As shown in Figure 7, the fluorescence signal of
Dir/PPy@TAA@PEF@FA in the tumor region was weak at 4 h, sensible at 12 h, and
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
strong at 24 h. Additionally, the tumor and main organs were dissected from mice
after 24 h and the strongest fluorescence intensity was observed in the tumor tissue,
indicating that the PPy@TAA@PEG@FA nanoparticles were stable during blood
circulation and were accumulated into the tumor with a long retention time. In
contrast, the biodistribution of PPy@TAA@PEG nanoparticles was relatively spread
out in mice, as observed by the relatively strong fluorescence intensity in tumor and
liver tissue, due to the passive nanoparticles accumulation in tumors by the EPR effect
and the liver being the main metabolism organ for elimination in mice. Thus, the
prepared nanoparticles showed superior tumor-targeting capability with negligible
accumulation in non-tumor regions.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
Figure 7. In vivo fluorescence imaging and biodistribution analysis of Dir loaded
PPy@TAA@PEG@FA and Dir loaded PPy@TAA@PEG nanoparticles in
tumor-bearing nude mice. (a) The time-dependent images of whole body showing the
distritution of nanoparitcles and ex vivo fluorescence images of major organs and
tumors after injection of nanoparticles at 24 h. &, the fluorescence images (upside)
correspond to the optical photographs (downside). The tumors are circled with black
line. (b) Quantitative biodistribution of Dir loaded PPy@TAA@PEG@FA and Dir
loaded PPy@TAA@PEG nanoparticles in nude mice at 24 h. *P <0.01; **P <0.05; &,
not significant, as compared with PPy@TAA@PEG group.
Dox Loaded Nanoparticles to Inhibit Tumor Growth in a Skov 3
Tumor-bearing Nude Mouse Model. The relative tumor volume and tumor weight
are shown in Figure 8 (a, b) and Supporting Information Figure S 9. All the
drug-treated groups were effective in preventing tumor growth compared to PBS
alone from the second therapy. The PPy@TAA@PEG@FA group exhibited the
greatest tumor inhibitory effect after 21 days treatment, with a relative tumor volume
reduction of 557.1%. The PPy@TAA@PEG@FA group resulted in a tumor weight of
0.2 g, showing the lowest tumor growth. This suggested a strong antitumor efficacy
and consistented with the in vitro cytotoxicity assay (Figure 6 (c)). Additionally, the
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
PPy@TAA@PEG group was also effective in tumor growth inhibition, but less than
the PPy@TAA@PEG@FA group. The free-Dox group only showed moderate
antitumor efficacy. Such differences demonstrate that PPy@TAA@PEG@FA and
PPy@TAA@PEG of 180 nm (hydrodynamic size) are ideal for passive accumulation
into tumor tissues via the EPR effect and that active FA targeting of
PPy@TAA@PEG@FA nanoparticles improved tumor particle accumulation.
The body weight of mice in the free-Dox group was unchanged along with
successive administration and the skin of mice was rough during the late period of
treatment (Figure 8 (c) and Figure S 9). Dox is an effective anticancer agent widely
used in clinical therapy. However, the high level of Dox in normal organs generates
toxic side effects, such as cumulative cardiotoxicity, myelosuppression, and
nephrotoxicity, leading to stop weight gain, as described in the above result.
Compared to the free-Dox group, the bodyweight of the PPy@TAA@PEG@FA and
PPy@TAA@PEG groups increased over the 21-day treatment, indicating that
Dox-loaded nanoparticles may effectively reduce the side effects of Dox. The large
tumors lead to bodyweight loss of mice in the PBS group (7.1% bodyweight loss from
initial weight) during the late period. The above data confirms that the appropriate
size (Figure 1 (a) and Table S 2), their effective encapsulation of Dox (Figure 5), and
low level systemic toxicity of the nanoparticles (Figure 6 (a)) make these
nanoparticles (PPy@TAA@PEG@FA) an effective formulation for tumor
suppression.
