ISSN (print): 1743 5889 | ISSN (online): 1748 6963

I.F.: 5.005 as per Thomson Reuters report July 2018 PreliminaryRCesearch Article
ommunication
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Biphasic magnetic nanoparticles–nanovesicle

hybrids for chemotherapy and self-controlled
hyperthermia
Aim: The aim was to develop magnetic nanovesicles for chemotherapy and self-controlled hyperthermia
that prevent overheating of tissues. Materials & methods: Magnetic nanovesicles containing paclitaxel and
a dextran-coated biphasic suspension of La0.75Sr0.25MnO3 and Fe3O4 nanoparticles (magnetic nanoparticles)
were developed. Results: Encapsulation efficiencies of magnetic nanoparticles and paclitaxel were 67 ± 5
and 83 ± 3%, respectively. Sequential release performed at 37°C for 1 h followed by 44°C for another 1 h
(as expected for intratumoral injection), showed a cumulative release of 6.6% (109.6 µg), which was above
the IC50 of the drug. In an alternating current magnetic field, the temperature remained controlled at 44°C
and a synergistic cytotoxicity of paclitaxel and hyperthermia was observed in MCF-7 cells. Conclusion: Magnetic
nanovesicles containing biphasic suspensions La0.75Sr 0.25MnO3 and Fe3O 4 nanoparticles encapsulating
paclitaxel have potential for combined self-controlled hyperthermia and chemotherapy.
Original submitted 15 May 2012; Revised submitted 7 March 2013

KEYWORDS: biphasic suspension n cancer n chemotherapy n magnetic nanovesicle Manashjit Gogoi1,

n self-controlled hyperthermia
Magnetic nanoparticle (MNP)-mediated hyper-
thermia is considered to be a potential candidate
for treating cancer [1]. In complex tumor masses,
oxygen-deprived hypoxic cells hardly respond to
radiation therapy [2–4], and chemotherapeutic
agents cannot reach the tumor sites in sufficient
amounts due to a lack of proper blood circulation
and high intratumoral pressure [5,6]. Under these
circumstances, hyperthermia can be effective in
treating cancer because it can kill tumor cells
by damaging the cellular structures [7,8]. Hyper­
thermia not only makes the cancer cells vulner-
able to radiation therapy [9], but also enhances
the bioavailability of drugs at tumor sites [10] by
increasing the blood supply to the tumor region.
Magnetic nano­vesicle-based hyperthermia has
been found to be effective in treating different
types of cancers in animal models [11,12]. Hyper-
thermia in combination with chemotherapy or
radiotherapy was reported to have improved the
treatment efficacy without additional systemic
toxicity [13]. Drug-loaded magnetic nano­vesicles
preferentially extravasate in tumor sites and
remain there via enhanced permeability and
retention effect, which reduces their side effects on
normal cells. Repeated doses of magnetic hyper-
thermia with cationic magnetic nano­vesicles
were successfully demonstrated to be effective in
treating tumor-bearing animals [11,12]. Pradhan
et al. reported the application of folate and
magnetic-targeted thermosensitive nanovesicles
Haladhar D Sarma2,
Dhirendra Bahadur3
for combined hyperthermia and chemotherapy
& Rinti Banerjee*1
1
Wadhwani Research Centre in
using doxorubicin [13]. However, therapeutic Biosciences & Bioengineering,
efficacy was limited due to low encapsulation of Department of Biosciences &
Fe3O4 nanoparticles (24 ± 6%) in the magnetic Bioengineering, Indian Institute of
Technology, Bombay,
vesicles. Drug-conjugated MNPs, magnetic Mumbai 400076, India
nano­vesicles and nano­hydrogels were reported 2
Radiation Biology & Health Sciences
Division, Bhabha Atomic Research
for combined hyperthermia and chemotherapy Centre, Mumbai 400085, India
application [14–16]. 3
Department of Metallurgical
Engineering & Materials Science,
Homogenous intratumoral distribution of Indian Institute of Technology,
magnetic nanomaterials is a challenging task and Bombay, Mumbai 400076, India
it determines the therapeutic efficacy of the hyper- *Author for correspondence:
Tel.: +91 222 576 7868
thermia treatment [17,18]. Fe3O4 nano­particles Fax: +91 222 572 3480
are extensively used in magnetic nano­vesicles. rinti@iitb.ac.in
One disadvantage of these Fe3O4 nano­particles
is their high Curie temperature (Tc; 580°C for
Fe3O4 and 477°C for Fe2O3 nano­particles, respec-
tively) [18], which causes generation of hot spots
during hyperthermia that leads to overheating of
tissues adjacent to tumor cells [19,20]. Application
of MNPs with a Tc of approximately 44°C can
be used to avoid overheating of tissues [20–23].
At the Tc, nanoparticles lose their magnetism
and become paramagnetic and, hence, further
heating is not possible. This process is called
self-­controlled hyperthermia. Kuznetsov et al.
reported the potential of Cu–Ni alloy nano­
particles in hyperthermia application [20]. MNPs
from the perovskite family with a Tc in the range
of 42–46°C have been explored for self-controlled
hyperthermia [21–23]. However, their studies were
limited to synthesis and a preliminary evaluation part of

