Accepted Manuscript

Title: þ-Cyclodextrin grafted polypyrrole magnetic

nanocomposites toward the targeted delivery and controlled
release of doxorubicin

Authors: Shasha Hong, Zengbo Li, Chenzhong Li, Chuan

Dong, Shaomin Shuang

PII: S0169-4332(17)32532-1
DOI: http://dx.doi.org/10.1016/j.apsusc.2017.08.201
Reference: APSUSC 37038

To appear in: APSUSC

Received date: 24-1-2017

Revised date: 15-8-2017
Accepted date: 24-8-2017

Please cite this article as: Shasha Hong, Zengbo Li, Chenzhong Li, Chuan Dong,
Shaomin Shuang, þ-Cyclodextrin grafted polypyrrole magnetic nanocomposites
toward the targeted delivery and controlled release of doxorubicin, Applied Surface
Sciencehttp://dx.doi.org/10.1016/j.apsusc.2017.08.201

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 β-Cyclodextrin grafted polypyrrole magnetic nanocomposites toward the targeted
delivery and controlled release of doxorubicin
Shasha Honga, Zengbo Lia, Chenzhong Lib, Chuan Donga, Shaomin Shuanga,*
a
College of Chemistry and Chemical Engineering, Institute of Environmental Science,
Shanxi University, Taiyuan 030006, PR China
b
Nanobioengineering/Bioelectronics Lab, Department of Biomedical Engineering,
Florida International University, Miami, USA
Graphical abstract

Highlights
• Facile synthesis of the novel nanocarrier with high drug loading content.
• Using polypyrrole for NIR light -responsive release
• Magnetic and receptor-targeting delivery of doxorubicin

Abstract
The Fe3O4@PPy-HA-β-CD nanocomposites as the novel nanocarrier were prepared
by grafting ethylenediamine derivative of β-CD to the surface of polypyrrole-coated
magnetic nanoparticles (Fe3O4@PPy) via using hyaluronan (HA) as the intermediate
linker. HA was also the efficient target ligand for CD44. The as-prepared drug carrier
was characterized by TEM, TGA, XRD, and VSM and used for the delivery of
doxorubicin hydrochloride (DOX) with the high loading content of 447 mg/g. The
multilayer Freundlich isotherm model was found to be a good fit for the loading of the
drug carrier for DOX. Significant NIR-triggered release of DOX was observed in a
weak acidic pH. And the release data in vitro was well described using the Retiger-
Pepper kinetic model. Furthermore, MTT assay and confocal microscopy against
Hep-G2 cells clearly illustrated that the drug carrier had no associated cytotoxicity and
could easily enter the cells. The release and accumulation of DOX were observed in
the cell nuclei. Thus, the DOX-loaded drug carrier killed the cancer cells efficaciously
and minimized adverse side effects due to its target effect. These results suggested the
as-prepared drug carrier would be of great potential for the controlled release and
targeted delivery of DOX.

Key words: magnetic nanocomposites; β-cyclodextrins; targeted delivery; controlled

release

1. Introduction

Currently, chemotherapy is an effective tool in cancer treatment. However, the

therapeutic efficiency of traditional chemotherapy drugs, like doxorubicin and
paclitaxel, is always restricted, due to nonspecific absorption and premature
degradation [1]. To avoid the side effect of anti-cancer drugs, the novel targeted drug
delivery systems (DDS) with high drug efficacy, desired stimuli-responsive controlled
release and marked biocompatibility are of the important concern.

In recent years, various carrier materials such as mesoporous silica [2]，carbon

nanotubes [3], magnetic nanoparticles (MNPs) [4] etc., have been introduced for
intracellular delivery of chemotherapeutics. Among them, Fe3O4 magnetic
nanoparticles (Fe3O4 NPs) have attracted considerable attention because of their larger
magnetic moments, excellent superparamagnetism, and biological degradability. And
more importantly, they could target drug at tumor sites with external magnetic field
under physiological conditions. However, Fe3O4 NPs get agglomerated easily and can
be oxidized in biological media due to their high surface area and magnetic dipole
interaction [5], which limits their further application. The chemical modification is an
effective method for the improvement of the stability of Fe3O4 NPs. Very commonly
used modifiers involve polymer [6], micelle [7], organic molecules[8] etc.. With the
development of research, attractive drug delivery systems have the tendency toward
dual-targeting function based on immobilization of targeting molecule on magnetic
nanoparticles [9]. To date, including but not limited to antibodies [10], aptamers [11],
and various ligands [12] have been used as targeting molecules. One favorable
candidate with targeting function is hyaluronan (HA) which is able to bind Cluster of
Differentiation 44 (CD44) receptors with high affinity. CD44 receptors are over
expressed in lymphoma, colorectal, melanoma, breast and lung tumor cells [13].