Tissue Distribution Studies in Tumor-bearing Mice. The total Dox levels in
tumor, heart, liver, spleen, lung, and kidney after free Dox or Dox-loaded
nanoparticles treatment are shown in Figure 8 (d). The tissue distribution
characteristics of Dox were significantly different compared with
Dox/PPy@TAA@PEF@FA, Dox/PPy@TAA@PEG, and free Dox group. As
anticipated, the total content of Dox in tumors for the Dox/PPy@TAA@PEG@FA
group was 1.8- and 3.12-fold higher than that of the Dox/PPy@TAA@PEG and
free-Dox groups, respectively. The biodistribution characteristics of the nanoparticles
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
were determined by the physicochemical properties of particle size and surface
modification. Generally, PEGylation of nanoparticles surfaces increased their
hydrophilicity and blocked electrostatic and hydrophobic interactions, resulting in
decreased liver, kidney, and spleen uptake. This was attributed to steric and hydration
repulsions of PEG[38]. Extensive accumulation was also observed in tumors in the
Dox/PPy@TAA@PEG group, likely due to passive accumulation into tumor tissues
via the EPR effect, suggesting that the appropriate particle size played a more
important role in improving the nanoparticles biodistribution profiles. Contrary to a
relatively strong fluorescence signal of Dir/PPy@TAA@PEG@FA and
Dir/PPy@TAA@PEG group in liver (Figure 7), the low Dox accumulation of
Dox/PPy@TAA@PEG@FA and Dox/PPy@TAA@PEG group in liver was attributed
to acidic-trigger release of Dox-loaded nanoparticles. Even though the Dox-loaded
nanoparticles were retained in liver and other organs, the Dox was not released
randomly. The free-Dox group displayed considerable Dox accumulation in all the
tissues but lung. Thus, in association with material characterizations (Figure 1), in
vitro cellular uptake (Figure 3 and 4), and drug release (Figure 5) results, a longer
circulation time was achieved by PEGylation, tumor accumulation of the
nanoparticles occurred by passive and FA targeting, and drug release was controlled
by the TAA guard.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
Figure 8. In vivo antitumor efficacy of nanoparticles in a Skov3 tumor-bearing mice
model. (a) Tumor volume changes of different groups according to time. *P <0.01;
**P <0.05, as compared with free-Dox group; #P <0.01; ##P <0.05, as compared with
PBS group. (b) Bodyweight shifts from different groups. *P <0.0001, as compared
#
with free-Dox group; P <0.0001, as compared with PBS group. (c) Tumor weight
after 21 days treatment. *P <0.0005; **P <0.05, as compared with free-Dox group; #P
##
<0.001, P <0.0005, as compared with PBS group. (d) Distribution profiles of total
&
Dox in tissues after intravenous administration of Dox-loaded nanoparticles n = 5; ,
not significant; *P <0.0001; **P <0.005, as compared with free-Dox group. All data
were expressed as mean ± SD, n = 5. (e) Hematoxylin and eosin stained with tumor
and heart sections. Nuclei were stained blue and cytoplasm were stained red, Scale
bars: 20 m.
Histological Analysis. Histological analysis also supported the excellent
therapeutic effect of the nanoparticles. As shown in Figure 8 (e), tumors treated with
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
free-Dox and PBS generally consisted of tightly packed tumor cells and some necrotic
regions as a result of fast tumor cell growth. However, extensive fragmentation and
nuclear shrinkage were observed in the Dox/PPy@TAA@PEG@FA and
Dox/PPy@TAA@PEG group tumors. This is attributed to the advantages of
nanoparticles: (1) the potential stability in blood circulation, (2) efficient delivery of
Dox into the tumor cells, and (3) the high intracellular drug concentration to inhibit
tumor cell growth. To evaluate the myocardial damage induced by the Dox treatments,
histological
changes
of
cardiomyocytes
were
observed.