doi:10.2217/NNM.13.90 © 2013 Future Medicine Ltd Nanomedicine (Epub ahead of print) ISSN 1743-5889
PRreliminary Communication
eview Authors Gogoi, Sarma, Bahadur & Banerjee
of the heating ability of nanoparticles and did
not coencapsulate the MNPs with drugs within
nanovesicles.
In this article, we report the use of pacli-
taxel-loaded thermosensitive magnetic nano­
vesicles containing a dextran-coated biphasic
suspension of nanoparticles for combined
self-­
c ontrolled cancer hyperthermia and
chemotherapy. Paclitaxel is an effective anti­
neoplastic agent, which basically polymerizes
the cellular microtubules, and thereby inhibits
the normal tubule dynamics required for cell
division and replication [24]. The lipid bilayer of
these nanovesicles consists of 1,2-distearol-sn-­
phosphatidylcholine (DSPC) and cholesterol.
DSPC is a thermosensitive lipid with a phase
transition temperature of 58°C, and cholesterol
is an important part of plasma membranes of
almost all eukaryotic cells. The addition of
cholesterol reduces the transition temperature
of saturated phospholipids such as DSPC and
broadens transition peak. At a high concentra-
tion of cholesterol (50 mol%), the gel-to-liquid
transition peak completely disappears [9,25]. In
our formulation, we used 34 mol% of chol­
esterol. The dextran-coated biphasic nano­
particle suspension contained La0.75Sr 0.25MnO3
(LSMO) and Fe3O4 nanoparticles in a ratio of
10:1. LSMO is a rare earth manganate, and
its Tc can be tuned to approximately 44°C by
controlling the amount of divalent Sr2+ ion
(x) (≤0.2  x  ≤0.4). It is expected that LSMO
nano­p articles will control the temper­ature
while Fe3O4 nanoparticles (possessing higher
heating ability) will provide sufficient heating
as well as make the biphasic suspension more
biocompatible. LSMO nanoparticles have a
low specific absorption rate or heating abil-
ity, whereas Fe3O4 nanoparticles have a higher
specific absorption rate, hence mixtures can be
used for temperature-tuned behavior.
One of the earlier studies from our group
reported that biphasic gels of LSMO and Al-
doped maghemite nanoparticles are suitable for
self-controlled hyperthermia [26]. To the best
of our knowledge, this is the first study report-
ing the application of a biphasic suspension of
nanoparticle-encapsulated magnetic nanovesicles
for cancer therapy. In fact, to date, there is no
report of LSMO nanoparticle-loaded magnetic
nanovesicles. The present study is focused on the
preparation of magnetic nanovesicle formulation,
characterization and its application for combined
chemotherapy and self-controlled hyper­thermia.
Dextran present in the biphasic suspension was
conjugated with fluorescein isothiocyanate
doi:10.2217/NNM.13.90 Nanomedicine (Epub ahead of print)
(FITC), a model fluorescent dye that enables the
magnetic nanovesicles formulation for in vitro
cellular imaging. Similarly, near infrared (NIR)
dyes may be conjugated for in vivo imaging.
The nanovesicle formulation was optimized for
temperature sensitivity and their potential for
chemotherapy and self-controlled hyperthermia
was evaluated.
Materials & methods
„„ Materials
Analytical grade chemicals were used through-
out the study without any further purification.
FeCl 2 .4H 2O, FeCl 3.6H 2O and sulforhoda-
mine B were procured from Sigma-Aldrich (MO,
USA) and La 2O3 was procured from Indian
Rare Earths Ltd (Cochin, India). DSPC was
purchased from Lipoid GmbH (Ludwigshafen,
Germany). Cholesterol was purchased from Loba
Chemicals (Mumbai, India). Anticancer drug
paclitaxel (purity: >99%) was supplied by Dabur
India Ltd (Ghaziabad, India). For cell culture,
human breast cancer cell line (MCF-7) and
mouse fibroblast cell line (L929) were purchased
from the National Centre for Cell Science (Pune,
India). Cells were maintained as a monolayer
culture in modified Eagle medium and DMEM
supple­mented with 10% fetal bovine serum and
1% antibiotic antimycotic solution, respectively,
at 37°C in a humidified incubator containing
5% CO2. Experiments were performed when
cells became 70–80% confluent.
Synthesis of Fe3O4 & LSMO nanoparticles
Fe3O4 nanoparticles were synthesized by the
coprecipitation method [15]. The black-colored
final solution was cooled down to room tem-
perature and particles were allowed to settle
down with the help of a magnet. Magnetically
separated particles were washed successively with
5% NH4OH solution, acetone and ultrapure
water (resistivity: 18.2 MWcm) three times with
each solvent, followed by an intermediate soni-
cation (at 20 kHz, 250 W; Vibronics Pvt Ltd,
Mumbai, India) of 5 min between two wash-
ing cycles. LSMO was synthesized using the
method reported in the literature [18,19] with
little modification. Briefly, La 2O3, SrCO3 and
MnCO3 were separately dissolved in nitric acid
and mixed with citric acid and ethylene glycol
in a ratio of 0.75[La 3+]:0.25[Sr2+]:[Mn 2+]:1.5[ci
tric acid]:2.25[ethylene glycol] and the pH was
adjusted to 9 by adding NH4OH. The mixture
was stirred and heated to 80–90°C to remove
water, and a pink-colored gel was formed. The
resulting gel was heated to 250°C, which led to
future science group
 Biphasic magnetic nanoparticles–nanovesicle hybrids
the formation of black dry powder after auto-
combustion and the dry powder was annealed
at 700 and 800°C for 1 h. The annealed powder
was then ground to a fine powder with a mor-
tar and pestle. The ground powder was initially
washed with ethanol followed by ultrapure water
to remove the impurities. The magnetic particles
were then dried in an oven.
Preparation of dextran-coated MNPs &
FITC conjugation
Dextran was dissolved in 0.5 M NaOH solu-
tion. LSMO nanoparticles (annealed at 700
and 800°C) were dispersed in 0.5 M NaOH
solution by sonication. The dextran-to-MNP
ratio was always kept at 5:1 (w/w). MNPs were
kept for sonication and the dextran solution was
added dropwise. After another 1 h of sonication,
the nanoparticles were centrifuged at 500 and
750 rpm to remove larger particles, and finally
centrifuged at 1000 rpm. Unbound dextran was
removed. Dextran-coated MNPs were resus-
pended in water and sonicated for 1 h to obtain
a stable magnetic fluid suspension. Similarly,
mixtures of LSMO and Fe3O4 nanoparticles in
1:0, 10:0.33 and 10:1 (w/w) ratios of the two
components were coated with dextran.
For the cellular internalization study, the
dextran­-coated biphasic suspension was conjug­
ated with FITC by slightly modifying the pro-
tocol described by Shiigi and Slomich [27]. For
this, the biphasic suspension was dialyzed against
0.15 M sodium chloride for 48 h, followed by
dialysis against 0.05 M bicarbonate-buffered
saline (pH 8.5) for 7 h. The biphasic suspension
was then dialyzed against a 100 µg/ml of FITC
in 0.05 M bicarbonate-buffered saline (pH 9.2)
for a further 14–16 h, and finally the reaction was
stopped by dialyzing against 0.02 M phosphate-
buffered saline (pH 7.4) for 2–3 h. It is note­
worthy to mention that all dialysis experiments
were performed at room temperature.
Characterization of MNPs
Crystallographic structures of Fe3O4 and LSMO
nanoparticles were analyzed by the X’Pert
Diffract­ometer (Philips/PANalytical, Almelo,
The Netherlands) with a Cu Ka radiation wave-
length of 1.54 Å. The mean crystal size of the
particles was calculated by using the Scherrer
equation [28]. Magnetic properties of Fe3O4 as
well as LSMO nanoparticles were measured
using a vibrating sample magnetometer (model
7410, Lake Shore, OH, USA). For this, the dry
powder of the nano­particles was placed inside
the sample holder and exposed to a magnetic
future science group
Preliminary Communication
field ranging from -20 to 20 kOe. The M-H
curves were recorded at room temperature. Mag-
netization values, measured in millielectromag-
netic units, were later normalized and expressed
in electromagnetic units per gram. In order to
determine the Tc, magnetization versus tempera-
ture measurements were carried out at 100 Oe.
The Tc value was calculated from a derivative
of the magnetization versus temperature curve.
In order to confirm the coating of dextran
on the LSMO and the biphasic suspension of
LSMO and Fe3O4 nanoparticles, Fourier trans-
form infrared (FTIR) analysis was performed
using the Magna 550 FTIR spectrophotometer
(Nicolet Instruments Corp., WI, USA). For this,
magnetic fluid was first frozen at -20°C and then
lyophilized. Lyophilized samples were then used
