Biomaterials 34 (2013) 9666e9677

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

PEGylated fullerene/iron oxide nanocomposites for photodynamic

therapy, targeted drug delivery and MR imaging
Jinjin Shi 1, Xiaoyuan Yu 1, Lei Wang, Yan Liu, Jun Gao, Jing Zhang, Rou Ma, Ruiyuan Liu,
Zhenzhong Zhang*
School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Recently, fullerene and fullerene derivatives owning to their highly enriched physical and chemical
Received 11 June 2013 properties have been widely explored for applications in many different fields including biomedicine. In
Accepted 19 August 2013 this study, iron oxide nanoparticles (IONPs) were decorated onto the surface of fullerene (C60), and then
Available online 10 September 2013
PEGylation was performed to improve the solubility and biocompatibility of C60-IONP, obtaining a multi-
functional C60-IONP-PEG nanocomposite with strong superparamagnetism and powerful photodynamic
Keywords:
therapy capacity. Hematoporphyrin monomethyl ether (HMME), a new photodynamic anti-cancer drug,
Fullerene
was conjugated to C60-IONP-PEG, forming a C60-IONP-PEG/HMME drug delivery system, which
Iron oxide
Bioimaging
demonstrated an excellent magnetic targeting ability in cancer therapy. Compared with free HMME,
Magnetic targeting remarkably enhanced photodynamic cancer cell killing effect using C60-IONP-PEG/HMME was realized
Photodynamic therapy not only in a cultured B16-F10 cells in vitro but also in an in vivo murine tumor model due to 23-fold
higher HMME uptake of tumor and strong photodynamic activity of C60-IONP-PEG. Moreover, C60-
IONP-PEG could be further used as a T2-contrast agent for in vivo magnetic resonance imaging. Our
work showed C60-IONP-PEG/HMME had a great potential for cancer theranostic applications.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Photodynamic therapy (PDT) has been developed in past de-
cades as an alternative method to the traditional treatment of
cancer and non-cancer diseases. It is a promising modality for the
management of various tumors and nonmalignant diseases, based
on the application of photosensitizer (PS) that is selectively local-
ized in the target tissue, activated by a specific wavelength of light
and resulted in photo damage and subsequent cell death [1e5].
Although PDT has emerged as a viable treatment option for early
stage cancer and an adjuvant for surgery in late-stage cancer, some
obstacles in clinical adoption of PDT still persist [4,6,7]. A key lim-
itation in PDT is the poor water solubility of many photosensitizers
and their tendency to aggregate under physiological conditions
[8,9]. In addition, accumulation and selective recognition of target
tissue is still not high enough for many clinical applications [10e12].
To improve current PDT, nanomedicine offers nano-agent stra-
tegies. Nanoparticles can increase the solubility of hydrophobic
drugs and offer the benefits of hydrophilicity and proper size to
* Corresponding author. Tel.: þ86 371 67781910; fax: þ86 371 67781908.
E-mail address: zhangzz08@126.com (Z. Zhang).
1
Jinjin Shi and Xiaoyuan Yu contributed equally to this work.
accumulate in the tumor tissue via the EPR effect [13,14]. Fullerene
(C60), the third allotrope of carbon after diamond and graphite, is
nanoscale carbon material with unique photo-, electro-chemical,
physical properties and low systemic toxicity, having shown
tremendous promise in target-specific delivery of drugs in the body
[15,16], and many reports about fullerene derivatives being used for
the delivery of anti-cancer drugs [17]. Recently, we reported a
polyethylenimine fullerene (C60-PEI) which was modified with
folic acid (FA) through an amide linker, then docetaxel was suc-
cessfully encapsulated onto the C60-PEI-FA nanoparticles, and this
C60 based drug delivery system afforded higher antitumor efficacy
without obvious toxic effects to normal organs owing to its pro-
longed blood circulation and 7.5-fold higher DTX uptake of tumor
[14]. Another great advantage of C60 is its potential in photody-
namic therapy (PDT) [18,19], the absorption of visible light com-
bined with an efficient intersystem crossing to a long-lived triplet
state which makes fullerenes generate reactive oxygen species
upon illumination and allow fullerenes to be photosensitizer (PS)
[20,21]. Due to the enormous PDT potential of fullerene, in recent
years there has been much interest in studying possible biological
activities of fullerenes with a view to using them in medicine [22].
Among the above researches, C60 was used either as a drug
delivery carrier or as a PS for PDT, and no study has taken the two