To realize controllable drug release ， choosing suitable modifying materials is

essential. Polypyrrole (PPy), an NIR-light-absorbing polymer, has been used to

modify Fe3O4 NPs for NIR-triggered drug release with enhanced antitumor efficacy in
vivo. In particular, PPy is also applied in photothermal therapy (PTT) due to its strong
NIR absorbance, good biocompatibility and outstanding stability [13]-15]. Wang et al.
[14] showed that DOX release from the Fe3O4@PPy-PEG-DOX nanocomposites
could be triggered by an external NIR laser. Subsequently, DOX-loaded
monodispersed spindle-like polypyrrole hollow nanocapsules was developed by Wang
et al. for pH-sensitive and NIR light-enhanced release of DOX under acidic pH [16].
β-Cyclodextrin (β-CD) as the modifying agent with a hydrophilic exterior and a
hydrophobic interior was able to form inclusion complexes with size suitable
molecule. Magnetic nanoparticles-graft-β-CD NPs for drug loading of 5-fluorouracil
[17], curcumin [18], docetaxel [19], ketoprofen [20] and doxorubicin [8] have been
reported involving in effective drug encapsulation and controlled release. These
findings trigger our research interests to engineer PPy-based nanocarriers for
simultaneous photothermal therapy and controlled drug delivery in one.
In the present work, we designed a novel combination strategy for the fabrication of
PPy-coated magnetic nanocomposites modified with β-CD and HA (Fe3O4@PPy-HA-

CD). As illustrated by Scheme 1, the drug carrier is composed of two steps. First, HA-
conjugated Fe3O4@PPy NPs were prepared using a facile surfactant-directed
chemical polymerization method and then ethylenediamine derivative of β-CD (en-β-
CD) was grafted onto the surface of Fe3O4@PPy-HA via an amidation reaction.

Afterward, a common anticancer drug doxorubicin was loaded into the cavity of en-β-
CD by host-guest interaction. The DOX-loaded Fe3O4@PPy-HA-CD NPs exhibited

both NIR-triggered sustainable release in the acid environment of tumors, as will be

demonstrated in detail below. Moreover, the cell uptake and cytotoxicity were
investigated on Hep-G2 cells to confirm the HA mediated targeted therapeutic
efficacy of Fe3O4@PPy-HA-CD/DOX complexes in vitro.

Scheme 1.

2. Experimental

2.1 Materials

FeCl3·6H2O (AR) and FeCl2·4H2O (AR) were procured from Tianjin Reagent
Factory. Ammonia (25-28% NH3 in water solution), N-hydroxysuccinimide (NHS),
pyrrole (98%), hyaluronic acid sodium salt (95%), 3-[3-dimethylaminopropyl]
carbodiimide hydrochloride (EDC), and sodium dodecyl sulfate (SDS, ACS) were
purchased from Aladdin Chemical Reagent Co. Ltd. China. β-CD was from
Guangdong Reagent Factory and re-crystallized twice from water and dried in
vacuum prior to use. Doxorubicin hydrochloride (DOX) was bought from Dalian
Mellon Biological Technology Co. Ltd. (Dalian, China). All chemicals were of
analytical grade and used as received. The deionized (DI) water was used in all
synthetic experiments.

2.2 Characterization

Transmission electron microscopy (TEM) analysis was done using a Tecnai G2 F20
S-Twin transmission electron microscope (FEI, USA) with an acceleration voltage of
200 kV. AFM experiments were performed at semicontact scanning mode by a SPA-
300HV atomic force microscope (AFM) (Seiko, Japan). Powder X-ray diffraction
patterns (XRD) were obtained from a D8 X-ray diffractometer (Bruker, Germany)

equipped with Cukα radiation (=0.15406nm). The chemical states of Fe

were confirmed by Amicus Budget X-ray photoelectron spectroscopy (XPS)
(Shimadzu, Japan). Magnetizations of samples were measured at room temperature
using a VersaLab vibrating sample magnetometer (VSM) (Quantum Design, USA).
Thermogravimetric analysis (TGA) was acquired to quantify the composition of

samples under a nitrogen atmosphere with a heating rate 10℃min-1 using a TGA Q50

thermo-gravimetric analyzer (TA Instruments, USA). All the fluorescence

measurements were performed on a F-4500 fluorescence spectrophotometer (Hitachi,
Japan). The UV-vis absorption intensity of DOX was measured at 480 nm on a
Double beam Spectrophotometer U-2900/2910 (Hitachi, Japan). UV-vis-NIR spectra
were carried out on a Lambda 950 UV-vis-NIR spectrophotometer (PerkinElmer,
USA). An optical-fiber-coupled power-tunable diode laser (MDL-Ⅲ-808nm-2.5W,

Changchun New Industries Optoelectronics Tech. Co., Ltd., China) was used for the
laser irradiation.

