Acta Biomaterialia 49 (2017) 402–413Contents lists available.docx

Acta Biomaterialia 49 (2017) 402–413
Contents lists available at ScienceDirect
Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat
Full length article
Programmed near-infrared light-responsive drug delivery
system for
combined magnetic tumor-targeting magnetic resonance imaging
and
chemo-phototherapy
http://dx.doi.org/10.1016/j.actbio.2016.11.035
1742-7061/� 2016 Acta Materialia Inc. Published by Elsevier
Ltd. All rights reserved.
⇑ Corresponding authors at: School of Pharmaceutical Sciences,
Zhengzhou
University, 100 Kexue Avenue, Zhengzhou 450001, China.
E-mail addresses: [email protected] (L. Hou), [email protected]
(Z. Zhang).
Qianhua Feng a,b,c, Yuanyuan Zhang a, Wanxia Zhang a,
Yongwei Hao a,b,c, Yongchao Wang a,b,c,
Hongling Zhang a,b,c, Lin Hou a,b,c,⇑ , Zhenzhong Zhang
a,b,c,⇑
a School of Pharmaceutical Sciences, Zhengzhou University,
100 Kexue Avenue, Zhengzhou 450001, China
b Collaborative Innovation Center of New Drug Research and
Safety Evaluation, Henan Province, Zhengzhou 450001, China
c Key Laboratory of Targeting Therapy and Diagnosis for
Critical Diseases, Henan Province, Zhengzhou 450001, China

a r t i c l e i n f o
Article history:
Received 15 July 2016
Received in revised form 3 November 2016
Accepted 15 November 2016
Available online 24 November 2016
Keywords:
Hollow mesoporous copper sulfide
Magnetic targeting
Controlled release
Theranostics
a b s t r a c t
In this study, an intelligent drug delivery system was developed
by capping doxorubicin (DOX)-loaded
hollow mesoporous CuS nanoparticles (HMCuS NPs) with
superparamagnetic iron oxide nanoparticles
(IONPs). Under near infrared (NIR) light irradiation, the
versatile HMCuS NPs could exploit the merits
of both photothermal therapy (PTT) and photodynamic therapy
(PDT) simultaneously. Herein, the mul-
tifunctional IONPs as gatekeeper with the enhanced capping
efficiency were supposed to realize ‘‘zero
premature release” and minimize the adverse side effects during
the drug delivery in vivo. More impor-
tantly, the hybrid metal nanoplatform
(HMCuS/[email protected]) allowed several emerging
exceptional
characteristics. Our studies have substantiated the hybrid
nanoparticles possessed an enhanced PTT
effect due to coupled plasmonic resonances with an elevated
heat-generating capacity. Notably, an effec-
tive removal of IONP-caps occurred after NIR-induced photo-
hyperthermia via weakening of the coordi-

nation interactions between HMCuS-NH2 and IONPs, which
suggested the feasibility of sophisticated
controlled on-demand drug release upon exposing to NIR
stimulus with spatial/temporal resolution.
Benefiting from the favorable magnetic tumor targeting
efficacy, the in vitro and in vivo experiments indi-
cated a remarkable anti-tumor therapeutic efficacy under NIR
irradiation, resulting from the synergistic
combination of chemo-phototherapy. In addition, T2-weighted
magnetic resonance imaging (MRI) con-
trast performance of IONPs provided the identification of
cancerous lesions. Based on these findings,
the well-designed drug delivery system via integration of
programmed functions will provide knowledge
for advancing multimodality theranostic strategy.
Statement of Significance
As we all know, a series of shortcomings of conventional
chemotherapy such as limited stability, rapid
clearing and non-specific tumor targeting ability remain a
significant challenge to achieve successful clin-
ical therapeutic efficiency in cancer treatments. Fortunately,
developing drug delivery system under the
assistance of multifunctional nanocarries might be a great idea.
For the first time, we proposed an intel-
ligent drug delivery system by capping DOX-loaded hollow
mesoporous CuS nanoparticles (HMCuS NPs)
with multifunctional IONPs to integrate programmed functions
including enhanced PTT effect, sophisti-
cated controlled drug release, magnetic targeting property and
MR imaging. The results showed HMCuS/
[email protected] could significantly enhance anti-tumor
therapeutic efficacy due to the synergistic com-
bination of chemo-phototherapy. By this delicate design, we
believe such smart and extreme versatile all-

in-one drug delivery platform could arouse broad interests in
the fields of biomaterials, nanotechnology,
and drug delivery system.
� 2016 Acta Materialia Inc. Published by Elsevier Ltd. All
rights reserved.
1. Introduction
Notwithstanding its pharmacological effect for cancer therapy,
conventional chemotherapy has been compromised by a series
of
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Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413 403
shortcomings such as limited stability, rapid clearing and non-
specific tumor targeting ability, which brought out serious side
effects [1,2]. In order to address this, developing drug delivery
sys-
tem under the assistance of nanocarries is an increasingly recog-
nized alternative method for cancer treatment [3,4]. Moreover,
allowing for the complexity and variability of cancers, it is
indis-
pensable to require synergistic combination of several
therapeutic
approaches in a coordinated way. From this viewpoint, realizing
multifunctional nanoparticles with distinct mechanisms is
particu-

larly anticipated to optimize cancer therapy.
Recently, copper sulfide nanoparticles (CuS NPs) have received
tremendous attention for their unique characteristics of NIR
local-
ized surface plasmon resonances (LSPR). Unlike other NIR
resonant
materials, which usually kill cancer cells relying on one single
prin-
ciple such as heat or reactive oxygen species (ROS) generation,
the
versatile CuS NPs could exploit the merits of both photothermal
therapy (PTT) and photodynamic therapy (PDT) simultaneously
[5,6]. Among numerous CuS nanomaterials reported so far [7,8],
hollow mesoporous CuS NPs (HMCuS NPs) were considered as
an
intelligent drug-delivery vehicle preferable to solid
nanoparticles
due to their uniform pore structure and high surface area for
drug
encapsulation [9–11]. Based on these advantages, a promising
paradigm combining chemotherapy with phototherapy
(including
PTT and PDT) based on HMCuS NPs was spontaneously
obtained
for synergistic cancer therapy. Nevertheless, with regard to
meso-
porous materials without gatekeeper modification, the
undesirable
premature drug leakage in circulation should be taken into
account
prior to their biomedical application [12,13]. Thus, it might be a
great copping strategy to cap the HMCuS NPs with a multifunc-
tional smart gatekeeper, which would remedy the drug leakage
defect to prevent any complications.

The FDA-approved superparamagnetic iron oxide nanoparticles
(IONPs), which featured inherent compatibility and high
magneti-
zation values, have been taken advantages in biomedical fields
related to drug delivery, diagnostics and hyperthermia therapy
[14–16]. Herein, IONPs with ultrafine particle sizes could act as
a
gatekeeper through capping onto HMCuS NPs to realize ‘‘zero
pre-
mature release”. In the meanwhile, the hybrid nanoplatform
makes
it possible to obtain some emerging exceptional characteristics.
According to our interparticle coupling effects on the surface
plas-
mon resonances of metallic complex structures could generate
highly enhanced local electromagnetic field and further enhance
the NIR absorption as reported in literatures such as Au-Fe3O4,
Au-CuS and r-GO-Au [17–20]. Encouragingly, our results
substanti-
ated that the hybrid [email protected] nanoparticles with
enhanced
SPR effect could generate elevated photothermal transduction
effi-
ciency by lower power laser irradiation in a short time. In
addition,
a quantity of IONP-caps could be remotely removed from the
sur-
face of HMCuS by NIR-induced photo-hyperthermia, probably
resulting from the weakening of the coordination interactions
between HMCuS-NH2 and IONPs [21]. Thus, it was more than
critical that the NIR stimulus would make progress on
controlled
on-demand drug release with spatial/temporal resolution.
Further-
more, the acknowledged intrinsic magnetic properties of IONPs
would endow the nanoplatform with magnetic targeted therapeu-

tic effect and T2-weighted MR imaging contrast performance
[22,23]. As a matter of course, the integration of IONPs on the
HMCuS NPs surface certainly would offer great advantages in
cancer treatment and diagnosis.
Herein, we proposed a programmed NIR-responsive drug deliv-
ery system for combined magnetic tumor targeting MR imaging
and chemo-phototherapy. As illustrated in Scheme 1, HMCuS
NPs
was utilized to encapsulate the chemotherapeutical drug doxoru-
bicin (DOX), and then capped with multifunctional IONPs to
inte-
grate programmed functions including enhanced PTT effect,
sophisticated controlled drug release, magnetic targeting
property
and MR imaging, which indicated a smart and extreme versatile
all-in-one drug delivery platform. In addition, hydrophilic PEG
modification allowed the nanoparticles for biocompatibility and
prolonged circulation characteristics [24,25]. The photothermal
effect under NIR irradiation was evaluated by using a thermal
cam-
era. Next, the tunable drug release upon exposing to NIR
stimulus
was explored on MCF-7 cells. Based on the noticeable magnetic
tumor targeting effect, it was envisioned that the synergistic
com-
bined chemo-phototherapy would significantly improve anti-
tumor therapeutic efficacy with minimal side effects. Besides,
the
MR imaging was also tested in vivo. By this delicate design,
such
a versatile hybrid nanoplatform of HMCuS/[email protected]
with
multi-functional characteristics will show great promising
poten-
tial in multimodality theranostic applications in cancer

treatment.
2. Experimental section
2.1. Materials
Doxorubicin hydrochloride (DOX HCl) was purchased from
Aladdin Reagent Database Inc. (Shanghai, China). PEG2000-
COOH,
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC HCl) and 1-Hydroxy-2,5-pyrrolidinedione (NHS) were
obtained from Sigma-Aldrich (Boston, MA, USA). All other
chemi-
cals acquired from the suppliers were at analytical grade. All
ani-
mal experiments were carried out in accordance with
institutional guidelines and approved by the local ethical
committee.
2.2. Synthesis of HMCuS/[email protected]
Synthesis of HMCuS NPs. Briefly, CuCl2 (8.5 mg), poly-
(vinylpyrrolidone) (PVP-K30, 240 mg) and hydrazine anhydrous
solution (6.4 lL) were added to 50 mL deionized water under
mag-
netic stirring at room temperature to form Cu2O nanoparitcles.
Subsequently, Na2S (64 mg) was quickly added into the above
solution. The mixture was maintained at 60 �C under strong
stir-
ring for 2 h. Then the resulting products (HMCuS NPs) were
puri-
fied by washing three times with water. To obtain amination
derivative of HMCuS, HMCuS NPs (1 mg/mL) was mixed with
2-aminoethanethiol (2 mg/mL) in PBS buffer under strong
stirring
for 24 h. As an end, the mixture was concentrated to obtain
HMCuS-NH2.