0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2013.08.049
 J. Shi et al. / Biomaterials 34 (2013) 9666e9677
advantages of C60 in one system up to now. Recently, many
nanostructured systems have been used to deliver hydrophobic PSs
to tumors, for example: liposomes [23,24], polymer nanoparticles
[25,26], polymer nanogels [27] and silica nanoparticles [28], but the
above nanoparticles have no biological activity to improve the
photodynamic efficacy of PDT. In this study, C60 based PDT nano-
composites have served as both PS carrier and an additional PS to
improve photodynamic efficacy.
Magnetic targeting, an attractive physical targeting technique, is
garnering substantial attention for drug delivery applications [29,30].
Here, the therapeutic agents to be delivered are either immobilized
on the surface or encapsulated into the magnetic micro- or nano-
particle carriers. These magnetic carriers, upon intravenous admin-
istration, concentrate at the tumor site using an external high-
gradient magnetic field [31,32]. After accumulation of the magnetic
carrier at the target tumor site in vivo, drugs are released from the
magnetic carrier and effectively taken up by tumor cells. Gallo et al.
developed magnetic chitosan microspheres containing oxantrazole
(MCM-OX) for the treatment of brain tumors. Compared to OX in
solution, there was at least a 100-fold increase in OX concentrations
in the brain after administration of MCM-OX [33]. Cheng, L. etc.
developed multi-functional nanoparticles (MFNPs) with highly in-
tegrated functionalities including upconversion luminescence,
superparamagnetism for magnetic targeting photothermal therapy
[34]. Huang, P. etc. designed and prepared Ce6 (photosensitizer)-
conjugated magnetic nanoparticles for magnetic targeting photody-
namic therapy [35]. Ma, X. etc. developed a functionalized graphene
oxide-iron oxide nanocomposite for magnetic targeting chemo-
therapy [36]. In this study, iron oxide nanoparticle (IONP) was linked
to C60 based drug delivery system in order to obtain a magnetic
targeting effect to B16-F10 cells and malignant tumor in mice models.
In this study, a C60-IONP nanocomposite is synthesized and
then functionalized by polyethylene glycol (PEG2000), giving C60e
IONPePEG with excellent stability in physiological solutions, and
then hematoporphyrin monomethyl ether (HMME), a new photo-
dynamic anti-cancer drug, was loaded onto C60-IONP-PEG through
a physical adsorption. Herein a magnetically targeted drug (HMME)
delivery system (C60-IONP-PEG/HMME) (Fig. 1) was developed and
Fig. 1. Scheme of C60-IONP-PEG/HMME and its biofunctions.
9667
characterized by transmission electron microscopy (TEM), dynamic
laser scattering (DLS), Fourier transmission infrared spectroscopy
(FT-IR), and vibrating sample magnetometer (VSM). The photody-
namic and magnet targeting efficacy of C60-IONP-PEG/HMME was
examined using B16-F10 cells and tumor-bearing mice models.
Furthermore, in vivo MR imaging of tumor-bearing mice using C60-
IONP-PEG/HMME is also realized. The C60-IONP-PEG/HMME
nanocomposite developed in this work may be a promising multi-
functional nano platform for cancer imaging, magnetic targeting
and PDT.
2. Materials and methods
2.1. Materials
Fullerene (C60, purity >95％) were purchased from Henan Fengyuan Chemicals
Co. Ltd. Hematoporphyrin monomethyl ether (HMME, purity >98％) was gotten
from Beijing Yi-He Biotech Co. Ltd. Diethyl bromomalonate, NaH, MeO-PEG(2000)-
NH2, FeCl3$6H2O, sodium acetate (NaOAc), ethylene glycol (EG), diethylene glycol
(DEG), N-(3-dimethylamino propyl-N0-ethylcar-bodiimide) hydrochloride
(EDC∙HCl) sodium dodecyl sulfate (SDS), and dimethyl sulfoxide (DMSO) were ob-
tained from SigmaeAldrich Co. LLC. Sulforhodamine B (SRB), DMEM cell culture
medium, penicillin, streptomycin, fetal bovine serum (FBS), and heparin sodium
were bought from Gibco Invitrogen. Other reagents were acquired from China Na-
tional Medicine Corporation Ltd. The dialysis bags (MWCO ¼ 10,000) were from
Spectrum Laboratories Inc.
2.2. Synthesis of C60-IONP-PEG
Diethyl bromomalonate (0.2 mL) dissolved in toluene (3 mL) was added drop
wise to a stirred anhydrous toluene solution (50 mL) containing C60 (50 mg) and
NaH (0.3 g). After stirring at room temperature under N2 for 5 h, the mixture was
concentrated to obtain malonate derivative of C60.
Malonate derivative of C60 (50 mg) and NaH (180 mg) were added to anhydrous
toluene (30 mL), and stirred at 80  C under N2 for 10 h, then concentrated HCl
(20 mL) was added to the reaction precipitate, followed by removing toluene. The
precipitate was dispersed in concentrated HCl, filtered and washed with H2O and
MeOH. The resulting solid was dissolved in MeOH and the insoluble products were
removed by filtering. After evaporation, the solid products were dried in vacuum at
50  C for 24 h.
In total, C60-COOH (50 mg), FeCl3$6H2O (270 mg), and sodium acetate (NaOAc,
750 mg) were dissolved in a mixture of ethylene glycol (EG, 0.5 mL) and diethylene
glycol (DEG, 9.5 mL). The resulting solution was then transferred to a Teflon-lined
stainless-steel autoclave, which was sealed and heated at 200  C for 10 h. The as-
prepared C60eIONP was washed several times with ethanol and deionized (D.I.)
water, and dried in vacuum for 12 h.
At last, 50 mg of MeO-PEG(2000)-NH2 was added to a solution of C60eIONP
(2 mg/mL, 10 mL) and ultrasonicated for 10 min. 5 mg of EDC∙HCl was added to the
solution. The reaction solution was stirred overnight at room temperature. The
resulting product (C60eIONPePEG) was purified by washing three times with D.I.
water through a membrane filter to remove unreacted PEG and other reagents, and
dried in vacuum for 12 h.
2.3. HMME adsorption on C60-IONP-PEG
C60-IONP-PEG (50 mg) was added to ethanol-water mixture (ethanol:
water ¼ 1:1, 50 mL) containing HMME (150 mg) and sonicated at room temperature
for 2 h. After evaporation to remove ethanol and water, the product was dispersed in
water (20 mL), and sonicated using an ultrasonic cell disruption system (400 W, 10
times), and then the nanosuspension was centrifuged to remove free HMME. The
entire process was carried out in the dark, and the resulting C60-IONP-PEG/HMME
nanosuspension was stored at 4  C in the dark until use.
2.4. Characterization
DLS (Zetasizer Nano ZS-90, Malvern, UK) and TEM (Tecnai G2 20, FEI) were used
for characterizing particle size, zeta potential and morphological of C60-IONP-PEG/
HMME, respectively. The optical properties of C60-IONP-PEG, and C60-IONP-PEG/
HMME were characterized using an ultra-violet-visible (UVevis) spectrometer
(Lambda 35, PerkineElmer, USA). FT-IR spectra were recorded on a Nicolet iS10