The
Dox/PPy@TAA@PEG@FA, Dox/PPy@TAA@PEG, and PBS groups showed no
significant morphological changes and had compact cardiomyocytes in an ordered
arrangement with clear structures. However, the free-Dox group exhibited disordered
myofibrillar structures, characterized by acute myocardial damage.
CONCLUSION
In summary, taking advantage of the template method, different size
PPy@TAA@PEG@FA nanoparticles were synthesized. The physicochemical
characteristics of the nanoparticles were determined by TEM, FT-IR, TGA, and DLS.
Flow cytometry and fluorescence imaging analyses showed that cellular uptake
exhibited size effect and targeting effect, and PPy@TAA@PEG@FA nanoparticles of
180 nm hydrodynamic size had higher Skov3 cell internalization. In vitro cytotoxicity
assays confirmed that Dox/PPy@TAA@PEG@FA nanoparticles induced Skov3
tumor cell death. In vivo studies demonstrated that Dox-loaded
PPy@TAA@PEG@FA nanoparticles exhibited the highest antitumor activity,
followed by PPy@TAA@PEG, compared to free Dox. Importantly, we successfully
developed and used the PPy model as a drug carrier. The nanoparticles design
combines the size effect as well as active targeting, opening up a new path to the
establishment of functionalized drug nanocarriers. Further improvements on the
nanoconstruction should focus on the drug loading capacity of the system.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
ACKNOWLEDGEMENTS
This project is financially supported by the National Natural Science Foundation of
China (No. 21276283, 21576295, 81201675 and 81572900) and the Open-End Fund
for the Valuable and Precision Instruments of Central South Univeristy
(CSUZC201725). We thank the MRI facility of Modern Analysis and Testing Center
of Central South University for technical support.
REFERENCES
[1] K. Riehemann, S. W. Schneider, T. A. Luger, B. Godin, M. Ferrari and H. Fuchs, Angew. Chem. Int. Ed.
2009, 48, 872-897.
[2] X. Lin, N. Zhao, P. Yan, H. Hu and F. J. Xu, Acta Biomater. 2015, 11, 381-392.
[3] G. A. Hughes, Nanomedicine 2005, 1, 22-30.
[4] Z. Y. Li, J. J. Hu, Q. Xu, S. Chen, H. Z. Jia, Y. X. Sun, R. X. Zhuo and X. Z. Zhang, J. Mater. Chem. B 2015,
3, 39-44.
[5] S. S. Han, Z. Y. Li, J. Y. Zhu, K. Han, Z. Y. Zeng, W. Hong, W. X. Li, H. Z. Jia, Y. Liu, R. X. Zhuo and X. Z.
Zhang, Small 2015, 11, 2543-2554.
[6] Y. Hu, J. Chen, X. Li, Y. Sun, S. Huang, Y. Li, H. Liu, J. Xu and S. Zhong, Nanotechnology 2017, 28,
375101.
[7] R. V. Chari, M. L. Miller and W. C. Widdison, Angew. Chem. Int. Ed. 2014, 53, 3796-3827.
[8] F. Xu, J. Liu, J. Tian, L. Gao, X. Cheng, Y. Pan, Z. Sun and X. Li, ACS Appl. Mater. Interfaces 2016, 8,
29906-29914.
[9] a) W. Zhang, Z. L. Yu, M. Wu, J. G. Ren, H. F. Xia, G. L. Sa, J. Y. Zhu, D. W. Pang, Y. F. Zhao and G. Chen,
ACS nano 2017, 11, 277-290; b) N. C. Fan, F. Y. Cheng, J. A. Ho and C. S. Yeh, Angew. Chem. Int. Ed.