2.3 Synthesis of Fe3O4

Nanosized magnetic particles were prepared based on the chemical coprecipitation

method [20]. The ferric and ferrous chlorides (molar ratio 2: 1) were dissolved in 150
mL DI water under a nitrogen atmosphere. As the solution was being heated to 80℃,

NH4OH solution (25-28%) was cautiously added drop by drop into the solution under
vigorous stirring. As the pH was maintained at about 10, the reaction was continued
for another 30 min at 80℃. The resulting suspension was cooled down to room

temperature and then washed several times with water and ethanol to remove
unreacted chemicals.

2.4 Synthesis of Fe3O4@PPy-HA

In a typical procedure, 100 mg of Fe3O4 NPs was added into 100 mL DI water that
contained 20 mg of SDS. The mixture was ultrasonicated for over 20 min to make
Fe3O4 NPs well dispersed, then vigorous stirring for about 2 h. 50 μL of pyrrole was
then added, after 10 min, 10mL of FeCl3·6H2O (44.8 mg/mL) that contained 10
mg/mL of HA was added dropwise to the above mixed solution to initiate
polymerization. Reaction was continued by further stirring for 20 h at room
temperature.The products using HA as the stabilizer (denoted as Fe3O4@PPy-HA)
were magnetically collected, and washed several times with DI water to remove
excess HA and other reagents. Black powder was obtained by lyophilization.

2.5 Modification of Fe3O4@PPy-HA with en-β-CD

Modification of Fe3O4@PPy-HA with en-β-CD was done according to the literature
method [21]. Briefly, 0.1g of dry Fe3O4@PPy-HA NPs were suspended in 80 mL DI
water and sonicated for 15 min. 95.08 mg of EDC and 14.28mg NHS was added and
the mixture was stirred in the dark at room temperature for 1 h. Finally, 10 mL of en-
β-CD solution (10 mg/mL ) was added and the reaction was continued for 24 h. The
suspension was then collected using a permanent magnet and washed with DI water to
remove any unreacted chemicals and dried using a lyophilizer.

2.6 Stability study of Fe3O4 and Fe3O4@PPy-HA-CD

In order to test the degradation stability of Fe3O4 and Fe3O4@PPy-HA-CD, the
samples were weighted and then incubated in phosphate buffered saline (PBS, pH=7.4)
at 37□ for 24, 48 and 72 h. After being separated and washed with distilled water, the
samples were completely dried at 30□ in a vacuum oven. Based on the sample weight
before and after degradation, the mass loss was calculated.
2.7 DOX loading and releasing experiments
As a routinely used water-soluble anticancer drug, DOX was chosen as a model
drug. The drug-loading capacity of the Fe3O4@PPy-HA-CD was investigated by
using UV-Vis spectroscopy. In brief, 5 mg of Fe3O4@PPy-HA-CD NPs were
dispersed in 10 mL PBS (pH 7.4) containing DOX with varying concentrations (40,
100, 150, 200, 300, 400, 500, 600 and 700 µg/mL). The suspension was shaken in
dark condition for 5 h for total adsorption equilibrium at 37□. Then the DOX loaded
Fe3O4@PPy-HA-CD samples were collected by a magnet and the supernatant was
measured by a UV-Vis spectrophotometer at 480 nm. The drug loading content, an
important parameter in the evaluation of drug loading capacity, was calculated from
the following equation:
(CO − C)V ASUS
qe = 2019-10-28 21:05:38
m
--------------------------------------------
rumus adsorpsi dari dox
Where C0 and C are the initial and final solution concentrations of DOX respectively,
V is the volume of DOX solution, and m is the mass of Fe3O4@PPy-HA-CD.
To study the release of DOX from DOX-loaded Fe3O4@PPy-HA-CD, two sets of
experiments with different pH (5.0 and 7.4) were performed at 37□. For each drug
release experiment, 3.0 mg of DOX loaded Fe3O4@PPy-HA-CD NPs was dispersed
in 10 mL PBS at a pH of 5.0 or 7.4 in a stoppered bottle and gently shaking it in a
water bath shaker under dark condition. Periodically, at predetermined time intervals,
the supernatant solution (2 mL) of each group were taken out by magnet separation
and an equal amount of fresh PBS was added at the same time to maintain the total
solution volume constant at 10mL. The supernatant was analyzed by fluorescence
spectroscopy to determine the amount of DOX released. The cumulative DOX release
was calculated using the formula:
Mt
Drug released(%) = × 100%
MO
Where Mt and M0 are the amount of drug released at time t and that of drug initially
loaded onto Fe3O4@PPy-HA-CD, respectively.
2.8 In vitro cytotoxicity assay
The vitro cytotoxicity was investigated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) assay using human hepatoma cell line (Hep-G2).
Hep-G2 cells were seeded in a 96-well plate and grown in Duibecco's modified
Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 50
mg/mL penicillin, and 50 mg/mL streptomycin at 37□ with 5% CO2. After incubation
period, the cells were treated with different concentrations of DOX (0.01, 0.1, 1 and
10 µg/mL), Fe3O4@PPy-HA-CD (0.107, 1.07, 10.7 and 107 µg/mL) and
Fe3O4@PPy-HA-CD/DOX (0.107, 1.07, 10.7 and 107 µg/mL). 48 hours post-
incubation, MTT solution was put into each well and cells were incubated for further
4 h incubation. Then an enzyme-linked immunosorbent assay (ELISA) reader was
used to measure the absorbance of suspension.
2.9 Cellular uptake assays
Confocal laser scanning microscope (CLSM，Olympus FV1000, Tokyo, Japan)
was used to observe the cellular uptake of the free DOX, Fe3O4@PPy-HA-CD, and
Fe3O4@PPy-HA-CD /DOX according to our previous reports [8]. Hep-G2 cells which
have been cultured in a confocal dish were incubated with free DOX and
Fe3O4@PPy-HA-CD/DOX (at equally DOX concentration with 10µg/mL) for 2h at
37□. Then the culture medium was removed and the cells were washed three times
with PBS to fully remove extracellular DOX. All images were visualized by CLSM
and analyzed with image analysis software.