spectrometer (Thermo). The relative amount of PEG linked to C60-IONP was tested
using a thermal gravimetric analysis (TGA, PerkinElmer) with the experimental
conditions of scanning from 25 to 800  C under nitrogen at a heating rate of
20  C/min. A vibrating sample magnetometer (VSM) was used for characterizing
the magnetic property of C60-IONP-PEG. The in vitro and in vivo T2-weighted MR
images were conducted on a 3-T clinical MRI scanner (SIEMENS).
9668 J. Shi et al. / Biomaterials 34 (2013) 9666e9677
2.5. Determination of HMME loading and release
C60-IONP-PEG/HMME nanosuspension was diluted with anhydrous ethanol and
sonicated to ensure that HMME was dissolved completely, and centrifuged to
separate C60-IONP-PEG and HMME to determine the amount of HMME loaded onto
C60-IONP-PEG. The concentrations of HMME were determined by high performance
liquid chromatography (HPLC, 1100 Agilent, USA) with the following conditions: an
Eclipse XDB-C18 column (150 mm  4.6 mm, 5.0 mm); mobile phase sodium acetate
solution (0.02 mol/L)/tetrahydrofuran 60:40; column temperature 40  C; fluores-
cence detector with the excitation and emission wavelengths set at 395 nm and
613 nm, respectively; flowrate 1.0 mL/min; and injection volume 20 mL.
For release study, C60-IONP-PEG/HMME and HMME samples, which had the
same HMME concentration, were placed into dialysis bags, which were dialyzed in
50 mL sodium dodecyl sulfate solution (SDS, 0.5%), respectively. The release assay
was performed at 37.0  0.5  C with a stirring rate of 100 r/min. 0.2 mL solution was
drawn from dialysis bag at various time points, being replaced by the same volume
of fresh SDS. The concentration of HMME released from C60-IONP-PEG into SDS
solution was quantified using HPLC under the above chromatographic conditions.
2.6. Cellular experiments
Cell culture. B16-F10 mice melanoma cell line was obtained from Chinese
Academy of Sciences Cell Bank (Catalog No. TCM36). Cells were cultured in normal
DMEM culture medium with 10% fetal bovine serum (FBS) and 1% penicillin/strep-
tomycin in 5% CO2 and 95% air at 37  C in a humidified incubator.
Cellular uptake. Intracellular uptake of C60-IONP-PEG/HMME and HMME were
performed with B16-F10 cells. B16-F10 cells were seeded at 5  104 cells per well on
glass cover slips in 6-well plates. When cells reached 70% confluence, they were
treated with HMME (10 mg/mL) and C60-IONP-PEG/HMME (HMME concentration:
10 mg/mL and C60-IONP-PEG concentration: 7.98 mg/mL) for 1, 2 and 3 h, respec-
tively. Cell nuclei were stained with DAPI for 30 min. After washing three times with
PBS, the cells were imaged by a laser confocal microscope (Olympus FV1100, Japan).
In vitro PDT treatment. To investigate the cancer cell phototoxicity of C60-IONP-
PEG, HMME and C60-IONP-PEG/HMME under 532 nm laser irradiation, B16-F10
cells were incubated with those three kinds of formulations at the same C60-IONP-
PEG and HMME concentrations (C60-IONP-PEG concentration: 7.98 mg/mL and
HMME concentration: 10 mg/mL) for 24 h, and then exposed to an 532 nm laser at
power density of 100 mW/cm2 or 300 mW/cm2 for 5 min. At last a standard cell
viability assay using SRB was conducted.
Intracellular ROS detection. ROS generation inside cells was detected using
DCFH-DA Reactive Oxygen Species Assay Kit. B16-F10 cells were seeded in confocal
dishes at a density of 5  104 cells/dish. Following incubation with C60-IONP-PEG,
HMME and C60-IONP-PEG/HMME for 24 h, DCFH-DA was loaded into the cells. After
30 min incubation, cells were washed twice with PBS and then exposed to 532 nm
irradiation for 5 min at the power density of 100 mW/cm2 or 300 mW/cm2. After
irradiation, fluorescence images of treated cells were acquired using a Fluorescence
Microscope (Zeiss LSM 510).
Magnetic drug targeting in vitro. For the magnetic drug targeting, C60-IONP-
PEG/HMME (HMME concentration: 10 mg/mL) was incubated with B16-F10 cells
for 2 h, with a magnet (magnetic field intensity: 0.3 T) placed under the center of
the culture dish, and DCFH-DA was loaded into the cells. After 30 min incubation,
cells were washed twice with PBS, and then exposed to 532 nm irradiation for
5 min at the power density of 300 mW/cm2. After irradiation, fluorescence im-
ages in red field and green field were acquired using a Fluorescence Microscope
(Zeiss LSM 510).
MR imaging of cells. B16-F10 cells were incubated with different concentrations
of C60-IONP-PEG for 3 h. The cells were washed with PBS, and then scanned under a
3-T clinical MRI scanner at room temperature to obtain the MR images.
2.7. In vivo experiments
Xenograft tumor mouse model. All animal experiments were performed under a
protocol approved by Henan laboratory animal center. Mice melanoma tumor
models were generated by subcutaneous injection of 1  106 B16-F10 cells in 0.1 mL
saline into the right shoulder of female C57 mice (18e22 g, Henan laboratory animal
center). The mice were used when the tumor volume reached 60e100 mm3 (w4
days after tumor inoculation).
In vivo PDT. For the in vivo antitumor experiments, the tumor-bearing mice
were divided into seven groups (six mice per group), minimizing the differences of
weights and tumor sizes in each group. The mice were administered with (1) saline
(0.1 mL), (2) 532 nm laser alone, (3) C60-IONP-PEG (4 mg/kg), (4) C60-IONP-PEG/
532 nm laser (4 mg/kg), (5) HMME/532 nm laser (5 mg/kg), (6) C60-IONP-PEG/
HMME/532 nm laser (HMME dose: 5 mg/kg, C60-IONP-PEG dose: 4 mg/kg) and (7)
C60-IONP-PEG/HMME/magnet/532 nm laser (HMME dose: 5 mg/kg, C60-IONP-PEG
dose: 4 mg/kg) in saline were intravenous injected into mice via the tail vein every 2
days, respectively and then the tumor regions were irradiated with 532 nm laser
(100 mW/cm2, 5 min) at 3 h post-injection. For the purpose of in vivo magnetic
targeting, a magnet was glued onto the tumor site of the mice. The mice were
observed daily for clinical symptoms and the tumor sizes were measured by a caliper
every other day and calculated as the volume ¼ (tumor length)  (tumor width)2/2.
After treatment for 10 days, the mice were killed to collect the tumors for H&E
staining.
Biodistribution studies. For biodistribution studies, the tumor-bearing mice
were fasted for 12 h before treatment, but had access to water ad libitum. The
experimental groups and control group were given C60-IONP-PEG/HMME, C60-
IONP-PEG/HMME/magnet or HMME, respectively (HMME dose: 5 mg/kg). In C60-
IONP-PEG/HMME/magnet group, a magnet was glued onto the tumor site of the
mice. After treatment for 0.5, 3 and 12 h, tissues/organs were collected, weighed,
and homogenized in buffer (acetonitrile to saline ratio, 1:1). HMME in tissues/organs