DOX loading. 1 mL of DOX in PBS (1 mg/mL) were added into
3 mL of HMCuS-NH2 in PBS (1 mg/mL). After stirring for 24
h, the
retrieved HMCuS/DOX was collected by centrifugation at
15,000 r/min for 5 min. The redundant DOX collected from
super-
natants was quantified by UV–vis spectroscopy measurements.
DOX loading capacity (LC) was calculated in the following
formula:
LC ¼ MDOX�prep � MDOX�supernatant
MDOX�prep
� 100%
Synthesis of IONPs and IONPs capping onto HMCuS/DOX. The
individual aqueous IONPs were synthesized by chemical co-
precipitation method [26]. FeCl2 (0.86 g) and FeCl3 (2.35 g)
were
added into 40 mL of deionized water and then the mixture was
heated at 60 �C under N2 atmosphere. Subsequently, aqueous
ammonia (5 mL) was slowly dropped into the above solution.
After
reaction for 30 min, citric acid (0.5 g/mL, 2 mL) was added.
The as-
prepared mixture was then maintained at 95 �C for 80 min and
cooled to room temperature under continuous stirring. The
result-
ing IONPs were collected with an external magnet. Finally, the
resulting product (HMCuS/[email protected]) was obtained by
mixing
IONPs and HMCuS/DOX (1:5) under stirring via noncovalent
interaction.

Scheme 1. Schematic representation of the synthesis of the drug
delivery system (HMCuS/[email protected]) for combining MR
imaging with chemo-phototherapy.
404 Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413
Conjugation of PEG to HMCuS/[email protected] In brief, a
mixture of
EDC HCl, NHS and PEG2000-COOH with molar ratio of 1:1:1
was dis-
solved in 20 mL of PBS, and the reaction was carried out for 30
min.
Then the mixed solution was added to HMCuS/[email protected]
in PBS
and stirred at room temperature for another 3 h. At last, the as-
prepared HMCuS/[email protected] was collected by
centrifugation.
According to the same manner, another product of
[email protected]
PEG NPs as blank vehicles for in vitro and in vivo experiments
was
also achieved without DOX loading.
2.3. Characterization
The morphology of the as-prepared samples was observed by
transmission electron microscopy (TEM, FEI Tecnai G20). N2
adsorption-desorption isotherms were recorded on a Micromerit-
ics ASAP2020 sorptometer (Micromeritics, USA). X-ray
diffraction
(XRD) measurements were conducted on an X-ray
diffractometer
(Model: XD-3X, Beijing, China). Ultra-violet-visible (UV–vis)
spec-
tra were recorded on an UV–vis spectrophotometer (Shimadzu).
FT-IR spectra were performed on a Nicolet iS10 spectrometer
(Thermo, USA). A vibrating sample magnetometer (VSM) was
uti-

lized for magnetic characterization of HMCuS/[email protected]
The leakage experiment was performed by incubating HMCuS/
DOX-PEG and HMCuS/[email protected] with the PBS (pH 7.4)
con-
taining 10% of FBS at 37 �C and monitoring leakage of free
DOX
continuously.
2.4. Photothermal effect of NIR on HMCuS/[email protected]
For photothemal effect, a continuous-wave NIR laser (Chang-
chun New Industries Optoelectronics Technology, China; wave-
length: 808 nm, power density: 2 W/cm2, spot size: 5 mm) was
used. Samples of different concentrations were irradiated by
808 nm laser for 3 min. And the photothermal images were
taken
by a thermal camera (FLIR, T330).
2.5. NIR light triggered release of DOX from
HMCuS/[email protected]
In vitro DOX release from HMCuS/[email protected] was per-
formed in PBS buffer at pH 7.4. In brief, 1.5 mg DOX loaded
nanoparticles were dialyzed in 100 mL PBS and shaken gently
at
37 �C. To evaluate NIR sensitivity of the formulation, samples
were
repeatedly exposed to a NIR irradiation (808 nm, 2 W/cm2, 3
min).
Subsequently, the released free DOX was taken at designed time
point and quantified by UV–vis spectra.
2.6. Cellular experiments
Cell culture. MCF-7 human breast cancer cells were cultured in
normal RPMI-1640 culture medium containing 10% fetal bovine
serum (FBS) and 1% penicillin/streptomycin at 37 �C in a
humidi-
fied atmosphere of 5% CO2 and 95% air. Cells were harvested
by
the use of trypsin and resuspended in fresh absolute medium

before plating.
NIR laser controlled DOX release in vitro. MCF-7 cells (5 �
104 -
cells per well) were co-cultured with HMCuS/[email protected]
(DOX concentration: 5 lg/mL) in 6-well plates. After the
internal-
ization of formations for 4 h, cells were exposed to a NIR laser
irra-
diation (808 nm, 2 W/cm2, 3 min). Then cell nucleus were
stained
with DAPI for another 30 min. At last, cell imaging was
conducted
by using confocal laser scanning microscopy (CLSM, Olympus
FV1100) after washing cells with PBS.
Intracellular ROS detection. DCFH-DA Reactive Oxygen
Species
Assay Kit was used to detected intracellular ROS generation.
MCF-7 cells were plated at 5 � 104 cells per well in a 6-well
plate.
Following incubation with free DOX (5 lg/mL),
[email protected]
(12 lg/mL) and HMCuS/[email protected] (17 lg/mL) for 4 h,
the
cells were washed three times with PBS and then DCFH-DA
was
entrapped into the cells. After 30 min incubation, the cells were
irradiated with a NIR laser (808 nm, 2 W/cm2) for 3 min. Then
flu-
orescence images of treated cells were obtained by using a
fluores-
cence microscope (Nikon Eclipse 50i, Japan).

In vitro cytotoxicity assay. MCF-7 cells were plated at
5 � 103 cells per well in 96-well plates and cultured for 24 h.
Then serial dilutions of different formulations at the indicated
concentrations were added to the culture medium. Subsequently,
the cells were irradiated with a NIR irradiation (808 nm, 2 W/
cm2, 3 min). After incubation for 24 h, cell samples were
treated
with a standard sulforhodamine B (SRB) assay to determine cell
viabilities.
Magnetic targeting effect in vitro. MCF-7 cells were placed into
6-well plates (5 � 104 cells per well) and then treated with
HMCuS/[email protected] (17 lg/mL) for 4 h with a magnet
(mag-
netic field intensity: 0.3 T) placed under the center of the
culture
dish. Then intracellular ROS were detected under NIR
irradiation
according to the method described above. Finally, cell imaging
in
green and red field was carried out by using a fluorescence
micro-
scope (Nikon Eclipse 50i, Japan).
MR imaging of cells. MCF-7 cells were treated with different
concentrations of HMCuS/[email protected] for 4 h, and then
were
rinsed with PBS for several times. Subsequently, T2-weighted
MR
images in vitro were collected on a 3-T clinical MRI scanner
(SIEMENS).
2.7. In vivo experiments
In vivo optical imaging. The near-infrared dye (IR783) was
used to mark the vehicle as a fluorescent probe. The tumor-

bearing mice received intravenous administration of HMCuS/
[email protected] (100 lg IR783/kg). The magnet group were
treated with an external magnet (magnetic field intensity:
0.3 T) laying above the tumor. The real-time optical imaging
was performed at the indicated time point (1, 3, 6, 8, 12 and
24 h) by using a noninvasive optical imaging system (Bruker,
Germany) with an excitation band pass filter at 730 nm and an
emission at 790 nm. Then mice were then sacrificed, with their
major organs (heart, liver, spleen, lung, kidney and tumor) har-
vested for ex vivo imaging.
MR imaging in vivo. The tumor-bearing mice were intra-
venously injected with HMCuS/[email protected] (13 mg/kg). A
magnet (magnetic field intensity: 0.3 T) was placed onto the
tumor
of the magnet group. At 6 h after injection, MR imaging was
per-
formed on a 3-T clinical MRI scanner.
In vivo anti-tumor activity. The tumor bearing mice were
assigned to seven groups randomly (n = 6 per group). The mice
were intravenously injected every other day with (1) saline, (2)
[email protected], (3) [email protected] + NIR laser, (4) DOX,
(5) HMCuS/[email protected], (6) HMCuS/[email protected] +
NIR
laser, (7) HMCuS/[email protected] + NIR laser + magnet (DOX
dose:
4 mg/kg, [email protected]: 9 mg /kg), respectively. The laser-
treated groups were irradiated with an 808 nm laser at a power
density of 2 W/cm2 for 0.5 min at 6 h after injection. And the
tumor
size was measured with a caliper and calculated by using the
fol-
lowing formula: Volume = (tumor length) � (tumor width)2/2.
After treatment, blood samples were collected for chemical
analy-

sis and tissues were excised for hematoxylin and eosin (H&E)
staining.
2.8. Statistical analysis
Quantitative data shown in this article were presented as the
mean ± SD and analyzed by using Student’s t test. A P-value
<0.05 was considered statistically significant.
3. Results and discussion
3.1. Synthesis and characterization of HMCuS/[email protected]
Highly uniform hollow mesoporous CuS NPs (HMCuS NPs)
with
spherical shape and good monodispersity were first synthesized
with an ion-exchanging process and characterized with TEM. As
depicted in Fig. 1A, a, the transparency core of HMCuS NPs
revealed
their hollow interior with particle size of 100 nm, while the
shell
with a thickness of about 20 nm exhibited distinct mesoporous
characteristics. Moreover, the EDS spectrum (Fig. S1)
illustrated a
strong correlation of Cu and S elements in HMCuS NPs. The
valence
states of HMCuS were determined by XPS measurement (Fig.
S2).
The doublet features of Cu 2p spectrum arose because of spin
orbit
splitting. As depicted in Fig. S2, the Cu 2p peaks at 933.6 and
954.1 eV were associated with Cu(II), while the peaks 932.6
and
952.5 eV were assigned to Cu(I). This indicated that Cu(I) was
also
existed in the products.
The features of hollow mesoporous CuS NPs, such as their large
surface area and great pore volume, made them candidates for

nanocarriers in which small DOX molecules could be loaded as
guest molecules inside the pores in doses sufficient for high
thera-
peutic efficacy [9,27]. Despite the high drug storage capacity
(336 lg/mg) of HMCuS/DOX, DOX leakage through mesoporous
without gatekeeper modification might take place during drug
delivery in vivo [12,13]. Herein, the individual aqueous IONPs
with
size ranging from 4 to 10 nm were prepared as gatekeepers for
drug controlled release (Fig. 1A, b), and the dynamic light
scatter-
ing analysis exhibited an average size of IONPs was 8.7 ± 0.6
nm
(Fig. S3). Afterwards, the outer surface of HMCuS/DOX was
capped
with multifunctional IONPs to obtain HMCuS/[email protected]
mainly
through two possible interactions between IONPs and HMCuS-
NH2: 1) the electrostatic interaction between the positively
charged HMCuS-NH2 (16.8 ± 1.4 mV) and negatively charged
IONPs
(�8.5 ± 0.8 mV) (Fig. S4); 2) the coordination interactions
between
the amino groups on the surface of HMCuS-NH2 and Fe atoms.
Nev-
ertheless, along with the increase of metal content in the hybrid
nanoplatform during this reaction, HMCuS/[email protected]
with poor
dispersion was not suitable for biological systems (Fig. 1B, b).
In
order to tackle this issue, hydrophilic PEG modification
endowed
the platform with biocompatibility and high aqueous solubility
of
5 mg/mL (Fig. 1B, c). Moreover, TEM images of HMCuS/
[email protected] (Fig. 1A, c) provided direct evidence of

