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Journal of Microencapsulation
Micro and Nano Carriers
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/imnc20
Anticancer potential of docetaxel-loaded cobalt
ferrite nanocarrier: an invitro study on MCF-7 and
MDA-MB-231 cell lines
Jnanranjan Panda , Bhabani Sankar Satapathy , Bidisha Mandal ,
Ramkrishna Sen , Biswajit Mukherjee , Ratan Sarkar & Bharati Tudu
To cite this article: Jnanranjan Panda , Bhabani Sankar Satapathy , Bidisha Mandal , Ramkrishna
Sen , Biswajit Mukherjee , Ratan Sarkar & Bharati Tudu (2021) Anticancer potential of docetaxel-
loaded cobalt ferrite nanocarrier: an invitro study on MCF-7 and MDA-MB-231 cell lines, Journal of
Microencapsulation, 38:1, 36-46, DOI: 10.1080/02652048.2020.1842529
To link to this article: https://doi.org/10.1080/02652048.2020.1842529
Published online: 30 Nov 2020.
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Anticancer potential of docetaxel-loaded cobalt ferrite nanocarrier: an
in vitro study on MCF-7 and MDA-MB-231 cell lines
Jnanranjan Panda
a
, Bhabani Sankar Satapathy
b
, Bidisha Mandal
a
, Ramkrishna Sen
c
, Biswajit Mukherjee
c
,
Ratan Sarkar
d
and Bharati Tudu
a
a
Department of Physics, Jadavpur University, Kolkata, India;
b
School of Pharmaceutical Sciences, Siksha ’O’Anusandhan (Deemed to
be University), Bhubaneswar, India;
c
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India;
d
Department of
Physics, Jogesh Chandra Chaudhuri College, Kolkata, India
ABSTRACT
Aim: To develop a biocompatible cobalt ferrite (CF-NP) nanodrug formulation using oleic acid
and poly (D,L-lactide-co-glycolic) acid (PLGA) for the delivery of docetaxel (DTX) specifically to
breast cancer cells.
Methods: The CF-NP were synthesised by hydrothermal method and conjugated with DTX in a
PLGA matrix and were systematically characterised using XRD, FE-SEM, TEM, DLS, FTIR, TGA,
SQUID etc. The drug loading, in vitro drug release, cellular uptake, cytotoxicity were evaluated
and haemolytic effect was studied.
Results: The CF-NP showed good crystallinity with an average particle size of 21nm and ferro-
magnetic nature. The DTX-loaded CF-NP (DCF-NP) showed 8.4% (w/w) drug loading with 81.8%
loading efficiency with a sustained DTX release over time. An effective internalisation and anti-
proliferative efficiency was observed in MCF-7 and MDA-MB-231 breast cancer cells and negli-
gible haemolytic effect.
Conclusion: The DCF-NP can have the potential for the effective delivery of DTX for breast can-
cer treatment.
ARTICLE HISTORY
Received 16 May 2020
Accepted 21 October 2020
KEYWORDS
Cobalt ferrite nanoparticles;
breast cancer; docetaxel;
cytotoxicity;
biocompatibility
1. Introduction
In recent times, magnetic nanoparticles have received
immense attention among the scientific community
owing to their distinct applications in magnetically
assisted wastewater treatment (Mandal et al. 2020),
biosensors (Rocha-Santos 2014), magnetic recording
media (Mohtasebzadeh et al. 2015) along with various
biomedical applications such as magnetic resonance
imaging (Wabler et al. 2014), hyperthermia (Mazario
et al. 2013), and drug delivery (Wang et al. 2012,
Tietze et al. 2015, Jauhar et al. 2016). Among various
types of magnetic nanoparticles, cobalt ferrite based
nanodrug delivery system has emerged as an attract-
ive drug delivery technique for improved cancer treat-
ment (Ansari et al. 2016, Dey et al. 2018). This is
because cobalt ferrite nanoparticles (CF-NP) have the
advantages of excellent biocompatibility, tuneable par-
ticle size, ease of surface functionalization, moderate
saturation magnetisation, large magnetic anisotropy
along with minimal toxicity (Ansari et al. 2016).
Recently, cancer has been identified as the most
fatal disease with an alarming mortality rate across the
globe. Based on the latest report from the
International Agency for Research on Cancer (IARC), in
2018, more than 18.1 million new cancer cases have
been diagnosed all over the world, with around 9.6
million deaths (Bray et al. 2018). Among all cancers,
breast cancer tops the list with the highest morbidity
and mortality counts among the women in western
countries. Even in India, breast cancer has secured 1st
rank among Indian females, with a mortality rate of
nearly 12.7 per 100,000 women (Malvia et al. 2017).
These figures signify the inefficiency of currently avail-
able treatment strategies to tackle the disease.