3. Results and discussion

3.1 Characterization of Fe3O4@PPy-HA-CD nanocomposites

The whole fabrication process of Fe3O4@PPy-HA-CD was illustrated in Scheme 1.

Bare Fe3O4 NPs were prepared by a chemical coprecipitation method. Subsequently,
the as-prepared Fe3O4 NPs were encapsulated with PPy layer, polymerized from

pyrrole monomers through oxidative polymerization using Fe3+ as the catalyst and
HA as the stabilizer. Meanwhile, due to charge-charge interactions between
negatively charged carboxyl groups of HA molecules and positively charged
polypyrroles, HA were incorporated on the surface of PPy NPs [22]. En-β-CD was
grafted onto the surface of Fe3O4@PPy-HA NPs via an amidation reaction between
surface carboxyl groups of HA and amino groups of en-β-CD in the presence of the
coupling reagent EDC. Consequently, a novel DDS was obtained and applied to
targeted delivery and controlled release of DOX. Introduction of carboxyl functional
groups and β-CD cavity on the surface of the final nanocomposites increased its
biocompatibility and ensured the better encapsulation of drug.
To judge the suitability of Fe3O4 and Fe3O4@PPy-HA-CD for clinical application,
their degradability and oxidation were examined in PBS (pH 7.4) for simulating the
inorganic part of blood plasma. The Fig. 1 shows the weight loss of the Fe3O4 NPs
before and after surface modification, which are immersed in PBS solution at 37□ for
periods of 24, 48 and 72 h, respectively. As shown in Fig. 1a, the synthesized Fe3O4
NPs exhibit a weight loss with positive percentage, which it is attributed to dissolution
of iron from magnetite. That means that the Fe3O4 is to some extent unstable and
sensitive to oxidation. And the oxidation of Fe3O4 involves the oxidation-reduction of
its surface in slightly basic PBS solution. During the oxidation of ferrous ions,
magnetite is transformed to maghemite (γ-Fe2O3) because of the migration of cations
through the lattice framework, which creats cationic vacancies to maintain the charge
balance [23]. After synthesized magnetite is coated, a weight loss with negative
percentage is observed. This phenomenon probably could be attributed to the presence
of hydroxyl and carboxyl groups on the β-cyclodextrins and hyaluronan, which
contribute to absorb water through the formation of hydrogen bonding. Additionally,
it can be seen from the TGA curves that the surface modified Fe3O4 NPs all have
obvious weight losses at 30-150□ (Fig. S1). Therefore, it’s better understanding of
the increase of weigh associated with the adsorption of water molecules on modified
Fe3O4 NPs. Our results are in agreement with the literature [24] and further approve
that the surface coating decrease the degradability and oxidation of Fe3O4 NPs and
improve the stability of Fe3O4 NPs.

Fig. 1

Surface morphology of the products was studied by transmission electron

microscope (TEM). Fig. 2 (a) and (b) showed TEM micrographs of Fe3O4 and
Fe3O4@PPy-HA-CD, respectively. It is clear that the synthesized Fe3O4 NPs are
spherical or quasi-spherical with relatively uniform size distribution, but also in some
areas somewhat aggregated particles are observed, more likely coming from the TEM
sample preparation process [25]. In addition, we observe that the Fe3O4 NPs are
surrounded by the grey colored polymer layer, resulting in a multicore-shell structure.
The particle size of Fe3O4 and Fe3O4@PPy-HA-CD calculated by Image J software
(Wayne Rasband, National Institutes of health, USA) is respectively 9.4 and 16.0 nm
(Fig. S2). Typical topographic atomic force microscopy (AFM) images of Fe3O4 (a)
and Fe3O4@PPy-HA-CD (b) have also been acquired, to evidence the coating shell
surrounding the iron core (Fig. S3). The area analyzed is 1 µm × 1 µm. It can be seen

in the surface images that the topography of the Fe3O4 is fairly regular，constituted by
spherical or ellipsoidal particles with 10.1 nm average diameter. By analysis of
several images, the overall size (magnetic core and polymer shell coating) is
estimated as 16.1 ± 2.1 nm, which is in close agreement with the size obtained by
TEM studies.