were determined by HPLC under the chromatographic conditions described in
Section 2.5.
In vivo MR imaging. For in vivo MR imaging, the tumor-bearing mice were
intravenously injected with C60-IONP-PEG (200 mL, 100 mg/mL), after injection for
3 h, MR imaging was conducted on a 3-T clinical MRI scanner.
2.7.1. Statistical analysis
Quantitative data are expressed as mean  SD and analyzed by use of Student’s t
test. P values < 0.05 were considered statistically significant.
3. Results and discussions
3.1. Synthesis and characterization of C60-IONP-PEG
The protocol for synthesis of C60-IONP-PEG was shown in Fig. 2.
The inherent hydrophobicity limits the use of C60, to overcome this
obstacle, carboxyl (-COOH) were introduced to the surface of C60,
C60-COOH was synthesized by Bingle cycloaddition reaction and
ester hydrolysis reaction according to the procedure of literature
with minor modifications (Fig. 2). C60 was water-insoluble, while
C60-COOH was stable in water over multiple weeks without sig-
nificant aggregation (Fig. 3A, a and b), implying successful intro-
duction of eCOOH onto C60. The results of FT-IR also showed new
peaks at C]O (w1716 cm1) and OeH (w3427 cm1) in the
spectrum of C60-COOH (Fig. 3B, b) compared with C60 (Fig. 3B, a),
suggesting that the functionalization of C60 with eCOOH was
successful [16]. The C60eIONP nanocomposite was synthesized by
chemical deposition of iron oxide nanoparticles onto water-soluble
C60 through a hydro thermal reaction (Fig. 2). During this reaction,
a part of carboxyl group in C60-COOH was reduced, so the FT-IR
spectrum of C60-IONP showed an obvious decrease in C]O and
OeH peaks (Fig. 3B, c) [37]. C60-IONP was not soluble in water
(Fig. 3A, c), thus could not be used in biological systems. To improve
the solubility and biocompatibility of C60-IONP, PEGylation was
performed via a condensation of carboxyl of C60-IONP and NH2
group of MeO-PEG(2000)-NH2. The resulting C60eIONPePEG
exhibited excellent stability in water (Fig. 3A, d) and various
physiological solutions including saline, cell medium and serum.
The successful PEGylation of C60eIONPe PEG was also evidenced
by its FT-IR spectrum, in which strong CeH (w1140 cm1), amideⅠ
(w1659 cm1), amideⅡ (1600 cm1) vibration peaks were clearly
seen (Fig. 3B, d).
The relative amount of PEG grafted onto the surface of C60-IONP
was tested by TGA. PEG degraded completely at about 500  C
(Fig. 3E), C60-IONP and C60-IONP-PEG showed about 6% and 35%
weight losses at 500  C, respectively, thus the relative amount of
PEG grafted onto C60-IONP was 29%.
We found that C60-IONP-PEG tend to form monodisperse ag-
gregates in the size range of 100e200 nm as confirmed by DLS and
TEM. The size and zeta potential of C60-IONP-PEG were
187  4.2 nm (Fig. 3C) and 33.4  2.3 mV (Fig. 3D), respectively.
The morphology of C60-COOH, IONP and C60eIONPePEG was
characterized by TEM. As can be seen from TEM images, C60-COOH
and C60-IONP-PEG both had a ball-like structure (Fig. S1, a and c),
while IONP was scattered in the vision (Fig. S1, b), suggesting IONP
was successfully deposited on C60-COOH. Fig. S1, d showed that
IONPs with diameters of 5e10 nm were deposited on C60 in the
C60-IONP-PEG sample.
 J. Shi et al. / Biomaterials 34 (2013) 9666e9677
Fig. 2. A schematic illustration of C60-IONP-PEG/HMME nanocomposite preparation.
Magnetic properties of C60eIONPePEG. Similar to IONP, C60-
IONP-PEG displayed strong magnetic property. Photos of C60-
IONP-PEG in water with and without a magnet clearly demon-
strated its excellent magnetic properties, when placed beside a
magnet, the C60-IONP-PEG sample was rapidly attracted by magnet,
leaving the solution colorless (Fig. 4A). The magnetization hysteresis
loop further indicated the superpara-magnetic nature of C60-IONP-
PEG (Fig. 4B). IONPs have been widely used as T2-contrast agent in
MR imaging [38,39], owing to the presence of IONPs inside C60-
IONP-PEG, the nanocomposites can act as a T2 contrast agent for
MR imaging. T2-weighted MR images (Fig. 4C) of C60eIONPePEG
solutions acquired on a 3-T MR scanner revealed the concentration-
dependent darkening effect. The transverse relaxivity (R2) of C60-
IONP-PEG was measured to be 156.28 mg mL1 s1 (Fig. 4D).
3.2. Preparation and characterization of C60-IONP-PEG/HMME
In this work, HMME, a new photodynamic anti-cancer drug, was
loaded onto C60-IONP-PEG through a physical adsorption. HMME
loaded onto C60-IONP-PEG was confirmed by a strong absorption
peak at around 395 nm over the background of C60-IONP-PEG
(Fig. 5B). As expected, a significant HMME absorption in the C60-
IONP-PEG/HMME sample was observed, likely due to the close
binding between HMME molecules and C60-IONP-PEG surface.
C60-IONP-PEG/HMME was stable in water, PBS buffer, cell culture
9669
medium and plasma of mice over multiple weeks without signifi-
cant aggregation (Fig. 5A). To determine the adsorption equilibrium
level of HMME loaded onto C60-IONP-PEG, different feed ratios of
HMME/C60-IONP-PEG were performed, indicating that HMME
loading efficiency increased from 14.8% to 134.4% (weight ratio of
HMME/C60-IONP-PEG) with the increased amount of HMME
(Fig. 5C). A HMME loading of w125% (HMME/C60-IONP-PEG
weight ratio of 5/1) was chosen for the following experiments. The
amount of HMME loaded onto C60-IONP-PEG was calculated to be
125.3 wt%. A lot of sp2-carbons were found in C60 molecules, and
HMME, a kind of porphyrin photodynamic therapy agent, has
delocalized p bonds, and this makes it easy to load onto C60
through pep stacking. The pep stacking between HMME and C60
allows such a high HMME loading capacity. Such a value of loading
was far beyond the other HMME delivery system, which were al-
ways below 100 wt% [40], indicating C60-IONP-PEG was a prom-
ising material for HMME delivery.
To investigate the release kinetics of active drug from
nanoparticle-drug system, we incubated the nanostructures in
sodium dodecyl sulfate solution (SDS, 0.5%). As seen in (Fig. 5D),
HMME release from C60-IONP-PEG was sustained over 120 h, in
contrast, the release of HMME group was very fast in SDS, sug-
gesting that interaction between C60-IONP-PEG and HMME plays a
critical role in the release of drug due to a dendritic structure of PEG
grafted onto C60-IONP.
9670 J. Shi et al. / Biomaterials 34 (2013) 9666e9677
Fig. 3. Characterization of fullerenes A) Photos of a) C60, b) C60-COOH, c) C60-IONP and d) C60-IONP-PEG in water; B) FT-IR spectrum of a) pristine C60, b) C60-COOH, c) C60-IONP
and d) C60-IONP-PEG; C) and D) Dynamic light scattering analysis of C60-IONP-PEG showing an average size distribution around 187 nm and a zeta potential around 33.4 mV; E)
TGA curves of C60-IONP, PEG and C60-IONP-PEG.