to analyze the characteristic FTIR peaks.
Upon application of an alternating magnetic
field, the temperature of the magnetic fluid
increases due to Néel and Brownian relaxations.
The specific absorption rate (SAR) of the MNP
suspensions were calculated from the tempera-
ture versus time curve. For this, 1 ml of mag-
netic nanovesicles was taken in a 15-ml falcon
tube and the tube was placed at the center of the
copper coil of the radiofrequency (RF) generator
(CLF-5000, Comdel, MA, USA) at a 423-kHz
frequency and field strength of 10 kA/m. Tem-
peratures of the magnetic material suspension
were measured with an alcohol thermometer.
The SAR was then calculated using Equation 1 [15]:
SAR = C^1/mM h (E quation 1)
where C is the specific heat capacity of the
solution in joules/gram-1/Kelvin-1. In all experi-
ments, specific heat capacity was taken as
4.18 J/g-K (value for water), and mM is the mass
fraction of magnetic material in the suspension.
In order to evaluate the biocompatibility of
dextran-coated LSMO and biphasic suspension
of nanoparticles, both the materials were incu-
bated in DMEM at 37°C for 48 h. The concen-
tration of materials was 10 mg/ml. After 48 h,
the materials were centrifuged at 8000 rpm for
15 min so that all of the MNPs were settled. The
supernatants containing leached-out extracts of
the nanoparticles were further diluted with com-
plete media and used for the bio­compatibility
study. For this, cells were seeded in a 96-well
microtiter plate (1 × 104 cells/200 µl per well
in one plate) in DMEM, supplemented with
10% fetal bovine serum and 1% antibiotic anti­
mycotic solution. After 24 h, old media was
discarded and the extracts of dextran-coated
www.futuremedicine.com doi:10.2217/NNM.13.90
PRreliminary Communication
eview Authors Gogoi, Sarma, Bahadur & Banerjee
MNPs at different concentrations of MNPs
ranging from 0.0156–5 mg/ml were mixed
with fresh media and added to the cells. The
cells were then incubated again for 48 h. Cell
viability was determined by a sulforhodamine B
assay [29].
Preparation of thermoresponsive
magnetic nanovesicles
Magnetic nanovesicles were prepared by the
thin-film hydration method [15]. For this,
DSPC:cholesterol (8:2 w/w) were taken in a
round-bottom flask and dissolved in a 2:1 chloro-
form and methanol mixture. The solvent was then
removed at 40°C by a rotary vacuum evaporator
(Superfit Continental Pvt Ltd, Mumbai, India)
to form a thin film. For drug-loaded magnetic
nanovesicles, the lipid-to-drug ratio was main-
tained at 10:1 (w/w). Hydration of the film was
performed with MNPs or FITC-tagged MNPs
for 1 h at 60°C. The lipid-to-MNP ratio was 4:1
(w/w). The nanovesicles thus formed were then
successively extruded through 400 and 100 nm
membranes using an extruder (Avanti Polar
Lipids, AL, USA) to make small unilamellar
nano­vesicles. Free MNPs and drugs were removed
by centrifuging the magnetic nanovesicle suspen-
sion at 1000 rpm for 10 min and 17000 rpm for
25 min, respectively.
Characterization of magnetic
nanovesicles
The size and zeta-potential of the magnetic
nanovesicles were determined by dynamic light
scattering (Brookhaven Instruments Corp., NY,
USA) and a zeta-potential analyzer (ZetaPALS,
Brookhaven Instruments Corp.). In order to
determine the encapsulation of MNPs in nano­
vesicles (Equation 2), magnetic nanovesicles were
dissolved in concentrated HCl and then fil-
tered through Whatman filter paper of pore
size 0.2 µm. MNP concentration in the filtered
solution was determined by inductively coupled
plasma atomic emission spectroscopy (ICP-AES).
% MNPs encapsulation =
Amount of MNP in formulation
100
Amount of MNP used during hydration #
(E quation 2)
Paclitaxel-encapsulation efficiency was deter-
mined by estimating the amount of unencap-
sulated drug present in the supernatant after
centrifugation of the nanovesicle sample at
17,000 rpm for 25 min. The amount of drug in
the supernatant was then subtracted from the
doi:10.2217/NNM.13.90 Nanomedicine (Epub ahead of print)
total amount of drug added during the process of
nanovesicle formation. Encapsulation efficiency
was then calculated using (Equation 3):
% drug encapsulation = 61 - ^ DS/DT h@ # 100
(E quation 3)
where, DS is the drug present in the super­
natant and DT is the total amount of drug used
during thin-film formation.
Drug-release studies
Initially taking calcein as a model drug, release
studies were performed according to the method
described by Anand et al., with nanovesicles hav-
ing different lipid ratios of DSPC:cholesterol
(i.e., 9:1, 8:2 and 7:3) [30]. Results showed that
DSPC to cholesterol in the ratio of 8:2 is the
most thermosensitive composition (data not
shown). Drug-release studies of paclitaxel-
loaded DSPC:cholesterol (8:2) nanovesicles
were carried out at 37 and 44°C in a magnetic
stirrer. In total, 1 ml of drug-loaded nano­vesicles
was put in a dialysis bag and placed in a beaker
containing 50 ml of release media containing
methanol:water in a 1:3 (volume:volume) ratio.
Media was stirred using a mechanical stirrer and
the temperature of the media was maintained
either at 37 or 44°C for all experiments. A total
of 2 ml of the sample was drawn at different
predetermined time points and substituted with
equal amounts of media to maintain the sink
conditions.
In another set of experiments, a release study
was carried out for 24 h at 37°C followed by a
release study at 44°C for another 1 h. The objec-
tive of this set of experiments was to mimic the
temperature changes expected on intravenous
injection in vivo. It was considered that nano­
vesicles take 24 h to reach the tumor site follow-
ing intravenous injection and then an alternating
magnetic field would be applied for another 1 h.
Similarly, one more set of sequential drug-release
studies was performed to mimic the temperature
changes expected in vivo, considering that mag-
netic nanovesicle formulation was administered
intratumorally where a drug-release study was
performed at 37°C for 1 h followed by a release
study at 44°C for another 1 h.
Cellular internalization of magnetic
nanovesicles
The cellular internalization study of FITC-tagged,
nanoparticle-loaded, DSPC:cholesterol (8:2) mag-
netic nanovesicles was performed with a MCF-7
cell line. Cells were seeded (1 × 105 cells/ml per
future science group
 Biphasic magnetic nanoparticles–nanovesicle hybrids
well) in 24-well micro­titer plates with or without
12-mm glass cover slips in DMEM supplemented
with 10% fetal bovine serum and 1% antibiotic
antimycotic solution. After 24 h of incubation,
old media was removed and 50 µl (50 µg MNPs)
of FITC-tagged, nanoparticle-loaded magnetic
nano­vesicles were added with fresh media. The
cells were then incubated for 3 h in a CO2 incu-
bator. After 3 h, the cells were washed with phos-
phate-buffered saline and fixed on the glass cover
slips with 10% formaldehyde solution. The glass
cover slips were mounted on glass slides and fixed
with glutaraldehyde. Confocal imaging of the cells
was performed with the IX81 con­focal microscope
(Olympus, Tokyo, Japan) and using FV-500 soft-
ware (Olympus). The cells were excited at 490 nm
and emission was captured at 530 nm.
The cells seeded without glass cover slips were
trypsinized and retrieved after washing out the
incubated FITC-tagged, nanoparticle-loaded
magnetic nanovesicles. Trypsinized cells were
centrifuged for 5 min at 2000 rpm to form pellets.
These cellular pellets were dissolved in concen-
trated HCl and MNP concentration in the filtrate
solutions were determined by ICP-AES (ARCOS
from M/s, SPECTRO, Kleve, Germany).
In vivo stability evaluation of magnetic
nanovesicles
In order to evaluate in vivo stability of mag-
netic nanovesicles, experiments were conducted
on female Swiss mice (bodyweight: 20–23 g;
6–8 weeks old) that were bred and reared in
the animal house facilities of Bhabha Atomic
Research Centre (Mumbai, India). The animal
experiment was conducted according to the rel-
evant national laws relating to animal experi-
mentations. For this study, animals were ran-
domly divided into three groups (n = 4). Mag-
netic nanovesicles with 2 and 1 mg MNPs were
administered into the animals of groups 1 and
2, respectively, through the tail vein, whereas no
injection was given to group 3 animals (control).
After 24 h, all of the animals were observed for
any toxicity and sacrificed, blood samples were
collected in glass vials containing 150 µl of 4%
