Journal of Controlled Release 272 (2018) 145–158

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Light/magnetic hyperthermia triggered drug released from multi-functional T

thermo-sensitive magnetoliposomes for precise cancer synergetic
theranostics
Yuxin Guoa, Yang Zhangb, Jinyuan Maa, Qi Lia, Yang Lic, Xinyi Zhoua, Dan Zhaoa, Hua Songa,
⁎ ⁎
Qing Chena, , Xuan Zhua,
a
Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of Pharmaceutical Sciences, Xiamen University, Xiamen, China
b
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics and Center for Molecular Imaging and Translational Medicine, School of Public Health,
Xiamen University, Xiamen, China
c
College of Materials, Xiamen University, Xiamen, China

A R T I C L E I N F O A B S T R A C T

Keywords: Precise delivery of antineoplastic drugs to specific tumor region has drawn much attention in recent years.
Thermo-sensitive Herein, a light/magnetic hyperthermia triggered drug delivery with multiple functionality is designed based on
Magnetoliposomes methotrexate (MTX) modified thermo-sensitive magnetoliposomes (MTX-MagTSLs). In this system, MTX and
Light/magnetic hyperthermia oleic acid modified magnetic nanoparticles (MNPs) can be applied in biological and magnetic targeting.
Triggered release
Meanwhile, lipophilic fluorescent dye Cy5.5 and MNPs are encapsulated into the bilayer of liposomes, which can
Magnetic/folate receptor targeting
Fluorescence/magnetic resonance imaging
not only achieve dual-imaging effect to verify the MTX-MagTSLs accumulation in tumor region, but also provide
an appropriate laser irradiation region to release Doxorubicin (Dox) under alternating magnetic field (AMF).
Both in vitro and in vivo results revealed that MTX-MagTSLs possessed an excellent targeting ability towards HeLa
cells and HeLa tumor-bearing mice. Furthermore, the heating effect of MTX-MagTSLs was amplified 4.2-fold
upon combination with AMF and local precise near-infrared laser irradiation (808 nm) (DUAL-mode) to rapidly
reach the phase change temperature (Tm) of MTX-MagTSLs in 5 min compared with either AMF or laser sti-
mulation alone, resulting in a significantly enhanced release of Dox at tumor region and precise cancer syner-
getic theranostics.

1. Introduction Folate-mediated receptor targeting is found to be promising in li-

Following decades of research in drug delivery systems, nanoscale
liposomal formulations is considered as one of the most advanced de-
livery systems for cytotoxic drugs [1,2]. Long circulating Dox-loaded
liposomes (Caelyx®, Schering-Plough; DOXIL®, Ortho Biotech), which
have been used in clinical cancer therapy for many years [3], is proved
to reduce cardiotoxic side effects [4] and improve pharmacokinetic
profile compared with free Dox due to the enhanced permeability and
retention effect (EPR) [5]. However, the therapeutic efficacy of lipo-
somal formulations is still undesired. Recent research reveal that it can
be further enhanced by either biological/physical targeting [6] or lo-
calized triggered drug release [7] from responsive liposomal formula-
tions to increase the drug concentration at the target site. Hence, tar-
geted and thermo-sensitive liposomes (TSLs) attract much attention due
to the capacity of releasing encapsulated molecules when the tem-
perature reach near Tm caused by the decomposition of TSLs [8].
posomal drug delivery for various cancer cells, including ovarian
[9–11], breast [12,13], kidney, lung, brain, endometrial [14,15], and
colon cancer cells [16], all of which overexpress folate receptor on the
surface of cell membranes. Besides acting the role of anticancer drug,
MTX also has a good targeting effect to folate receptor-overexpressed
cancer cells such as human cervical carcinoma (HeLa) cells, due to the
similar structure with folate [17–20]. MTX can be connected to phos-
pholipid molecules through amide reaction for functionalization of
TSLs, which introduce folate receptor-mediated active targeting
strategy in the treatment of cancer [21]. Although several targeting
strategies have been developed in recent decades, the folate receptor-
mediated active targeted delivery is still limited by the insufficient drug
release at tumor site, which can be improved by local temperature-
triggered drug release from the TSLs [22–24].
MNPs, in particular iron oxides, is approved by Food and Drug
Administration for in vivo usage due to their low toxicity and good

⁎
Corresponding authors.
E-mail addresses: chenqing@xmu.edu.cn (Q. Chen), zhuxuan@xmu.edu.cn (X. Zhu).