IONPs dis-
tribution on the outer surface of HMCuS due to an evident
coating
morphology by forming an uniform and dense layer, though
PEG
modification barely influenced the structure. As depicted in
Fig. S5, the dynamic light scattering analysis showed an
average
size of HMCuS/[email protected] was 124.5 ± 3.8 nm, which
was
consistent with the TEM results. The surface charge of HMCuS/
[email protected] was further examined to be around �
20.3 ± 2.1 mV (Fig. S4), which was beneficial for individual
disper-
sion and long blood circulation property of the nanoparticles
[28].
In this system, the conjugating ratio of IONPs and PEG were
inves-
tigated. The amount of modified IONPs was about 11.8% with
an
ICP-MS quantitative analysis of Fe. Then TGA was performed
to
determine the amount of organic PEG grafted onto the
nanoparti-
cles (Fig. S6). Pure PEG degraded completely at about 617 �C,
[email protected] and [email protected] showed about 33.9%
and
42.5% weight losses at 617 �C, respectively, thus the relative
Fig. 1. Characterization of the nanoplatform. (A) TEM images
of HMCuS NPs (a), IONPs (b), HMCuS/[email protected] (c)
and HMCuS/[email protected] after NIR irradiation
(808 nm, 2 W/cm2, 10 min) (d); (B) The stability of
HMCuS/DOX (a), HMCuS/[email protected] (b) and

HMCuS/[email protected] (c) dispersed in PBS buffer; (C)
Leakage of DOX from
HMCuS/DOX-PEG and HMCuS/[email protected] stored at 37
�C; (D) X-ray diffraction (XRD) pattern; (E) FT-IR spectra; (F)
UV–vis absorption spectra; (G) N2 sorption isotherms
and pore size distribution plots of HMCuS,
HMCuS/[email protected] and HMCuS/[email protected] after
NIR irradiation (808 nm, 2 W/cm2, 10 min).
amount of PEG grafted onto nanoparticles was about 8.6%. The
loading efficiency of DOX on HMCuS with or without IONPs
were
measured to be 336 and 118 lg/mg, respectively. And the gate-
keeper of IONPs with admirable capping efficiency should be
responsible for the higher loading capacity of HMCuS/
[email protected] Then the capping efficiency of IONPs was
further
assessed by leakage experiment (Fig. 1C). In the case of
HMCuS/
DOX-PEG group, about 31.4% of DOX leakage was observed
after
7 days of storage. While little DOX leaked (5.2%) in the
formation
of HMCuS/[email protected] and IONPs. Thus, the gatekeeper
of
IONPs should be responsible for the admirable capping
efficiency
and was supposed to minimize premature drug release during
the drug delivery in vivo.
Subsequently, to obtain more insight into the nanoparticles,

various characterization of [email protected] without drug were
carried out to verify the successful IONPs capping and PEG
modifi-
cation. According to the XRD patterns in Fig. 1D, the three
strong
peaks of as-prepared individual HMCuS [(102), (103) and
(110)]
matched exactly those of the covellite CuS (JCPDS No. 06-
0464)
while peaks of IONPs [(311), (511) and (440)] matched those of
face-centered cubic Fe3O4 (JCPDS No. 75-1609) [29,30]. The
sam-
ples of [email protected] showed the typical patterns of both
CuS
and Fe3O4, which could be indexed to the successful
conjugation
of the two. Then the reaction was further supported by FT-IR
results in Fig. 1E. The appearance of strong N–H peak at
1640 cm�1 substantiated the formation of amination derivative
of
HMCuS (HMCuS-NH2). Additionally, the new peak at 570 cm
�1
owing to of Fe–O lattice mode of IONPs and peak at 1650
cm�1
of the characteristic –NH–CO– stretching vibration further
revealed the successful IONPs capping and PEG modification,
respectively. Next, the optical property of [email protected] was
investigated. As Fig. 1F showed, both bare HMCuS and IONPs
exhibited broad absorption throughout the visible and NIR
region.
The strong absorption of HMCuS in NIR region (k = 700–1000
nm)
was consistent with previous reports due to SPR arising from p-

type carriers [31]. And the absorption band of IONPs was
mainly
attributable to the transitions from atomic 3d states of the Fe2+
and Fe3+ ions to the 4s band with an onset near 2 eV [17].
More-
over, it was noteworthy that [email protected] showed a remark-
able enhancement of absorption in NIR region, together with a
redshift of adsorption edge in comparison with IONPs as well as
HMCuS alone. Such phenomenon could be explained by the fact
Fig. 2. Photothermal heating effect and in vitro release profiles.
(A) Infrared thermal im
after irradiation by an 808 nm laser for 3 min; (B) The
photothermal heating curves of H
laser; (C) Release curves of DOX from
HMCuS/[email protected] with or without NIR irra
that surface plasmon coupling effect in hybrid metallic
nanoparti-
cles generated highly enhanced local electromagnetic field, thus
extending beyond the resonant plasmon excitation energy and
fur-
ther increasing effective absorption cross sections [32,33].
Encouraged by the enhanced absorption capacity of our mate-
rial in NIR region as mentioned above, we moved on to
investigate
its photothermal effect. As depicted in Fig. 2A, the heat-
generating
capacity of HMCuS (4T = 29.7 �C, 100 lg/mL) was higher than
that
of IONPs (4T = 8.2 �C, 20 lg/mL) after NIR irradiation for 3
min,
and this was in sharp contrast to that of PBS (4T = 2.1 �C).
Surpris-
ingly, the temperature of HMCuS/[email protected] (200 lg/mL,
containing 100 lg/mL HMCuS and 20 lg/mL IONPs) increased

by
even 43.0 �C, indicating a favorable photothermal transduction
efficiency of IONPs capped HMCuS owing to the local field
enhanced absorption intensity. Even though the formation sub-
10 nm [email protected] core-shell nanoparticles have been
reported
by Tian et al., their local field enhancement of PTT was rarely
explored [34]. Then Fig. 2B revealed that the photothermal
effect
of HMCuS/[email protected] occurred in both concentration-
and
time-dependent manners. Then the photothermal conversion
effi-
ciency of HMCuS/[email protected] was measured. Briefly, NPs
dis-
persions were continuously illuminated by an 808 nm laser with
a power of 2 W/cm2 until the temperature reached a plateau.
The
irradiation source was then switched off while the temperature
was monitored to determine the rate of heat transfer from the
sys-
tem (Fig. S7). Following a previously reported procedure (See
also
the detailed calculation of photothermal conversion efficiency
in
the Supplementary data) [35], the photothermal conversion effi-
ciency (g) of HMCuS/[email protected] was calculated to be
42.12%. This value was higher than that of recently reported
Cu9S5 NCs (25.7%) [36]. Therefore, such extraordinary
photother-
mal effect of HMCuS/[email protected] suggested it could serve
as
ages of centrifuge tubes with PBS, HMCuS NPs, IONPs and
MCuS/[email protected] with different concentrations after
irradiation by an 808 nm

diation.
Fig. 3. Magnetic properties of HMCuS/[email protected] (A)
Magnetization loops of
IONPs and HMCuS/[email protected], the inset showed that the
PEG was rapidly attracted to one side of the cuvette after
adding external magnetic
field for 5 min; (B) T2-weighted MR photographs of
HMCuS/[email protected] in
aqueous solution with different Fe concentrations; (C)
Corresponding relaxation
rate r2 (1/T2) of HMCuS/[email protected] as a function of Fe
concentration.
a promising photothermal agent for thermal ablation in cancer
therapy.
A previous study explained that copper ions leaking from cop-
per sulfide nanocrystals under NIR irradiation would be
subjected
to similar redox reactions with surrounding medium primarily
via
a modified Haber-Weiss cycle [37], and then proved the
enhanced
�OH levels (up to 83.5%) generation under NIR irradiation
compared
to the no-laser-treated group by electron spin resonance spec-
troscopy (ESR) [5]. To further provide the evidence of the PDT
effect in this system, the �OH generation by
PEG was measured by degradation reaction of the methylene
blue

(MB) molecules due to the quenching of �OH in a cell-free
experi-
ment. The UV–vis spectra in Fig. S8 showed that the
characteristic
absorption peaks of MB at 290 and 680 nm decreased rapidly
trea-
ted with the NPs in an irradiation time-dependent manner,
which
demonstrated the �OH generation under NIR irradiation.
According
to the above results, we believe the HMCuS/[email protected] as
photodynamic agents would offer the opportunity for PDT to
attain
a more effective antitumor nanotherapy.
Besides, the TEM image after NIR irradiation was also taken,
with results exhibiting an appreciable removal of IONPs to a
cer-
tain extent from the outer surface of HMCuS (Fig. 1A, d). To
the best
of our knowledge, the coordinate bonds are commonly
considered
to be comparable to, but weaker than covalent bonds [38]. Most
interestingly, heating up the system gradually leads to thermally
induced cleavage of coordinate bond [39]. And this phenomenon
has been reported in a published literature [21], which
substanti-
ated the photo-hyperthermia induced by Pd nanosheets as pho-
tothermal agents resulted in coordinate bond cleavage. Thus, the
removal of IONPs-caps could be probably interpreted by the
fact
that the photothermal effect weakened the coordination between
HMCuS-NH2 and IONPs under NIR irradiation. Next, the NIR-
induced removal of IONPs was further demonstrated by nitrogen
adsorption-desorption isotherms (Fig. 1G). HMCuS itself
displayed

a type of V isotherm with a well-defined hysteresis loop
revealing
well-developed mesoporous characteristics. As listed in Table
S1,
the specific Brunauer–Emmett–Teller (BET) surface area of
HMCuS
was measured to be 155.8 m2/g and the average pore size was
around 5.1 nm, allowing small drug molecules, DOX (size of
1.53–1.19 nm), to spread into the hollow interior through the
mesoporous shells. In contrast, after DOX loading and IONPs
cap-
ping, the striking decreases in the surface area (22.3 m2/g) and
pore size (almost to zero) were observed, which indicated an
unex-
ceptionable pore blocking to realize ‘‘zero premature release”.
Whereas, the surface area and pore size of
PEG after NIR irradiation increased to 96.4 m2/g and 3.2 nm,
respectively, mainly due to the effective removal of IONPs-caps
triggered by NIR stimulus.
In light of these desirable properties of
under NIR irradiation, it was interesting to explore its drug
release
behavior. Fig. 2C showed that the no-laser-treated group
displayed
a sustained-release property along with the time progress and
only
a small amount of DOX (24.4%) released within 14 h. By
contrast, a
burst release of DOX occurred with NIR irradiation and an
increased release amount about 57.8% was observed, indicating
a
NIR-responsive controlled drug release profile in an impulsive
manner. On the basis of the NIR-induced removal of IONP-caps

