Integrative Biology

View Article Online

PAPER View Journal

Biocompatibility and therapeutic evaluation of

magnetic liposomes designed for self-controlled
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.

Cite this: DOI: 10.1039/c6ib00234j

cancer hyperthermia and chemotherapy†
Manashjit Gogoi,a Manish K. Jaiswal,b Haladhar Dev Sarma,c
Dhirendra Bahadur *b and Rinti Banerjee*a

Magnetic liposome-mediated combined chemotherapy and hyperthermia is gaining importance as an

Received 29th November 2016,
Accepted 11th April 2017
DOI: 10.1039/c6ib00234j
rsc.li/integrative-biology
effective therapeutic modality for cancer. However, control and maintenance of optimum hyperthermia
are major challenges in clinical settings due to the overheating of tissues. To overcome this problem, we
developed a novel magnetic liposomes formulation co-entrapping a dextran coated biphasic suspension
of La0.75Sr0.25MnO3 (LSMO) and iron oxide (Fe3O4) nanoparticles for self-controlled hyperthermia and
chemotherapy. However, the general apprehension about biocompatibility and safety of the newly
developed formulation needs to be addressed. In this work, in vitro and in vivo biocompatibility and
therapeutic evaluation studies of the novel magnetic liposomes are reported. Biocompatibility study of
the magnetic liposomes formulation was carried out to evaluate the signs of preliminary systemic
toxicity, if any, following intravenous administration of the magnetic liposomes in Swiss mice. Therapeutic
efficacy of the magnetic liposomes formulation was evaluated in the fibrosarcoma tumour bearing mouse
model. Fibrosarcoma tumour-bearing mice were subjected to hyperthermia following intratumoral injection
of single or double doses of the magnetic liposomes with or without chemotherapeutic drug paclitaxel.
Hyperthermia (three spurts, each at 3 days interval) with drug loaded magnetic liposomes following single
dose administration reduced the growth of tumours by 2.5 fold (mean tumour volume 2356  550 mm3)
whereas the double dose treatment reduced the tumour growth by 3.6 fold (mean tumour volume 1045 
440 mm3) compared to their corresponding control (mean tumour volume 3782  515 mm3). At the end
of the tumour efficacy studies, the presence of MNPs was studied in the remnant tumour tissues and vital
organs of the mice. No significant leaching or drainage of the magnetic liposomes during the study was
observed from the tumour site to the other vital organs of the body, suggesting again the potential of the
novel magnetic liposomes formulation for possibility of developing as an effective modality for treatment of
drug resistant or physiologically vulnerable cancer.

Insight, innovation, integration

This study deals with the development of a thermosensitive magnetic liposomes formulation for combined chemotherapy and self-controlled hyperthermia.
We are also reporting the biocompatibility study of the magnetic liposomes formulation. This study is unique because the liposomes contain a biphasic
suspension of Fe3O4 and La0.75Sr0.25MnO3 (LSMO) nanoparticles. Biocompatibility study of liposomes prepared for combined chemotherapy and self-controlled
hyperthermia containing a biphasic suspension of magnetic nanoparticles is reported for the first time. This is one of the earliest reports of therapeutic
evaluation with magnetic liposomes developed for self-controlled hyperthermia and chemotherapy.

a
Nanomedicine Laboratory, Department of Biosciences and Bioengineering,
Indian Institute of Technology-Bombay, Mumbai-400076, India. 1. Introduction
E-mail: rinti@iitb.ac.in; Fax: +91 22 2572 3480; Tel: +91 22 2576 7868
b
Department of Metallurgical Engineering and Materials Science, Indian Institute of Success of chemotherapy in treating solid tumour is limited by
Technology-Bombay, Mumbai-400076, India. E-mail: dhirenb@iitb.ac.in;
severe side effects of chemotherapeutic agents and the unique
Fax: +91 22 2572 3480; Tel: +91 22 2576 7632
c
Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre,
tumour environments that cause drug resistance. The chaotic
Mumbai-400085, India and complex vasculatures developed in and around the tumour,
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ib00234j and their abnormal interstitial properties1 prevent effective

This journal is © The Royal Society of Chemistry 2017 Integr. Biol.