The size and zeta potential of C60-IONP-PEG/HMME were
163.8  3.7 nm (Fig. 6A) and 33.63  2.1 mV (Fig. 6B), respectively.
TEM images (Fig. 6C) indicated that C60-IONP-PEG/HMME had a
uniform size and a ball-like structure. Compared with C60-IONP-
PEG, the size of C60-IONP-PEG/HMME were a little smaller, this is
probably due to the longtime of ultrasonic dispersion in prepara-
tion of C60-IONP-PEG/HMME.
3.3. Intracellular PDT and ROS production
Cytotoxicity of the C60-IONP-PEG is an important consideration
for materials intended for in vivo application. Toward this goal,
assessment of cell viability of B16-F10 cancer cells was examined.
Incubation with C60-IONP-PEG showed that there was minimal
decrease in cell viability (<10%), even when the cells were incubated
with 100 mg/mL of C60-IONP-PEG for 24 h (Supporting Information,
Fig. S2). This cytotoxicity study demonstrated that C60-IONP-PEG
itself possesses low toxicity without light irradiation.
In order to investigate the enhanced PDT efficiency of C60-IONP-
PEG/HMME in cancer cells, B16-F10 cells were incubated with C60-
IONP-PEG, HMME and C60-IONP-PEG/HMME at the same concen-
trations of HMME (10 mg/mL) and C60-IONP-PEG (7.98 mg/mL) for
24 h and then exposed to a 532 nm laser at a power density of
100 mW/cm2 or 300 mW/cm2 for 5 min. Standard SRB assay was
carried out to determine relative viabilities of cells after PDT
treatment. As seen from Fig. 7A, a laser power density-dependent
cytotoxicity of all groups was shown. According to the result, we
can clearly see that C60-IONP-PEG exhibit a relatively small
cytotoxic to B16-F10 cells, while C60-IONP-PEG/532 nm laser group
greatly enhanced the cytotoxic, indicating C60-IONP-PEG is a
promising photodynamic agent for cancer therapy. An advantage of
photodynamic therapy is that can be positioned in the treatments
[41,42], so for the in vivo antitumor study, we just irradiated the
tumor site, and that would reduce the side effects on normal tissues
and organs. Remarkably enhanced cancer cell killing effect was
observed for B16-F10 cells treated by C60-IONP-PEG/HMME after
laser exposure, in comparison to those treated with C60-IONP-PEG
or HMME at the same C60-IONP-PEG and HMME concentrations
(Fig. 7A).
The level of intracellular reactive oxygen species (ROS) was an
important indicator for PDT, therefore, intracellular ROS induced by
C60-IONP-PEG/HMME under the 532 nm laser irradiating was also
determined. ROS productions were observed in B16-F10 cells
incubated with C60-IONP-PEG/HMME by using DCFH-DA fluores-
cent probe (Fig. 7B). As shown in Fig. 7B, green fluorescence of
DCFH was observed in cancer cells incubated with C60-IONP-PEG,
HMME or C60-IONP-PEG/HMME at the same presence of HMME
(10 mg/mL) and C60-IONP-PEG (7.98 mg/mL) for 24 h and then
exposed to a 532 nm laser at a power density of 100 mW/cm2 or
300 mW/cm2 for 5 min, whereas control cells or without laser
irradiating showed negligible DCFH fluorescence. Greener fluores-
cence of DCFH was observed for B16-F10 cells treated by C60-IONP-
PEG/HMME after laser exposure, in comparison to those treated
with C60-IONP-PEG or HMME at the same C60-IONP-PEG and
HMME concentrations, indicating that C60-IONP-PEG/HMME
greatly improved the PDT efficacy of HMME.
 J. Shi et al. / Biomaterials 34 (2013) 9666e9677
Fig. 4. Magnetic properties of C60-IONP-PEG A) Photos of C60-IONP-PEG in water with and without a magnet; B) Magnetization loops of C60-IONP-PEG and IONP; C) T2-weighted
MR images of C60-IONP-PEG solutions at different concentrations; D) T2 relaxation rates (R2) of C60-IONP-PEG solutions at different concentrations.
3.4. Cellular uptake
To further explore the difference in uptake of drug-
nanostructure system by B16-F10 cells, we tracked HMME inter-
nalization into the cells through colocalization of HMME signal (red
fluorescence). Red fluorescence of HMME and blue emission of
DAPI for cell nucleus staining can be simultaneously observed in
B16-F10 cells at different incubation time (Fig. 8A). To semi-
quantify HMME in cells at different incubation time, Image J 4.0
was used to analyze the cellular uptake of C60-IONP-PEG/HMME
and HMME by area of integrated optical density (AOI) of fluores-
cence image (Fig. 8B). Cells incubated with C60-IONP-PEG/HMME
for different periods of time clearly revealed the time-dependent
cellular uptake of the nanocomposite, and the uptake of C60-
IONP-PEG/HMME was faster than that of HMME (Fig. 8), and
more HMME signal was found in nuclei of B16-F10 cells at 3 h in
C60-IONP-PEG/HMME group than that of HMME alone. This
distinction in uptake could explain the difference in susceptibility
of C60-IONP-PEG/HMME and HMME to B16-F10 cells.
3.5. Magnetic drug targeting in vitro
We utilized the magnetic property of C60-IONP-PEG/HMME for
magnetic targeted drug delivery. B16-F10 cells were incubated with
C60-IONP-PEG/HMME for 2 h at 37  C in the presence of a magnetic
field (magnetic field intensity: 0.3 T) (Fig. S3, A). After cells were
washed to remove free nanoparticles, fluorescence images revealed
high uptake of C60-IONP-PEG/HMME for cells close to the magnet,
while in areas around 1 cm from the magnet, rather low HMME
9671
signals were observed inside cells (Fig. S3, B and C). After exposing
the cells under 532 nm laser (300 mW/cm2, 5 min), DCFH-DA
fluorescent probe was added to determine the ROS productions,
green fluorescence of DCFH was observed (Fig. S3, D and E). There
was only trace green fluorescence in cells around 1 cm from the
magnet, while the cells close to the magnet showed strong fluo-
rescence of DCFH. The above results showed that IONPs encapsu-
lated in nanocomposite could be used for magnetic targeted drug
delivery.
3.6. In vitro MRI
To evaluate the diagnostic potential of the C60-IONP-PEG as a
MRI contrast agent, B16-F10 cells after incubation with different
concentrations of C60-IONP-PEG for 3 h was investigated on a
clinical 3.0 T MRI scanner, clearly showing that T2-weighted MRI
images of the cells incubated with C60-IONP-PEG presented a sig-
nificant negative contrast enhancement in comparison to that of
the control cells (Fig. S4). Meanwhile, the dose-dependent MRI
signal intensity of B16-F10 cells (Fig. S4) displays a significant MRI
signal intensity decrease with the increase of the incubation con-
centration of C60-IONP-PEG, revealing the capability of C60-IONP-
PEG as an effective T2 MRI contrast agent for cell labeling.
3.7. In vivo cancer PDT
To investigate in vivo PDT efficacy of C60-IONP-PEG/HMME,
comparative efficacy studies were conducted. The B16-F10 tumor-
bearing mice were divided into 7 groups and were treated
9672 J. Shi et al. / Biomaterials 34 (2013) 9666e9677