as mentioned above, it was not surprised that the NIR stimulus
could trigger the encapsulated DOX release from nanoparticles.
Given the fact that the normal body temperature was about
37 �C, far lower to cleave the coordinate bond, few IONPs may
detached from CuS during the delivery process. Therefore, the
attachment of IONPs on HMCuS would be stable during the
drug
delivery in vivo, and the drug would release in a burst manner
at
the tumor site under a locally applied external NIR irradiation.
In
this sense, it was plausible to believe that such smart
controllable
on-demand cargo delivery (HMCuS/[email protected]) with
spatial/
temporal resolution was expected to minimize the adverse side
effects together with enhancing antitumor efficacy in vivo.
The magnetic properties of HMCuS/[email protected] were then
evaluated. As shown in Fig. 3A, similar to IONPs, the S-shape
mag-
netization loop of HMCuS/[email protected] exhibited typical
super-
paramagnetic characteristic. The lower saturation magnetization
(Ms) of HMCuS/[email protected] (44.8 emu/g) than IONPs
(69.1 emu/g) might be mainly due to the presence of the
nonmag-
netic components (HMCuS, PEG and DOX etc.). Meanwhile, it
was
clearly seen that HMCuS/[email protected] could be rapidly
manip-
ulated and controlled by an external magnetic field, showing an

effective magnetic response. Then, the magnetic resonance
signal
enhancing capability of HMCuS/[email protected] was further
assessed by T2-weighted MR images (Fig. 3B), revealing a
remark-
able concentration-dependent darkening effect. With the high
transverse relaxivity (r2 = 29.5 mM
�1 s�1) (Fig. 3C), HMCuS/
[email protected] as a promising T2 MRI contrast agent merited
its
usage for theranostic biomedical applications.
Fig. 4. In vitro cell experiments. (A) Confocal images of MCF-7
cells incubated with HMCu
in MCF-7 cells of control group (a), DOX (b), [email protected]
(c) and HMCuS/[email protected]
NIR irradiation (808 nm, 2 W/cm2, 3 min), the free DOX group
used an equivalent DOX c
MCF-7 cells treated with different HMCuS/[email protected]
concentrations; (E) Photo of
HMCuS/[email protected] treated MCF-7 cells in the presence
of a magnet (right). DCFH fo
1, 2, 3 represented above the magnet, the edge of the magnet
and outside the magnet, re
figure legend, the reader is referred to the web version of this
article.)
3.2. Cellular experiments
Keeping in mind the appreciable cell-free in vitro NIR-
responsive controlled release profile of
HMCuS/[email protected],
it was highly urgent to test its practicability in MCF-7 cells.
Confo-
cal laser scanning microscopy (CLSM) images of HMCuS/
[email protected] in Fig. 4A showed that an insignificant DOX
fluo-

rescence intensity was observed in the perinuclear cytoplasm
owing to the fluorescence quenching effect of metallic
nanomate-
rials [40]. While a stronger red fluorescence accumulated in
nucleus after NIR irradiation in comparison with the no-laser-
treated group as expected. These results provided direct
evidence
of NIR-responsive controlled DOX release in vitro, which was
required to realize higher cytotoxicity to tumor cells. In
addition,
S/[email protected] without or with NIR irradiation; (B)
Detection of intracellular ROS
NP-PEG (d); (C) Cytotoxicity assays of MCF-7 cells after
different treatments under
oncentration to the HMCuS/[email protected] group; (D) T2-
weighted MR images of
the cell culture plate in the presence of a magnet (left) and
fluorescence images of
r ROS detection (green) and released DOX fluorescence (red)
were recorded. Position
spectively. *p < 0.05, **p < 0.01. (For interpretation of the
references to colour in this
the level of intracellular ROS induced by HMCuS as
photosensitizer
was further investigated by using DCFH-DA fluorescent probe.
For
[email protected] group (Fig. 4B, c), a negligible fluorescence
of
DCFH was observed without NIR irradiation, while the laser-
treated group displayed an enhanced green fluorescence with
sig-
nal increasing by almost seven times (Fig. S9). Generally, The

ROS
generation by HMCuS under NIR irradiation could be explained
by
the fact that copper ions leaking from HMCuS would be
subjected
to similar redox reactions with surrounding medium primarily
via
a modified Haber-Weiss cycle [5]. Besides, DOX group also
exhib-
ited a dim fluorescence intensity (Fig. 4B, b). And it was also
note-
worthy that HMCuS/[email protected] displayed the highest
fluorescence intensity under NIR irradiation, indicating a great
deal
of ROS generation (Fig. 4B, d). To the best of our knowledge,
ROS
certainly would induce irreversibly oxidation damage to DNA
and proteins, as well as cause strong pro-inflammatory and pro-
apoptotic effects to some extend [41]. From this viewpoint,
ROS
generation by HMCuS/[email protected] would offer the
opportu-
nity for PDT to attain a more effective antitumor nanotherapy.
Next, in vitro cytotoxic effects of various formulations were
evaluated on MCF-7 cells by SRB assay in this section. It was
found
that the cell viability of carrier was unaffected even at high
concen-
trations up to 500 lg/mL (Fig. S10), suggesting that
[email protected]
PEG possessed good biocompatibility for biological
applications.
Under such circumstances, a NIR irradiation was conducted and
a
concentration-dependent cytotoxicity induced by

[email protected]
PEG was observed with a decreased cell viability of 61.7 ±
1.9% at
24 lg/mL (Fig. 4C), which could be explained by the effective
pho-
totherapy (enhanced PTT as well as PDT) of the hybrid
platform. In
addition, [email protected] as a photosensitizer and drug carrier
simultaneously could offer a platform for combined chemo-
phototherapy. As illustrated in Fig. 4C, all groups displayed the
concentration-dependent cytotoxicity. It was of great
importance
to note that only [email protected] under NIR irradiation (pho-
totherapy) or DOX (chemotherapy) alone could not realize the
ideal therapeutic efficacy. Nevertheless, the cell viability of
HMCuS/[email protected] ([email protected] concentration:
24 lg/mL) under NIR irradiation declined significantly to
7.8 ± 1.3%, considerably lower than that of DOX (42.2 ± 2.5%)
or
[email protected] (61.7 ± 1.9%) group. Thus, the synergistic
com-
bination of chemo-phototherapy with enhanced cytotoxic effect
occurred and was bound to improve the therapeutic index of
drugs
with high efficiency. Moreover, the enhanced DOX release upon
exposing to NIR stimulus was also responsible for the
prominent
cell-killing effect.
In the end, the magnetic properties of HMCuS/[email protected]
were further investigated in vitro. In agreement with
Fig. 4B and A, green or red fluorescence for ROS detection and
DOX release under NIR irradiation, respectively, were detected
in
Fig. 4E. Strong fluorescence in cells was observed above the
magnet

(position 1) in the whole vision, while the cells outside the
magnet
(position 3) showed a dim fluorescence intensity. At the edge of
the
magnet (position 2), both red fluorescence of released DOX and
green fluorescence of DCFH had obvious demarcation in the
half
vision. The above results demonstrated the promising potential
of HMCuS/[email protected] in magnetic targeted theranostic
appli-
cations. Meanwhile, cells treated with HMCuS/[email protected]
displayed a significant negative contrast enhancement with the
increase of the [Fe] concentration (Fig. 4D), successfully
suggesting
it as an promising T2 MRI contrast agent for cell labeling.
3.3. In vivo experiments
Prior to investigating the anti-tumor efficacy, the in vivo
biodis-
tribution of nanoparticles labeled with the near-infrared dye
(IR783) would be made clear in tumor-bearing mice by a non-
invasive optical imaging technique (Fig. 5A, a). And semi-
quantitative biodistribution analysis of the tumor site was per-
formed over time (Fig. S11). Compared to IR783 group, evident
flu-
orescence signals of HMCuS/[email protected] were widely
distributed throughout the whole body within 3 h, indicating
their
prolonged circulation features due to PEG modification. Intrigu-
ingly, the fluorescence intensity preferentially accumulated at
the
tumor region as a function of time and reached a maximum at
6 h post-injection, probably resulting from the enhanced perme-
ability and retention effect (EPR effect). When a magnet was
glued

onto the tumor site, elevated fluorescence signals were observed
at
the tumor site and persisted for more than 24 h post-injection,
demonstrating the favorable magnetic tumor targeting
efficiency.
Next, the inspiring tumor targeting efficiency was further con-
firmed by ex vivo fluorescence imaging (Fig. 5A, b) and
analysis
(Fig. 5B). The fluorescence intensity of
+ magnet group at the tumor region was �4.8 and �1.7 times as
compared to that of free IR783 and HMCuS/[email protected]
group, respectively. Because of the combined function of
prolonged
circulation characteristics and tumor targeting ability (EPR
effect
and magnetic tumor targeting effect), this [email protected]
based drug delivery system should play an important role in its
therapeutic performance. Moreover, considering the maximum
accumulation time of fluorescence, [email protected] mediated
phototherapy or MR imaging would be conducted at 6 h after
injection.
Furthermore, the DOX distribution of each group was investi-
gated after exposure to a NIR irradiation at 6 h post-injection
(Fig. S12). Compared to the DOX group,
group exhibited a superior tumor targeting ability and NIR-
responsive controlled drug release behavior, which was
consistent
with the in vivo optical imaging results. In the meanwhile, the
rel-
atively high drug distribution of nanoparticles in liver and lung
could be probably interpreted by the sessile macrophages
present
there [42]. Furthermore, Fig. S12 showed that

PEG group could significantly decreased the distributions of
DOX in
heart, even though higher fluorescence signals of NPs in heart
was
found in optical imaging. Thus the distribution of IR873 could
not
represent the distribution behavior of DOX completely.
As compared with optical imaging modality, MR imaging with
high spatial resolution is one of the most widely used clinical
imag-
ing tools in cancer diagnosis and prognosis [43]. In light of this,
the
in vivo T2-weighted MRI contrast performance of the
nanoparticles
was carried out as a proof-of-concept trial (Fig. 5C). In the case
of
HMCuS/[email protected] group, an appreciable darkening
effect in
the tumor site was observed in comparison with the control
group.
What’s more, the T2-weighted signals in tumor region of
magnet-
treated group were extraordinarily lower than HMCuS/
[email protected] group, showing a 2.5-fold reduction in the
signal
intensity due to magnetic targeting effect (Fig. 5D), which was
in
consistent with the in vivo optical imaging data. Reasonably,
the
contrast enhancing effect of HMCuS/[email protected] as an
MRI
contrast agent offered compelling chances for multimodality
ther-
anostic applications in cancer treatments.