Fig. 2

Fig.3 shows the XRD patterns of the resulting products. All the diffraction peaks
occurred at 2 of 30.1°, 35.5°, 43.2°, 53.6°, 57.1° and 62.8°, which can be indexed to

(220), (311), (400), (422)，(511), and (440) planes of the magnetite structure，
respectively. The average crystal size of Fe3O4 particles is calculated to be about 12
nm using the data derived from the (311) plane, according to the Debye-Scherrer’s

formula [D=ｋ/(βcos), whereｋ, , β,  are the shape factor of value of 0.9, the

wavelength of the X-ray radiation, the half width of XRD diffraction lines and the
half diffraction angle of 2]. Although the black color of the sample should suggest
the presence of magnetite, it is difficult to differentiate magnetite, Fe3O4 (JCPDS, 19-
0629), and maghemite, γ-Fe2O3 (PDF card 25-1402) structure, due to the similarity of
their XRD patterns [26]. In order to verify the sample, XPS spectrum of bare Fe3O4
NPs has been measured, because of its sensitivity to Fe2+/Fe3+ ions. As can be seen in
Fig. S4, the main peaks centered at around 285 eV, 530 eV and 725 eV represent the
binding energies of C1s, O1s and Fe2p, respectively. The existence of carbon
elements was caused by ethanol and gas molecules, absorbed by the surface of the
sample. Higher resolution spectra of the Fe region (inset Fig. S4) exhibits two wide
peaks of Fe 2p3/2 (711.5 eV) and Fe 2p1/2 (725.3 eV), which are mainly ascribed to Fe-
O bonds and correspond with the literature value of magnetite (Fe3O4) [27]. The
absence of a visible satellite peak hence further demonstrates that the sample is Fe3O4,
rather than γ-Fe2O3.

Fig. 3

Fig. 4 shows the UV-vis-NIR absorption spectra of PPy, Fe3O4@PPy-HA and

Fe3O4@PPy-HA-CD NPs. It can be seen that a broad NIR absorption band from 700
to 1200 nm appears for PPy, which is characteristic of the bipolaronic metallic state of
doped polypyrrole, making it a potential PTT agent. Encouragingly, both
Fe3O4@PPy-HA and Fe3O4@PPy-HA-CD nanocomposites exhibited high optical
absorption in the NIR region with a peak at ∼1100 nm. This may allow the products
to generate heat when irradiated by a NIR laser. And it makes the materials become
excellent candidates for NIR-triggered drug release and thermal therapy.

Fig. 4

In order to investigate the magnetic properties of the synthesized nanocomposites,

the magnetic measurements were performed using a vibrating sample magnetometer
at room temperature. The results are presented in Fig. 5 and summarized in Table 1. It
is clearly seen that all the samples have coercivity (Hc) and remanence (Mr),
indicating ferromagnetic properties [26]. A decrease in the saturated magnetization
(Ms) value is observed when Fe3O4 NPs are coated with non-magnetic materials. The

Ms value of Fe3O4, Fe3O4@PPy-HA and Fe3O4@PPy-HA-CD is 68.5 emu g-1 、 36.2

emu g-1 and 9.2 emu g-1, respectively. Compared with the bare Fe3O4 NPs, the Ms of
the Fe3O4@PPy-HA-CD nanocomposites is obviously decreased because of a low
mass fraction of the Fe3O4 magnetic substance, which is attributed to the diamagnetic
contribution of the thick shell. The Ms of Fe3O4 NPs tends to decrease with the
increasing thickness of non-magnetic materials, similar behavior is also observed in
core-shell superparamagnetic iron oxide nanoparticles [28].
Fig. 5
Table 1
The Fe3O4, Fe3O4@PPy-HA and Fe3O4@PPy-HA-CD were measured by TGA
analysis, at the heating rate of 10℃/min in nitrogen flow. In Fig. 6, the total weight

loss for Fe3O4 was about 2.8%, which was attributed to the loss of the adsorbed
solvent (water and ethanol) and dehydration of the surface -OH groups. TGA analysis
of Fe3O4@PPy-HA showed the weight loss of 46% in a broad temperature range
between 150 and 800℃ which is attributed to degradations of polypyrrole and
hyaluronan molecules. Fe3O4@PPy-HA-CD and Fe3O4@PPy-HA showed the same
degradation pattern and both degraded in more than one step because of the
degradation of organic component. Moreover, it can be seen that the
Fe3O4@PPy-HA-CD had a major decomposition with the weight loss of about 49%,
indicating about 3% surface grafting of Fe3O4@PPy-HA by β-CD.