EDTA solution and evaluated for packed cell
volume (PCV) and agglomeration of MNPs.
Cytotoxicity study of MNPs
& hyperthermia
For in vitro cytotoxicity study, MCF-7 cells
were seeded in two 96-well microtiter plates
(1 × 104 cells/200 µl per well in one plate) in
modified Eagle medium supplemented with 10%
fetal bovine serum and 1% antibiotic antimycotic
future science group
Preliminary Communication
solution. After a 24-h incubation, old media was
discarded and magnetic nanovesicles were diluted
in complete media at different concentrations of
drug ranging from 50 to 600 nM. After adding the
magnetic nanovesicles, cells were incubated again
for 48 and 72 h. Cell viability was then determined
according to the aforementioned method.
In vitro hyperthermia with magnetic nano­
vesicles was performed in the MCF-7 cell line
under an alternating magnetic field. For this,
12 × 105 cells were divided equally and put
into two sterile polypropelyne centrifuge tubes
(15 ml; Tarsons Ltd, Kolkata, India) with
complete media, and centrifuged at 2000 rpm
for 5 min to form pellets. The media was dis-
carded and magnetic nanovesicles with/without
112 nM of the drug in complete medium were
added to the pellets and cells were resuspended.
A total of 6 × 105 cells suspended in complete
media were taken in another 15-ml sterile poly-
prolyne tube. All three tubes were placed in
the copper coil of a RF generator (CLF-5000)
at a fixed frequency of 423 kHz and a field of
10 kA/m one by one. During the experiment,
the temperature was 44 ± 0.5°C for 15 min. The
cells were then seeded in a 24-well plate with
six replicates and incubated for 48 h. For the
control group, 6 × 105 cells were also seeded in
a 24-well plate with six replicates and incubated
for 48 h. The viable cells were then counted by
the trypan blue dye exclusion method using a
hemocytometer.
Valeriote’s method was used to evaluate the
combined effect of magnetic hyperthermia and
chemotherapy [13]. If A, B and (A + B) represent
the percentage cell viability after treatment A,
B and the combination of treatment A and B,
respectively, the combined effects were defined
as: synergistic: (A + B) < (A × B)/100; additive:
(A + B) = (A × B)/100; subadditive: (A × B)/100
< (A + B) < A, if A < B; interference: A < (A + B)
< B if A < B; and antagonistic: B < (A + B), if
A < B.
„„ Statistical analysis
All experiments were repeated at least three times
and results were expressed as mean ± stand-
ard deviation. Statistical analysis was carried
out using t-tests (paired t-test with unequal
variances). An event is considered as significant
when p < 0.05 (with 95% CI).
Results
„„ Characterization of MNPs
X-ray diffraction results (Supplementary Figure  S1;
see online at www.futuremedicine.com/doi/
www.futuremedicine.com doi:10.2217/NNM.13.90
PRreliminary Communication
eview Authors Gogoi, Sarma, Bahadur & Banerjee
suppl/10.2217/NNM.13.90) confirmed the
formation of Fe3O4 and LSMO nanoparticles.
The size of Fe3O4 and LSMO nanoparticles were
10 and 25 nm, respectively. Vibrating sample
magnetometer studies showed that saturation
magnetization of LSMO nanoparticles annealed
at 700°C was 15.34 emu/g and its Tc was 48.6°C
(Figure 1). As we increased the annealing tempera-
ture, the magnetization values as well as the Tcs
of magnetic materials increased. The saturation
magnetization and Tc of LSMO nanoparticles
annealed at 800°C were 21 emu/g and 59.7°C,
respectively (Supplementary Figure S2).
Dextran-coated MNPs were lyophilized and
FTIR was studied. Dextran had peaks at 3400,
2927 and 1649 cm-1 due to the presence of O-H,
C-H and C-C bonds, respectively, as shown in
Supplementary Figure  S3. In dextran-coated MNPs,
the corresponding peaks were at 3404, 2921 and
1632 cm-1. From the FTIR results, it is clear that the
peaks in dextran due to O-H, C-H and C-C bonds
are shifted. This indicates that there is interaction
between dextran and the MNPs as the magnetic
particles become coated with dextran.
A transmission electron microscope
image of biphasic suspension confirmed that
A 25
Magnetization
20
15
(emu/g)
10
5
0 Field (kOe) 10
-20 -10 -5
-10
-15
-20
-25
B 2.5
Magnetization (emu/g)
2 0.00 × 10-4
1.5
1 emu/g
dm/dT (emu/°C)
0.5
0 -4.00 × 10-4
25 35 45 55 65 75 85 95 105 115
Temperature (°C)
Figure 1. Magnetic properties of La0.75Sr0.25MnO3 nanoparticles. (A) M-H loop
recorded at room temperature for La0.75Sr0.25MnO3 nanoparticles calcined at 700 and
800°C. (B) Curie temperature of La0.75Sr0.25MnO3 nanoparticles calcined at 700°C.
dm/dT: First order derivative of magnetization with respect to temperature.
doi:10.2217/NNM.13.90 Nanomedicine (Epub ahead of print)
nano­particles are uniformly dispersed (Figure 2A).
The size of nanoparticles is in the range of
10–30 nm. It was confirmed from the x-ray dif-
fraction results that the smaller particles were
Fe3O4 nano­particles, while larger-sized parti-
cles were LSMO nano­particles. Annealing at a
high temperature led to a formation of bridges
among the LSMO particles and, therefore, the
actual size of particles became more than the
size determined by x-ray diffraction [18]. A bio-
compatibility study of extracts of LSMO and
biphasic nanoparticle suspension was performed
in a L929 cell line (Figure 2B). Extracts of both
the materials were incubated with cells for
48 h. Cell viability for the extract of dextran-
coated LSMO was 81% after 48 h of incuba-
tion, whereas for the extract of dextran-coated
biphasic nanoparticle suspension it was 82%.
Results of a SAR experiment showed that the
suspension of LSMO nanoparticles annealed
at 700°C caused an increase in temperature of
up to 33.8°C in 15 min, whereas during the
same time period, temperature due to sus-
pension of LSMO nanoparticles annealed at
800°C rose to 37.6°C, which might result in
prolonged exposure of the animal model dur-
ing in vivo experiments (Supplementary Figure S4).
The addition of Fe3O4 nanoparticles increased
the temperature within a short span of time.
Hence, Fe3O4 nanoparticles were added in dif-
ferent amounts and the SAR of these biphasic
suspensions were evaluated. Finally, a biphasic
suspension of LSMO to Fe3O 4 nanoparticles
(10:1) was selected for further experiments.
20
This biphasic suspension reached 42.4°C in
700°C 4.5 min. The SAR value of magnetic nano­
800°C vesicles was 8.9 W/g and it took 13 min to raise
the temperature to 44°C.
„„ Characterization of magnetic
1.00 × 10-4 nanovesicles
The mean hydrodynamic diameter of
DSPC:cholesterol magnetic nanovesicles was
dm/dT (emu/°C)
-1.00 × 10-4 225 ± 45 nm and its zeta-potential was -41 ± 5 mV.
A transmission electron microscope image of
-2.00 × 10-4 the magnetic nanovesicles shows that the size of
the nanovesicles is in the range of 180–200 nm
-3.00 × 10-4
(Figure 3A). Figure 3B shows the diffraction pattern
of magnetic nanovesicles. The rings and bright
spots indicate the presence of MNPs within the
nano­vesicles. Figure 3C shows the transmission elec-
tron microscope image of a blank nanovesicle and
Figure 3D is its corresponding diffraction pattern.
The paclitaxel and MNP encapsulation effi-
ciency of magnetic nanovesicles were 83 ± 3 and
67 ± 5%, respectively.
future science group
 Biphasic magnetic nanoparticles–nanovesicle hybrids Preliminary Communication
Drug release from DSPC:cholesterol (8:2) A
nanovesicles
A calcein-release study showed that
DSPC:cholesterol (8:2) nanovesicle formulation
was more thermosensitive than the other two for-
mulations (DSPC:cholesterol 9:1 and 7:3 w/w).
Therefore, the paclitaxel release was carried out
with DSPC:cholesterol (8:2) nanovesicle formula-
tion at 37 and 44°C. The paclitaxel release from
this formulation was 3.9 (33.54 µg) and 5.2%
(44.7 µg; p < 0.05), at 37 and 44°C, respec-
tively, as shown in Figure 4A . In order to mimic the
expected in vivo temperature change on the intra-
venous injection, a sequential paclitaxel release
with DSPC:cholesterol (8:2) nanovesicles was
performed at 37°C for 24 h, followed by release at
200 nm
44°C for another 1 h. The cumulative drug release
at the end of the study was 8.5% (120.7 µg) and,
thus, higher than those carried out only at 44°C. B 120
The drug-release profile is shown in Figure 4C. The BS
drug-release profile during the transition of tem- LSMO
100
perature is shown in Figure 4D. At the end of the sec-
ond sequential release study of sequential release at
80
Cell viability (%)