https://doi.org/10.1016/j.jconrel.2017.04.028
Received 24 November 2016; Received in revised form 6 April 2017; Accepted 13 April 2017
Available online 23 April 2017
0168-3659/ © 2017 Published by Elsevier B.V.
Y. Guo et al.
biocompatibility. It is applied widely in biomedical fields [25,26]. For
instance, MNPs can be used in magnetic targeting, thermal therapy, and
magnetic resonance imaging (MRI) [27,28], which allow to monitor the
biodistribution in vivo non-invasively. Magnetic TSLs (MagTSLs, mag-
netic nanoparticles encapsulated within TSLs) appear to realize mag-
netocaloric mediated triggered drug release based on Brown and Neel
relaxations [29,30]. In order to achieve a high magnetocaloric con-
version efficiency, MNPs are embedded into the hydrophobic bilayer to
lead heat dissipating directly into the surrounding bilayer and bring the
temperature across the Tm of the liposomes to achieve drug release
effect [31].
In addition, it is reported that MagTSLs can convert radiofrequency
irradiation energy into heat energy under AMF to trigger drug release
when the temperature reach near the Tm of TSLs [32,33]. However,
such a magnetocaloric way still suffers from several disadvantages,
including the injure of the health tissues and the low heating efficiency
which brings inefficient drug release, both of which could impede the
clinical translation of the magnetocaloric therapy. In recent study, it is
demonstrated that magnetocaloric effect of iron oxide-based nanocubes
can be significantly enhanced when in combination with laser treat-
ment [34]. Thus, we assume that the heating effect of MagTSLs can also
be increased appreciably under the simultaneous stimulation of AMF
and laser, achieving a better enhanced drug release effect as well as
reducing the side effects.
Incorporating both antineoplastic drugs and imaging agents within
a multi-functional carrier combined with active targeting, followed by
delivering and releasing the right drug to the right location at the right
time based on certain mechanism, which can achieve precise cancer
synergetic theranostics [35–37]. Herein, we design the MTX modified
TSLs encapsulating MNPs/Cy5.5 in lipid bilayer and Dox in hydrophilic
lumen, combining biological/physical targeting, light/magnetic medi-
ated triggered drug release, and fluorescence/MR imaging for the
cancer therapy. A conceptual scheme for MTX-MagTSLs targeting to
tumor site and releasing Dox locally is shown in Fig. 1. Magnetic and
folate receptor targeting effect support and accelerate MTX-MagTSLs
targeting to tumor tissue from intravenous injection in nude mouse,
then proceed cell uptake and accelerate drug release at tumor site under
the simultaneous application of AMF and laser followed by working
finally in nuclear to cause tumor cell killing accurately.
To the best of our knowledge this is the first report committed to
precise medicine about using AMF and near-infrared laser irradiation
(808 nm) synchronously to stimulate the rapid release of drug from
multi-functional MagTSLs with dual-mode targeting and dual-mode
imaging, which is expected to improve therapeutic efficacy, reduce the
side effects of Dox, and realize the precise cancer synergetic ther-
anostics.
2. Materials and methods
2.1. Materials
Dipalmitoylphosphatidylcholine (DPPC), 1,2-diacyl-SN-glycero-3-
phosphoethanolamine-N-[methoxy(poly(ethyleneglycol))-2000] (DSPE-
MPEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ami
no(polyethylene glycol)-2000] (DSPE-PEG2000-NH2), and N-hydro-
xysuccinimide (NHS) were purchased from Shanghai Ponsure
Biotechnology (Shanghai, China). Cholesterol (Chol) was obtained from
Shanghai Advanced Vehicle Technology Co. Ltd. (Shanghai, China).
Octadecanamine (SA) was obtained from Sigma-Aldrich. Doxorubicin
(Dox) was from Beijing Huachunlianbo Technology Co., Ltd. (Beijing,
China). Iron (III) acetylacetonate was purchased from Alfa Aesar.
Methotrexate (MTX) was purchased from Bio Basic Inc. (Markham,
Ontario, Canada). Dicyclohexylcarbodiimide (DCC) was purchased
from J&K Chemical Scientific Co. Ltd. (Beijing, China). Cy5.5 ester was
obtained from AAT Bioquest. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazo-lium bromide (MTT) was obtained from Life Technologies
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Journal of Controlled Release 272 (2018) 145–158
(Carlsbad, CA). Dimethyl sulfoxide (DMSO) was brought from Aladdin.
HeLa cells and A549 cells were provided by American Type Culture
Collection (ATCC). Dulbecco's minimum essential medium (DMEM)
was obtained from GE Healthcare Life Sciences HyClone Laboratories.
Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang
Biological Technology Co., Ltd. (Hangzhou, China). Penicillin and
streptomycin were from Beyotime Biotechnology (Shanghai, China). All
other chemicals were analytical grade and were used without further
purification. The dialysis bags (MWCO = 3500) were from BIOSHARP
(Hefei, China).
2.2. Synthesis and characterization of DSPE-PEG2000-MTX
DSPE-PEG2000-MTX conjugate was synthesized by a previously de-
scribed method [21] with minor modification. Briefly, 16.4 mg of MTX,
4.2 mg of NHS, and 7.4 mg of DCC were dissolved in 2 mL of DMSO in a
round-bottomed flask, and reacted for 6 h under nitrogen atmosphere
protection to activate the carboxyl group of MTX. Then, 20 mg of DSPE-
PEG2000-NH2 was added to the round-bottomed flask, which was dis-
solved in 1.5 mL of DMSO containing 6 μL of TEA. The reaction mixture
was carried on stirring for an additional 72 h under nitrogen atmo-
sphere protection, and filtered to remove the precipitate after reaction
mixture diluting into 2 times with water. The reaction progress was
monitored by thin layer chromatography (TLC) according to the re-
duced quantity of DSPE-PEG2000-NH2/MTX and increased amount of
DSPE-PEG2000-MTX. The prepared product was dialyzed against PBS
and DI water for 48 h respectively to completely remove excess MTX
and byproducts, and then lyophilized for 24 h to obtain solid DSPE–-
PEG2000–MTX. The impurities were further monitored by TLC to detect
whether been removed completely or not after dialysis. The final pro-
duct was analyzed by mass spectrometry (MS) and Fourier transform
infrared spectrometry (FT-IR). MS was performed with a matrix assisted
laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF-
MS), using DSPE-PEG2000-NH2 for comparison. FT-IR was determined
on a Nicolet Avatar370 Fourier Transform Infrared Spectrometer, using
DSPE-PEG2000-NH2 and MTX for comparison.
2.3. Preparation of magnetic nanoparticles (MNPs)
1 mM of Iron (III) acetylacetonate was added into 9 mL of benzyl
alcohol, and then reacted under 200 °C for 10 h after adding 1.5 mL of
oil amine and 1.5 mL of oleic acid quickly, followed by centrifuging
5 min with the speed of 10000 rpm. After removing of supernatant, the
oleic acid-coated iron oxide NPs were dispersed in 10 mL of chloroform
for storage.
2.4. Preparation of Dox loaded MTX-MagTSLs
MTX-MagTSLs were prepared by thin film dispersion method. In this
process, 15 mg of total lipids composed of DPPC:Chol:SA:DSPE-
MPEG2000:DSPE-PEG2000-MTX (mol ratio 67:17:13:1:2), containing
5 mg of MNPs and 100 μg of Cy5.5, were dissolved in 10 mL of
chloroform in a round bottom flask. Then the chloroform was evapo-
rated using a rotator evaporator (N-1100, TOKOY RIKAKIKAI CO.LTD)
at 50 °C to form a thin film on walls of eggplant type flask, starting at
0.05 MPa for 10 min, then increased to 0.9 MPa for 30 min, then the
flask was frozen at − 20 °C for one night. For loading Dox with an
ammonium sulfate gradient loading method [38], 250 mM of ammo-
nium sulfate solution was added into the flask, followed by hydration at
55 °C (above the Tm of MTX-MagTSLs) for 50 min while rotating at
110 rpm at atmospheric pressure using the rotary evaporator, followed
by ultrasound for 30 min and being extrude through 450 nm, 200 nm,
and 100 nm polycarbonate membranes using an extruder (Avanti Polar
Lipids, USA) for size control. The prepared MTX-MagTSLs were dia-
lyzed against PBS for 2 h. 1 mg of Dox was then added into the sus-
pension to incubate at 40 °C for 30 min. Unencapsulated Dox was
Y. Guo et al. Journal of Controlled Release 272 (2018) 145–158

Fig. 1. Schematic illustration of realizing theranostic functionalities using MTX-MagTSLs. The multi-functional MTX-MagTSLs can be targeted to tumor site under constant magnetic field
(CMF) and folate receptor targeting, followed by triggering Dox release under light/magnetic hyperthermia simulation synchronously to achieve the effect of drug treatment. The process
can be monitored by fluorescence and MR imaging.

removed by gel filtration using Sephadex G50 in a HR 10/30 column
(GE Healthcare) with phosphate buffered saline (PBS) as eluent at a
flow rate of 1 mL/min.
2.5. Payload and encapsulation efficiency
After addition of methyl alcohol for demulsification, the Dox con-
centration of MTX-MagTSLs was determined by EnVision Multilabel
Reader (EnVision, USA) under 490/550 nm [39]. A calibration curve
was used for quantification by calculating the Dox concentration of a
dilution series of free Dox in PBS (0.1–5 μg·mL− 1 of Dox concentra-
tion). The Dox encapsulation and loading efficiency was calculated
using the following formula [40]:
Weight Dox of encapsulation
Dox encapsulation efficiency (%) =
Initial weight Dox
Weight Dox of encapsulation
Dox loading efficiency (%) =
Initial weight Total
The lipid concentration of liposomes was determined using phos-
phorus molybdenum blue spectrophotometry as described previously
with minor modification [41] after the liposomes destruction with
chloroform and perchloric acid, then converted to phospho-molybdic
acid by the addition of ammonium molybdate followed by being re-
duced to a blue colored complex by ascorbic acid during heating at
65 °C for 20 min, which was determined colorimetrically at 819 nm
using an ELISA reader (Multiskan™ Go, Thermo Fisher Scientific,
America). A calibration curve was used for quantification by calculating
the lipid concentration of a dilution series of monopotassium phosphate
solution (0.4–2 μg·mL− 1 of phosphorus concentration). The con-
centration of iron of MTX-MagTSLs was measured by Inductively cou-
pled plasma mass spectrometry (7500CE, Agilent Technologies, USA)
after being treated with aqua regia for dissolving iron and diluted with
2% dilute nitric acid. A calibration curve was used for quantification by