Fig. 5. Characterization of C60-IONP-PEG/HMME A) Photos of C60-IONP-PEG/HMME in a) water, b) PBS buffer, c) plasma of mice and d) cell culture for two weeks; B) UV spectrum
of 1) C60-IONP-PEG and 2) C60-IONP-PEG/HMME in water; C) HMME loading at different feeding amounts of HMME; D) HMME release profiles. Data were presented as
mean  standard deviation (n ¼ 3).

Fig. 6. Characterization of C60-IONP-PEG/HMME A) and B) Dynamic light scattering analysis of C60-IONP-PEG showing an average size distribution around 163 nm and a zeta
potential around 33.6 mV; C) TEM image of C60-IONP-PEG/HMME.
 J. Shi et al. / Biomaterials 34 (2013) 9666e9677 9673

Fig. 7. Photodynamic therapy of cancer cells A) Relative viabilities of B16-F10 cells treated with C60-IONP-PEG, HMME and C60-IONP-PEG/HMME with or without laser irradiation
(532 nm, 100 mW/cm2 or 300 mW/cm2, 5 min); B) Detection of intracellular reactive oxygen production (ROS) by DCFH-DA staining in B16-F10 cells incubated with C60-IONP-PEG/
HMME a) Control cells; b) C60-IONP-PEG; c) HMME and d) C60-IONP-PEG/HMME. Data were presented as mean  standard deviation (n ¼ 6).