Paper
permeation of drugs to the tumour cells and minimize their
anticancer activity.2,3 The highly acidic and oxygen deprived hypoxic
environments within the tumour mass reduce the effectiveness of
drugs like doxorubicin that are basic in nature and/or utilize oxygen
free radicals for anticancer action.4 In the case of solid tumour, a
substantial portion of tumour cells are in quiescent state and they
do not divide in the early stage of tumour formation.5 Therefore, the
presence of these dormant cells further compromises the upshot of
treatment with chemotherapeutic agents which are effective against
rapidly dividing cells.6 Under such circumstances, hyperthermia in
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
combination with chemotherapy can serve as an effective modality
for treating cancers because hyperthermia can directly damage
cellular structures7,8 and then chemotherapeutic drugs can act on
the damaged tumour cells more effectively or vice versa resulting
in improved net outcome. Experimental evidence suggests that
hyperthermia helps in extravasation of drug loaded nanoparticles
to tumour tissues and enhances the bioavailability of drugs at
tumour sites.9 Hyperthermia following intratumoral injection of
magnetic nanoparticles (MNPs) can be used to minimize the
toxicities related to systemic administration of MNPs. Clinical
trials have been reported with Fe3O4 nanoparticle mediated
hyperthermia.10–12 Magnetic liposomes have been used for cancer
hyperthermia13–21 due to their biocompatibility and ability to carry
therapeutic molecules to the tumour sites. Both MNPs10–12,22–29
and magnetic liposomes13–21,30,31 with or without drugs have been
explored for cancer therapy under a wide range of hyperthermic
conditions. In most of these studies, iron oxide based nano-
structures were used, which raised the temperature on the
tumour surface to 45 1C or above leading to cell death by
necrosis16,17,23,24 and generation of heat shock proteins that in
turn resulted in heat resistance.24 These treatment strategies
were harsh due to frequent application of hyperthermia as high
as nine times within 15 days23 and the temperature at the
surface of tumour reaching as high as 45 1C.16,17 Uniform
distribution of MNPs within the tumour mass is a big challenge
and it determines the therapeutic efficacy of hyperthermia.17
Non-uniform distribution of MNPs leads to generation of hot
spots which damage the normal tissues adjacent to the tumour.
In order to prevent collateral damage of normal tissue during
hyperthermia, tumour temperature needs to be controlled by
switching off/on the magnetic field frequently. To overcome this
problem, we engineered magnetic liposomes for alternating
magnetic field (AMF) induced self-controlled hyperthermia
and chemotherapy. In our earlier study,14 we reported development
and in vitro evaluation of paclitaxel loaded thermosensitive
magnetic liposomes containing a dextran coated biphasic
suspension of LSMO and Fe3O4 nanoparticles in the MCF-7
cell line. In order to avoid overheating of tissues, a fine balance
in the ratio of MNPs is essential for controlling the temperature
of the liposomes so that the tumour temperature does not
shoot beyond 44 1C even after continuous application of AMF.
LSMO nanoparticles were extensively used for self-controlled
hyperthermia.28,29,32–37 Pollert et al.33 reported the synthesis of
LSMO nanoparticles for self-controlled hyperthermia. Prasad
et al.34 studied the feasibility of application of a biphasic gel of
La1xSrxMnO3 and maghemite for hyperthermia application.34
Integr. Biol.
View Article Online
Integrative Biology
But their study was limited to evaluation of heating ability of
biphasic gels. Dextran-coated La0.7Sr0.3MnO3 nanoparticles
have been demonstrated to be effective in treating melanoma
bearing C57BL/6J mice.29 Nevertheless, a large number of
nanoparticles are reported, they are not evaluated for their
potential health hazards. Therefore, in vivo biocompatibility
study of these magnetic liposomes is warranted for furthering
the lead on specific formulation.
Here we report in vitro and in vivo biocompatibility of
magnetic liposomes containing a biphasic suspension of LSMO
and iron oxide nanoparticles. In vivo biocompatibility study was
done in 6–8 week old Swiss mice. Biochemical and haematological
parameters of blood collected at different time points following
intravenous injection of magnetic liposomes were evaluated. After
evaluating the biocompatibility of these magnetic liposomes, their
therapeutic efficacy was evaluated in a fibrosarcoma cell line as
well as in the corresponding tumour bearing animal model.
Fibrosarcoma is a type of malignancy, originating from fibroblastic
cells and collagen fibres. Surgical resection with adequate margin
offers good local control of the tumour, but inadequately margined
resection or erroneous excision of lesion leads to higher morbidity
with limited functional outcomes and increased rate of recurrence.38
It is more or less resistant to radiotherapy;39 however local
hyperthermia in combination with chemotherapy was found
to be more effective than chemotherapy alone.40
We also report ICP-AES and VSM analysis of MNPs in the
vital organs of the animals following intratumoral injection of
magnetic liposomes. To the best of our knowledge, this is the
first study reporting the in vivo evaluation of drug loaded
magnetic liposomes containing paclitaxel and a biphasic suspension
of LSMO and Fe3O4 nanoparticles for biocompatibility, combined
chemotherapy and self-controlled hyperthermia as well as bio-
distribution of MNPs.
2. Materials and methods
2.1. Materials
FeCl24H2O and FeCl36H2O were procured from Sigma Aldrich
(St. Louis, MO, USA). La2O3 was procured from Indian Rare
Earths Ltd (Cochin, India). 1,2-Distearol-sn-phosphatidylcholine
(DSPC) was purchased from Lipoid (Ludwigshafen, Germany).
Anticancer drug paclitaxel (purity 499%) and Taxol (6 mg ml1),
a marketed formulation of anticancer drug paclitaxel, were
supplied by Dabur India Ltd (Ghaziabad, India).
For cell culture, fibrosarcoma cells (HT-1080) were purchased
from the National Centre for Cell Science (Pune, India). Cells were
maintained as monolayer culture in DMEM supplemented with
10% fetal bovine serum and 1% antibiotic-antimycotic solution at
37 1C in a humidified incubator containing 5% CO2. Experiments
were performed when cells became 70–80% confluent.
2.2. Preparation and characterization of magnetic liposomes
containing a biphasic suspension
A dextran coated biphasic suspension of MNPs and magnetic
liposomes were prepared and characterized according to the
This journal is © The Royal Society of Chemistry 2017

Integrative Biology
protocols reported earlier.14 Encapsulation of MNPs in liposomes
was determined using ICP-AES (Arcos, M/S. Spectro, Germany). In
order to determine the encapsulation efficiency of MNPs, magnetic
liposomes were dissolved in concentrated HCl and then filtered
through a Whatman filter paper of 0.2 micron pore size. MNP
concentration in the filtered solution was determined by ICP-AES.
Then MNP encapsulation was determined using eqn (1):
% MNP encapsulation
Amount of MNPs in formulation (1)
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
¼  100
Amount of MNPs used during hydration
The heating ability of magnetic liposomes or the specific
absorption rate (SAR) value was determined experimentally
using eqn (2).13 For this, magnetic liposomes with different
known concentrations of MNPs were made up to 100 ml volume
and put in 1.5 ml micro-centrifuge tubes. Then the tubes were
placed under an AMF (250 kHz frequency and 27.9 kA m1 field
strength) generated by a water-cooled and Teflon coated cylindrical
copper coil (diameter 6.5 cm, 4 turns) connected to a radio
frequency generator (Ameritherm, USA) one by one. 100 ml of
magnetic liposomes was used for SAR studies as this is the
maximum volume of magnetic liposomes that could be injected
into the tumour. The temperature profile of magnetic liposome
solutions was recorded using an optical fibre probe (OEM 650,
LumaSense, USA) connected to a computer.
1 dT
SAR ¼ C   (2)
m dt
where C, m, and dT/dt are the specific heat capacity of water,
the mass fraction of the magnetic material in the sample and the
initial slope of the temperature versus time curve respectively.
2.3. In vitro biocompatibility of magnetic liposomes in L929
cells
Biocompatibility study was carried out with the extract of
magnetic liposomes in the mouse fibroblast (L929) cell line
according to the protocol mentioned in an earlier paper14
with little modifications. Magnetic liposomes with a MNP
concentration of 10 mg ml1 were dispersed in DMEM and
incubated for 48 h at 37 1C. Then liposomes were removed by
centrifugation at 17 000 rpm; the supernatant was further
diluted with fresh media at different concentrations ranging
from 0.16–5 mg ml1 of MNPs. The diluted supernatant at
these concentrations was incubated with cells. After 48 h, cell
viability was determined by sulphorhodamine B (SRB) assay.
Table 1 Grouping of animals for biocompatibility study
Treatment group
Control (no magnetic liposomes treatment)
MD1 (magnetic liposomes treatment dose 1 with 1 mg of MNPs)
MD2 (magnetic liposomes treatment dose 2 with 2 mg of MNPs)
Total mice
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
2.4. In vitro cytotoxicity and AMF induced hyperthermia
studies in the HT-1080 cell line
In vitro cytotoxicity study of magnetic liposomes was carried out
in the fibrosarcoma (HT-1080) cell line according to the proto-
col reported previously.14 In this experiment, Taxol was used as
a positive control. The concentrations of drug in the magnetic
liposomes and Taxol groups were in the range of 50–400 nM.
10  105 HT-1080 cells were equally divided and put in two
sterile 15 ml polypropylene centrifuge tubes (Tarsons Ltd,
Kolkata, India), with complete media and centrifuged at 2000 rpm
for 5 min to form pellets. Then the medium was discarded and
magnetic liposomes with or without 147 nM drug in complete
medium were added to the pellets and cells were resuspended.
5  105 cells suspended in complete media were taken in another
sterile tube. All three tubes were placed one by one at the centre of
the water cooled copper coil of the RF generator and a field of
27.9 kA m1 and 250 kHz frequency was applied. During the
experiment, a uniform temperature of 43.5 1C was obtained for
30 min. Then cells were seeded in a 24 well plate with 5 replicates and
incubated for 48 h. For the control group, 5  105 cells were seeded
with 5 replicates and incubated for 48 h. Viable cells were counted
by the trypan blue dye exclusion method using a haemocytometer.
2.5. In vivo biocompatibility study
Experiments were conducted on female Swiss mice (20–23 g
body weight, 6–8 weeks old), bred and reared in the animal
facility of Bhabha Atomic Research Centre (BARC, Mumbai,
India). Experimental animals were maintained in a temperature
and humidity controlled room on a 12 h light and dark cycle
with proper food and water supply. All animal experiments were
conducted with permission (approval number was BAEC-9/11)
and supervision of Institutional Animal Ethics Committee of
Bhabha Atomic Research Centre, Mumbai, India, which is
constituted under provision of national law on animal welfare,
i.e. ‘‘The Prevention of Cruelty to Animals Act, 1960’’. For the
biocompatibility study, the animals were randomly divided into
3 treatment groups (n = 5), viz. (i) control (no magnetic lipo-
somes treatment), (ii) MD1 (magnetic liposomes treatment dose
1 with 1 mg of MNPs) and (iii) MD2 (magnetic liposomes
treatment dose 2 with 2 mg of MNPs). Animals from the
experimental groups were sacrificed at different time points as
shown in Table 1. Magnetic liposomes were administered into
the animals through tail vein. Magnetic liposomes were not
injected in the control animals.
Animals were then sacrificed at 1 h, 48 h and 7 days post-
administration of magnetic liposomes. Blood samples were
Number of animals sacrificed at different time points
1h 48 h 7d
4 — 4
4 4 4
4 4 4
32
Integr. Biol.