37°C for 1 h followed by 44°C for 1 h (as expected

for intratumoral injection), the cumulative drug
release was 6.6% (109.6 µg; shown in Figure 4B). 60

Cellular internalization of magnetic

nanovesicles
Therapeutic effects of drug-loaded nano­particles
are demonstrated to be dependent on the cellu-
lar internalization and sustained retention by the
malignant cells [31,32]. Although there are a num-
ber of differences between in vitro and in vivo pro-
cesses, in vitro experiments can provide some basic
ideas regarding the intracellular movement and
accumulation of nanoparticles within the cells.
In vitro cellular internalization studies were car-
ried out with FITC-tagged biphasic nanoparticle
suspension-loaded magnetic nanovesicles. Cellular
uptake of magnetic nanovesicles was qualitatively
and quantitatively determined by using con­focal
microscopy of FITC-tagged biphasic nanoparticle
suspension-loaded magnetic nanovesicles in
MCF-7 cell lines and ICP-AES (Figure 5). ICP-AES
results suggested that 49% of the total MNPs were
internalized after 3 h of incubation (Figure  5E).
Moreover, these FITC-tagged biphasic nano­
particle suspension-loaded magnetic nanovesicles
could also be used for in vitro cellular imaging.
In vivo stability evaluation of magnetic
nanovesicles
In order to evaluate the stability of the mag-
netic nanovesicles in plasma and to evaluate the
feasibility of intravenous administration, the
40
20
0
Control 0.16 0.31 0.63 1.25 2.50 5.00
Dose of MNPs (mg/ml)
Figure 2. Characterization of biphasic suspension. (A) Transmission electron
microscopy image of BS; and (B) cell viability of dextran-coated BS and LSMO
nanoparticles in the L929 cell line.
BS: Biphasic suspension; LSMO: La0.75Sr0.25MnO3; MNP: Magnetic nanoparticle.
levels of hemoglobin, mean corpuscular volume
and PCV were evaluated (shown in Figur e  6).
The hemoglobin level in the control group was
15.5 ± 0.4 and 14.8 ± 0.2 g/dl for group 1 and
15.4 ± 0.6 g/dl for group 2 animals. No sig-
nificant change was observed in hemoglobin
levels, mean corpuscular volume or PCV on
administration.
In vitro cytotoxicity of magnetic
nanovesicles
A cytotoxicity study of paclitaxel-loaded mag-
netic nanovesicles was carried out by incubat-
ing for 48 and 72 h. Results of the cytotoxicity
studies are shown in Figure 7. It was observed that
for 48-h treatment groups, the percentage cell