Fig. 6

3.2 Loading and release properties of DOX

The variation of drug loading content with time was measured (Fig.7) to study the
drug loading process. From the figure it is clear that the drug loading content
increased with time and attained equilibrium in about 5 h. As illustrated in Fig.8, the
equilibrium adsorption capacity of DOX onto Fe3O4@PPy-HA-CD NPs increased
with increasing initial DOX concentration. The highest equilibrium adsorption
capacity reached 447 mg/g under the optimum conditions (pH 7.4 and a DOX
concentration of 0.7 mg/mL), which was ascribed to the formation of a β-CD/DOX
host-guest inclusion complex. In addition, to gain more insight into the interaction
between the drug and DDS, adsorption equilibrium data was fitted with the Langmuir
model and Freundlich model. Langmuir isotherm is a theoretical model normally used

to analyze monolayer sorption on a uniform surface [29], and can be expressed in

linear form:
Ce Ce 1
= +
q e q m q m KL
Where qe (mg/g) is the equilibrium adsorption capacity of DOX, qm (mg/g) is the
maximum adsorption capacity, Ce (mg/L) is the liquid phase equilibrium
concentration of DOX and KL (mL/mg) is the Langmuir adsorption constant related to
the energy of adsorption. On the other hand, the Freundlich isotherm assumes that

adsorption occurs over a heterogeneous surface in multilayer, with a non-uniform

distribution of heat [30]. The linear form of Freundlich equation can be represented
as:
1
logqe = logK f + logCe
n
Where Kf (mg/g) is the Freundlich constants indicating loading capacity and n related
to the intensity of adsorption. Table 2 shows the relative parameters obtained from
Langmuir and Freundlich models. According to the results, the Freundlich model with
correlation coefficient R2 = 0.9953 fits experimental data slightly better than the

Langmuir isotherm model with correlation coefficient R2=0.9370. Thus, the

Freundlich model is more suitable to describe the drug loading process, which
suggests that the DOX adsorption belongs to multilayer adsorption process.
Meanwhile, the value of 1/n is also found to be smaller than 1, indicating that the drug
loading process of DOX on Fe3O4@PPy-HA-CD is favorable. The loaded drug by the
nanocomposites may be attributed to the following reasons: (a) the cavities of β-CD
can package DOX molecules to form stable host-guest inclusion complexes. (b) There
are also a lot of carboxyl groups on the surface of hyaluronan shell that can interact
with DOX molecules by electrostatic interactions. Therefore, DOX molecules can be
encapsuled effectively by Fe3O4@PPy-HA-CD nanocomposites.

Fig. 7

Fig. 8

Table 2

3.3 In vitro drug release

The DOX release curves from Fe3O4@PPy-HA-CD/DOX were investigated in two

different pH conditions 5.0 and 7.4 at 37□ and the results are presented in Fig.9. Both
the systems showed a constant sustained drug release pattern as expected. The DOX

was released very slowly in the neutral system (pH 7.4) and only about 12% of the
loaded DOX was released after 116 h. However, in the weak acid system (pH 5.0),
DOX was released very quickly and 27.0% of DOX were released in 24 h. Upon
prolonging the incubation time to 116 h, about 34% of the total bound DOX was
released. The elevated released amount of DOX at low pH (i.e., pH 5.0) was
attributed to the weakened hydrophobic interactions between DOX and β-CD in β-
CD/DOX inclusion complexes and DOX molecules tend to be more hydrophilic.
Meanwhile, the DOX molecules were not only entrapped in β-CD cavities, but were
also absorbed by the hyaluronan shell due to electronic action between surface

carboxyl groups of HA and amino groups of DOX. Addition of H+ could weaken the
electrostatic interaction to stimulate drug release from the DOX loaded
nanocomposites due to the neutralization of carboxyl groups of HA [31]. Based on the
tumor acidic microenvironment and intracellular acidic endosomes and lysosomes,
such a pH-sensitive release of DOX can reduce the undesired drug release during
transportation in the blood circulation system and enhance the therapeutic anticancer
effect. To gain more insight into the mechanism underlying DOX release from
Fe3O4@PPy-HA-CD NPs, the in vitro release data was analyzed by using Zero-order,
First-order, Higuchi and Ritger-Peppas models and the results were displayed in Table
3. Compared with other release kinetic models, Ritger-Peppas model showed the

highest correlation coefficient (R2). The resulting exponent n values in Ritger-Peppas

model were 0.2335 (pH 7.4) and 0.2674 (pH 5.0), within the limiting value of 0.5
(Fig.10), clearly indicating a Fickian release behaviour [32]. And drug release from
the systems was also influenced by drug diffusion.
To examine whether the NIR photothermal effect of PPy could be utilized to induce

the DOX release, we then explored the release kinetics of DOX from the Fe3O4@PPy-
HA-CD/DOX composite with an 808 nm NIR laser. Fe3O4@PPy- HA-CD/DOX was