Paper
collected in glass vials treated with 150 ml of 0.2% EDTA solution
and sent for determining the parameters related to complete
blood count and serum biochemistry on a day-to-day basis.
2.5.1. Analysis of biochemical parameters. The level of
glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic
transaminase (GPT) was measured using a fully automatic
biochemistry analyzer, FA200 (Clindiag Systems BVBA, Belgium)
to evaluate liver function. Kidney function was evaluated by
determining blood urea nitrogen (BUN) and creatinine using
a semiautomatic biochemistry analyzer, ARX-100 (Micro Lab
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
Instruments, Mumbai, India).
2.5.2. Complete blood counts. Complete blood counts
(CBC) and other blood parameters were determined by using
a semi-automated haematology analyzer, BC 2300 (Shenzhen
Mindray Bio-Medical Electronics Co. Ltd, Shenzhen, China)
and differential leukocyte count (DLC) was done by examining
blood smear microscopically.
2.6. In vivo therapeutic evaluation of magnetic liposomes and
their biodistribution
Female Swiss mice (6–8 weeks old), bred and reared at the
laboratory animal facility of Bhabha Atomic Research Centre,
Mumbai, India, were used for in vivo experiments. The animals
were housed in appropriate isolated cages, at 22 1C and on a
12 h light/dark cycle with sterile rodent food and acidified water
ad libitum. Tumours were transplanted into the left femoral
region of hind legs of the mice by injecting 106 fibrosarcoma cells
suspended in 100 ml of PBS. Following tumour cell transplantation,
the animals were observed daily for growth of tumour.
On 12 day of tumour transplantation, animals with uniform
tumour size (B8–15 mm diameter) were selected and randomly
divided into 6 groups (n = 5) as follows: (i) control: no treatment
was given, (ii) TX: treated with marketed paclitaxel (Taxol) at
38.4 mg kg1 dose intratumorally, (iii) MSD: treated with
magnetic liposomes single dose, (iv) PSD: treated with paclitaxel
loaded magnetic liposomes single dose, (v) MDD: treated with
magnetic liposomes double dose and (vi) PDD: treated with
paclitaxel loaded magnetic liposomes double dose. Grouping of
animals for hyperthermia study is shown in Table 2. Animals
were given the first dose of treatment on the 12th day after
tumour transplantation and it was denoted as day 1 of therapy.
After injecting the magnetic liposomes intratumorally, animals
of groups MSD, PSD, MDD and PDD were anaesthetised with
intraperitoneal injection of ketamine-HCl (50–80 mg kg1) and
xylazine-HCl (5–10 mg kg1) and exposed to AMF (350 Oe and
Table 2 Grouping of animals for therapeutic evaluation of magnetic liposomes
Scheduled day of treatment and sacrifice of the experimental animals
Treatment 1st dose injected (12 d from Day of sacrifice (from
Sl. no. group (n = 5) tumour transplantation) (d) start of treatment) (d)
1 Control — 12
2 TX 1 12
3 MSD 1 16
4 PSD 1 16
5 MDD 1 —
6 PDD 1 —
Integr. Biol.
View Article Online
Integrative Biology
frequency 250 kHz) for 30 min. The magnetic field was generated
by a water-cooled cylindrical copper coil connected to a RF
generator. Animals of groups MSD and PSD were subjected to
hyperthermia thrice over a period of 8 days and animals of
groups MDD and PDD were subjected to hyperthermia five times
within the initial 16 days. The temperature at different points on
the surface of the tumour was measured using an optical probe at
regular intervals. Tumour size of animals was measured using a
Vernier calliper at different time points. Tumour volume was
calculated using the following formula:
p
Tumour volume ¼ ðminor dimensionÞ2 ðmajor dimensionÞ (3)
6
Animals of control and Taxol groups were sacrificed on the
12th day, animals of MSD and PSD groups were sacrificed on
the 16th (due to tumour burden 43700 mm3) day, whereas
animals of groups MDD and PDD were sacrificed on the 22nd
day (Table 1). After sacrificing the animals, their vital organs,
viz. lung, liver, heart, brain, spleen, and kidney, and tumour
were excised and used for determining the amount of MNPs.
Treatment related toxicity was evaluated by measuring the body
weight of mice at predetermined time points. Loss of body
weight by more than 20% was regarded as a sign of significant
systemic toxicity as described in the earlier literature.41 The
other signs monitored for toxicity include fur roughening and
shedding, and decreased animal activity.
2.7. Statistical analysis
All the data are expressed as mean  S.D. (standard deviation)
with n = 5 for hyperthermia and biodistribution studies.
Statistical differences are computed by Student’s t-test and
p r 0.05 was used as the criteria to determine significant
differences.
3. Results
3.1. Characterization of magnetic liposomes
The mean hydrodynamic diameter and zeta potential of magnetic
liposomes were 225  45 nm and 41  5 mV respectively. MNP
and paclitaxel encapsulation efficiencies were 67  5% and 83 
3% respectively. For liposomes prepared using 10 mg of lipids,
the amounts of encapsulated MNPs and drug were 1.675 mg and
0.83 mg respectively. The SAR value of magnetic liposomes was
17.4 W g1. The temperature profile of magnetic liposomes is
shown in Fig. S1 (ESI†). The SAR of magnetic liposomes was
2nd dose injected (from Hyperthermia: Day of sacrifice (from
start of treatment) (d) no. of spout start of treatment) (d)
— 0 —
— 0 —
— 3 —
— 3 —
11 5 22
11 5 22
This journal is © The Royal Society of Chemistry 2017