147
Y. Guo et al.
calculating the iron concentration of a dilution series of ferric chloride
hexahydrate solution in 2% dilute nitric acid (5–120 ng·mL− 1 of iron
concentration). Then the encapsulation efficiency of MNPs was calcu-
lated by using the final quality of MNPs of MTX-MagTSLs (converted by
the final quality of iron) divided by the initial amount of MNPs (5 mg of
the drying MNPs). The concentration of MTX of MTX-MagTSLs was
measured by UV-VIS spectrophotometer (UV-2550, SHIMADZU, Japan)
due to its absorption to specific wavelengths of ultraviolet [21] after
demulsification and dilution with methanol. A calibration curve was
used for quantification by calculating the MTX concentration of a di-
lution series of free MTX solution in methanol (1–25 μg·mL− 1 of MTX
concentration).
2.6. Characterization
X-ray diffraction (XRD, Rigaku Ultima IV) was used to determine
the crystallinity and phase composition of MNPs. Magnetic properties of
the MNPs and MTX-MagTSLs was measured with a vibrating sample
magnetometer (VSM). The T2-weighted MR images in vitro were con-
ducted on a 7-T MRI scanner (Varian, Palo Alto, CA, USA). To evaluate
the stability of MTX-MagTSLs at 4 °C, the size distribution and zeta
potential of MTX-MagTSLs were determined using a Malvern zetasizer
(Malvern instrument, Worcestershire, UK) apparatus in a week, and the
changes of the encapsulation and loading efficiency of Dox of MTX-
MagTSLs were measured as well within 7 days. Each experiment was
done in triplicate. The morphology of the MNPs and MTX-MagTSLs
were characterized by transmission electron microscopy (TEM, Tecnai
G2 Spirit, FEI, Hong Kong) and scanning electron microscopy (SEM,
Zeiss SIGMA, Germany). The elemental analysis of MTX-MagTSLs was
detected by energy dispersive spectrometer (EDS, X-MaxN, Oxford, UK).
The Tm of different lipids compositions was measured by differential
scanning calorimetry (VP-Capillary, Malvern instrument, UK) between
10 °C and 90 °C at a heating rate of 1 °C/min.
2.7. Specific absorption rate (SAR) measurement
After measuring the absorption spectra of MTX-MagTSLs with a
series of iron concentration (ranging from 2.5 to 15 mM) to investigate
its absorbing ability to 808 nm near-infrared light, the heating ability of
MNPs and MTX-MagTSLs with 13.93 mM of iron were measured by SAR
based time-dependent calorimetric measurements under AMF
(500 kHz, 20 kA·m− 1, the sample was positioned in the center of a 10-
turn circular copper coil with a 8 cm outer diameter), 808 nm near-
infrared laser irradiation (0.8 W/cm2, the sample was placed just below
the laser source), and DUAL-mode (Laser 0.8 W/cm2, AMF 500 kHz,
20 kA·m− 1) for 5 min respectively. The PBS and TSLs were measured in
negative control mode under the same condition. The impacts of the
concentration of iron (ranging from 2.5 to 15 mM) and the laser power
(0.3, 0.8, and 1.2 W/m2) to heat absorption capacity of MTX-MagTSLs
were investigated with the same mode of treatment as well. An alter-
nating magnetic field was produced by an AMF generator (SPG-10-II,
Shenzhen double-Power Technology Co. Ltd. China). Near-infrared
laser irradiation (808 nm) was produced by Stone Laser (STL 808CF-
10W, China). The temperature of the sample was monitored by a
thermocouple thermometer (TES-1310, Taiwan). The SAR value was
calculated using the following equation [42]:
△T m
SAR = CP × V
△t mNP
where CP is the heat capacity of the medium, ΔT/Δt is the experimen-
tally observed heating rate, mV is the mass of the suspension, mNP is the
iron content of the suspension.
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Journal of Controlled Release 272 (2018) 145–158
2.8. In vitro release study
To determinate the release of Dox from MTX-MagTSLs with
13.93 mM of iron, a cellulose membrane (molecular weight cut-off of
3500 kDa, Solarbio, China) was mounted between the donor and re-
ceptor compartments. The donor medium was consisted of 500 μL of
free Dox solution or MTX-MagTSLs formulation with the same con-
centration of Dox (0.182 mg·mL− 1). The receptor medium consisted of
200 mL of PBS (10 mM, pH 7.4). During the dialysis, the temperature
was kept at 37 °C and 45 °C respectively. At pre-determined time in-
tervals, the amount of the released Dox was determined by EnVision
Multilabel Reader (EnVision, USA) under 490/550 nm from 0 to 24 h.
2.9. Hemolysis assay
1 mL of fresh collected whole blood was centrifuged for 5 min at
4 °C with 10000 rpm to isolate red blood cells, then it was washed three
times with the isotonic icy PBS and suspended in 10 mL of icy PBS.
0.5 mL of the prepared solution above was added to 0.5 mL of MagTSLs
at different phospholipid concentration (between 25 and 400 μg·mL− 1)
suspended in PBS, followed by an intensive mixing and retaining for 1 h
at 37 °C, then centrifuged 5 min with 3000 rpm. The absorbance value
of supernatant liquid containing hemoglobin was determined at
560 nm. A similar volume of PBS was used as negative control and de-
ionized (DI) water as the positive control. The percentage of hemolysis
was calculated using the formula: hemolysis value (%) =
(Asample − Anegative control) / (Apositive control − Anegative control) × 100%.
2.10. Pharmacokinetic study
0.5 mL of blood was drawn from eyes of tumor-free healthy SD rats
after intravenous administration of free Dox and MTX-MagTSLs (Dox
dose: 2 mg·kg− 1) at 5, 15, 30, 60, 120, 180, 240, 300, 420, 540, and
1440 min, respectively, followed by centrifuging 10 min at 10000 rpm.
Then 0.1 mL of supernatant was added into 0.3 mL of acid ethanol
solution (12.5 mL of concentrated hydrochloric acid mixed with
250 mL of dehydrated alcohol then added into distilled water as to
500 mL). A calibration curve was used for quantification by calculating
the Dox concentration of a dilution series of free Dox in acidic ethanol
prepared above. Each group comprised six rats. The Dox concentration
of the solution was determined by EnVision Multilabel Reader
(EnVision, USA) under 490/550 nm after mixing by vortexing. The
pharmacokinetic parameters were calculated by fitting the blood drug
concentrations to a non-compartment model using WinNonlin
Professional Edition Version 2.1 (Pharsight Corporation, Mountain
View, California).
2.11. Cell culture
HeLa (human cervical carcinoma) and A549 (human lung adeno-
carcinoma) cell lines were cultured in DMEM supplemented with 10%
fetal calf serum and 100 μg/mL of streptomycin/penicillin at 37 °C in a
humidified atmosphere of 5% CO2.
2.12. Cellular uptake
The cells were seeded at the density of 1 × 105 cells per well in the
6-well plates with complete DMEM. The cells were incubated at 37 °C in
5% CO2 for 24 h. The free Dox and MTX-MagTSLs (10 μg·mL− 1 at Dox-
eq. dose) were added into HeLa cells and incubated further for 1 and
4 h. The cells were washed with PBS, fixed with 4% paraformaldehyde
and stained with DAPI. The fluorescence intensity of cells was observed
using a Leica TCS SP5 LCSM (Leica Microsystems, Mannheim,
Germany). Additionally, to investigate the targeting ability of MTX-
MagTSLs against HeLa cells, MagTSLs and MTX-MagTSLs (10 μg·mL− 1
at Dox-eq. dose) were added into HeLa and A549 cells respectively, and
Y. Guo et al.
incubated further for 4 h. To investigate the impact of AMF and laser on
the drug release, MTX-MagTSLs (10 μg·mL− 1 at Dox-eq. dose) were
added into HeLa cells and incubated further for 1 h after AMF (500 kHz,
20 kA·m− 1, 5 min) and DUAL-mode (0.8 W/cm2, 500 kHz, 20 kA·m− 1,
5 min) respectively.
2.13. Flow cytometry
To measure the internalization of free Dox, MagTSLs, and MTX-
MagTSLs in the absence of external stimulation of physical conditions,
and MTX-MagTSLs under AMF (500 kHz, 20 kA·m− 1, 5 min), near-in-
frared laser irradiation (808 nm, 0.8 W/cm2, 5 min), and DUAL-mode
(0.8 W/cm2, 500 kHz, 20 kA·m− 1, 5 min) respectively, the HeLa and
A549 cells were incubated in 6-well plates at a density of 1 × 105 cells
per well for 24 h at 37 °C in 5% CO2, followed by being cultured with
free Dox, MagTSLs, and MTX-MagTSLs (10 μg·mL− 1 at Dox-eq. dose)
for 1 h or 4 h. The wells cultured with MTX-MagTSLs were treated with
no stimulation, AMF (500 kHz, 20 kA·m− 1, 5 min), 808 nm near-in-
frared laser irradiation (0.8 W/cm2, 5 min), and DUAL-mode (Laser
0.8 W/cm2, AMF 500 kHz, 20 kA·m− 1, 5 min) respectively. After the
incubation for the prescribed period, the cells were washed with cold
PBS, harvested by 0.25% trypsin-EDTA, centrifuged with 1000 rpm for
5 min and resuspended in PBS. The fluorescence intensity of the cells
was assessed using a Beckman Coulter ALTRA Epics flow cytometer
(Beckman Coulter, CA, USA).
2.14. Cytotoxicity study
The MTT assay was used to evaluate the cytotoxicity of the Dox-free
MagTSLs and Dox-free MTX-MagTSLs against HeLa cell, MTX-MagTSLs
against HeLa cell and A549 cells, and MTX-MagTSLs against HeLa cells
under AMF (500 kHz, 20 kA·m− 1), 808 nm near-infrared laser irradia-
tion (0.8 W/cm2), and DUAL-mode (Laser 0.8 W/cm2, AMF 500 kHz,
20 kA·m− 1) respectively. The cells were seeded into 96-well plates at a