Fig. 8. A) Confocal images of B16-F10 cells incubated with a) HMME and b) C60-IONP-PEG/HMME for 1, 2, and 3 h; B) Uptakes of HMME and C60-IONP-PEG/HMME in cancer cells.
Data were presented as mean  standard deviation (n ¼ 3).
9674 J. Shi et al. / Biomaterials 34 (2013) 9666e9677

according to protocols as summarized in method Section 2.7. The
changes of relative tumor volume as a function of time were plotted
in (Fig. 9A). After 10 days treatment, control group showed a rela-
tive tumor volume (V/V0) of 11.27  0.94, 532 nm alone group and
C60-IONP-PEG vehicle showed (V/V0) of 10.96  0.49 and
11.33  1.12, suggesting that 532 nm laser alone and C60-IONP-PEG
without irradiating would not affect the tumor growth. HMME/
532 nm laser and C60-IONP-PEG/532 nm laser resulted in V/V0 of
6.45  0.81 and 5.96  0.79, the tumor-bearing mice treated with
C60-IONP-PEG/HMME/532 nm laser and C60-IONP-PEG/HMME/
magnet/532 nm laser achieved (V/V0) of 2.72  0.55 and
1.69  0.42. C60-IONP-PEG/HMME/magnet/532 nm laser had tu-
mor growth inhibition (TGI) of 85.0%, it is significantly more
effective than the other therapeutic groups (p < 0.05). Compared
with C60-IONP-PEG, mice treated with C60-IONP-PEG/532 nm
laser, the tumor volume was greatly reduced, this is a successful
application that C60 was used as a photosensitizer in PDT to ach-
ieve in vivo tumor treatment efficacy. Remarkably enhanced PDT
effect was observed for tumor-bearing mice treated by C60-IONP-
PEG/HMME after laser exposure, in comparison to those treated
with C60-IONP-PEG or HMME at the same C60-IONP-PEG and
HMME doses. Because of the magnetic targeting property of C60-
IONP-PEG/HMME, when a magnet was glued to the top of the tu-
mor, more C60-IONP-PEG/HMME would go to the tumor site than
non-magnetic group, so the PDT efficacy of C60-IONP-PEG/HMME
with a magnet was higher than that of non-magnetic group (Fig. 9).
The growth of tumor tissue was successfully suppressed by C60-
IONP-PEG/HMME/magnet/532 nm laser. This high therapeutic ef-
ficacy originates from the high HMME and C60 accumulation in
tumor tissue.
Allowing for high toxicity usually leads to weight loss, body
weight of the mice for all groups were measured during the
treatments, and no weight loss was observed (Fig. 9B), implying
that the toxicity of treatments were not obvious.
Histological analysis of tumor tissue in different treatment
groups at day 10 post-treatment (Fig. 9C) reveals that no damage
was found in tumors of control, 532 nm laser alone and C60-IONP-
PEG mice. However, markedly increased apoptotic and necrotic

Fig. 9. In vivo PDT treatments A) Tumor growth of mice in different treatment groups within 10 days. *, P < 0.05, C60-IONP-PEG/HMME/532 nm laser versus C60-IONP-PEG/HMME/
magnet/532 nm laser; B) Changes of body weight of mice in different groups during treatment; C) H&E stained tumor tissues harvested from the mice with different treatments, 1e
7: control, 532 nm laser, C60-IONP-PEG, C60-IONP-PEG/532 nm laser, HMME/532 nm laser, C60-IONP-PEG/HMME/532 nm laser and C60-IONP-PEG/HMME/magnet/532 nm laser.
Data were presented as mean  standard deviation (n ¼ 6).
 J. Shi et al. / Biomaterials 34 (2013) 9666e9677 9675

tumor cells were observed in all PDT treatment groups, and

furthermore a large amount of cell death in tumor tissue was
observed in the mice treated with C60-IONP-PEG/HMME/magnet/
532 nm laser.
Recently, a number of groups including our group have used
carbon nanotubes and graphene, sisters of C60, for the delivery of
photodynamic therapy agents. Huang, P. etc. developed a folic
acid-conjugated graphene loaded a PDT drug Ce6 as effective drug
delivery system in targeting PDT [43]. Tian, B. etc. developed a
Ce6 delivery system based on graphene oxide (GO) for potential
multi-functional cancer therapies [44]. Our group used hyaluronic
acid modified carbon nanotubes (CNTs) as a HMME delivery for
tumor targeting PDT [45]. C60 has the intrinsic singlet oxygen
generation ability while CNTs and GO do not have, the above
nanoparticles have no biological activity to improve the photo-
dynamic efficacy of PDT. So in the carbon nanomaterials, C60
should be the most appropriate for the delivery of photodynamic
therapy agents.

3.8. Biodistribution

To understand tumor treatment efficacy of various HMME

formulations (HMME, C60-IONP-PEG/HMME, and C60-IONP-PEG/
HMME/magnet), the biodistribution of HMME in tumor and
various main organs was investigated. It was observed that sig-
nificant differences for biodistributions of HMME in the three
formulations (Fig. 10). C60-IONP-PEG/HMME and C60-IONP-PEG/
HMME/magnet showed noticeable HMME activity in blood at
30 min or 3 h after injection, whereas HMME levels in the blood
were much lower in the HMME group (P < 0.001), indicating that
C60-IONP-PEG/HMME significantly increased the blood circulation
time of HMME in vivo. Differences in biodistributions of HMME
were the most obvious at 3 h after injection, C60-IONP-PEG/
HMME and C60-IONP-PEG/HMME/magnet both with high HMME
levels in the RES organs (liver/spleen) were observed (Fig. 10).
Importantly, C60-IONP-PEG/HMME/magnet afforded much higher
HMME uptake in tumor than HMME and C60-IONP-PEG/HMME
(P < 0.05). The HMME level in tumor of C60-IONP-PEG/HMME/
magnet group was higher than that of in HMME group and C60-
IONP-PEG/HMME group by 14- and 1.6-fold, respectively, at 3 h
after injection and by 23- and 2.6-fold higher, respectively, at 12 h
after injection (Fig. 10). The ability of higher drug delivery effi-
ciency to tumor by C60-IONP-PEG/HMME/magnet was striking
and directly responsible for the higher tumor suppression efficacy
than the other formulations in PDT. Fig. 10. Biodistribution in tumor-bearing mice injected with HMME, C60-IONP-PEG/
HMME, and C60-IONP-PEG/HMME with a magnet in tumor site at 30 min, 3 and 12 h
after injection (i.v.). *, P < 0.05, C60-IONP-PEG/HMME versus C60-IONP-PEG/HMME/
3.9. In vivo MRI magnet. Data were presented as mean  standard deviation (n ¼ 6).