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PRreliminary Communication
eview Authors Gogoi, Sarma, Bahadur & Banerjee
A AC magnetic fields were shown in Figure 8B. The
B combination of hyperthermia and chemo­therapy
2 nm-1
C Discussion
200 nm
D chemotherapy. The thermosensitive nanovesicles
1000 nm
2 nm-1
Figure 3. Transmission electron micrograph and diffraction patterns of
nanovesicles. (A) Transmission electron microscopy images of magnetic liposomes
containing biphasic suspension of La0.75Sr0.25MnO3 and iron oxide nanoparticles in
10:1 ratio. The size of magnetic liposomes is in the range of 180 to 250 nm.
(B) The diffraction pattern of magnetic liposomes; (C) a transmission electron
microscopy image of a blank liposome; and (D) a diffraction pattern of a blank
liposome.
viability sharply reduced for magnetic nano­
vesicles until 200 nM and did not change sig-
nificantly even after increasing the dose. The
IC50 value of paclitaxel-loaded magnetic nan-
ovesicles was determined using the method men-
tioned in our earlier paper [15], and the value was
112 nM (95.64 µg). Therefore, a hyper­thermia
experiment was performed using magnetic nano­
vesicles containing 112 nM paclitaxel with a
48-h incubation period. After a 72-h incubation
period, cell viability was less than 20% for all
the concentrations of paclitaxel-loaded magnetic
nanovesicles.
In vitro hyperthermia under an
alternating current magnetic field
MCF-7 cells were subjected to hyperthermia
under an alternating current (AC) magnetic
field with bare and drug-loaded magnetic nano­
vesicles containing 112 nM paclitaxel. On the
other hand, control cells were not subjected to
any treatment. Hyperthermia experiments were
conducted at 44°C temperature for 15 min and
the result was presented in Figure 8. Temperature
profiles during hyperthermia experiments and
doi:10.2217/NNM.13.90 Nanomedicine (Epub ahead of print)
results showed that there was no effect of an
AC magnetic field on the cells. It was observed
that the cell viability for cells treated with mag-
netic nanovesicles mediating hyperthermia was
74.2%. Interestingly, this value (74.2%) reduced
to 24.3%, when the cells were subjected to a
(drug: 112 nM). The result suggested that a
combination of hyperthermia and chemotherapy
act synergistically on the MCF-7 cell line. Con-
sequently, the efficacy of treatment improved
due to the combined effect of these treatments.
We developed novel biphasic MNP–nanovesicle
hybrids for self-controlled hyperthermia and
coencapsulated a biphasic suspension consisting
of Fe3O4 and LSMO nanoparticles and pacli-
taxel. The biphasic suspension was optimized
for self-controlled hyperthermia. LSMO nano-
particles with a low SAR value regulated the
overall temperature gained by the suspension,
whereas Fe3O4 nanoparticles were responsible for
speeding up the heating process. A fine balance
between both the nanoparticles is necessary to
achieve a hyperthermia temperature within 1 h.
The possibility of using a biphasic gel of
LSMO and Al-substituted maghemite nano-
particles for self-controlled hyperthermia has
been successfully demonstrated in our earlier
work [26]; however, the work was limited to the
evaluation of heating rates and did not evaluate
the encapsulation within a biocompatible lipid
nanovesicle. Concerns regarding the toxicity of
LSMO nanoparticles have largely limited their
biomedical applications. The cellular responses
to LSMO nanoparticles have been largely unex-
plored. In the present study, the encapsulation
of biphasic suspensions of Fe3O4 nanoparticles
and LSMO within thermosensitive nano­vesicles
has been achieved. An in vitro biocompatibility
experiment with L929 cell lines showed that the
extract of the biphasic suspension did not show
any toxicity up to a 5 mg/ml concentration. Fur-
thermore, no toxic effects were observed on intra-
venous in vivo administration. The study paves
the way for exploration of further biomedical
applications of hybrid MNPs for self-controlled
hyperthermia.
Phospholipids, such as DSPC, with high tran-
sition temperature are required for maintaining
a gel state in the body. The inclusion of choles-
terol in DSPC liposomes at various molar ratios
has been reported to affect the clearance rates
future science group
 Biphasic magnetic nanoparticles–nanovesicle hybrids Preliminary Communication
A 6 B 7 37°C 44°C

Transition period
5 6
Cumulative drug release (%)

Cumulative drug release (%)

5
4
4
3
3
2
2

1 Release at 44°C 1
Release at 37°C

0 0
0 10 20 30 40 50 60 70 0 30 60 90 120 150
Time (min) Time (min)

C 10 37°C D 10
37°C 44°C

Transition period
9
Cumulative drug release (%)
Cumulative drug release (%)

8
9
7

5 8

3
7
2

0 6
0 5 10 15 20 25 30 23 23.5 24 24.5 25 25.5
Time (min) Time (min)

Figure 4. Drug release studies from nanovesicles under different physiological conditions.
(A) Drug release from magnetic liposomes at 37 and 44°C. (B) Sequential drug release (i.e., in order
to mimic the expected temperature change in vivo following intratumoral injection) was performed at
37°C for 1 h followed by a release study at 44°C for another 1 h. (C) Sequential drug release (i.e., in
order to mimic the expected temperature change in vivo following intravenous injection) was
performed at 37°C for 24 h followed by a release study at 44°C for another 1 h. (D) Enlarged image
of drug-release profile showing the transition period between 37 and 44°C.