Encouraged by the notable tumor accumulation of
[email protected] described in imaging data, we moved on to
evaluate the anti-tumor efficacy of our system in tumor-bearing
mice. The changes of relative tumor volumes (V/V0) after
various
treatments were recorded as a function of time in Fig. 5E. The
[email protected] treatment exhibited no decent tumor regres-
sion with tumor volumes increasing rapidly. While exposed to a
NIR irradiation, the tumor growth was partially inhibited to
some
extent with V/V0 of 4.01 ± 0.28. And a moderate growth
inhibition
effect with V/V0 of 3.13 ± 0.21 was observed in the case of
DOX
group. Unfortunately, neither purely phototherapy nor
chemother-
apy at the study endpoint could ideally clean up the tumors. In
Fig. 5. In vivo experiments. (A) (a) Time-dependent in vivo
optical imaging of tumor bearing mice in different treatment
groups, the tumor regions were marked by red circle; (b)
Ex vivo optical imaging of the dissected organs at 24 h post-
injection. Note: (I) free IR783; (II) HMCuS/[email protected];
(III) HMCuS/[email protected] + magnet. (B)
Biodistribution of IR783 in each tumor and organ; (C) In vivo
T2-weighted MR images of tumor-bearing mice (top row) and
tumor site (bottom row) under different treatments: (a)
Control group, (b) HMCuS/[email protected] and (c)
HMCuS/[email protected] + magnet, the tumor regions were
marked by red ellipse; (D) Average MRI signal intensity of
tumor
measured from MR images shown in (C); (E) Tumor growth
curves in different treatment groups of tumor-bearing mice; (F)

H&E stained tumor tissues harvested from different
groups of mice: (a) Control group, (b) [email protected], (c)
[email protected] + NIR, (d) DOX, (e)
HMCuS/[email protected], (f) HMCuS/[email protected] + NIR
and (g) HMCuS/
[email protected] + NIR + magnet. *p < 0.05, **p < 0.01. (For
interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
contrast, the HMCuS/[email protected] under NIR irradiation
reduced the tumor growth at the initial level with V/V0 of
0.97 ± 0.15. Impressively, the tumors were almost completely
sup-
pressed without recurrence with V/V0 of 0.41 ± 0.13 after
adding
the magnetic field, displaying an unexceptionable therapeutic
effect. Pathology changes of tumors were further investigated
through H&E staining to reveal the therapeutic mechanism.
Fig. 5F showed that typical pathological characteristics such as
intact shape and tight arrangement were observed in the control
and [email protected] treated group. As expected, HMCuS/
[email protected] group under NIR irradiation and magnetic
field
exhibited the most serious malignant necrosis with intercellular
blank and fragmentation to a significant extent, although
apparent
cell apoptosis in view was also found in other groups.
Such striking anti-tumor therapeutic efficacy with multi-
mechanisms for HMCuS/[email protected] + NIR + magnet
group

might be contributed to the following factors: 1) the prolonged
cir-
culation characteristics and favorable magnetic tumor targeting
ability reinforced the significant accumulation in tumor site; 2)
the combination of chemo-phototherapy inevitably resulted in a
remarkably synergistic therapeutic effect for cancer treatment.
Moreover, the elevated temperature by PTT effect might
enhance
the reactivity of ROS for tumor apoptosis according to a
previous
report [5]; 3) the enhanced DOX release upon exposing to NIR
stimulus was also responsible for the higher therapeutic effect.
In spite of the excellent anti-tumor efficacy of the multifunc-
tional drug delivery system, the detailed long-term toxicity
in vivo should be put into consideration. As can be seen, no
obvious
body weight decrease was observed during the treatment
(Fig. S13). Then blood biochemistry studies of tumor-bearing
mice
were performed. As depicted in Fig. S14, both the liver function
marker AST and the kidney function marker BUN were
measured
to be normal, suggesting no obvious hepatic and kidney disorder
of mice after the treatment. Comparatively, there was a
noticeable
elevation of CK and LDH associated with the physiological
status of
heart in DOX group, while biochemical parameters in other
groups
remained normal levels. This finding implied that the injection
of
DOX possibly caused certain long-term toxic effect to heart.
Then
the histological analysis (Fig. S15) of heart tissue in DOX
group

showed a serious cardiotoxicity with extensive muscle fiber
break-
age and cell nucleus gather. By contrast, rare structural
disturbance
in heart was found in HMCuS/[email protected] group. Hence,
all the
results from anticancer activity and systematic toxicity studies
implied that HMCuS/[email protected] with remarkable tumor
tar-
geting efficiency could dramatically reduce side effect of DOX
to
other organs and was considered a promising candidate for
cancer
treatments.
In our study, this system have demonstrated their abilities to
enhance therapeutic efficacy and reduce unwanted side effects
in
the experiments. What’s more, there were many other reported
theranostics nano-agents such as IONPs decorated MoS2
nanosheets for imaging guided photothermal therapy which had
shown a number of advantages compared with conventional
chemotherapy [44]. However, there are still many challenges
ahead toward further clinical applications. (i) The potential long
term toxicity of the nanomaterials. Although no obvious
toxicities
of most nanoagents were observed in the tested dose ranges, it
could still be tough for clinical use. Thus, the development of
bio-
compatible and biodegradable nanoagents for photothermal ther-
apy has a much higher clinical value. (ii) The limited light
penetration depth of NIR light. At present, phototherapy has
made
effective treatment for superficial cancerous lesions but
generally
failed to treat deep localized tumor. Encouragingly,

phototherapy
in areas with tumor invasion following the tumor removal as an
additional intraoperative treatment might be an alternative
approach [45]. (iii) The development of multifunctional
nanocarri-
ers that enable different therapeutic mechanisms for cancer
com-
bination therapy may bring great opportunities to the new
generation of cancer therapy [46].
4. Conclusions
In summary, the well-designed magnetic tumor targeted and
NIR-responsive drug delivery system
(HMCuS/[email protected])
developed here offered compelling highlights for multimodality
theranostic applications in cancer treatments. Encouragingly,
the
enhanced absorption of IONPs capped HMCuS NPs in NIR
region
could generate elevated photothermal transduction efficiency in
short time. With the spatiotemporally control of NIR
irradiation,
on-demand DOX release taken place due to the photo-
hyperthermia. Moreover, the unexceptionable therapeutic
efficacy
and MR imaging in vitro and in vivo of this versatile
nanoplatform
will open exciting opportunities for theranostic biomedical
appli-
cations. Such versatile HMCuS/[email protected], which
confirmed
significant advantages in unexceptionable therapeutic efficacy
and MR imaging in vitro and in vivo, held a great promising in
can-
cer diagnosis and therapy.
Acknowledgements

This work was supported by Grants from the National Natural
Science Foundation of China (81573364 and 81572991),
Science
and Technology Project of Henan Province (162102310510) and
Outstanding young Talent Research Fund of Zhengzhou
University
(51099255).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.actbio.2016.11.
035.
References
[1] L. Hou, X. Yang, J. Ren, Y. Wang, H. Zhang, Q. Feng, Y.
Shi, X. Shan, Y. Yuan, Z.
Zhang, A novel redox-sensitive system based on single-walled
carbon
nanotubes for chemo-photothermal therapy and magnetic
resonance
imaging, Int. J. Nanomed. 11 (2016) 607–624.
[2] Y. Hao, L. Wang, Y. Zhao, D. Meng, D. Li, H. Li, B. Zhang,
J. Shi, H. Zhang, Z.
Zhang, Y. Zhang, Targeted imaging and chemo-phototherapy of
brain cancer by
a multifunctional drug delivery system, Macromol. Biosci. 15
(2015) 1571–
1585.
[3] Y. Hao, B. Zhang, C. Zheng, R. Ji, X. Ren, F. Guo, S. Sun,
J. Shi, H. Zhang, Z. Zhang,
L. Wang, Y. Zhang, The tumor-targeting core-shell structured
DTX-loaded
[email protected] nanoparticles for chemo-photothermal therapy

and X-ray imaging, J.
Control. Release 220 (2015) 545–555.
[4] J. Shi, Z. Chen, L. Wang, B. Wang, L. Xu, L. Hou, Z.
Zhang, A tumor-specific
cleavable nanosystem of PEG-modified [email protected] hybrid
aggregates for radio
frequency-controlled release, hyperthermia, photodynamic
therapy and X-ray
imaging, Acta Biomater. 29 (2016) 282–297.
[5] S. Wang, A. Riedinger, H. Li, C. Fu, H. Liu, L. Li, T. Liu,
L. Tan, M.J. Barthel, G.
Pugliese, F.D. Donato, M.S. D’Abbusco, X. Meng, L. Manna, H.
Meng, T.
Pellegrino, Plasmonic copper sulfide nanocrystals exhibiting
near-infrared
photothermal and photodynamic therapeutic effects, ACS Nano
9 (2015)
1788–1800.
[6] K. Ding, J. Zeng, L. Jing, R. Qiao, C. Liu, M. Jiao, Z. Li, M.
Gao, Aqueous synthesis
of PEGylated copper sulfide nanoparticles for photoacoustic
imaging of
tumors, Nanoscale 7 (2015) 11075–11081.
[7] M. Zhou, J. Li, S. Liang, A. Sood, D. Liang, C. Li, CuS
nanodots with ultrahigh
efficient renal clearance for positron emission tomography
imaging and
image-guided photothermal therapy, ACS Nano 9 (2015) 7085–
7096.
[8] Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, S.
Yang, J. Wang, J. Wang, J. Hu,

Hydrophilic flower-like CuS superstructures as an efficient 980
nm laser-
driven photothermal agent for ablation of cancer cells, Adv.
Mater. 23 (2011)
3542–3547.
[9] K. Dong, Z. Liu, Z. Li, J. Ren, X. Qu, Hydrophobic
anticancer drug delivery by a
980 nm laser-driven photothermal vehicle for efficient
synergistic therapy of
cancer cells in vivo, Adv. Mater. 25 (2013) 4452–4458.
http://refhub.elsevier.com/S1742-7061(16)30631-6/h0005

[10] L. Guo, D. Yan, D. Yang, Y. Li, X. Wang, O. Zalewski, B.
Yan, W. Lu, Combinatorial
photothermal and immuno cancer therapy using chitosan-coated
hollow
copper sulfide nanoparticles, ACS Nano 8 (2014) 5670–5681.
[11] Q. Feng, Y. Zhang, W. Zhang, X. Shan, Y. Yuan, H.
Zhang, L. Hou, Z. Zhang,
Tumor-targeted and multi-stimuli responsive drug delivery
system for near-
infrared light induced chemo-phototherapy and photoacoustic
tomography,
Acta Biomater. 38 (2016) 129–142.
[12] J. Shi, Z. Chen, B. Wang, L. Wang, T. Lu, Z. Zhang,
Reactive oxygen species-
manipulated drug release from a smart envelope-type
mesoporous titanium
nanovehicle for tumor sonodynamic-chemotherapy, ACS Appl.
Mater.