Integrative Biology
studied for 1 h. The results showed that the maximum temperature
achieved was 43.5 1C.
3.2. In vitro biocompatibility study in the L929 cell line
The magnetic liposomes contain a dextran coated biphasic
suspension of LSMO and Fe3O4 nanoparticles in a ratio of
9 : 1. In our study, the extract of magnetic liposomes was used
for in vitro biocompatibility study using the L929 cell line. The
results showed (Fig. 1) that cell viability was more than 90% for
an extract concentration up to 5 mg ml1. At extract concentration
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
5 mg ml1, cell viability was 94.2  1.3% even after 48 h of
incubation. The cytotoxicity is substantially reduced after using the
extract of magnetic liposomes. Addition of Fe3O4 nanoparticles to
LSMO nanoparticles, coating the MNPs with dextran and then
encapsulation with lipids improve the biocompatibility of MNPs.
3.3. In vitro cytotoxicity and hyperthermia of magnetic
liposomes in the HT-1080 cell line
Magnetic liposomes and Taxol were incubated with HT-1080 cells
for 48 h or 72 h. For the 48 h incubation period, IC50 of magnetic
liposomes formualation containing 147 nM paclitaxel in the
HT-1080 cell line was 147 nM.
During in vitro hyperthermia, magnetic liposomes containing
147 nM paclitaxel were used and the AMF was applied for 30 min.
The results (Fig. 2) showed that AMF did not have any significant
effect on cell viability. Hyperthermia with magnetic liposomes
(without drug) resulted in cell viability of 69  3% while cell
viability was reduced to 52  4.3% due to the treatment with
magnetic liposomes containing 147 nM paclitaxel. Combination
of hyperthermia and chemotherapy reduced the cell viability
to 28  2.4%.
3.4. In vivo biocompatibility study
3.4.1. Analysis of biochemical parameters. The level of
GOT and GPT was determined to evaluate the function of liver
(Fig. 3). For treatment group MD1 (magnetic liposome dose 1)
the level of GOT increased significantly to 284  60.3 U l1 ( p o
0.05) in 1 h and then came down to 186  27 U l1 over a period
of one week. The GOT level for the control group of mice was
Fig. 1 In vitro biocompatibility study of extract of magnetic liposomes on
the L929 cell line (results are expressed as mean  sd, n = 5).
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
Fig. 2 In vitro hyperthermia study: cell viability following treatment with
ac magnetic field (AMF), magnetic liposomes with hyperthermia, paclitaxel
loaded magnetic liposomes (147 nm ptx), and paclitaxel loaded magnetic
liposomes with hyperthermia (147 nm ptx within magnetic liposomes).
AMF at field 27.9 kA m1 (350 Oe) and 250 kHz frequency was applied for
30 min.
173  20.3 U l1. For treatment group MD2 (magnetic liposome
dose 2) the level of GOT marginally increased to 222 
74.3 U l1 at the end of 1 h and remained at 208  26 U l1
till the end of the experiment on the 7th day. The levels of GPT
were significantly higher ( p o 0.05) than the control value in
1 h for animals of both the groups. The corresponding values
came down to 36  8.5 U l1 and 28  2.2 U l1 respectively on
the 7th day. The value of GPT was 30  7 U l1 for the mice of
control group.
The levels of creatinine and BUN were determined to evaluate
the condition of the kidney. The levels of creatinine remained
almost stable within the range of 0.28  0.05 mg dl1 to 0.3 
0.06 mg dl1 for animals of dose 1 and dose 2 groups during the
experimental period. The value of creatinine for the control group
was 0.28  0.07. For animals subjected to dose 1, the levels of
BUN were 24.5  3.21 mg dl1 after 1 h and 26  0.38 mg dl1 on
the 7th day. Similarly for animals of dose 2, the levels of BUN
were 24  4.3 mg dl1 after 1 h and 24  2.3 mg dl1 on the 7th
day. During this period the BUN value was 20.8  4 mg dl1 for
the control group of animals.
3.4.2. Complete blood counts. The levels of haemoglobin
and RBCs (Fig. 4) were almost equal to their respective control
values, i.e. 15  1 mg dl1 and (8.3  0.5)  106 cmm1 for
animals of both MD1 and MD2 groups throughout the experi-
mental period. WBC and platelet levels remained high for both
treatment groups of mice till the end of the experiment. For
both MD1 and MD2 groups of animals, the levels of MCV and
PCV were as stable as for the control group (Fig. S2, ESI†).
Neutrophil percentage remained high for both groups of
animals; the neutrophil level for the control group was 39.4 
6.4%. There was a transient change in % lymphocytes observed
during the period of experiment. The lymphocyte level for
control animals was 59  7%. The blood parameters did not
change significantly following intravenous administration of
magnetic liposomes, suggesting the colloidal stability of the
magnetic liposomes formulation; the magnetic liposomes did
not aggregate in blood or cause any harm to the components
of blood.
Integr. Biol.
 View Article Online

Paper Integrative Biology

Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.

Fig. 3 Variations in serum biochemistry parameters in mice of experimental groups MD1 and MD2 following intravenous administration of magnetic
liposomes containing 1 mg and 2 mg of MNPs respectively: (A) GOT, (B) GPT, (C) creatinine, (D) BUN (results are expressed as mean  sd, n = 5. *p o 0.05
with respect to control. h = hour, d = days).

3.5. In vivo therapeutic evaluation of magnetic liposomes and
their biodistribution
In vivo hyperthermia was applied by placing the tumour bearing
mice inside a horizontal water cooled coil connected to a RF
generator. During the in vivo hyperthermia experiment, the
temperature at the surface of tumour was measured at regular
intervals using a highly sensitive fibre optic sensor. The tem-
perature on the tumour surface reached 40–41 1C within 10 min
which rose to a maximum of 43 1C. During hyperthermia,
animals were air cooled with a table top fan. The maximum
temperature recorded on the skin of the animals other than the
area of the tumour was 37 1C. This suggests that magnetic
liposomes were heating the tumour; there was no heating
due to hyperthermia outside the tumour region. Tumour
temperature reached saturation at 20 min and remained
constant (at 42.5  0.5 1C) till the end of the experiments.
Since the maximum temperature attained in vitro was 43.5 1C
with these magnetic liposomes formulation, it is suggested that
the temperature inside the tumour would be in the range of
42 to 43.5 1C depending upon the availability of MNPs.