of pure DSPC liposomes in vivo. At 20 mol% cells and reduce the entry of nanovesicles into
cholesterol, the circulation half-life was found the tumor tissue [34–36]. For example, Hong et al.
to be approximately 30 min, compared with showed that the plasma area under the curve for
seconds for pure DSPC vesicles. The inclusion doxorubicin-loaded PEGylated nanovesicles was
of 30 mol% cholesterol further extended the approximately twofold more than doxorubicin
half-life of the vesicles to 5 h. The cholesterol entrapped in non-PEGylated DSPC:cholesterol
effect on circulation time plateaued at 30 mol%, nanovesicles [37]. However, at the end of 72 h,
without further benefits of higher amounts of the concentration of doxorubicin entrapped
cholesterol addition [33]. in non-PEGylated DSPC:cholesterol nano­
Sterically stabilized PEGylated nanovesicles vesicle in tumor tissue was 1.44-times more
can escape from opsonization by plasma proteins than doxorubicin in PEGylated nanovesicles.
and are characterized by a long blood circulation These results suggest that non-PEGylated nan-
time. However, the presence of large molecules, ovesicles composed of DSPC and cholesterol
such as polyethylene glycol, on the surface of are suitable for accumulation at cancerous sites.
nanovesicles may minimize the interaction with Such nano­vesicles have advantages of being

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PRreliminary Communication
eview Authors Gogoi, Sarma, Bahadur & Banerjee

A B

50 µm 50 µm

C D

E 60
internalization (%)

50
40
30
20
MNP

10
0 50 µm 50 µm
Control Treated

Figure 5. Confocal laser scanning microscopy images showing the cellular internalization of
magnetic liposomes in the MCF-7 cell line. (A) Fluorescence images of treated cells and
(B) merged image treated cells. (C) Fluorescence image of control cells and a (D) differential
interference contrast image of control cells. (E) The quantitative estimation of MNPs in cells.
MNP: Magnetic nanoparticle.

thermo­sensitive, lower in cost and easier to
manufacture than PEGylated nanovesicles.
In our study, during in vivo evaluation, mag-
netic nanovesicles containing biphasic suspen-
sion up to 2 mg of MNPs was administered
intravenously and the level of hemoglobin, mean
corpuscular volume and PCV percentage did
not change significantly, suggesting the colloidal
stability of the magnetic nanovesicles. The mag-
netic nanovesicles neither formed any aggregate
in blood, nor caused any acute toxic effects.
During the hyperthermia experiment the
maximum temperature achieved was 44°C.
This showed that the temperature of the mag-
netic nanovesicles was stabilized at 44°C without
any overheating. The temperature of magnetic

A 18 B 60 C 50

15 50 40
Hemoglobin (mg/dl)

12 40
30
PCV (%)
MCV (fl)

9 30
20
6 20
10
3 10

0 0 0
Control 24 h Control 24 h Control 24 h
Time point Time point Time point

Control Group 1 Group 2

Figure 6. The in vivo stability evaluation of magnetic liposomes. (A) Hemoglobin level, (B) MCV level and (C) PCV percentage.
MCV: Mean corpuscular volume; PCV: Packed cell volume.

doi:10.2217/NNM.13.90 Nanomedicine (Epub ahead of print) future science group

 Biphasic magnetic nanoparticles–nanovesicle hybrids Preliminary Communication
nano­vesicles did not increase beyond this tempera- A 120
ture, even after applying an alternating magnetic
field due to the sharp decrease in saturation mag- 100
netization value in the vicinity of the Tc. The tem-

Cell viability (%)

80
perature is well below their actual Tc (i.e., 48.6°C),
this is due to the dead mass of dextran and lipids. 60
The magnetic nanovesicles have high MNP
and drug encapsulation efficiencies. High encap- 40
sulation of the drug and MNPs are necessary 20
for achieving a better therapeutic index with a
lesser amount of delivery systems. In most lit- 0
erature, the encapsulation efficiency of MNPs 0 50 100 200 250 400 500 600
Concentration of PTX (nM)
has been low when coencapsulated with an
anticancer drug. The other approach used to
B 120
increase this efficiency has been to functional-
ize the surfaces of MNPs and then conjugate the 100
drug [38]. Generally, the conjugation chemistry is Cell viability (%)

quite complex and the method of cleavage of the 80

drug to allow release of the drug at the target site
needs to be carefully controlled. Further asso-
ciation of the drug on the surface, rather than
within the nanocarrier, may influence the bio­
distribution of MNPs [39] by altering the physical
and/or surface characteristics of the bare MNPs
(e.g., hydrodynamic size, stability and magneti-
zation) [40–43]. Magnetic nanovesicles can play an
important role in this regard, and we formulated
DSPC:cholesterol magnetic nanovesicles with-
out compromising the encapsulation efficiencies
of MNPs and the drug, as well as the original
physiological properties of MNPs.
In vitro cellular internalization showed that
magnetic nanovesicles were internalized by the
cells. The panel for control cells was dark, sug-
gesting that there was negligible autofluorescence
from the cells during the in vitro experiment,
suggesting the feasibility of using these mag-
netic nanovesicles for in vitro optical imaging.
However, interference due to auto­fluorescence
and photobleaching cannot be ruled out during
in vivo applications. In that case, NIR-sensitive
dyes may be used instead of FITC, which have
very low interference from the tissue auto­
fluorescence. Moreover, problems such as pho-
tostability, photobleaching and low quantum
yield can be overcome by using the NIR dyes or
NIR dye-encapsulated nano­particles for tumor
detection and imaging [44].
Considering the fact that hyperthermia
treatment can be applied continuously for a
maximum of 1 h, drug release studies were lim-
ited to 1 h. The percentage of cumulative drug
release was low due to the hydrophobic nature
of paclitaxel, but was sufficient for a therapeutic
effect as it was found to be higher than the IC50
of paclitaxel. In order to mimic temperature
60
40
20
0
0 50 100 200 250 400 500 800
Concentration of PTX (nM)
PTX Magnetic liposomes
Figure 7. Comparative cytotoxicity studies of magnetic liposomes and
marketed taxol formulation in MCF-7 cells. (A) 48-h incubation period and
(B) 72-h incubation period.
PTX: Paclitaxel.
changes expected in vivo following systemic
administration of magnetic nano­vesicles, a
sequential drug-release experiment was per-
formed where a release study was carried out
at 37°C for 24 h, followed by a release study
at 44°C for another 1 h. The cumulative drug
release during sequential release increased to
8.5% (i.e., 120.7 µg). However, the drug that
is released at 37°C can lead to systemic toxicity.
In case of a second sequential release study,
where it was considered that the AC magnetic
field would be applied after 1 h of intratumoral
injection of magnetic nanovesicles, the cumula-
tive drug release was 6.6% (109.6 µg), and the
entire amount was released within the tumor
mass. Systemic toxicity due to the drug would be
avoided. Hence, intratumoral administration of
magnetic nanovesicles was preferred to systemic
administration for self-controlled hyperthermia
and drug delivery. It was expected that upon
intratumoral injection that the drug would act as
a depot within the nanovesicles and continue to
release slowly within the tumor mass. Achieving