density of 5000 cells per well in 100 μL of complete DMEM, followed by
24 h of incubation, then added with Dox-free MagTSLs at different
phospholipid concentrations (25, 50, 100, 200, and 400 μg/mL) for
24 h to study the toxicity of Dox-free MagTSLs, free MTX and Dox-free
MTX-MagTSLs which contained the same MTX concentrations with
MTX-MagTSLs (2.5–10 μg·mL− 1 at Dox-eq, 0.6–2.4 μg·mL− 1 at MTX-
eq.) for 24 h to investigate the toxicity of MTX, free Dox/TSLs/
MagTSLs/MTX-TSLs/MTX-MagTSLs at different Dox concentrations
(2.5, 5, and 10 μg·mL− 1) for 24 h respectively to investigate the toxicity
of MTX-MagTSLs, and MTX-MagTSLs at several concentrations (2.5, 5,
and 10 μg·mL− 1 at Dox-eq.) for 12 h under no stimulation, AMF
(500 kHz, 20 kA·m− 1, 5 min), near-infrared laser irradiation (808 nm,
0.8 W/cm2, 5 min), and DUAL-mode (0.8 W/cm2, 500 kHz, 20 kA·m− 1,
5 min) respectively after 2 h incubation to inspect the toxicity of MTX-
MagTSLs under different conditions. The medium in each well was
replaced by 100 μL of (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide dye (MTT) solution (500 μg/mL in DMEM), followed
by incubating for 4 h at 37 °C and 5% CO2. Then, the medium was re-
moved and added with 100 μL of dimethyl sulfoxide (DMSO) per well.
Absorbance was measured by using a spectrophotometer (Biotek, USA)
at 490 nm, the relative cell viability (%) was calculated by
(Abssample − Absbackground) / (Abscontrol − Absbackground) × 100%.
2.15. Xenograft tumor mouse model
All female BALB/c nude mice (16–18 g) were supplied by Xiamen
University Laboratory Animal Center. The HeLa tumor models were
generated by subcutaneous injection of 2 × 106 cells in 0.1 mL of PBS
into the right shoulder, and the mice were used when the tumor volume
reached 60–100 mm3. All animal experiments were performed under a
protocol approved by Xiamen University Laboratory Animal Center.
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Journal of Controlled Release 272 (2018) 145–158
2.16. In vivo fluorescence imaging
To evaluate the synergetic tumor targeting effect by using MTX and
CMF through the targeting capability of the TSLs, MagTSLs, MTX-TSLs,
MTX-MagTSLs, and MTX-MagTSLs/CMF, the HeLa tumor-bearing nude
mice were randomly divided into six groups (three mice per group),
minimizing the difference of weights and tumor sizes in each group.
0.2 mL of PBS, TSLs, MagTSLs, MTX-TSLs, and MTX-MagTSLs at
2 mg·kg− 1 (Dox-eq. dose) were injected into the mice intravenously
respectively to investigate the tumor targeting efficiency. Two groups
were injected with MTX-MagTSLs, and one of group was fixed with a
10 mm diameter circular magnet on skin surface of tumor (MTX-
MagTSLs/CMF). All mice were used for fluorescence imaging of Cy5.5
using a Maestro™ in vivo imaging system (Cambridge Research &
Instrumentation, Woburn, MA, USA) at 4, 8, and 12 h after injection.
The tumor and major organs (heart, liver, spleen, lung, and kidney)
were excised at 12 h post-injection, followed by washing with 0.9%
NaCl for the ex vivo imaging.
2.17. In vivo MRI
To examine the suitability of MTX-MagTSLs for in vivo MRI and its
dual-targeting (magnetic and folate receptor) ability, HeLa tumor-
bearing nude mice were intravenously injected with 0.2 mL of MTX-
MagTSLs at 2 mg·kg− 1 (Dox-eq. dose) for in vivo MRI. The mice in MTX-
MagTSLs/CMF group were fixed with a 10 mm diameter circular
magnet on skin surface of tumor (taken away before MRI), and the mice
in control group were not fixed the magnet (without CMF). After in-
jection for 0, 4, 8, and 12 h, the pseudo-color processing T2-weighted
MR images were conducted on a 7-T MRI scanner (Varian, Palo Alto,
CA, USA).
2.18. In vivo anticancer effect
HeLa tumor-bearing nude mice were randomly divided into seven
groups and treated as follows: PBS, free Dox, MagTSLs, MTX-MagTSLs,
MTX-MagTSLs/CMF, MTX-MagTSLs/CMF/AMF, and MTX-MagTSLs/
CMF/AMF/Laser. Each group comprised six mice. The mice were in-
jected intravenously with different formulation at 2 mg·kg− 1 (Dox-eq.
dose) every two days. In addition, the MTX-MagTSLs/DUAL group was
added to complementally verify dual-targeting ability for comparison
with the MTX-MagTSLs/CMF/DUAL group. The weight of every mice
was recorded every two days. The tumor sizes were measured using a
caliper every two days and calculated as volume = (tumor
length) × (tumor width)2 / 2, until the animals were given a eu-
thanasia on day 15. Recording each group's survival situation as well.
For histological examination, the heart, liver, spleen, lung, kidney, and
tumor harvested from all groups were fixed in 10% buffered formalin,
then embedded in paraffin, sectioned, stained with hematoxylin/eosin
(H&E), and examined by digital microscopy (D-35578 Wetzlar, Leica
Microsystems CMS GmbH, German).
2.19. Data analysis
Quantitative data were expressed as means ± standard deviation
(SD). The analysis of variance was performed to determine the sig-
nificance level among the tested groups and p values < 0.05 were
considered to be statistically significant.
3. Results and discussion
3.1. Synthesis and characterization of DSPE-PEG2000-MTX
Carboxyl group of MTX was activated and then reacted with amine
group of DSPE-PEG2000-NH2 to form stable amide linkage. After that,
excessive amounts of MTX and other impurities were removed by
Y. Guo et al.
dialysis using PBS buffer/water. The reaction and purification process
were monitored by TLC. The peak of DSPE-PEG2000-MTX in the mass
spectra (Fig. S1A) shifted a molecular weight of approximately 454
(consistent with the molecular weight of MTX) to right. The infrared
spectrum of DSPE-PEG2000-MTX (Fig. S1B) gave peaks at 1637 cm− 1
for C]O stretching vibration absorption peak, 1486 cm− 1 for NeH
bending vibration absorption peak, and 1398 cm− 1 for CeN stretching
vibration absorption peak, derived from the amide bond in structure of
DSPE-PEG2000-MTX. These results confirmed the successful synthesis of
DSPE-PEG2000-MTX which could be incorporated into TSLs later for
targeting to tumor cells via the folate receptor.
3.2. Synthesis and characterization of MNPs and MTX-MagTSLs
MNPs were synthesized using a high temperature decomposition
method. Then MTX-MagTSLs were prepared by the thin film dispersion
method using MNPs and TSLs, followed by encapsulating Dox using
ammonium sulfate gradient loading method. The concentration of lipid,
iron, and Dox of MTX-MagTSLs were 3.12 mg·mL− 1, 13.93 mM, and
0.182 mg·mL− 1 respectively. The encapsulation efficiency of MNPs in
MTX-MagTSLs was 87.6 ± 1.83%. The encapsulation efficiency and
loading efficiency of Dox were 72.74 ± 1.68% and 3.64 ± 0.08%
respectively. The MTX-MagTSLs was schematically illustrated in
Fig. 2A. Hydrophobic MNPs/Cy5.5 was encapsulated in lipid bilayer
Fig. 2. The characterization of MNPs and MTX-MagTSLs. (A) Schematic illustration of MTX-MagTSLs. (B) TEM image of MNPs. (C) TEM image of MTX-MagTSLs. (D) UV–Vis absorption
spectra of free MTX, Mag-TSLs, and MTX-MagTSLs in methanol. (E) Absorbance at 298 nm as a function of MTX concentration. (F) Particle size distribution of MTX-MagTSLs. (G) Zeta
potential distribution of MTX-MagTSLs. (H) The changes of size and zeta potential of MTX-MagTSLs within a week at 4 °C. (I) The changes of encapsulation efficiency and loading
efficiency of Dox in MTX-MagTSLs within a week at 4 °C.
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Journal of Controlled Release 272 (2018) 145–158
and hydrophilic Dox in hydrophilic lumen of MTX modified TSLs. As
observed in TEM (Fig. 2B), the MNPs were uniformly scattered and
showed a diameter of about 4 nm, which was in agreement with the
conclusion that a threshold maximum NPs diameter of 6.5 nm for in-
corporation of NPs into lipid bilayers [31]. The morphology of MTX-
MagTSLs was characterized by TEM (Fig. 2C) and SEM (Fig. S2). It
could be seen that MTX-MagTSLs had a uniform size with oleic acid-
coated iron oxide NPs scattered in the phospholipid bilayer, suggesting
that magnetic nanoparticles were embedded in the lipid membrane.
The elemental analysis (Fig. S2) showed the content of major elements.
Fig. 2D showed the UV–Vis spectra of the free MTX, Mag-TSLs, and
MTX-MagTSLs in methanol. The free MTX and MTX-MagTSLs gave
peaks at around 300 nm deriving from the structure of MTX [43]. MTX
methanol solution revealed a good linear relationship between its
concentration and absorption value at the maximum absorption wa-
velength in the range of 1–25 μg·mL− 1 (Fig. 2E), which calculated the
final concentration of MTX of MTX-MagTSLs (42.7 μg·mL− 1) after de-
mulsification and dilution with methanol. It has been found that MTX-
MagTSLs tended to form monodisperse aggregates in the size of
107.5 ± 1.19 nm primitively (Fig. 2F) with almost no change after a
week (112.3 ± 2.19 nm) as confirmed by DLS, and the zeta potential
also presented near 16.8 ± 1.2 mV primitively (Fig. 2G) with almost
no change after a week (Fig. 2H). Additionally, the encapsulation and
loading efficiency of Dox in MTX-MagTSLs decreased lowly within a
Y. Guo et al. Journal of Controlled Release 272 (2018) 145–158