Fig. 4 Variations in blood parameters in mice of experimental groups MD1 and MD2 following intravenous administration of magnetic liposomes
containing 1 mg and 2 mg of MNPs respectively: (A) haemoglobin, (B) RBC, (C) WBC, (D) platelet (results are expressed as mean  sd, n = 5; h = hour,
d = days).

Integr. Biol. This journal is © The Royal Society of Chemistry 2017

 View Article Online

Integrative Biology Paper

The mean tumour volume of animals of treatment groups

MSD (treated with magnetic liposomes single dose) and PSD
(treated with paclitaxel loaded magnetic liposomes single dose)
was reduced to 2356  551 mm3 and 1809  996 mm3 respectively
on the 16th day of treatment, whereas the volume of tumour in the
control group was 5867  378 mm3. Taxol was not found to be
effective against the fibrosarcoma tumour. The volume of tumour
in the Taxol treated (TX) group was 4511  209 mm3 on the 12th
day of treatment. In MDD (treated with magnetic liposomes
double dose) and PDD (treated with paclitaxel loaded magnetic
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.

liposomes double dose) treatment groups magnetic liposomes

with or without drug were administered intratumorally twice
(i.e. on 12 d and 23 d of tumour transplantation) and the mice were
subjected to hyperthermia five times at 3 day interval within the
span of the 16 days from i.e. 12 to 23 d of tumour transplantation.
In comparison to the control group, the tumour volumes of
animals (Fig. 5) of hyperthermia and combined hyperthermia
and chemotherapy groups were regressed significantly ( p r 0.05).
The mean tumour volumes of double dose magnetic liposomes
mediated hyperthermia treated animals of MDD and PDD groups
were 2254  430 mm3 and 1046  440 mm3 respectively on 22 d of
treatment (i.e. 34 d of tumour transplantation), whereas the tumour
volume in the corresponding control group reached 3782 
515 mm3 on 12 d of treatment or 24 d of tumour transplantation
itself. The difference in tumour volume between the animals of
MDD and PDD was significant ( p r 0.05).
The body weights of control and TX group of animals were
increased during the experimental period. But the body weights
of hyperthermia and combined therapy groups did not change
much during the same period (Fig. 6). For double dose groups,
the body weights of hyperthermia and combination groups of
animals were found to reduce initially as a result of the treatment. Fig. 5 Results of in vivo hyperthermia experiments: (A) tumour growth
However, as the treatment progressed the animals started gaining profile of experimental animals following treatment with paclitaxel (TX),
weight. magnetic liposomes single dose (MSD) and paclitaxel loaded magnetic
liposomes single dose (PSD); (B) tumour growth profile of experimental
VSM is an indirect way of measuring the amount of MNPs
animals following treatment with magnetic liposomes double dose (MDD)
present in different organs. It measures the magnetic moment and paclitaxel loaded magnetic liposomes double dose (PDD); and (C)
which in turn gives the amount of MNPs present in the sample. temperature profile during hyperthermia application. Tu_Temp1, Tu_Temp2,
In the present study, the highest magnetization value from the Tu_Temp3, Tu_Temp4 and Tu_Temp5 represent tumour surface temperature
M–H loop of the samples was used to quantify the amount of and Bd_Temp represents average temperature measured on the body surface
other than the tumour site during each spout of hyperthermia application at
MNPs present in the different organs of the mice. The highest
different time intervals. Experimental animals were subjected to each spout of
magnetization value was obtained from the organs of the mice hyperthermia at 3 d intervals under AMF 27.9 kA m1 (350 Oe) and 250 kHz
subjected to double dose of combination therapy and the value frequency for 30 min.
was approximately 0.05 emu g1 for tumour tissue. In the case
of spleen, the value was 0.02 emu g1 (Fig. S3, ESI†). ICP-AES
values showed (Fig. 7) similar trends. It was observed from the ICP- nanoparticles was developed for combined cancer chemotherapy
AES data that the maximum amount of MNPs was accumulated in and self-controlled hyperthermia and evaluated both in vitro and
tumour tissue. Amounts of MNPs accumulated in tumour were in vivo. LSMO consists of La, Sr and Mn, and Bhayani et al.32
161  20.9 and 126.2  16.3 mg g1 of dry tumour mass from MSD reported that LSMO structure is very stable and no toxicity was
and PSD groups respectively. The corresponding values from MDD observed due to the leaching of Mn. They reported that bare
and PDD were 340.7  12.5 and 368.9  15 mg g1 of dry tumour LSMO nanoparticles at a concentration of 20 mg ml1 can reduce
mass respectively. the cell viability to as low as 20% which can be significantly
improved by coating with dextran or BSA (bovine serum albumin).
4. Discussion In our study, the extract of magnetic liposomes was used for in vitro
biocompatibility study using the L929 cell line. The results
A novel magnetic liposomes formulation containing paclitaxel showed (Fig. 1) that cell viability was more than 90% for an
and a dextran coated biphasic suspension of LSMO and Fe3O4 extract concentration up to 5 mg ml1. At extract concentration

This journal is © The Royal Society of Chemistry 2017 Integr. Biol.