The excellent biocompatibility and high in vitro MRI contrast

performance of C60-IONP-PEG inspired us to pursue their appli-
cability for in vivo trials. Female C57 mice bearing B16-F10 tumors
were intravenously injected with C60-IONP-PEG (200 mL, 100 mg/
mL) for 3 h and imaged by a 3-T clinical MR scanner. An obvious
darkening effect in the tumor, liver spleen and kidney was
observed in T2-weighted MR images (Fig. 11A). The darkening of
the MR images in the mouse organs after injection was also
confirmed quantitatively, as shown in Fig. 11B. The signal in-
tensities of liver, spleen and kidney were reduced remarkably after
the administration of C60-IONP-PEG. After injection of C60-IONP-
PEG with a magnet glued onto the tumor for 3 h, a greater increase
was shown in the signal intensities of liver, spleen and kidney than
that of without the magnet (Fig. 11B), while the magnet glued
group exhibited a decrease in the signal intensities of tumor
(Fig. 11B), proving that the magnetic targeting property of C60-
IONP-PEG existed in vivo. Consequently, these results suggest
that C60-IONP-PEG can act as a suitable negative (T2) contrast
agent in MRI applications.
4. Conclusion
In summary, a fullerene-based multi-functional magnetic
nanocomposite, C60-IONP-PEG was developed. The nanoscale
C60-IONP-PEG showed neglectable toxicity, and could serve not
only as a powerful PDT and MR imaging agent but also as a
magnetic targeted drug delivery carrier. A new PDT drug HMME
was loaded onto C60-IONP-PEG with high loading efficacy to form
an enhanced PDT drug delivery system. In the in vitro and in vivo
studies, C60-IONP-PEG/HMME showed excellent PDT efficacy,
magnetic targeting property and MRI ability, indicating that there
is a great potential of C60-IONP-PEG/HMME for cancer theranostic
applications.
9676 J. Shi et al. / Biomaterials 34 (2013) 9666e9677
Fig. 11. A) In vivo T2-weighted MRIimages 1) control, 2) 3 h after injection of C60-IONP-PEG and 3) 3 h after injection of C60-IONP-PEG with a magnet in tumor site; B) the relative
signal intensity in liver, spleen, kidney and tumor after administration of C60-IONP-PEG with or without a magnet. *, P < 0.05, C60-IONP-PEG versus C60-IONP-PEG/magnet. Data
were presented as mean  standard deviation (n ¼ 3).
Acknowledgments
The work is supported by grants from the National Natural
Science Foundation of China (Nos.3097/3660). We wish to thank
Ms. Yanxia Cao (Material Department, Zhengzhou University) for
her technical assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2013.08.049.
References
[1] Gomaa I, Ali SE, El-Tayeb TA, Abdel-kader MH. Chlorophyll derivative medi-
ated PDT versus methotrexate: an in vitro study using MCF-7 cells. Photo-
diagnosis Photodyn Ther 2012;9:362e8.
[2] Bozzini G, Colin P, Betrouni N, Nevoux P, Ouzzane A, Puech P, et al. Photo-
dynamic therapy in urology: what can we do now and where are we heading?
Photodiagnosis Photodyn Ther 2012;9:261e73.
[3] Tian G, Ren W, Yan L, Jian S, Gu Z, Zhou L, et al. Red-emitting upconverting
nanoparticles for photodynamic therapy in cancer cells under near-infrared
excitation. Small 2013;9:1929e38.
[4] Cui S, Yin D, Chen Y, Di Y, Chen H, Ma Y, et al. In vivo targeted deep-tissue
photodynamic therapy based on near-infrared light triggered upconversion
nanoconstruct. ACS Nano 2013;7:676e88.
[5] Huang P, Lin J, Wang X, Wang Z, Zhang C, He M, et al. Light-triggered thera-
nostics based on photosensitizer-conjugated carbon dots for simultaneous
enhanced-fluorescence imaging and photodynamic therapy. Adv Mater
2012;24:5104e10.
[6] Obaid G, Chambrier I, Cook MJ, Russell DA. Targeting the oncofetal Thomsene
Friedenreich disaccharide using jacalin-PEG phthalocyanine gold nano-
particles for photodynamic cancer therapy. Angew Chem Int Ed Engl 2012;51:
6158e62.
[7] Juarranz A, Jaen P, Sanz-Rodriguez F, Cuevas J, Gonzalez S. Photodynamic
therapy of cancer. Basic principles and applications. Clin Transl Oncol
2008;10:148e54.
[8] Celli JP, Spring BQ, Rizvi I, Evans CL, Samkoe KS, Verma S, et al. Imaging and
photodynamic therapy: mechanisms, monitoring, and optimization. Chem
Rev 2010;110:2795e838.
[9] Detty MR, Gibson SL, Wagner SJ. Current clinical and preclinical photosensi-
tizers for use in photodynamic therapy. J Med Chem 2004;47:3897e915.
[10] Benachour H, Seve A, Bastogne T, Frochot C, Vanderesse R, Jasniewski J, et al.
Multifunctional peptide-conjugated hybrid silica nanoparticles for photody-
namic therapy and MRI. Theranostics 2012;2:889e904.
[11] Bovis MJ, Woodhams JH, Loizidou M, Scheglmann D, Bown SG, Macrobert AJ.
Improved in vivo delivery of m-THPC via pegylated liposomes for use in
photodynamic therapy. J Control Release 2012;157:196e205.
[12] Barth BM, I Altinoglu E, Shanmugavelandy SS, Kaiser JM, Crespo-Gonzalez D,
DiVittore NA, et al. Targeted indocyanine-green-loaded calcium phosphosili-
cate nanoparticles for in vivo photodynamic therapy of leukemia. ACS Nano
2011;5:5325e37.
[13] Maeda H, Matsumura Y. EPR effect based drug design and clinical outlook for
enhanced cancer chemotherapy. Adv Drug Deliv Rev 2011;63:129e30.
[14] Shi J, Zhang H, Wang L, Li L, Wang H, Wang Z, et al. PEI-derivatized fullerene
drug delivery using folate as a homing device targeting to tumor. Biomaterials
2013;34:251e61.
[15] Montellano A, Da Ros T, Bianco A, Prato M. Fullerene C(6)(0) as a multifunc-
tional system for drug and gene delivery. Nanoscale 2011;3:4035e41.
[16] Shi J, Wang Z, Wang L, Wang H, Li L, Yu X, et al. Photodynamic therapy of a 2-
methoxyestradiol tumor-targeting drug delivery system mediated by Asn-
Gly-Arg in breast cancer. Int J Nanomedicine 2013;8:1551e62.
[17] Chaudhuri P, Paraskar A, Soni S, Mashelkar RA, Sengupta S. Fullerenol-cyto-
toxic conjugates for cancer chemotherapy. ACS Nano 2009;3:2505e14.
[18] Fan J, Fang G, Zeng F, Wang X, Wu S. Water-dispersible fullerene aggregates as
a targeted anticancer prodrug with both chemo- and photo-dynamic thera-
peutic actions. Small 2013;9:613e21.
[19] Chen Z, Ma L, Liu Y, Chen C. Applications of functionalized fullerenes in tumor
theranostics. Theranostics 2012;2:238e50.
[20] Zhen M, Zheng J, Ye L, Li S, Jin C, Li K, et al. Maximizing the relaxivity of Gd-
complex by synergistic effect of HSA and carboxylfullerene. ACS Appl Mater
Interfaces 2012;4:3724e9.
[21] Liu J, Ohta S, Sonoda A, Yamada M, Yamamoto M, Nitta N, et al. Preparation of
PEG-conjugated fullerene containing Gd3þ ions for photodynamic therapy.
J Control Release 2007;117:104e10.
[22] Milanesio ME, Alvarez MG, Rivarola V, Silber JJ, Durantini EN. Porphyrin-
fullerene C60 dyads with high ability to form photoinduced charge-separated
state as novel sensitizers for photodynamic therapy. Photochem Photobiol
2005;81:891e7.
[23] Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin VP, et al.
Vascular permeability in a human tumor xenograft: molecular size depen-
dence and cutoff size. Cancer Res 1995;55:3752e6.
[24] Charrois GJ, Allen TM. Rate of biodistribution of STEALTH liposomes to tumor
and skin: influence of liposome diameter and implications for toxicity and
therapeutic activity. Biochim Biophys Acta 2003;1609:102e8.
 J. Shi et al. / Biomaterials 34 (2013) 9666e9677
[25] van Nostrum CF. Polymeric micelles to deliver photosensitizers for photody-
namic therapy. Adv Drug Deliv Rev 2004;56:9e16.
[26] Bugaj AM. Targeted photodynamic therapyea promising strategy of tumor
treatment. Photochem Photobiol Sci 2011;10:1097e109.
[27] Schmitt F, Lagopoulos L, Kauper P, Rossi N, Busso N, Barge J, et al. Chitosan-based
nanogels for selective delivery of photosensitizers to macrophages and improved
retention in and therapy of articular joints. J Control Release 2010;144:242e50.
[28] Roy I, Ohulchanskyy TY, Pudavar HE, Bergey EJ, Oseroff AR, Morgan J, et al.
Ceramic-based nanoparticles entrapping water-insoluble photosensitizing
anticancer drugs: a novel drug-carrier system for photodynamic therapy. J Am
Chem Soc 2003;125:7860e5.
[29] Riegler J, Wells JA, Kyrtatos PG, Price AN, Pankhurst QA, Lythgoe MF. Targeted
magnetic delivery and tracking of cells using a magnetic resonance imaging
system. Biomaterials 2010;31:5366e71.
[30] Kato T, Nemoto R, Mori H, Abe R, Unno K, Goto A, et al. Magnetic microcap-
sules for targeted delivery of anticancer drugs. Appl Biochem Biotechnol
1984;10:199e211.
[31] Zhang J, Misra RD. Magnetic drug-targeting carrier encapsulated with ther-
mosensitive smart polymer: core-shell nanoparticle carrier and drug release
response. Acta Biomater 2007;3:838e50.
[32] Fu A, Wilson RJ, Smith BR, Mullenix J, Earhart C, Akin D, et al. Fluorescent
magnetic nanoparticles for magnetically enhanced cancer imaging and tar-
geting in living subjects. ACS Nano 2012;6:6862e9.
[33] Hassan EE, Gallo JM. Targeting anticancer drugs to the brain. I: enhanced brain
delivery of oxantrazole following administration in magnetic cationic mi-
crospheres. J Drug Target 1993;1:7e14.
[34] Cheng L, Yang K, Li Y, Zeng X, Shao M, Lee ST, et al. Multifunctional nano-
particles for upconversion luminescence/MR multimodal imaging and
magnetically targeted photothermal therapy. Biomaterials 2012;33:2215e22.
[35] Huang P, Li Z, Lin J, Yang D, Gao G, Xu C, et al. Photosensitizer-conjugated
magnetic nanoparticles for in vivo simultaneous magnetofluorescent imaging
and targeting therapy. Biomaterials 2011;32:3447e58.
9677
[36] Xinxing Ma HT, Yang Kai, Feng Liangzhu, Cheng Liang, Shi Xiaoze,
Li Yonggang, et al. A functionalized graphene oxideeiron oxide nano-
composite for magnetically targeted drug delivery, photothermal therapy, and
magnetic resonance imaging. Nano Res 2012;5:199e212.
[37] Sun HM, Cao LY, Lu LH. Magnetite/reduced graphene oxide nanocomposites:
one step solvothermal synthesis and use as a novel platform for removal of
dye pollutants. Nano Res 2011;4:550e62.
[38] Shi X, Gong H, Li Y, Wang C, Cheng L, Liu Z. Graphene-based magnetic plas-
monic nanocomposite for dual bioimaging and photothermal therapy. Bio-
materials 2013;34:4786e93.
[39] Girard OM, Ramirez R, McCarty S, Mattrey RF. Toward absolute quantification
of iron oxide nanoparticles as well as cell internalized fraction using multi-
parametric MRI. Contrast Media Mol Imaging 2012;7:411e7.
[40] Cai H, Gu Y, Zeng J, Li SR, Wang Y, Shi DW, et al. Effect of HMME-PDT on
hyperplastic scar in rabbit ear model. Zhonghua Zheng Xing Wai Ke Za Zhi
2007;23:425e7.
[41] Zhao T, Shen X, Li L, Guan Z, Gao N, Yuan P, et al. Gold nanorods as dual photo-
sensitizing and imaging agents for two-photon photodynamic therapy.
Nanoscale 2012;4:7712e9.
[42] Gandra N, Abbineni G, Qu X, Huai Y, Wang L, Mao C. Bacteriophage bio-
nanowire as a carrier for both cancer-targeting peptides and photosensitizers
and its use in selective cancer cell killing by photodynamic therapy. Small
2013;9:215e21.
[43] Huang P, Xu C, Lin J, Wang C, Wang X, Zhang C, et al. Folic acid-conjugated
graphene oxide loaded with photosensitizers for targeting photodynamic
therapy. Theranostics 2011;1:240e50.
[44] Tian B, Wang C, Zhang S, Feng L, Liu Z. Photothermally enhanced photody-
namic therapy delivered by nano-graphene oxide. ACS Nano 2011;5:7000e9.
[45] Shi J, Ma R, Wang L, Zhang J, Liu R, Li L, et al. The application of hyaluronic
acid-derivatized carbon nanotubes in hematoporphyrin monomethyl ether-
based photodynamic therapy for in vivo and in vitro cancer treatment. Int J
Nanomedicine 2013;8:2361e73.