Fig. 3. Magnetic properties of MNPs and MTX-MagTSLs.

(A) XRD patterns of MNPs. (B) Magnetization loops of
MNPs and MTX-MagTSLs. (C) T2-weighted MR images of
MTX-MagTSLs at different iron concentrations. (D) T2 re-
laxation rates of MTX-MagTSLs at different iron con-
centrations.

week, the overall amplitude of which were < 5% and 0.3% respectively
(Fig. 2I). All results illustrated the good stability of MTX-MagTSLs.
As shown in the XRD spectrum of MNPs (Fig. 3A), the presence of
diffraction peaks at 30.2, 35.5, 43.2, 53.4, 57.0, and 62.7 coincided
well with the 〈220〉, 〈311〉, 〈400〉, 〈422〉, 〈511〉, and
〈440〉 facets of γ-Fe2O3 respectively [44], which indicated that the
formed γ-Fe2O3 nanocrystals loaded in MTX-TSLs are typically cubic
spinel structure. The MNPs and MTX-MagTSLs displayed good magnetic
properties. The magnetization hysteresis loop indicated the super-
paramagnetic nature of oleic acid-coated iron oxide NPs and MTX-
MagTSLs (Fig. 3B), in which the superparamagnetism of oleic acid-
coated iron oxide NPs was much higher than that of MTX-MagTSLs. The
reason was that oleic acid-coated iron oxide NPs embedded in the lipid
membrane would reduce the magnetic properties to a certain extent.
The MTX-MagTSLs could act as a T2 contrast agent for MRI, which
revealed the concentration dependent darkening effect (Fig. 3C). It
showed smaller concentration of MTX-MagTSLs which gave brighter
T2-weighted images. The transverse relaxivity (R2) of the MTX-
MagTSLs was measured to be 60.06 mM− 1 s− 1 (Fig. 3D).
The DSC thermogram (Fig. 4) of MTX-MagTSLs with a series of Fig. 4. Differential scanning calorimetric thermogram of liposomes with a series of
phospholipid components showed different main phase transition peaks phospholipid components.
and peak width respectively. It was observed that the liposomes com-
posed of DPPC showed a sharp phase transition at around 41 °C which
was correlated to its NIR photothermal effect. The decrease of gradual
was in agreement with previously published results [8,32], addition of
absorption was mainly due to the charge transfer and ligand transitions
cholesterol parts broadened the phase transition peak between 37 and
of iron [34]. The absorbance of MTX-MagTSLs at 808 nm increased
47 °C. Further addition of DSPE-MPEG2000 and DSPE-MPEG2000-MTX
linearly with the iron concentration (ranging from 2.5 to 15 mM)
caused sharper phase transition peak and shifted peak to higher tem-
(Fig. 5B). The heating temperature of PBS (Fig. 5C) and TSLs (Fig. 5D)
perature. The main phase transition peaks of MTX-MagTSLs shifted to
induced by different conditions have a minor increase in 5 min, in-
lower temperature when composed of more proportion of DPPC. To
dicated that there was almost no influence on the heating ability of
distinguish between Tm and normal body temperature, the proportion
MTX-MagTSLs under either AMF or laser. Fig. 5E and Fig. 5F showed
of phospholipid components was determined finally as DPPC:Chol:-
the time course of the temperature measured in MNPs and MTX-
SA:DSPE-MPEG:DSPE-PEG2000-MTX = 67:17:13:1:2 in mol ratio, with
MagTSLs suspension with the same iron concentration (13.93 mM)
Tm at around 45 °C, indicated that the prepared MTX-MagTSLs could
under different conditions respectively, from which the SAR values
improve drug release until 40 °C.
were calculated. Intriguingly, it could be calculated that MTX-MagTSLs
had an obviously higher SAR value under DUAL-mode (i.e. 621.5 W/g)
3.3. Measurement of heating ability of MTX-MagTSLs than that under both AMF (i.e. 171.6 W/g) and laser (i.e. 160.4 W/g)
condition, while MNPs had a relatively higher SAR value (i.e. 414.5 W/
Absorption spectra of MTX-MagTSLs were shown in Fig. 5A, which g) than MTX-MagTSLs under the laser, but lower value than MTX-