Paper
Fig. 6 Body weight of mice measured during the experiment. (A) Body
weight of mice treated with Taxol (TX), magnetic liposomes single dose
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
(MSD) and paclitaxel loaded magnetic liposomes single dose (PSD);
(B) body weight of mice treated with magnetic liposomes double dose
(MDD) and paclitaxel loaded magnetic liposomes double dose (PDD).
Fig. 7 ICP-AES estimation of MNPs present in different organs of mice
treated with hyperthermia and combined therapy.
5 mg ml1, cell viability was 94  1.3% even after 48 h of incubation
with the cells. The cytotoxicity is substantially reduced after using
the extract of magnetic liposomes. Addition of Fe3O4 nanoparticles
to LSMO nanoparticles, coating the MNPs with dextran and then
encapsulation with lipids improved the biocompatibility of MNPs.
Paclitaxel is one of the most potent anticancer drugs and it
inhibits the replication of tumour cells by polymerizing the
stable microtubules in the late G2 mitotic phase of the cell
cycle.13 In the current Taxol formulation, paclitaxel is dissolved
in a 50 : 50 (v/v) mixture of Cremophor EL (polyethoxylated
castor oil) and dehydrated alcohol which causes serious or fatal
hypersensitivity in humans. In order to minimize the Cremophor
EL associated toxicity, premedication with dexamethasone and
antihistamines is used clinically. So, there is a strong urge for
developing a safer and better paclitaxel formulation. Paclitaxel
was encapsulated in liposomes and was found to show promising
results both in vitro and in vivo.42,43 In our in vitro cytotoxicity
studies, magnetic liposomes have shown almost similar effects as
Taxol on the HT-1080 cell line whereas in vivo results showed that
magnetic liposomes were superior to paclitaxel formulation due
to their minimal toxicity and better anti-tumour activity. During
the in vitro hyperthermia experiment, the temperature of the
magnetic liposomes did not exceed 43.5 1C, suggesting the
Integr. Biol.
View Article Online
Integrative Biology
suitability of magnetic liposomes for self-controlled hyperthermia.
In the hyperthermia experiments, we incorporated paclitaxel in
magnetic liposomes at a concentration equal to its IC50 value
without any alteration of the hyperthermic property of the MNPs.
The combination of paclitaxel and hyperthermia was found to act
synergistically and killed 72.3% of HT-1080 cells within 30 min.
This is a preliminary cytotoxicity study, encouraging the
possible in vivo applications of these magnetic liposomes.
In vivo therapeutic evaluation cum biodistribution studies were
carried out following intratumoral injection. But the lipid
composition of these magnetic liposomes was reported to be
thermosensitive and have long blood circulation time and
hence suitable for hyperthermia and drug delivery applications
through systemic administration also.14 In order to study
biocompatibility, the magnetic liposomes were administered
intravenously to assess toxicity due to their systemic exposure.
The magnetic liposomes are not or least exposed to other
organs directly during intratumoral administration, and hence
these liposomes were administered intratumorally during ther-
apeutic efficacy study to assess their maximum therapeutic index.
In the biocompatibility experiment, the levels of GOT and
GPT were marginally changed following the administration of
magnetic liposomes and came down to normal level towards
the end of the experiment. This transient surge in enzyme
activities seems to be the general inflammatory response of the
body. Jain et al.44 reported similar results following administration
of oleic acid–pluronic coated Fe3O4 nanoparticles. The increase in
GOT levels by three times the upper limit of the normal range
(range 39–262 U l1) represents abnormal liver function.45,46 The
changes in our studies were significantly below this limit, which
indicates the normalcy in liver functions.
The serum creatinine level depends on the glomerular
filtration rate (GFR).47 If the creatinine level doubles, the GFR
is considered to have been halved. The transient change in the
levels of creatinine and BUN following administration of magnetic
liposomes was within permissible limits. This indicated the
normal kidney functions and reflected the structural integrity of
the kidney.
In the haematological studies, the levels of haemoglobin,
RBCs, WBCs, platelets, lymphocytes, and neutrophils, mean
cell volume (MCV), packed cell volume (PCV), etc. were estimated.
Haemoglobin is a red coloured globular protein present in the
RBCs and is responsible for transport of oxygen round the body.
An inadequate level of haemoglobin in blood causes weakness,
pallor and sometimes breathlessness. The level of haemoglobin
in blood was normal, which indicated that the animals were not
suffering from any blood haemoglobin related disorder. RBCs are
the most common type of blood cells and their common function
is to transport oxygen within the body. The normal level of RBCs
in the MD1 and MD2 groups indicated that the experimental
animals were not suffering from RBC related disorders. WBCs are
part of body’s immune system and they defend the body from
both infectious diseases and foreign substances. Since the
magnetic liposomes are foreign substances, it was quite natural
to have elevated WBC levels. Platelets also engulf foreign bodies
and kill them by phagocytosis. The level of platelets was
This journal is © The Royal Society of Chemistry 2017

Integrative Biology
expected to increase during the course of experiment. Lympho-
cytes and neutrophils are two different types of WBCs responsible
for body’s immune system. So, it was natural following
administration of magnetic liposomes to have elevated levels
of lymphocytes and neutrophils as they raise antibodies to fight
against foreign substances. MCV and PCV of the blood cells did
not change with administration of magnetic liposomes into
mice; this indicated that the size and shape of RBC were not
affected due to the magnetic liposomes. In short, it can be
concluded from these observations that the magnetic liposomes
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
were found to be fairly biocompatible and harmless to the body.
Reliability of the results of an in vivo experiment largely
depends on the suitability of the experimental model and the
ability of the model system to withstand the applied treatment
protocol with minimum or no intervention in its normal
physiology. Considering the frailty of the experimental models
and their vulnerability to the plausible stress inflicted by the
treatment, we adopted a milder treatment protocol compared
to the ones reported often in the literature. The treatment
parameters such as frequency of dosing (maximum two), shot
and interval of hyperthermia (maximum five shots at 72 h
interval each), and the conditions for hyperthermia adopted
in the study were milder than the ones commonly reported in
the literature.17,18,23,24 These assume significance in extending
better leverage and flexibility to choose a judicious experimental
protocol for an elaborate preclinical and toxicity evaluation of the
formulation. In a recent study, the tumour bearing mice were
subjected to hyperthermia under AMF nine times following
intratumoral injection of magnetic hydroxyapatite suspension
within a period of 15 days23 and the tumour surface temperature
reached 45–46 1C. These parameters seem to be harsh considering
the anatomical and physiological features of the mice. In contrast,
we applied an AMF of 27.9 kA m1 at a frequency of 250 kHz and a
reasonable break of 48 or 72 h was given between two successive
treatments maintained at a lower temperature of 43 1C which is
preferable.
The temperature plot showed the highest gain in temperature
on the 8th day and drop in temperature during subsequent
treatments. During in vivo hyperthermia, the dose of Taxol was
38.4 mg kg1 body weight of the experimental animal. This
38.4 mg kg1 dose value was calculated as mice equivalent dose
based on the adult human (weighing 60 kg) therapeutic dose
of 175 mg m2. The maximum tolerated dose of paclitaxel is
50 mg kg1 body weight.48 The growth of tumour was not
significantly inhibited upon application of Taxol. This showed
that fibrosarcoma is resistant to paclitaxel. This result was in
agreement with earlier investigation where it was confirmed that
the response rate of soft tissue sarcoma due to paclitaxel mono-
therapy was very low, i.e. 12.5%.49 The animals of control and
TX (paclitaxel) groups were sacrificed on the 12th day due to
excessive growth of tumour volume. Combined hyperthermia and
chemotherapy effectively inhibited the tumour growth and
thereby prolonged the effective survival of the animals of PSD
and PDD groups.
We had recorded the body weight of the experimental
animals as an indicator of normal physiological functioning.
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
There was a mild degree of undesirable effects observed in the
MDD and PDD groups as evidenced by the reduction in body
weights of animals until eight days. As treatment progressed,
the body weight of the animals, however, increased and
reached normal levels suggesting that the adverse effect due
to the treatment was transitory.
VSM data were recorded using the tumour and other organs of
sacrificed mice at the end of each experiment. The magnetization
value obtained from the VSM curves of different organs of treated
mice suggested that the maximum amount of MNPs was confined
in tumour tissues. This indicated that MNPs administered through
intratumoral injection did not move to other organs and hence the
adverse effect of MNPs would not be observed in the other organs
of mice. The same conclusion can be drawn from ICP-AES data
also. ICP-AES data suggested that next to the tumour, the spleen
absorbs the highest amount of MNPs per unit dry mass; however,
the amount of MNPs was not sufficient to produce enough
magnetic moment during VSM measurements. The deposition
of MNPs in the spleen may be due to the inherent properties of the
magnetic liposomes formulation as well as their size. Similar
results were reported in earlier reports also.30,31 In the beginning
of the treatment, the MNPs present per unit volume of tumour
were sufficient to raise the temperature for hyperthermia treat-
ment and the treatment retarded the growth rate of tumour,
but the tumours were growing at a slower pace. So, eventually
the MNPs present per unit volume of tumour became very low,
and hence VSM signals received from tumours were weak and
large standard deviations were observed.
In order to perform the ICP-AES experiments the entire
tumour was processed and used. So, the ICP-AES data had less
standard deviation. Both VSM and ICP-AES data demonstrated
that MNPs were intact in the tumours only. There were apparently
no adverse effects due to hyperthermia and chemotherapy on
the vital organs. This result was in congruence with earlier study
reported by Yanase et al.30 This is one of the detailed studies
showing the therapeutic effect of self-controlled hyperthermia
and chemotherapy along with the biodistribution of MNPs.
These novel magnetic liposomes mediated anticancer activities
through self-controlled hyperthermia and release of chemother-
apeutic drug. The results of the study strongly suggest the
potential of the liposomes containing paclitaxel and the biphasic
suspension of LSMO and Fe3O4 nanoparticles in the effective
inhibition of growth of experimental tumours with minimal or
no toxicity implying the worthiness of elaborate therapeutic
evaluation in suitable human xenograft models and detailed
preclinical studies.
The effect of magnetic hyperthermia on survival will be an
important additional end point for determining the success of
the treatment. The primary study end point following diagnosis
of first tumour recurrence and secondary overall survival endpoint
following primary tumour diagnosis were determined in a clinical
study using magnetic iron-oxide nanoparticles combined with
external beam radiotherapy on patients with recurrent glio-
blastoma multiforme.50 In this line, further studies evaluating
graded doses and their effects on survival are planned and will
help in translation of the technology.
Integr. Biol.