incubated in 10mL of PBS (pH 5.0) and then irradiated by the 808 nm laser (1W/cm2)
for 5 min. For each measurement, aliquots of each 2mL supernatant were removed for
analysis of drug release, and the same volume of fresh PBS was added back for the
later drug release experiments. The amount of released DOX in the supernatant was
determined by a fluorescence spectrophotometer at desired time intervals. As shown

in Fig.9, the amount of cumulative released DOX from Fe3O4@PPy-HA-CD/DOX

was NIR-light irradiation enhancement whether at pH 7.4 or at pH 5.0. For example,

the cumulative percentage release of DOX increased markedly from 25.8% to 48.7%
in pH 7.4 PBS, and increased from 8.5% to 27.9% in pH 7.4 PBS for a period of 24 h
with NIR irradiation. The increased drug release upon NIR-light irradiation could be
attributed to heat stimulative dissociation of the hydrophobic interaction between
DOX and β-cyclodextrin and also the electrostatic interaction between DOX and HA.

In addition, all groups showed a sustained release of DOX, which suggested that the
Fe3O4@PPy-HA-CD nanocomposites could be used as an efficient drug delivery

system for encapsulation

of the hydrophilic drugs. These results demonstrated that the Fe3O4@PPy-HA-CD
was one kind of effective pH-sensitive and NIR-triggered drug-release vehicle, and
could minimize the side effect of the drug.
Table 3
Fig. 9

Fig. 10

3.4 In vitro cytotoxicity study

To evaluate in vitro anticancer activities, free DOX, DOX-loaded Fe3O4@PPy- HA-

CD NPs with excess HA, DOX-loaded Fe3O4@PPy-HA-CD NPs, and the blank
nanocomposites were investigated against Hep-G2 cell lines at different drug
concentrations for 48 h. As shown in Fig.11, cell viability showed a drug concentration-

dependent manner against both free DOX and DOX-loaded composite nanoparticles.
However, DOX-loaded Fe3O4@PPy-HA-CD NPs showed lower cytotoxicity than that

of free DOX. For example, the viability of Hep-G2 cells was 48% for Fe3O4@PPy-HA-
CD/DOX and 21% for free DOX with 1 µg/mL DOX. That can be ascribed to the slow

release of DOX from the nanocomposites at the same DOX concentration [33]. In
addition, the cell viability of the Fe3O4@PPy-HA-CD was higher than 90% at all
concentrations, which showed that the Fe3O4@PPy-HA-CD NPs had a good

biocompatibility and could be used as bio-safe delivery carriers for anticancer agents.
Meanwhile, higher cytotoxicity for DOX-loaded nanocomposites than that in the
presence of free excess were obtained, showing that excess HA restrained the
high affinity between the Hep-G2 cell membrane and
Fe3O4@PPy-HA-CD drug delivery system and proving that HA-nanocomposites were
taken up by the cell via a receptor mediated endocytosis pathway.

Fig.11

3.5 In vitro cellular uptake study

To investigate the intracellular uptake efficiency and the HA targeting ability,

DOX-loaded NPs were evaluated using CLSM. As shown in Fig.12, the obvious red
fluorescence of DOX could be clearly observed in the cytoplasm of Hep-G2 cells
after 2 h of incubation with Fe3O4@PPy-HA-CD/DOX or free DOX at a drug dosage
of 10 µg/mL, which implied that a large quantity of DOX released from either
Fe3O4@PPy-HA-CD/DOX or DOX·HCl groups had entered the tumor cells and
accumulated in cytoplasm. By contrast, after incubation with Fe3O4@PPy-HA-CD/
DOX and excess amount of free HA for 2h, weak red fluorescence was found in
cytoplasm. Therefore, it could be concluded that HA selectively binds to CD44,
resulting in the reduced availability of CD44 on cancer cells for Fe3O4@PPy-HA-CD/
DOX nanocomposites, which were internalized into the cells via receptor mediated
endocytosis. Moreover, the HA-modified nanocomposites might have potential as
anti-tumor drug carriers for cancer therapy.
Fig.12

4. Conclusions

In summary, a novel multifunctional drug delivery platform based on Fe3O4@PPy

core-shell structured nanocomposites was facilely constructed and exhibited
satisfactory properties such as high drug loading content, good biocompatibility, NIR-
triggered drug release behavior from in vitro experiments. HA modification of the
shell provided the molecule targeting, and the iron oxide nanoparticles core can also

be utilized to magnetic-guided drug delivery. Both MTT assay and Cellular uptake
assays demonstrated that DOX-loaded Fe3O4@PPy-HA-CD could effectively kill
Hep-G2 cells. Hence, we believe that the presented Fe3O4@PPy-HA-CD
nanocomposites have promising potential for targeted cancer therapy, MR imaging
and other biomedical applications. Meanwhile, further translation of this core-shell
structured nanocomposites to clinical applications should accomplish the following
important features: (i) the good biocompatibility with blood cells to guarantee the
successful intravenous administration of drug-loaded nanocomposites; (ii) the high
targeted efficiency of HA-modified magnetic nanocomposites to the tumor; (iii) the
excellent activity of HA targeting moiety after forming physical crosslinking by the
electrostatic interaction and other non-covalent interactions; and (iv) the increased
stability in biological media.