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Fig. 5. Heating capacity of PBS, TSLs, MNPs, and MTX-MagTSLs under different conditions. (A) Absorption spectra of MTX-MagTSLs at different iron concentrations [Fe] = 2.5–15 mM.
(B) Absorbance at 808 nm as a function of iron concentration. (C) Temperature increase of PBS in Laser (0.8 W/cm2), AMF (500 kHz, 20 kA·m− 1), and DUAL-mode (Laser 0.8 W/cm2,
AMF 500 kHz, 20 kA·m− 1) for 5 min respectively. (D) Temperature increase of TSLs in Laser (0.8 W/cm2), AMF (500 kHz, 20 kA·m− 1), and DUAL-mode (Laser 0.8 W/cm2, AMF 500 kHz,
20 kA·m− 1) for 5 min respectively. (E) Temperature increase of MNPs (CFe = 13.93 mM) under Laser (0.8 W/cm2), AMF (500 kHz, 20 kA·m− 1), and DUAL-mode (Laser 0.8 W/cm2, AMF
500 kHz, 20 kA·m− 1) for 5 min respectively. (F) Temperature increase of MTX-MagTSLs (CFe = 13.93 mM) under Laser (0.8 W/cm2), AMF (500 kHz, 20 kA·m− 1), and DUAL-mode (Laser
0.8 W/cm2, AMF 500 kHz, 20 kA·m− 1) for 5 min respectively. (G) Temperature increase of MTX-MagTSLs at different iron concentrations (2.5–15 mM) under DUAL-mode (Laser 0.8 W/
cm2, AMF 500 kHz, 20 kA·m− 1) for 5 min. (H) Temperature increase of MTX-MagTSLs with different NIR-laser powers under DUAL-mode (Laser 0.3–1.2 W/cm2, AMF 500 kHz,
20 kA·m− 1) for 5 min.

3.4. Doxorubicin release study in vitro

In in vitro release study, the comparative free Dox

(CDox = 0.182 mg·mL− 1) and Dox released from MTX-MagTSLs
(CDox = 0.182 mg·mL− 1, CFe = 13.93 mM) profiles following 24 h in-
cubation in PBS at 37 and 45 °C respectively could be seen in Fig. 6. Dox
released from MTX-MagTSLs at 37 °C in PBS was about 44% but around
83% at 45 °C in 24 h. In contrast, free Dox gave a burst release within
an hour, and was about 97% in PBS in 24 h. The above results showed
that the Dox released from MTX-MagTSLs could be controlled by ad-
justing the temperature, which indicated that a significant enhanced
Dox release could be realized with simultaneous application of AMF
and laser according to SAR results.

Fig. 6. Dox released profiles (CDox = 0.182 mg·mL− 1, CFe = 13.93 mM) in PBS (pH 7.4) 3.5. In vivo pharmacokinetics
at 37 °C and 45 °C (mean ± SD, n = 3).
No notable hemolytic analysis effects of Dox-free MagTSLs were
MagTSLs under DUAL-mode (i.e. 567.0 W/g). It is speculated that observed at phospholipid concentrations between 25 and 400 μg·mL− 1
photothermal effect of MNPs would reduce when encapsulated in li- (< 2% hemolysis) (Fig. S3), which confirmed good blood compat-
posomes. There had never been a coverage about an appreciable en- ibility. In the pharmacokinetics investigation of free Dox and MTX-
hancement of drug release under DUAL-mode stimulation for those MagTSLs, the results showed that Dox plasma concentration in both the
have either poor magnetocaloric effect or photothermal effect based free Dox and the MTX-MagTSLs group was highest at the completion of
multi-functional drug delivery so far, so the speculation is that general injection and then decreased, while the decreasing trend of the free Dox
magnetocaloric effect (due to small size of embedded MNPs) [45], in- group was faster noticeably than the MTX-MagTSLs group after in-
crease outstandingly under laser excitation for the reason that the travenous administration (Fig. S4). Compared to free Dox, the area
transition radiation of electron energy levels of MNPs and the change of under the curve (AUC) of MTX-MagTSLs (3.95 ± 0.46 h μg·mL− 1) was
spin states lead to the enhancement of Brownian and Néel relaxation, about four times greater than that of free Dox
which need further investigation on principle. SAR values were de- (1.01 ± 0.13 h μg·mL− 1). The elimination rate of MTX-MagTSLs and
termined as a function of laser power (ranging from 0.3 to 1.2 W/m2) free Dox were 60.49 ± 5.67 L h− 1 kg− 1 and 244.85 ±
−1 −1
and iron concentrations (ranging from 2.5 to 15 mM). The SAR values 19.18 L h kg , respectively. The elimination half-life (t1/2) of
increased with increasing laser power (Fig. 5G) and iron concentrations MTX-MagTSLs (32.36 ± 2.33 h) was approximately four times longer
(Fig. 5H). Tumor cells were generally found to be more heat-sensitive than that of free Dox (8.17 ± 1.03 h), indicating that MTX-MagTSLs
and vulnerable compared to normal cells between 42 and 47 °C [46,47]. increased the blood circulation time of Dox in vivo markedly. These
Therefore, 0.8 W/m2 was selected for the subsequent experiments. results could be explained by the great stability of the prepared long
circulating TSLs at normal body temperature, and regarded as a re-
ference in in vivo animals treatment cycle (2 days) later as well.

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3.6. In vitro cellular uptake
The folate receptor-mediated targeting ability for MTX-MagTSLs
was studied against HeLa and A549 cells. Both Dox and Cy5.5 (as
markers of MTX-MagTSLs) were used as fluorescent dyes to investigate
the uptake of MagTSLs and MTX-MagTSLs in cells by LCSM. Compared
with free Dox and MagTSLs, the enhanced intracellular internalization
of MTX-MagTSLs at Dox-eq. and Cy5.5-eq. dose in HeLa cells in 1 and
4 h (Fig. 7A and B) indicated high selectivity of MTX-MagTSLs. In ad-
dition, the cellular uptake of MTX-MagTSLs by HeLa cells (folate re-
ceptor-positive) increased more than that of MTX-MagTSLs by A549
cells (folate receptor-negative), and there was significant difference in
cellular uptake of MTX-MagTSLs towards these two cell models
(Fig. 7B). All these results provided strong evidence that MTX could use
to be an effective selective targeted molecule, which would improve
targeting efficiency and reduce side effects. Furthermore, the cellular
uptake of the MTX-MagTSLs treated by DUAL-mode increased signally
more than being influenced by either no additional condition or single
AMF treating condition (Fig. 7C), which demonstrated that the si-
multaneous application of AMF and laser could enhance uptake of
MTX-MagTSLs in cancer cells and accelerate intracellular Dox release
markedly. The enhanced uptake and drug release using only AMF was
caused by two reasons. Firstly, the broad phase transition peak of MTX-
MagTSLs (Fig. 4) leaded to the phospholipid bilayer of MTX-MagTSLs
with increased permeability gradually before achieving the tempera-
ture of the phase transition peak value under AMF. Secondly, the
measured temperature in the experiment represented the macroscopical
temperature of the whole NPs suspension. MNPs embedded in MTX-
MagTSLs generated heat under AMF, followed by passing heat to sur-
roundings to cause solvent warming up, which means the microcosmic
local temperature of MTX-MagTSLs was higher than the whole NPs
suspension [48]. Therefore, although the temperature ascensional
range of MTX-MagTSLs under AMF was approximately 5.2 °C in 5 min
(Fig. 5F), it could still partly promote the release of Dox, and achieve
enhanced uptake and drug release in HeLa cells to a certain extent.
were used to assess the targeting efficacy of MTX and verify the toxicity
of MNPs. On the one hand, there was no significant difference of two
cell lines viability treated with TSLs and MagTSLs respectively, which
was in consistent with good safety features of the MNPs in Fig. 8A. On
the other hand, the cytotoxicity of MTX-MagTSLs against HeLa cells
was higher than treated with free Dox, and MTX-MagTSLs induced
more potent cytotoxicity than MagTSLs against HeLa cells, but smaller
trend against A549 cells. Such a cytotoxicity difference revealed that
MTX could be used to enhance cytotoxic effect selectively ascribed to
folate receptor targeting ability and intrinsic toxicity, which was in
agreement with the previous reports [21]. The enhanced cytotoxicity of
free MTX and Dox-free MTX-MagTSLs compared with Dox-free
MagTSLs demonstrated certain toxicity of MTX at 1.2 and 2.4 μg·mL− 1
of MTX concentration (Fig. 8C). In addition, the Dox-free MTX-MagTSLs
showed a little more cytotoxicity than free MTX due to the better
membrane permeability [49,50]. Furthermore, compared with either
single AMF or laser treating, the enhanced cytotoxicity of MTX-
MagTSLs under DUAL-mode demonstrated that the simultaneous ap-
plication of AMF and laser could lead to enhance cytotoxicity of Dox
due to the acceleration of Dox release and uptake markedly (Fig. 8D).
All these cell viability results were consistent with the results of early