Paper
5. Conclusions
We developed a magnetic liposomes formulation composed of
DSPC/cholesterol and containing a biphasic suspension of
LSMO and Fe3O4 nanoparticles for combined self-controlled
hyperthermia and chemotherapy. Biocompatibility of these
magnetic liposomes was studied in vitro and in vivo. In vivo
study showed that no significant changes were observed in the
liver and kidney functions as well as in blood parameters
following intravenous administration of magnetic liposomes
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
up to a concentration of 2 mg of MNPs. This suggests that the
magnetic liposomes may be used for in vivo therapeutic applications.
This is a preliminary study; further elaborate studies are required
before clinical applications.
Therapeutic efficacy of these magnetic liposomes for self-
controlled hyperthermia and drug delivery was evaluated both
in vitro and in vivo. No adverse effects were observed due to
hyperthermia and chemotherapy following intratumoral injection
and maximum intratumoral accumulation of MNPs was achieved
over a period of 22 days. The study suggests the feasibility of
exploring other MNPs apart from iron oxide nanoparticles alone,
along with anticancer drugs to cause increased tumor cytotoxicity,
with a controlled rise of temperature without overheating or any
systemic toxicity.
Acknowledgements
We thank Centre for Research in Nanotechnology and Nano-
sciences for using their instrumentation facilities. The financial
support provided by Department of Information Technology
and Department of Science and Technology is gratefully
acknowledged.
References
1 M. J. Paszek, N. Zahir, K. R. Johnson, J. N. Lakins, G. I.
Rozenberg, A. Gefen, C. A. Reinhart-King, S. S. Margulies,
M. Dembo, D. Boettiger, D. A. Hammer and V. M. Weaver,
Cancer Cell, 2005, 8, 241–254.
2 O. Tredan, C. M. Galmarini, K. Patel and I. F. Tannock,
J. Natl. Cancer Inst., 2007, 99, 1441–1454.
3 A. Bhattacharya, K. Toth, R. Mazurchuk, J. A. Spernyak,
H. K. Slocum, L. Pendyala, R. Azrak, S. Cao, F. A. Durrani
and Y. M. Rustum, Clin. Cancer Res., 2004, 10, 8005–8017.
4 J. A. Kellen, in Reversal of Multidrug Resistance in Cancer, ed.
J. A. Kellen, CRC Press, Boca Raton, 1993, pp. 69–92.
5 S. Rockwell and C. S. Hughes, Cell Proliferation, 1994, 27,
153–163.
6 I. F. Tannock, Cancer Metastasis Rev., 2001, 20, 123–132.
7 M. Gogoi, M. K. Jaiswal, R. Banerjee and D. Bahadur, in
Magnetic Nanoparticles: From Fabrication to Clinical Applications,
ed. N. Thanh, CRC Press, Boca Raton, 2012, pp. 479–498.
8 A. Hervault and N. T. K. Thanh, Nanoscale, 2014, 6, 11553–11573.
9 G. Kong, R. D. Braun and M. W. Dewhirst, Cancer Res., 2001,
61, 3027–3032.
10 B. Thiesen and A. Jordan, Int. J. Hyperthermia, 2008, 24, 467–474.
Integr. Biol.
View Article Online
Integrative Biology
11 F. K. H. van Landeghem, K. Maier-Hauff, A. Jordan, K.-T.
Hoffmann, U. Gneveckow, R. Scholz, B. Thiesen, W. Bruck
and A. von Deimling, Biomaterials, 2009, 30, 52–57.
12 M. Johannsen, U. Gneveckow, K. Taymoorian, B. Thiesen,
N. Waldofner, R. Scholz, K. Jung, A. Jordan, P. Wust and
S. A. Loening, Int. J. Hyperthermia, 2007, 23, 315–323.
13 P. Kulshrestha, M. Gogoi, D. Bahadur and R. Banerjee,
Colloids Surf., B, 2012, 96, 1–7.
14 M. Gogoi, H. D. Sarma, D. Bahadur and R. Banerjee, Nano-
medicine, 2014, 9, 955–970.
15 P. Pradhan, J. Giri, F. Rieken, C. Koch, O. Mykhaylyk,
M. Doblinger, R. Banerjee, D. Bahadur and C. Plank,
J. Controlled Release, 2010, 142, 108–121.
16 A. Ito, K. Tanaka, H. Honda, S. Abe, H. Yamaguchi and
T. Kobayashi, J. Biosci. Bioeng., 2003, 96, 364–369.
17 Y. Shido, Y. Nishida, Y. Suzuki, T. Kobayashi and N. Ishiguro,
J. Bone Jt. Surg., Br. Vol., 2010, 92, 580–585.
18 M. Yoshida, Y. Watanabe, M. Sato, T. Maehara, H. Aono,
T. Naohara, H. Hirazawa, A. Horiuchi, S. Yukumi, K. Sato,
H. Nakagawa, Y. Yamamoto, H. Sugishita and K. Kawachi,
Int. J. Cancer, 2010, 126, 1955–1965.
19 S. A. Shah, M. U. A. Khan, M. Arshad, S. U. Awan, M. U. Hashmi
and N. Ahmad, Colloids Surf., B, 2016, 148, 157–164.
20 L. Willerding, S. Limmer, M. Hossann, A. Zengerle, K. Wachholz,
T. L. M. ten Hagen, G. A. Koning, R. Sroka, L. H. Lindner and
M. Peller, J. Controlled Release, 2016, 222, 47–55.
21 R. Di Corato, G. Béalle, J. Kolosnjaj-Tabi, A. Espinosa,
O. Clément, A. K. A. Silva, C. Ménager and C. Wilhelm,
ACS Nano, 2015, 9, 2904–2916.
22 L. Pradhan, R. Srivastava and D. Bahadur, Acta Biomater.,
2014, 10, 2976–2987.
23 C.-H. Hou, S.-M. Hou, Y.-S. Hsueh, J. Lin, H.-C. Wu and
F.-H. Lin, Biomaterials, 2009, 30, 3956–3960.
24 A. Ito, H. Honda and T. Kobayashi, Cancer Immunol. Immunother.,
2006, 55, 320–328.
25 M. K. Jaiswal, M. Gogoi, H. D. Sarma, R. Banerjee and
D. Bahadur, Biomater. Sci., 2014, 2, 370–380.
26 M. K. Jaiswal, M. De, S. S. Chou, S. Vasavada, R. Bleher, P. V
Prasad, D. Bahadur and V. P. Dravid, ACS Appl. Mater.
Interfaces, 2014, 6, 6237–6247.
27 Q. Yuan, R. Venkatasubramanian, S. Hein and R. D. K. Misra,
Acta Biomater., 2008, 4, 1024–1037.
28 R. Haghniaz, R. D. Umrani and K. M. Paknikar, Int.
J. Nanomed., 2015, 10, 1609–1623.
29 R. Haghniaz, R. D. Umrani and K. M. Paknikar, Int.
J. Nanomed., 2016, 11, 1779–1791.
30 M. Yanase, M. Shinkai, H. Honda and T. Wakabayashi, Jpn.
J. Cancer Res., 1998, 89, 463–469.
31 A. Ito, M. Fujioka, T. Yoshida, K. Wakamatsu, S. Ito, T. Yamashita,
K. Jimbow and H. Honda, Cancer Sci., 2007, 98, 424–430.
32 K. R. Bhayani, S. N. Kale, S. Arora, R. Rajagopal, H. Mamgain,
R. Kaul-Ghanekar, D. C. Kundaliya, S. D. Kulkarni, R. Pasricha,
S. D. Dhole, S. B. Ogale and K. M. Paknikar, Nanotechnology,
2007, 18, 345101.
33 E. Pollert, K. Knı́žek, M. Maryško, P. Kašpar, S. Vasseur and
E. Duguet, J. Magn. Magn. Mater., 2007, 316, 122–125.
This journal is © The Royal Society of Chemistry 2017