2012, 51, 8806-8810; c) S. A. Kularatne, V. Deshmukh, M. Gymnopoulos, S. L. Biroc, J. Xia, S. Srinagesh,
Y. Sun, N. Zou, M. Shimazu, J. Pinkstaff, S. Ensari, N. Knudsen, A. Manibusan, J. Y. Axup, C. H. Kim, V. V.
Smider, T. Javahishvili and P. G. Schultz, Angew. Chem. Int. Ed. 2013, 52, 12101-12104; d) S. K. Jones, V.
Lizzio and O. M. Merkel, Biomacromolecules 2016, 17, 76-87.
[10] M. C. Planca-wessels, G. C. Booth, A. J. Convertine, B. B. Lundy, G. Y. Berguig, M. F. Press, P. S.
Stayton and O. W. Press, Oncotarget 2016, 7, 9561-9575.
[11] C. O. Evans, P. Reddy, D. J. Brat, E. B. O'Neill, B. Craige, V. L. Stevens and N. M. Oyesiku, Cancer Res.
2003, 63, 4218-4224.
[12] J. A. Ledermann, S. Canevari and T. Thigpen, Ann. of Oncol. 2015, 26, 2034-2043.
[13] a) L. Yan, B. Zhao, X. Liu, X. Li, C. Zeng, H. Shi, X. Xu, T. Lin, L. Dai and Y. Liu, ACS Appl. Mater.
Interfaces 2016, 8, 6834-6840; b) Z. F. Zhou, F. Zhang, J. G. Wang, Q. C. Chen, W. C. Yang, W. Z. Yang, N.
He, Y. Y. Jiang, F. Chen and J. J. Liu, ACS Biomater. Sci. Eng. 2016, 2, 1572-1581.
[14] a) N. Hosseini Nassab, D. Samanta, Y. Abdolazimi, J. P. Annes and R. N. Zare, Nanoscale 2017, 9,
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
143-149; b) D. Uppalapati, B. J. Boyd, S. Garg, J. Travas-Sejdic and D. Svirskis, Biomaterials 2016, 111,
149-162.
[15] Q. Li, C. Yu, R. Gao, C. Xia, G. Yuan, Y. Li, Y. Zhao, Q. Chen and J. He, Biosens. Bioelectron. 2016, 80,
674-681.
[16] S. H. Bauquier, K. J. McLean, J. L. Jiang, R. C. Boston, A. Lai, Z. Yue, S. E. Moulton, A. J. Halliday, G.
Wallace and M. J. Cook, Macromol. Biosci. 2017, 17.
[17] Y. Wang, Y. Xiao and R. Tang, Chem. Eur. J. 2014, 20, 11826-11834.
[18] M. Zhang, T. Wang, L. Zhang, L. Li and C. Wang, Chem. Eur. J. 2015, 21, 16162-16171.
[19] D. Samanta, J. L. Meiser and R. N. Zare, Nanoscale 2015, 7, 9497-9504.
[20] P. M. George, D. A. LaVan, J. A. Burdick, C. Y. Chen, E. Liang and R. Langer, Adv. Mater. 2006, 18,
577-581.
[21] G. Chen and C. Mao, Nanoscale 2016, 8, 10026-10029.
[22] J. Zhang, Y. Li, F. F. An, X. Zhang, X. Chen and C. S. Lee, Nano Lett. 2015, 15, 313-318.
[23] Q. Zhang, F. Liu, K. T. Nguyen, X. Ma, X. Wang, B. Xing and Y. Zhao, Adv. Funct. Mater. 2012, 22,
5144-5156.
[24] a) S. Y. Li, L. H. Liu, H. Z. Jia, W. X. Qiu, L. Rong, H. Cheng and X. Z. Zhang, Chem. Commun. 2014,
50, 11852-11855; b) C. Y. Sun, S. Shen, C. F. Xu, H. J. Li, Y. Liu, Z. T. Cao, X. Z. Yang, J. X. Xia and J. Wang,
J. Am. Chem. Soc. 2015, 137, 15217-15224; c) S. S. Han, Z. Y. Li, J. Y. Zhu, K. Han, Z. Y. Zeng, W. Hong, W.
X. Li, H. Z. Jia, Y. Liu, R. X. Zhuo and X. Z. Zhang, Small 2015, 11, 2543-2554.
[25] a) N. C. Fan, F. Y. Cheng, J. A. Ho and C. S. Yeh, Angew. Chem. Int. Ed. 2012, 51, 8806-8810; b) W.