3.7. Flow cytometry

Flow cytometry was performed to confirm the targeting efficiency of

MTX-MagTSLs as a quantitative analysis of the cellular uptake. The
intracellular fluorescence intensity of Dox was enhanced as the in-
cubation time extended from 1 h (Fig. S5A) to 4 h (Fig. S5B) in HeLa
cells. The fluorescence intensity treated with free Dox was higher than
the MagTSLs group but lower than the MTX-MagTSLs group, indicated
that MTX had improved targeting efficacy. The intracellular fluores-
cence intensity of Dox was enhanced as the incubation time extended
from 1 h (Fig. S5C) to 4 h (Fig. S5D) in A549 cells, and there was no
obvious difference of fluorescence intensity between being treated with
MagTSLs and MTX-MagTSLs in A549 cells compared with in HeLa cells,
indicating the good folate receptor-mediated targeting efficacy of MTX-
MagTSLs. Furthermore, the MTX-MagTSLs under DUAL-mode exhibited
higher cellular uptake than either no additional condition or single
AMF treating for processing (Fig. S5E). The quantitative results were in
good agreement with the qualitative results (see Fig. 7), giving further
confirmation of improved cellular uptake efficiency of MTX-MagTSLs
and enhanced Dox release treated with AMF and laser simultaneously.

3.8. In vitro cell viability

HeLa and A549 cells were chosen to evaluate the cytotoxicity of
MTX-MagTSLs. No significant inhibiting effect against HeLa cells was
demonstrated by Dox-free MagTSLs at phospholipid concentrations
between 25 and 400 μg·mL− 1 (Fig. 8A), which indicated Dox-free
MagTSLs had almost no toxicity to HeLa cells within this concentration
range. All the Dox loaded groups presented dose-dependent cytotoxicity
against both HeLa and A549 cells (Fig. 8B). Then these two cell lines
Fig. 7. Qualitative cellular Dox uptake of the MTX-MagTSLs. (A) HeLa cells were treated
with free Dox and MTX-MagTSLs respectively under 1 and 4 h. (B) HeLa and A549 cells
were treated with MTX-MagTSLs respectively, and HeLa cells were treated with MagTSLs
under 4 h. (C) HeLa cells were treated with MTX-MagTSLs under no stimulation, AMF
(500 kHz, 20 kA·m− 1, 5 min), and DUAL-mode (0.8 W/cm2, 500 kHz, 20 kA·m− 1, 5 min)
respectively.

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3.9. In vivo fluorescence and MR imaging

To evaluate the targeting capability of TSLs, MagTSLs, MTX-TSLs,

Fig. 7. (continued)
uptake and flow cytometry experiments, verified that MTX-MagTSLs
could be used as effective targeted anti-tumor agents at the cellular
level, and should be further investigated in vivo.
MTX-MagTSLs, and MTX-MagTSLs/CMF, the above materials were in-
jected intravenously into the HeLa tumor-bearing nude mice. In vivo
biodistribution was investigated by a non-invasive near-infrared (NIR)
optical imaging technique. The TSLs and MagTSLs group presented
weak fluorescence signal in tumor region compared with the MTX-TSLs
and MTX-MagTSLs group due to the lacking of folate receptor-mediated
targeting (Fig. 9A). The MTX-MagTSLs/CMF group presented a stronger
fluorescence signal in tumor region over 8 h compared with other
groups, which revealed that the amount of MTX-MagTSLs at tumor site
in the MTX-MagTSLs/CMF group was higher than groups with single
targeting (MTX-MagTSLs and MTX-TSLs) or without targeting
(MagTSLs and TSLs). Furthermore, the fluorescence signal of tumor
harvested from mice of all groups at 12 h post-injection from strong to
weak was as follows, MTX-MagTSLs/CMF > MTX-MagTSLs ≈ MTX-
TSLs > MagTSLs ≈ TSLs (Fig. 9B), which verified the dual role of
magnetic and biological targeting ability of MTX-MagTSLs under CMF.
The MTX-MagTSLs/CMF group demonstrated a much higher accumu-
lation at the tumor site and lower accumulation in other major organs
(Table 1), which clearly indicated the in vivo targeting ability of MTX-
MagTSLs/CMF and verified the dual role of magnetic and biological

Fig. 8. Relative cell viability of free Dox and different formulations at a series of either phospholipid or Dox concentrations. (A) HeLa cells were incubated with Dox-free MagTSLs at
different phospholipid concentrations (25, 50, 100, 200, and 400 μg/mL) for 24 h. (B) A549 cells (folate receptor-negative) and HeLa cells (folate receptor-positive) were incubated with
free Dox/TSLs/MagTSLs/MTX-TSLs/MTX-MagTSLs at different Dox concentrations (2.5, 5, and 10 μg/mL) for 24 h respectively. (C) HeLa cells were incubated with Dox-free MagTSLs,
free MTX, and Dox-free MTX-MagTSLs at different MTX concentrations (0.6, 1.2, and 2.4 μg/mL) for 24 h respectively. (D) HeLa cells were incubated with MTX-MagTSLs under no
stimulation, Laser (0.8 W/cm2, 5 min), AMF (500 kHz, 20 kA·m− 1, 5 min), and DUAL-mode (0.8 W/cm2, 500 kHz, 20 kA·m− 1, 5 min) (mean ± SD, n = 6). *p < 0.05, **p < 0.01,
***p < 0.001.