Integrative Biology
34 N. K. Prasad, K. Rathinasamy, D. Panda and D. Bahadur,
J. Biomed. Mater. Res., Part B, 2008, 85, 409–416.
35 N. K. Prasad, L. Hardel, E. Duguet and D. Bahadur, J. Magn.
Magn. Mater., 2009, 321, 1490–1492.
36 K. R. Bhayani, J. M. Rajwade and K. M. Paknikar, Nanotechnology,
2013, 24, 15102.
37 N. D. Thorat, S. V Otari, R. M. Patil, V. M. Khot, A. I. Prasad,
R. S. Ningthoujam and S. H. Pawar, Colloids Surf., B, 2013,
111, 264–269.
38 J. Duart-Clemente, M. San-Julián, R. Martı́nez-Monge and
Published on 25 April 2017. Downloaded by Kings College London on 02/06/2017 06:22:48.
S. Martı́n-Algarra, Rev. Esp. Cir. Ortop. Traumatol., 2008, 52,
21–26.
39 M. S. Lemson, H. J. Mud, A. J. Wijnmaalen and R. W. M. Giard,
Breast, 2016, 5, 368–371.
40 R. D. Issels, L. H. Lindner, J. Verweij, P. Wust, P. Reichardt,
B.-C. Schem, S. Abdel-Rahman, S. Daugaard, C. Salat,
C.-M. Wendtner, Z. Vujaskovic, R. Wessalowski, K.-W. Jauch,
H. R. Dürr, F. Ploner, A. Baur-Melnyk, U. Mansmann,
W. Hiddemann, J.-Y. Blay and P. Hohenberger, Lancet
Oncol., 2016, 11, 561–570.
41 H. L. Wong, A. M. Rauth, R. Bendayan and X. Y. Wu, Eur.
J. Pharm. Biopharm., 2007, 65, 300–308.
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
42 M. C. Woodle, Adv. Drug Delivery Rev., 1995, 16, 249–265.
43 A. Sharma and R. M. Straubinger, Pharm. Res., 1994, 11,
889–896.
44 T. K. Jain, M. K. Reddy, M. A. Morales, D. L. Leslie-Pelecky
and V. Labhasetwar, Mol. Pharmaceutics, 2008, 5, 316–327.
45 D. R. Dufour, in Tietz Textbook of Clinical Chemistry and
Molecular Diagnostics, ed. C. A. Burtis, E. R. Ashwood and
D. E. Bruns, Elsevier Science Publishers, St. Louis, MO,
5th edn, 2012, pp. 1637–1694.
46 E. D. Olfert, B. M. Cross and A. A. McWilliam, Guide to the
Care and Use of Experimental Animals, Canadian Council on
Animal Care, Ottawa, 2nd edn, 1993.
47 P. B. Sanghani, Human Anatomy and Physiology, Tata
McGrow Hill Education (P). Ltd, New Delhi, 2012.
48 U. Vanhoefer, S. Cao, A. Harstrick, S. Seeber and Y. M. Rustum,
Ann. Oncol., 1997, 8, 1221–1228.
49 A. Milano, G. Apice, E. Ferrari, F. Fazioli, V. de Rosa, A. S. de
Luna, R. V. Iaffaioli and F. Caponigro, Crit. Rev. Oncol.
Hematol., 2006, 59, 74–84.
50 K. Maier-Hauff, F. Ulrich, D. Nestler, H. Niehoff, P. Wust,
B. Thiesen, H. Orawa, V. Budach and A. Jordan, J. Neuro-
Oncol., 2011, 103, 317–324.
Integr. Biol.