Interfaces 7 (2015) 28554–28565.
[13] S. Giri, B.G. Trewyn, M.P. Stellmaker, V.S.Y. Lin,
Stimuli-responsive
controlled-release delivery system based on mesoporous silica
nanorods
capped with magnetic nanoparticles, Angew. Chem. Int. Ed. 44
(2005)
5038–5044.
[14] Q. Gan, X. Lu, Y. Yuan, J. Qian, H. Zhou, X. Lu, J. Shi, C.
Liu, A magnetic, reversible
pH-responsive nanogated ensemble based on Fe3O4
nanoparticles-capped
mesoporous silica, Biomaterials 32 (2011) 1932–1942.
[15] L. Wang, P. Zhang, J. Shi, Y. Hao, D. Meng, Y. Zhao, Y.
Yin, D. Li, J.
Chang, Z. Zhang, Radiofrequency-triggered tumor-targeting
delivery
system for theranostics application, ACS Appl. Mater.
Interfaces 7
(2015) 5736–5747.
[16] S. Shen, S. Wang, R. Zheng, X. Zhu, X. Jiang, D. Fu, W.
Yang, Magnetic
nanoparticle clusters for photothermal therapy with near-
infrared irradiation,
Biomaterials 39 (2015) 67–74.
[17] B. Wang, S. Qu, Absorption spectra and near-electric field
enhancement effects
of Au- and Ag-Fe3O4 dimers, Appl. Surf. Sci. 292 (2014) 1002–
1008.
[18] S. Lakshmanan, X. Zou, M. Hossu, L. Ma, C. Yang, W.

Chen, Local field enhanced
Au/CuS nanocomposites as efficient photothermal transducer
agents for
cancer treatment, J. Biomed. Nanotechnol. 8 (2012) 883–890.
[19] H. Moon, D. Kumar, H. Kim, C. Sim, J. Chang, J. Kim, H.
Kim, D. Lim, Amplified
photoacoustic performance and enhanced photothermal stability
of reduced
graphene oxide coated gold nanorods for sensitive photo
acoustic imaging,
ACS Nano 9 (2015) 2711–2719.
[20] D. Lim, A. Barhoumi, R. Wylie, G. Reznor, R. Langer, D.
Kohane, Enhanced
photothermal effect of plasmonic nanoparticles coated with
reduced graphene
oxide, Nano Lett. 13 (2013) 4075–4079.
[21] W. Fang, S. Tang, P. Liu, X. Fang, J. Gong, N. Zheng, Pd
nanosheet-covered
hollow mesoporous silica nanoparticles as a platform for the
chemo-
photothermal treatment of cancer cells, Small 8 (2012) 3816–
3822.
[22] N. Lee, D. Yoo, D. Ling, M. Cho, T. Hyeon, J. Cheon, Iron
oxide based
nanoparticles for multimodal imaging and magnetoresponsive
therapy,
Chem. Rev. 115 (2015) 10637–10689.
[23] J. Park, G. Maltzahn, L. Zhang, M. Schwartz, E. Ruoslahti,
S. Bhatia, M. Sailor,
Magnetic iron oxide nanoworms for tumor targeting and
imaging, Adv. Mater.

20 (2008) 1630–1635.
[24] G. Prencipe, S. Tabakman, K. Welsher, Z. Liu, A.
Goodwin, L. Zhang, J. Henry, H.
Dai, PEG branched polymer for functionalization of
nanomaterials with
ultralong blood circulation, J. Am. Chem. Soc. 131 (2009)
4783–4787.
[25] S. Kooijmans, L. Fliervoet, R. Meel, M. Fens, H. Heijnen,
P. Henegouwen, P.
Vader, R. Schiffelers, PEGylated and targeted extracellular
vesicles display
enhanced cell specificity and circulation time, J. Control.
Release 224 (2016)
77–85.
[26] Y. Sahoo, A. Goodarzi, M. Swihart, T. Ohulchanskyy, N.
Kaur, E. Furlani, P.
Prasad, Aqueous ferrofluid of magnetite nanoparticles:
fluorescence labeling
and magnetophoretic control, J. Phys. Chem. B 109 (2005)
3879–3885.
[27] L. Han, Y. Zhang, X. Chen, Y. Shu, J. Wang, Protein-
modified hollow copper
sulfide nanoparticles carrying indocyanine green for
photothermal and
photodynamic therapy, J. Mater. Chem. B 4 (2016) 105–112.
[28] X. Guo, C. Shi, G. Yang, J. Wang, Z. Cai, S. Zhou, Dual-
responsive polymer
micelles for target-cell-specific anticancer drug delivery, Chem.
Mater. 26
(2014) 4405–4418.
[29] Z. Zha, S. Zhang, Z. Deng, Y. Li, C. Li, Z. Dai, Enzyme-

responsive copper sulphide
nanoparticles for combined photoacoustic imaging, tumor-
selective
chemotherapy and photothermal therapy, Chem. Commun. 49
(2013) 3455–
3457.
[30] P. Kucheryavy, J. He, V. John, P. Maharjan, L. Spinu, G.
Goloverda, V.
Kolesnichenko, Superparamagnetic iron oxide nanoparticles
with variable
size and an iron oxidation state as prospective imaging agents,
Langmuir 29
(2013) 710–716.
[31] X. Tan, X. Pang, M. Lei, M. Ma, F. Guo, J. Wang, M. Yu,
F. Tan, N. Li, An efficient
dual-loaded multifunctional nanocarrier for combined
photothermal and
photodynamic therapy based on copper sulfide and chlorin e6,
Int. J. Pharm.
503 (2016) 220–228.
[32] J. Lee, E. Shevchenko, D. Talapin, Au-PbS core-shell
nanocrystals: plasmonic
absorption enhancement and electrical doping via intra-particle
charge
transfer, J. Am. Chem. Soc. 130 (2008) 9673–9675.
[33] E. Cho, C. Kim, F. Zhou, C. Cobley, K. Song, J. Chen, Z.
Li, L. Wang, Y. Xia,
Measuring the optical absorption cross sections of Au-Ag
nanocages and Au
nanorods by photoacoustic imaging, J. Phys. Chem. C 113
(2009) 9023–9028.

[34] Q. Tian, J. Hu, Y. Zhu, R. Zou, Z. Chen, S. Yang, R. Li, Q.
Su, Y. Han, X. Liu, Sub-
10 nm [email protected] core-shell nanoparticles for dual-modal
imaging and
photothermal therapy, J. Am. Chem. Soc. 135 (2013) 8571–
8577.
[35] D. Roper, W. Ahn, M. Hoepfner, Microscale heat transfer
transduced by surface
plasmon resonant gold nanoparticles, J. Phys. Chem. C 111
(2007) 3636–3641.
[36] I. Kriegel, C. Jiang, J. Rodriguez-Fernandez, R. Schaller,
D. Talapin, E. Como, J.
Feldmann, Tuning the excitonic and plasmonic properties of
copper
chalcogenide nanocrystals, J. Am. Chem. Soc. 134 (2012) 1583–
1590.
[37] M. Kadiiska, P. Hanna, L. Hernandez, R. Mason, In vivo
evidence of hydroxyl
radical formation after acute copper and ascorbic acid intake:
electron spin
resonance spin-trapping investigation, Mol. Pharmacol. 42
(1992) 723–729.
[38] Z. Xu, Mechanics of metal-catecholate complexes: the
roles of coordination
state and metal types, Sci. Rep. UK 3 (2013).
[39] M. Konopka, R. Turansky, J. Reichert, H. Fuchs, D. Marx,
I. Stich,
Mechanochemistry and thermochemistry are different: stress-
induced
strengthening of chemical bonds, Phys. Rev. Lett. 100 (2008).

[40] Z. Wang, Z. Chen, Z. Liu, P. Shi, K. Dong, E. Ju, J. Ren,
X. Qu, A multi-stimuli
responsive gold nanocage-hyaluronic platform for targeted
photothermal and
chemotherapy, Biomaterials 35 (2014) 9678–9688.
[41] X. Fu, M. Yang, M. Cao, D. Li, X. Yang, J. Sun, Z. Zhang,
L. Mao, S. Zhang, F. Wang,
F. Zhang, C. Fan, B. Fan, Strategy to suppress oxidative
damage-induced
neurotoxicity in PC12 cells by curcumin: the role of ROS-
mediated DNA
damage and the MAPK and AKT pathways, Mol. Neurobiol. 53
(2016) 369–378.
[42] R. Patil, J. Yu, S. Banerjee, Y. Ren, D. Leong, X. Jiang, M.
Poper, B. Tsui, D.
Kraitchman, H. Mao, Probing in vivo trafficking of
polymer/DNA micellar
nanoparticles using SPECT/CT imaging, Mol. Ther. 19 (2011)
1626–1635.
[43] M. Mertens, S. Koch, P. Schuster, J. Wehner, Z. Wu, F.
Gremse, V. Schulz, L.
Rongen, F. Wolf, J. Frese, V. Geshé, M. Zandvoort, P. Mela, S.
Jockenhoevel, F.
Kiessling, T. Lammers, USPIO-labeled textile materials for
non-invasive MR
imaging of tissue-engineered vascular grafts, Biomaterials 39
(2015) 155–163.
[44] T. Liu, S. Shi, C. Liang, S. Shen, L. Cheng, C. Wang, X.
Song, S. Goel, T. Barnhart,
W. Cai, Z. Liu, Iron oxide decorated MoS2 nanosheets with
double PEGylation
for chelator-free radio labeling and multimodal imaging guided

photothermal
therapy, ACS Nano 9 (2015) 950–960.
[45] Q. Chen, H. Ke, Z. Dai, Z. Liu, Nanoscale theranostics for
physical stimulus-
responsive cancer therapies, Biomaterials 73 (2015) 214–230.
[46] L. Cheng, C. Wang, L. Feng, K. Yang, Z. Liu, Functional
nanomaterials for
phototherapies of cancer, Chem. Rev. 114 (2014) 10869–10939.