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PRreliminary Communication
eview Authors Gogoi, Sarma, Bahadur & Banerjee

A self-controlled magnetic vesicles on intravenous

120
administration would depend on the percentage
100 of the drug-loaded magnetic nanovesicles reaching
the tumor site.
Cell viability (%)

80
In the hyperthermia experiments, we chose
60 the dose of drug in magnetic nanovesicles
equal to its IC50 value. The combination of
40
paclitaxel and heat killed 75.7% cells within
20 15 min. Hyperthermia can kill the cells by
irreversibly damaging the cellular complexes
0 required for DNA synthesis and repair. The
Control AMF Heat 112 nM PTX Heat +
112 nM PTX extent of cell killing may vary depending
Treatment upon the temperature of treatment and the
duration of experiment. In an in vitro study,
B 50 Prasad et al. demonstrated that the cytotoxic
effect of magnetic hyperthermia is time and
40 temperature dependent [47]. They showed
that magnetic hyperthermia killed the cells
Temperature (°C)

30 by irreversibly damaging the cytoskeleton.

20
10
Magnetic liposomes
Only AMF
0 ferent modalities are used in combination to
0 10 20 30 40
Time (min)
Figure 8. Cellular toxicity of MCF-7 cell line during hyperthermia experiments.
(A) A hyperthermia experiment was performed under an AMF with bare and drug-
loaded magnetic liposomes (112 nM paclitaxel). During the experiment, the field
strength was constantly maintained. The AMF group of cells were exposed to only
the AMF. (B) The temperature profile during hyperthermia experiment as well as
only for the AMF.
AMF: Alternating current magnetic field; PTX: Paclitaxel.
sustained drug release is very important in can-
cer therapy as it prevents cancer from relapsing
and developing resistance against the drug [45,46].
While there are advantages of a regional therapy,
intratumoral injection also has the disadvantage
of being invasive, depending on the location of
the tumor, and may cause an uneven distribution.
Moreover, it is worth mentioning that the cumula-
tive drug releases for both the sequential release
studies were more than the IC50 of magnetic nano­
vesicles formulation (95.64 µg). Our preliminary
study suggests that there was no aggregation of
the magnetic vesicles on intravenous administra-
tion. However, the half-life of the magnetic vesi-
cles in plasma on systemic administration and
the biodistribution needs to be determined. It
is expected that even a 10% localization of the
magnetic vesicles at the tumor site would allow a
sufficient temperature rise within 1 h and a drug
release above the IC50. However, the success of the
In our study, upon validating the results of
hyperthermia and chemotherapy with Veleri-
ote’s method, it was found that the combined
effect of chemotherapy and hyperthermia was
synergistic in nature. In cancer therapy, dif-
achieve a synergistic effect and improve the
therapeutic index. This helps in reducing the
dose of individual therapeutic modalities and,
hence, the side effects of individual modal-
ity’s can be minimized. Pradhan et al. [13] and
Kulshrestha et al. [15] showed the synergistic
effect of hyperthermia and chemotherapy in
different cancer cell lines but did not have
any self-control in the hyperthermia as only
Fe3O4 nanoparticles were entrapped in those
nanovesicles. Ito et al. demonstrated that the
combined effect of 4-S -cysteaminylphenol
treatment and hyperthermia on B16 melanoma
cells was additive in nature [48]. This may be
due to the nature of the action of the drug and
duration of hyperthermia. In the present study
the effects of hyperthermia and drug delivery
were found to be synergistic. This suggests the
potential application of DSPC:cholesterol-
based thermosensitive magnetic nanovesicles
encapsulating biphasic suspensions of LSMO
and Fe3O4 nanoparticles for chemotherapy and
self-controlled hyperthermia.
Conclusion & future perspective
A novel magnetic nanovesicle containing pacli-
taxel and a biphasic suspension of Fe3O4 and
LSMO nanoparticles were prepared by a thin-
film hydration method. To the best of our
knowledge, this is the first magnetic nanovesicle
prepared from a biphasic magnetic nanohybrid

doi:10.2217/NNM.13.90 Nanomedicine (Epub ahead of print) future science group

 Biphasic magnetic nanoparticles–nanovesicle hybrids
for combined chemotherapy and self-controlled
hyperthermia. Dextran-coated biphasic nano-
particle suspension was conjugated with FITC,
and coencapsulated with paclitaxel within the
thermosensitive magnetic nano­vesicles. The IC50
of this magnetic nanovesicle was 112 nM. The
combined effect of hyperthermia and chemo-
therapy was synergistic in nature while con-
trolling the temperature at 44°C. The biphasic
nanoparticle nanovesicle hybrid has the potential
for combined self-­controlled hyper­thermia and
chemotherapy.
Acknowledgements
The authors thank the Centre for Research in Nanotech­
nology and Nanoscience for using their instrumentation
facilities.
Preliminary Communication
Financial & competing interests disclosure
The financial support provided by the Department of
Information Technology and the Nanomission, Department
of Science and Technology are gratefully acknowledged. The
authors have no other relevant affiliations or financial
involvement with any organization or entity with a finan­
cial interest in or financial conflict with the subject matter
or materials discussed in the manuscript apart from those
disclosed.
No writing assistance was utilized in the production of
this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate insti­
tutional review board approval or have followed the princi­
ples outlined in the Declaration of Helsinki for all animal
experimental investigations.

Executive summary
Need for self-controlled hyperthermia and chemotherapy
ƒƒ Magnetic materials that can prevent overheating of tissues are required for combined hyperthermia and temperature-triggered drug
delivery.
ƒƒ Magnetic biphasic suspension of La0.75Sr0.25MnO3 and iron oxide nanoparticles were developed for self-controlled hyperthermia and
chemotherapy.
Results of encapsulation and proof of principle
ƒƒ Encapsulation efficiencies of paclitaxel and magnetic nanoparticles were 83 ± 3 and 67 ± 5%, respectively.
ƒƒ Magnetic nanovesicles coencapsulating La0.75Sr0.25MnO3, iron oxide nanoparticles and paclitaxel were biocompatible.
ƒƒ The IC50 of magnetic nanovesicles in the MCF-7 cell line was 112 nM.
ƒƒ Combined self-controlled hyperthermia and chemotherapy in an AC magnetic field showed synergistic effects in cytotoxicity in MCF-7
cell lines and maintained the temperature at 44°C.
Conclusion
ƒƒ Magnetic nanovesicles containing La0.75Sr0.25MnO3 and iron oxide nanoparticles and coencapsulating paclitaxel have potential for
self-controlled hyperthermia and trigger responsive drug delivery.

nn Synthesis and application of magnetic hyperthermia. Crit. Rev. Oncol. Hematol.

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