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Fig. 9. In vivo imaging of MTX-MagTSLs. (A) In vivo fluorescence images of the HeLa tumor-bearing nude mice at 4, 8, and 12 h after intravenous injection of PBS, MTX-MagTSLs, MTX-
MagTSLs/CMF, MagTSLs, TSLs, and MTX-TSLs respectively. (B) Ex vivo fluorescence images of the tumor excised from HeLa tumor-bearing nude mice treated with PBS, MTX-MagTSLs/
CMF, MagTSLs, MTX-MagTSL, TSLs, and MTX-TSLs at 12 h post-injection respectively. (C) In vivo pseudo-color processing T2-weighted MR images of MTX-MagTSLs with a magnet at 0, 4,
8, and 12 h post-injection. (D) In vivo pseudo-color processing T2-weighted MR images of MTX-MagTSLs without a magnet at 0, 4, 8, and 12 h post-injection. The arrow indicated the
tumor sites.

targeting of MTX. and 12 h after the intravenous injection of MTX-MagTSLs with a

To examine the suitability of MTX-MagTSLs for in vivo MRI, HeLa magnet at the tumor position (Fig. 9C). A sustained attenuation of the
tumor-bearing nude mice were further established for in vivo MRI T2 contrast signal at the tumor position was detected from 0 to 8 h after
evaluation using a 7-T MRI scanner. MR images were acquired for 4, 8, MTX-MagTSLs injection, indicating the effective accumulation of MTX-

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Table 1 was in consistent with in vitro anti-cancer effect of those. The result
In vivo biodistribution. demonstrated remarkable magnetic/MTX targeting function and in-
The fluorescence intensities of heart, liver, spleen, lung, kidney, and tumor excised
crease of Dox release under DUAL-mode. In addition, except for the free
from HeLa tumor-bearing nude mice treated with TSLs, MagTSLs, MTX-TSLs, MTX-
MagTSL, and MTX-MagTSLs/CMF at 12 h post-injection respectively. Dox group with relatively high toxicity, the body weights in other
groups had no significant changes in two weeks (Fig. 10B). The survival
Heart Liver Spleen Lung Kidney Tumor results of mice correlated to the therapy response were observed in
Fig. 10C. Compared to the free Dox and the MagTSLs group, the MTX-
TSLs 1.57E 6.57E 1.49E 1.51E 8.28E + 07 3.57E
+ 07 + 08 + 08 + 08 + 07 MagTSLs/CMF/AMF/Laser group showed improved survival sig-
MagTSLs 1.55E 6.66E 1.46E 1.49E 8.27E + 07 3.59E nificantly. Furthermore, the MTX-MagTSLs/DUAL group was added to
+ 07 + 08 + 08 + 08 + 07 verify dual-targeting ability compared with the MTX-MagTSLs/CMF/
MTX-TSLs 1.09E 5.35E 1.09E 9.01E 7.19E + 07 7.63E DUAL group. The tumor inhibition efficiency of MTX-MagTSLs/CMF/
+ 07 + 08 + 08 + 07 + 07
DUAL was obviously higher than MTX-MagTSLs/DUAL (Fig. S6A), the
MTX-MagTSLs 1.10E 5.28E 1.16E 8.97E 7.16E + 07 7.67E
+ 07 + 08 + 08 + 07 + 07 mice in both of two groups showed no significant body weight changes
MTX-MagTSLs/CMF 1.07E 4.62E 9.36E 4.47E 6.83E + 07 1.29E (Fig. S6B). The survival results of mice in MTX-MagTSLs/DUAL group
+ 07 + 08 + 07 + 07 + 08 was worse than the mice in MTX-MagTSLs/CMF/DUAL group due to
the insufficient targeting at tumor site and larger toxicity to other or-
gans under AMF/Laser triggering drug release without magnetic tar-
geting effect (Fig. S6C). All results suggested that MTX-MagTSLs/CMF/
MagTSLs at the tumor site via both magnetic targeting and folate re- AMF/Laser were highly effective for improving the therapeutic effects
ceptor-mediated targeting effect. The maximum decreased MRI signal of Dox while decreasing its associated toxicity.
intensities at tumor site was achieved at 8 h post-injection, varying Histological analysis by H&E staining of heart, liver, spleen, lung,
10.72% compared with the initiative value, followed by a gradual re- kidney, and tumor with different treatments at day 14 post-treatment
covery of MRI signal after 8 h post-injection, hinting the MTX-MagTSLs (Fig. 11) expressed that cell necrosis, lysis, and fragmentation occurred
could act as suitable negative (T2) contrast agents in MRI applications. in tumor tissue, while tight arrangement and intact shape in other tis-
As control, MR images of MTX-MagTSLs without a magnet at 0, 4, 8, sues when treated with MTX-MagTSLs under DUAL-mode. There was
and 12 h post-injection revealed a sustained attenuation of the T2 appearing an opposite result in the free Dox group and no obvious
contrast signal at the tumor position (Fig. 9D), and there was a rela- difference between other groups, which confirmed that combining AMF
tively lower MRI signal intensities attenuation (5.69%) compared with and laser could efficiently accelerate Dox release at tumor site, thereby
the MTX-MagTSLs/CMF group at tumor site, suggesting the less effec- achieved an optimal anti-tumor efficacy in vivo.
tive accumulation of MTX-MagTSLs in the tumor region without the
magnetic targeting effect. The metabolites of tumor including a large
amount of lactic acid from mainly anaerobic oxidation were different 4. Conclusions
from normal tissues (Warburg effect), which could cause the oppres-
sion, inflammation, the change of permeability, and the surrounding MTX modified thermo-sensitive magnetoliposomes, which consist of
edema under the development stage of tumor [51–53]. As a result of the DPPC:Chol:SA:DSPE-PEG2000:DSPE-PEG2000-MTX, has been developed
different composition and volume of distributive tissue fluids in the for precise cervical cancer therapy. The MTX-MagTSLs showed ex-
inflammatory mature tumor compared with other tissues, the mice cellent targeting ability, temperature sensitivity, and strong respon-
appeared a little strong signals in tumor before the administration due siveness to AMF and laser processing simultaneously, which improved
to more inherent hydrogen protons in the tumor region. Dox uptake and release into HeLa cells significantly, and also improved
tumor cell killing effects. Hence, both light/magnetic hyperthermia
3.10. In vivo anti-tumor efficacy triggered drug release and magnetic/MTX active targeting synergisti-
cally increased cytotoxicity to tumor cells and tissues while leading to a
The anti-tumor effects of MTX-MagTSLs in different groups were reduction of side effects compared with free Dox. Furthermore, it could
evaluated by measuring the tumor volume, weight, and survival si- be monitored in real time by fluorescence and MR imaging throughout
tuation of HeLa tumor-bearing nude mice. As shown in Fig. 10A, the the drug treatment. Therefore, this multi-functional liposomes can be
inhibition efficiency of different groups from high to low was shown as used as the potential carriers for precise cancer related diagnoses and
follows, MTX-MagTSLs/CMF/AMF/Laser > MTX-MagTSLs/CMF/ treatments.
AMF > MTX-MagTSLs/CMF > MTX-MagTSLs > MagTSLs, which

Fig. 10. In vivo anti-tumor activity. (A) Tumor growth curves, (B) changes of body weight, and (C) Kaplan-Meier survival curves of HeLa tumor-bearing nude mice after intravenous
injection of PBS, free Dox, MagTSLs, MTX-MagTSLs, MTX-MagTSLs/CMF, MTX-MagTSLs/CMF/AMF, and MTX-MagTSLs/CMF/DUAL at a Dox dose of 2 mg/kg every treatment (2 days)
within 14 days (mean ± SD, n = 6). *p < 0.05, ***p < 0.001.

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Fig. 11. H&E stained tissues harvested from the mice with different treatments. (A) Control, (B) free Dox, (C) MagTSLs, (D) MTX-MagTSLs, (E) MTX-MagTSLs/CMF, (F) MTX-MagTSLs/
CMF/AMF, and (G) MTX-MagTSLs/CMF/DUAL.
Acknowledgements
The research was financially supported by Social Development
Guidance Project of Fujian Province (No. 2014Y0074), Project of
Xiamen Science and Technology Bureau (3502Z20163004), and
Natural Science Foundation of Fujian Province (No. 2015J01348).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jconrel.2017.04.028.
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