Chen, J. Ouyang, H. Liu, M. Chen, K. Zeng, J. Sheng, Z. Liu, Y. Han, L. Wang, J. Li, L. Deng, Y. N. Liu and S.
Guo, Adv. Mater. 2017, 29; c) K. Han, W. Y. Zhang, J. Zhang, Q. Lei, S. B. Wang, J. W. Liu, X. Z. Zhang and
H. Y. Han, Adv. Funct. Mater. 2016, 26, 4351-4361.
[26] J. J. Hu, L. H. Liu, Y. Z. Li, R. X. Zhuo and X. Z. zhang, J. Mater. Chem. B 2016, 4, 1932-1940.
[27] a) Y. Wang, J. Chen, C. Zhou, L. Zhou, Y. Kong, H. Long and S. Zhong, Electrochim. Acta 2014, 115,
269-276; b) J. Chen, Y. Kang, C. Li, H. Chen, L. Sun, Y. Wang and S. Zhong, J. Electrochem. Soc. 2016, 164,
B66-B73.
[28] a) D. E. Applequst, G. F. Fanta and B. W. Henrikson, The Journal of Organic Chemistry 1958, 23,
1715-1716; b) S. F. Marrian, Chem. Rev. 1948, 43, 149-202.
[29] R. Banerjee, S. Maiti and D. Dhara, Green Chem. 2014, 16, 1365-1373.
[30] Y. He, M. Lin, Z. Li, X. Liang, G. Li and J. C. Antilla, Org. Lett. 2011, 13, 4490-4493.
[31] S. Bose, T. Kuila, M. E. Uddin, N. H. Kim, A. K. T. Lau and J. H. Lee, Polymer 2010, 51, 5921-5928.
[32] Q. Zhang, F. Liu, K. T. Nguyen, X. Ma, X. Wang, B. Xing and Y. Zhao, Adv. Funct. Mater. 2012, 22,
5144-5156.
[33] a) S. Sharma, R. W. Johnson and T. A. Desai, Biosens. Bioelectron. 2004, 20, 227-239; b) C.
Malitesta, I. Losito, L. Sabbatini and P. G. Zambonin, J. Electron Spectrosc. Relat. Phenom. 1995, 76,
629-634.
[34] P. Kingshott, H. Thissen and H. J. Griesser, Biomaterials 2002, 23, 2043-2056.
[35] C. O. Evans, P. Reddy, D. J. Brat, E. B. O'Neill, B. Craige, V. L. Stevens and N. M. Oyesiku, Cancer res.
2003, 63, 4218-4224.
[36] X. Fu, Y. Li, A. Alvero, J. Li, Q. Wu, Q. Xiao, Y. Peng, Y. Hu, X. Li, W. Yan, K. Guo, W. Zhou, Y. Wang, J.
Liu, Y. Zhang, G. Mor, J. Wen and G. Yin, Oncotarget 2016, 7, 80633-80654.
[37] Y. Yuan, D. A. Nymoen, H. P. Dong, O. Bjorang, I. M. Shih, P. S. Low, C. G. Trope and B. Davidson,
Hum. pathol. 2009, 40, 1453-1460.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702945
Chemistry - A European Journal
[38] a) K. Knop, R. Hoogenboom, D. Fischer and U. S. Schubert, Angew. Chem. Int. Ed. 2010, 49,
6288-6308; b) J. M. Rabanel, P. Hildgen and X. Banquy, J. Controlled Release 2014, 185, 71-87.
This article is protected by copyright. All rights reserved.