http://refhub.elsevier.com/S1742-7061(16)30631-
6/h0230Programmed near-infrared light-responsive drug
delivery system for combined magnetic tumor-targeting
magnetic resonance imaging and chemo-phototherapy1
Introduction2 Experimental section2.1 Materials2.2 Synthesis of
HMCuS/[email protected]2.3 Characterization2.4 Photothermal
effect of NIR on HMCuS/[email protected]2.5 NIR light

triggered release of DOX from HMCuS/[email protected]2.6
Cellular experiments2.7 In vivo experiments2.8 Statistical
analysis3 Results and discussion3.1 Synthesis and
characterization of HMCuS/[email protected]3.2 Cellular
experiments3.3 In vivo experiments4
ConclusionsAcknowledgementsAppendix A Supplementary
dataReferences
Journal of Controlled Release 243 (2016) 303–322
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Review article
Hybrid protein-inorganic nanoparticles: From tumor-targeted
drug
delivery to cancer imaging
Ahmed O. Elzoghby a,b,⁎, Ayman L. Hemasa c, May S. Freag
a,d
a Cancer Nanotechnology Research Laboratory (CNRL), Faculty
of Pharmacy, Alexandria University, Alexandria 21521, Egypt
b Department of Industrial Pharmacy, Faculty of Pharmacy,
Alexandria University, Alexandria 21521, Egypt
c Department of Pharmacy, School of Medicine, Faculty of
Health, University of Tasmania, Hobart, Tasmania, Australia
d Department of Pharmaceutics, Faculty of Pharmacy,
Alexandria University, Alexandria 21521, Egypt
⁎ Corresponding author at: Cancer Nanotechnology Res
E-mail address: [email protected] (A.O. E
http://dx.doi.org/10.1016/j.jconrel.2016.10.023
0168-3659/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t
a r t i c l e i n f o
Article history:
Received 17 August 2016
Accepted 23 October 2016
Available online 26 October 2016
Recently, a great interest has been paid to the development of
hybrid protein-inorganic nanoparticles (NPs) for
drug delivery and cancer diagnostics in order to combine the
merits of both inorganic and protein nanocarriers.
This review primarily discusses the most outstanding advances
in the applications of the hybrids of naturally-oc-
curring proteins with iron oxide, gadolinium, gold, silica,
calcium phosphate NPs, carbon nanotubes, and quan-
tum dots in drug delivery and cancer imaging. Various
strategies that have been utilized for the preparation of
protein-functionalized inorganic NPs and the mechanisms
involved in the drug loading process are discussed.
How can the protein functionalization overcome the limitations
of colloidal stability, poor dispersibility and tox-
icity associated with inorganic NPs is also investigated.
Moreover, issues relating to the influence of protein hy-
bridization on the cellular uptake, tumor targeting efficiency,
systemic circulation, mucosal penetration and skin
permeation of inorganic NPs are highlighted. A special
emphasis is devoted to the novel approaches utilizing the
protein-inorganic nanohybrids in combined cancer therapy,
tumor imaging, and theranostic applications as well
as stimuli-responsive drug release from the nanohybrids.
© 2016 Elsevier B.V. All rights reserved.
Keywords:
Nanohybrids
Protein nanoparticles
Inorganic nanoparticles

Tumor targeting
Anti-cancer drug delivery
Cancer imaging
Theranostics
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 304
2. Hybridization strategies . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
2.1. Chemical conjugation. . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 305
2.2. Desolvation-chemical crosslinking . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
2.3. In situ coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 305
2.4. Spray-drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . 306
2.5. Protein template-directed biomimetic synthesis. . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
2.6. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 307
3. Drug loading mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
3.1. Covalent bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 307
3.2. Physical entrapment . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 307
3.2.1. Desolvation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . 307
3.2.2. Hydrophobic interaction . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 307
3.2.3. Electrostatic attraction . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . 308

3.2.4. Soaking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . 308
4. Impacts of protein functionalization . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
4.1. Improved biocompatibility and reduced toxicity. . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
4.1.1. Reducing the release of free metal ions. . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
4.1.2. Hiding the residual toxic capping agents . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
4.1.3. Enhancing the water dispersibility. . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 308
4.1.4. Reducing the immunotoxicity . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 308
earch Laboratory (CNRL), Faculty of Pharmacy, Alexandria
University, Alexandria 21521, Egypt.
lzoghby).
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4.1.5. Improved renal excretion . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 309
4.1.6. Reducing the thrombogenic activity . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
4.2. Prolonged circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 309
4.3. Improved colloidal stability . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
4.4. Altered skin permeation. . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 310
4.5. Enhanced targeting efficiency . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
4.5.1. Small molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . 310
4.5.2. Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . 310
4.5.3. Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . 311
5. Pharmaceutical applications of hybrid protein-inorganic
nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
311
5.1. Combinatorial cancer therapy . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
5.1.1. Combined chemotherapy and magnetic targeting . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
5.1.2. Combined chemotherapy and magnetic hyperthermia . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
5.1.3. Combined chemotherapy and photothermal therapy (PTT)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
5.1.4. Combined chemotherapy and photodynamic therapy . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
5.1.5. Combined photothermal and photodynamic therapy. . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
5.1.6. Combined nanophotothermolysis and protein targeting . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

5.1.7. Combined chemo- and radio-therapy. . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
5.2. Cancer imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 313
5.2.1. Improved MR contrast imaging . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 313
5.2.2. Enhanced fluorescence imaging . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 313
5.2.3. Dual imaging modality . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 315
5.2.4. Multimodal imaging modality . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 315
5.2.5. Blood-pool angiography. . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 315
5.3. Cancer nano-theranostics . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
5.4. Stimuli-responsive drug release . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
5.4.1. Magneto-responsive drug release . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 317
5.4.2. pH-responsive drug release . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 317
5.4.3. Enzyme-responsive drug release . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 318
5.4.4. Thermo-responsive drug release . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 318
5.4.5. Multi-stimuli-responsive drug release . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
6. The physicochemical properties of nanohybrids . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
6.1. Particle size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . 319

6.2. Surface charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . 319
7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 319
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 320
1. Introduction
Nanocarriers provide endless opportunities in the area of anti-
can-
cer drug delivery. Therefore, the research in the area of Cancer
nanomedicines has been progressively enlarged since the early
2000s.
Nanoparticles offer the possibility to entrap poorly soluble anti-
cancer
drugs rendering them suitable for parenteral administration and
modify
their blood circulation and tissue distribution by their
preferential accu-
mulation at the tumor site. Furthermore, some nanosystems have
been
reported to overcome multi-drug resistance [1–3]. A great
interest has
been paid to the design of nanocarriers for therapeutic drug
delivery
with simultaneous imaging capability for monitoring of their
biodistribution, the “nano-theranostics”. Nanoparticulate
contrast
agents are more advantageous to the conventional small contrast
ones
by exhibiting more prolonged systemic circulation in addition to
its ca-
pability for modification with targeting ligands to confer tissue-
specific
contrasting properties [4,5].

Inorganic NPs show optimal drug delivery characteristics, such
as
availability, biocompatibility, inertness and stability [5,6].
Compared to
conventional polymer or lipid nanocarriers, the unique optical
and mag-
netic characteristics of inorganic NPs make them potential
theranostic
carriers for cancer therapy and imaging [6]. On the other hand,
protein
nanocarriers have been developed as drug delivery devices due
to
their safety, biodegradability, non-antigenicity, and
significantly high
drug binding potential [7]. For tumor-targeted delivery, protein
nanocarriers can offer the following features: (a) Prolonged
systemic
circulation due to lower uptake by the reticuloendothelial
system
(RES) thus facilitating passive drug accumulation at tumor
tissues by
the enhanced permeation and retention (EPR) effect. (b)
Proteins
contain multiple functional groups (e.g. NH2, COOH, and OH)
available
for drug conjugation or ligand-mediated tumor-targeting [8]. (c)
Some
proteins such as albumin and lactoferrin can enhance tumor
targeting
of anti-cancer drugs via interaction with specific receptors over-
expressed on tumor cells. Abraxane®, albumin-bound paclitaxel
(PTX)
NPs, showed enhanced drug accumulation in solid tumors via
binding
to albondin gp60 and SPARC (secreted protein, acidic, rich in

cysteine)
receptors. (d) Some other proteins such as casein demonstrated
ex-
traordinary cancer cell penetration ability comparable to cell-
penetrat-
ing peptides [9,10]. (e) β-Lactoglobulin, gelatin, and elastin
showed pH-
, enzyme-and thermo-responsive drug release, respectively so
they can
be exploited in development of stimuli-responsive nanosystems.
(f)
Some plant proteins such as zein and gliadin can be used for
controlled
delivery of poorly soluble anti-cancer drugs by virtue of their
hydropho-
bicity [7].
Recently, a great effort has been devoted to the
functionalization of
inorganic NPs with proteins to combine the merits of both
inorganic
and protein nanocarriers and to mitigate the pitfalls of inorganic
NPs.
In addition to the reported toxicity of some inorganic NPs, their
high
chemical stability may hinder their metabolism resulting in their
long-
time accumulation in the body [11]. It was reported that
PEGylated
quantum dots (QDs) could reside in the body for two years [12].
More-
over, inorganic NPs have a high tendency for aggregation due to
their
small size. Besides, hydrophobic capping agents such as oleic
acid or
oleylamine are usually coating the surface of inorganic

nanocarriers
thus hindering their aqueous dispersibility. These obstacles can
impair
the clinical utility of inorganic NPs for therapeutic or
diagnostic pur-
poses [11,12]. One way to overcome the pitfalls of inorganic
NPs is to
hybridize them with lipids, polysaccharides, and proteins.
Among
305A.O. Elzoghby et al. / Journal of Controlled Release 243
(2016) 303–322
them, proteins offered various advantages especially in tumor-
targeted
drug delivery and cancer imaging [4–6]. Therefore, this article
reviews
the advanced applications of protein-inorganic nanohybrids in
drug de-
livery to tumors and cancer cell imaging.
2. Hybridization strategies
Various techniques were successfully employed for
development of
hybrid protein-inorganic NPs including covalent conjugation
and phys-
ical entrapment via desolvation, coating or spray-drying. Other
methods
such as biomimetic synthesis, salting out and electrospinning
may also
be involved.
2.1. Chemical conjugation