Acknowledgements

This work was supported by the National Natural Science Foundation of China
(21475080 and 21575084) and the Shanxi Province Hundred Talents Project.
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Figures

Fig. 1. Weight loss of Fe3O4 (a) and Fe3O4@PPy-HA-CD (b) after 24, 48 and 72 h in
the PBS solution.

Fig. 2. TEM images of (a and b) Fe3O4 nanoparticles, (c and d) Fe3O4@PPy-HA-CD

nanocomposites at two different magnifications.

Fig. 3. XRD patterns of the samples: Fe3O4 (a), Fe3O4@PPy-HA (b), Fe3O4@PPy-
HA-CD (c).
Fig. 4. UV-vis-NIR absorption spectra of PPy (a), Fe3O4@PPy-HA (b) and Fe3O4@
PPy-HA-CD (c).

Fig. 5. Room-temperature magnetization curves of Fe3O4 (a), Fe3O4@PPy-HA (b) and

Fe3O4@PPy-HA-CD (c).

Fig. 6. TGA curves of Fe3O4 (a), Fe3O4@PPy-HA (b) and Fe3O4@PPy-HA-CD (c),
respectively.

Fig. 7. Time course of DOX loading onto the Fe3O4@PPy-HA-CD.

Fig. 8. Adsorption isotherm for the Fe3O4@PPy-HA-CD drug-loading process.

Fig. 9. In vitro drug release profiles of DOX from Fe3O4@PPy-HA-CD/DOX in PBS

buffer at pH values of 7.4 and 5.0 with or without laser irradiation at 808 nm (1W cm-
2
).
Fig. 10. Ritger-Peppas plots for release of DOX from Fe3O4@PPy-HA-CD/DOX in
PBS buffer at pH values of 7.4 and 5.0 with or without laser irradiation at 808 nm (1
Wcm-2).

Fig. 11. The in vitro cell viability of Hep-G2 cells incubated with free DOX,
Fe3O4@PPy-HA-CD, DOX-loaded Fe3O4@PPy-HA-CD and DOX-loaded Fe3O4@
PPy-HA-CD in the presence of excess free HA at different concentrations for 48 h.
Fig. 12. Confocal laser scanning microscopy images of Hep-G2 cells incubated for
2 h at 37 °C with free DOX, Fe3O4@PPy-HA-CD, and DOX-loaded Fe3O4@PPy-HA-
CD/DOX in the presence or absence of free-HA. Scale bar = 20 µm.

Scheme 1. Illustration of the synthesis of Fe3O4@PPy-HA-CD nanoplatform.

Table 1 The magnetic parameters (coercivity (Hc), saturation magnetization (Ms) and
remanence (Mr)) of Fe3O4, Fe3O4@PPy-HA and Fe3O4@PPy-HA-CD.

Hc (Oe) Ms (emu/g) Mr (emu/g)

Fe3O4 95.5 68.5 7.2

Fe3O4@PPy-HA 66.4 36.2 2.8

Fe3O4@PPy-HA-CD 34.0 9.2 0.3

Table 2 Langmuir and Freundlich isotherm constants for DOX sorption.

Langmuir Freundlich
KLqmCe
qe =
1 + KLC qe = KfCe1/n

qm = 1569.58 mg/g Kf = 102.835 mg/g

KL = 0.607 mL/mg n = 1.123
R2 = 0.9370 R2 = 0.9953

Table 3 Fitting results of drug-release model of Fe3O4@PPy-HA-CD NPs.

pH Model Equations R2
Zero-order Qt = 0.0761t + 5.1202 0.8344
7.4 -4
First-order ln(100-Qt) = -8.2764×10 t + 4.5526 0.8454
1/2
Higuchi Qt = 0.8498t + 3.7205 0.9435
Ritger-Peppas lnQt = 0.2335lnt + 1.4038 0.9742
Zero-order Qt = 0.1698t + 17.507 0.6689
7.4+hν First-order ln(100-Qt) = -0.0022t + 4.411 0.7061
Higuchi Qt = 2.0933t1/2 + 13.660 0.8404
Ritger-Peppas lnQt = 0.2168lnt + 2.5874 0.9331
Zero-order Qt = 0.2260t + 15.5474 0.7183
5.0 First-order ln(100-Qt) = -0.0030t + 4.4348 0.7612
Higuchi Qt = 2.6197t1/2 + 11.0123 0.8802
Ritger-Peppas lnQt = 0.2674lnt + 2.4178 0.9441
Zero-order Qt = 0.343t + 31.43 0.745
5.0+hν First-order ln(100-Qt) = -0.0064t + 4.2242 0.8358
Higuchi Qt = 4.218t1/2 + 23.31 0.8969
Ritger-Peppas lnQt = 0.2222lnt + 3.1570 0.9746