By virtue of their chemical structure, proteins can be
successfully
conjugated to the surface of inorganic NPs via covalent bonding
be-
tween the protein reactive moieties (such as carboxyl and amino
groups) and the functional groups conferred by the capping
agent of
NPs. Carbodiimide coupling is the most commonly used
reaction for
protein-NP conjugation via amide or ester bond formation.
Arginine
was employed to decorate the surface of iron oxide NPs
(IONPs) to pro-
vide free amine groups giving an opportunity for the amide
bond forma-
tion with the carboxylic groups of bovine serum albumin (BSA)
[13].
Similarly, the carboxylic groups of both gadolinium diethylene
triamine
pentaacetic acid (Gd-DTPA) complexes and thioglycolic acid
functional-
ized CdTe quantum dots (TGA-QDs) were conjugated via
carbodiimide
coupling to the amino groups of HSA and BSA, respectively
[14,15].
The use of ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochlo-
ride (EDC) as a linker to conjugate TGA-QDs to BSA is found
to be com-
parable to bifunctional crosslinkers e.g. glutaraldehyde while
avoiding
their toxicity. Alternatively, to increase the protein amino
groups for
better conjugation efficiency, albumin could be cationized by
replacing
the side chain carboxylic groups with ethylenediamine. Then,

amino
groups of the cationized albumin were linked to the surface
carboxylic
groups of the citrate-capped magnetic NPs thus elaborating
albumin-
magnetic nanohybrids [16]. Thus, the conjugation efficiency has
been
improved to be 28% cationic albumin coupled to MNPs as
demonstrated
by Bradford assay.
A common linker; succinic anhydride could also be used to
conju-
gate mesoporous silica NPs (MSNs) to the proteins; gelatin,
BSA, and ly-
sozyme leading to fabrication of MSN-protein nanohybrids [17].
In this
approach, the surface of amino-functionalized MSNs was first
covalent-
ly decorated with succinic anhydride molecules producing
carboxylated
MSNs. Then, the proteins were immobilized via their amino
groups onto
the surface of carboxylated MSNs by carbodiimide coupling. It
was
found that albumin and gelatin were coupled to MSNs in higher
Fig. 1. Schematic diagram of the desolvation technique for
preparation of BSA-FeNi3 nanohybrid
(modified from ref. [22]).
amounts compared to lysozyme, most likely because of their
higher mo-
lecular weight.
Another technique; Thiol-maleimide coupling was also utilized
to
link thiolated proteins to the surface of maleimide-derivatized

inorganic
NPs. Thiolated transferrin (Tf) was successfully attached to
PEG–
maleimide activated HSA-Gd-DTPA NPs with 84% Tf binding
efficiency
[18]. Another conjugation method involves the formation of
Schiff-
base bonds between residual aldehyde groups on the surface of
inor-
ganic NPs and amine groups of the proteins [19]. By treating
amine-
modified MSNs with glutaraldehyde, the aldehyde-
functionalized
MSNs were reacted with gelatin allowing the construction of
gelatin co-
rona on the surface of MSNs.
2.2. Desolvation-chemical crosslinking
The hydrophilic nature of proteins makes it possible to prepare
pro-
tein nanohybrids by organic solvent-induced desolvation from
aqueous
protein solution entrapping the inorganic NPs. In this technique,
inor-
ganic NPs were added as nuclei to the aqueous protein solution
prior
to desolvation by adding either ethanol or acetone thus
producing sta-
ble core-shell composite NPs. The abundant amino groups on
the pro-
tein surface enable further crosslinking using dialdehydes such
as
glutaraldehyde for enhanced stabilization of the developed
nanohybrids. Fe3O4 and FeNi3 NPs were encapsulated within
folate-con-

jugated HSA [20], and BSA [21] nanospheres prepared via
desolvation,
respectively. Since albumin is negatively charged above its pI
(isoelec-
tric point; 4.8), BSA molecules were coated onto FeNi3 NPs
with its an-
ionic carboxylate group. Upon adding ethanol, the attached BSA
molecules were denatured and fixedon the FeNi3 NPs exterior,
which
lead to the formation of stable core-shell FeNi3-BSA
nanohybrids. Simi-
larly, gold nanorods (AuNRs) were simultaneously encapsulated
with
paclitaxel (PTX) into HSA NPs by acetone desolvation
procedure
(Fig. 1). Albumin could strongly bind the surface of gold that is
coordi-
nated by amines and sulfur-containing moieties in albumin [22].
How-
ever, the use of chemical crosslinkers such as glyoxal and
glutaraldehyde was avoided to prevent quenching of the
nanocluster lu-
minescence [23]. In this technique, the protein coating acts as a
protec-
tive layer physically encapsulating the inorganic NPs, imparting
stability
and biocompatibility.
2.3. In situ coating
In this strategy, inorganic NPs were neither entrapped within
pro-
tein NPs via desolvation nor covalently bonded to the protein.
Instead,
the inorganic NPs were only coated with a protein layer via
either elec-

trostatic or hydrophobic interaction. Being charged
macromolecules
(above or below their pI), proteins can interact electrostatically
with
the oppositely-charged surface-capped inorganic NPs forming a
protein
s (modified from ref. [21]), TEM image of AuNR encapsulated
into HSA NPs by desolvation
Image of Fig. 1
306 A.O. Elzoghby et al. / Journal of Controlled Release 243
(2016) 303–322
shell around the NPs. The cationic poly(allylamine
hydrochloride)-coat-
ed AuNPs bind electrostatically to the negatively charged HSA
(above its
pI ~4.7) [24]. HSA binding was concluded from the decrease in
positive
zeta potential (from +42 to +20 mV) and the increase in
hydrodynam-
ic diameter (from 77 to 112 nm). Similarly, the electrostatic
interaction
between negatively charged CdTe QDs and cationic Lysozyme
(Lys) (at
pH below its pI ~10) was utilized for preparation of QDs-loaded
Lys-
carboxymethyl cellulose coacervates. Then, the samples were
heated
at 80 °C for 30 min to induce thermal denaturation of Lys
forming
more stable nanogels [25]. It was found that the electrostatic
complex-
ation between QDs and Lys has significantly potentiated the
fluores-

cence intensity of QDs. This could be attributed to the enlarged
size
and light scattering of the nanogel complex. In another
investigation,
hybrid gelatin-anionic ammonium heptamolybdate (AHM), as
model
anti-cancer polyoxometalate NPs, were developed via
electrostatic
complexation between negatively charged AHM and the cationic
frac-
tion of zwitterionic gelatin [26,27]. Additional stabilization of
the
formed complex may be attributed to the repulsion offered by
the an-
ionic fraction of gelatin. i.p. injection of gelatin-AHM NPs with
high
HM content (70%)into hepatoma-bearing mice demonstrated a
superior
antitumor efficacy in comparison with free AHM [27].
Proteins may be layer-by-layer assembled onto the surface of
inor-
ganic NPs via electrostatic interaction. Positively charged BSA
layer at
pH 4 (below its pI) were adsorbed onto the negatively charged
nitric
oxide (NO)-releasing S-nitroso silica NPs (SNO-SiNPs) for
controlling
NO release. BSA binding has reduced the surface charge of bare
SNO-
SiNPs from −40 mV to become nearly neutralized/slightly
positive
with a size increase from 200 nm to 300 nm [28]. The
polyanionic
drug, suramin acted as a linker between cationic BSA layers
where the

positively charged amino acids (lysine, arginine, and histidine)
of BSA
molecules form salt bridges with negatively charged sulfonate
groups
of suramin [28].
Hydrophobic interaction between the hydrophobic amino acids
within the protein sequence and the hydrophobic capping agent
of
the NPs could also be utilized as a hybridization mechanism.
Based on
the hydrophobic interaction between hydrophobic amino acids
of gela-
tin and the surface hydrophobic oleylamine molecules,
oleylamine-
coated IONPs could be transferred from chloroform to water by
gelatin
encapsulation (with the particle diameter increased to 178 nm)
[29].
In another approach, the hydrophobicity of gelatin could be
increased
via grafting a hydrophobic moiety (hexanoyl anhydride) thus
facilitated
the hydrophobic interaction with the adsorbed oleic acid on
magnetic
nanocrystallites [30]. Upon self-assembly, the hydrophobized
gelatin
provoked aggregation of the IONPs to compose the core thus
producing
hybrid amphiphilic gelatin–iron oxide (AGIO) NPs.
In ligand exchange approach, a compatible ligand replaces the
con-
ventional hydrophobic capping layer of inorganic NPs (e.g.
oleic acid
or oleylamine) to improve the NP hydrophilicity [4,11]. Being

consid-
ered as amphiphilic copolymers composed of hydrophilic and
hydro-
phobic amino acids, proteins form a coating layer around
inorganic
nanocarriers via hydrophobic interaction with the oleic acid
residue
Fig. 2. Schematic diagram of ligand exchange techniq
while exposing their hydrophilic fraction to the aqueous media.
Howev-
er, this conventional exchange technique requires harsh reaction
condi-
tions (heating or sonication) which can easily denature proteins.
A
novel exchange-encapsulation method was recently developed to
transfer the hydrophobic IONPs to the aqueous phase to be
capped
with casein (CN-IONPs) [31]. In this process, glucose was first
oxidized
and polymerized into oligosaccharides which then partially
replace
oleic acid followed by encapsulation of IONPs within CN
micelles to ob-
tain water-soluble CN-IONPs (Fig. 2). The encapsulation
resulted in
swelling of micelles with a size increase from 38 nm to 142 nm
and
made the micelles respond to external magnetic field. Similarly,
the hy-
drophobic trioctylphosphine oxide (TOPO)-coated CdSe QDs
were con-
verted into hydrophilic QDs via a ligand-exchange method [32].
TOPO
was exchanged with the hydrophilic ligand 11-
mercaptoundecanoic
acid then hydrophilic QDs were entrapped into gelatin NPs via

desolvation.
2.4. Spray-drying
In this technique, hybrid inorganic-protein nanocomposite
spheres
could be prepared by encapsulating inorganic NPs into protein
matrix
by spray-drying-ultrasonic atomization. Then, heating the
spray-dried
protein-inorganic NPs (150–160 °C) above their denaturation
tempera-
ture leads to thermal crosslinking of the hybrid nanocomposites
thus
enhancing their stability without need for chemical crosslinkers.
Using
this technique, the fluorescence of spray-dried QD-BSA
nanocomposites
could be controlled by changing the QD size. Thus, multi-
fluorescent
NPs may be obtained via incorporation of different-sized QDs
into indi-
vidual BSA nanospheres [33]. The same technique was utilized
to embed
gold selenium nanostructures together with the photosensitizer
zinc
phthalocyanine (ZnPc) into BSA nanocomposite spheres [34].
This tech-
nique offered many advantages including rapid and low-cost
large-scale
production of hybrid inorganic-protein nanocomposites.
Moreover, this
method produces nanoscale spheres (most of them are b600 nm)
with
narrow size distribution. The use of ultrasonic atomizer is
advantageous

as it produces smaller aqueous droplets (b5 μm) than those
formed by
the rotary atomizer (up to several hundred micrometers)
resulting in
large inorganic-protein microscale spheres with a broad size
distribu-
tion [33,34]. Finally, this preparation technique is safe with no
residual
organic solvent.
2.5. Protein template-directed biomimetic synthesis
Biomimetic techniques are highly recommended for the
fabrication
of inorganic nanocarriers due to the mild conditions utilized in
their
preparation. Biomolecules, such as polysaccharides, lipids,
proteins,
and nucleic acids have been explored as templates for
manufacturing
of nanomaterials. Among them, the stabilizing properties of
proteins en-
able their use as templates for biomimetic synthesis of inorganic
NPs
under mild conditions [35]. BSA was used as a stabilizer for the
biomi-
metic preparation of GdNPs. Then, the tyrosine residues of BSA
were
ue for preparation of casein-coated IONPs [31].
Image of Fig. 2
307A.O. Elzoghby et al. / Journal of Controlled Release 243
(2016) 303–322
completely iodinated via chloramine-T method to form

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