Multimodal treatment strategies including a combin-
ation of surgery, radiation therapy, chemotherapy
have failed to improve treatment outcomes in meta-
static breast cancer cases. Conventional chemotherapy
has the limitation due to the non-selective distribution
of anticancer drugs between cancer cells and healthy
cells, which in turn produces severe toxic effects
CONTACT Bharati Tudu bharati.tudu@jadavpuruniversity.in Department of Physics, Jadavpur University, Kolkata 700032, India; Ratan Sarkar
dr.ratansarkar@jogeshchaudhuricollege.org Department of Physics, Jogesh Chandra Chaudhuri College, Kolkata 700033, India
ß2020 Informa UK Limited, trading as Taylor & Francis Group
JOURNAL OF MICROENCAPSULATION
2021, VOL. 38, NO. 1, 36–46
https://doi.org/10.1080/02652048.2020.1842529
including bone marrow depression inside the body
(Maji et al. 2014, Pourjavadi et al. 2018). Therefore, the
main challenge of cancer treatment is the site-specific
delivery of toxic anticancer drugs with minimal side-
effect on healthy tissues and organs. In the last few
years, various researches on therapies based on mag-
netic nanoparticles have been carried out as an
improved, alternative approach, where the cytotoxic
anticancer drug(s) loaded in magnetic nanoparticles
can be targeted to the specific tumour site using an
external magnetic field with minimal side-effects
(Wang et al. 2012, Panda et al. 2019).
CF-NP show an inverse spinel structure which is
represented as AB
2
O
4
, where A represents Co and B
represents Fe. In this structure, all the oxygen atoms
are arranged in a cubic, closely packed manner. Here,
Co
2þ
ions are occupied in the octahedral (B) sites,
whereas Fe
3þ
ions are equally distributed in octahe-
dral and tetrahedral sites (Goodarz Naseri et al. 2010,
Ansari et al. 2016). Various synthesis techniques such
as hydrothermal, co-precipitation and micro-emulsion
have been proposed to synthesise cobalt ferrite nano-
particles. Compared to other synthesis routes, the
hydrothermal technique has been found as a simple
and environment-friendly approach for preparing
nanoparticles of uniform size distribution, and, hence,
for our study, this technique has been selected for the
synthesis of cobalt ferrite nanoparticles.
In our study, we have taken docetaxel (DTX) as a
model anticancer drug. Though it is a semisynthetic
derivative of paclitaxel (PTX), it possesses fewer side
effects than PTX. DTX has shown impressive anticancer
efficacy against different types of cancers such as
lung, breast, head/neck, prostate, and brain cancer
(Satapathy et al. 2016, Panda et al. 2019). Though a
wide variety of nanocarrier platforms have been inves-
tigated to enhance the anticancer effect of DTX for
breast cancer therapy, an optimised magnetic-medi-
ated targeted delivery system of DTX for the improved
treatment of breast cancer is yet to be standardised at
the pre-clinical stage. Recent studies over the past few
years have reported the targeted delivery of some
chemotherapeutic drugs like doxorubicin (DOX),
methotrexate (MTX), etc. using different magnetic car-
riers. Dey et al. (2018) have reported the therapeutic
application of CF-NP as an anticancer drug carrier that
can be used for breast cancer treatment . Ansari et al.
(2016) have synthesised cobalt ferrite nanoparticles
and investigated their anticancer potential against the
MCF-7 cells . Mohapatra et al. (2011) developed folic
acid decorated superparamagnetic cobalt ferrite nano-
carriers for cervical cancer treatment. In another report
by Wang et al. (2018) drug loading and release study
of L-cysteine coated DOX-loaded cobalt ferrite nano-
particles was carried out. Similarly, Fan et al. (2018)
proposed silica-coated cobalt ferrite nanoparticles
could be used as a targeted nanocarrier for delivery of
DOX to the Hela cell line. But, the anticancer efficacy
and cellular uptake of DTX-loaded cobalt ferrite nano-
particles on both human epidermal growth factor
receptor HER (þve) and HER (–ve) breast cancer cell
lines have not been reported yet.
To fill up this gap, this study was aimed to develop
an oleic acid stabilised cobalt ferrite based nanocarrier
for breast cancer cell specific delivery of the DTX in a
sustained manner. The formulation was fabricated in the
smaller nano-size range (30 nm) to achieve effective
cancer cell permeation. In the preparation steps, few
parameters were optimised to obtain the formulation
having desired physicochemical properties. The as-syn-
thesised cobalt ferrite nanoparticles were investigated by
various characterisation techniques viz. X-ray diffraction
(XRD), Superconducting Quantum Interference Device
(SQUID), field emission electron microscope (FE-SEM),
Transmission electron microscope (TEM), Dynamic light
scattering (DLS), Fourier transform infra-red spectra (FTIR)
and Thermogravimetric analysis (TGA). Different formula-
tions with DTX-loaded cobalt ferrite nanoparticles were
prepared by varying the ratio of DTX drug and polymer.
The best formulation was selected and investigated for
in vitro drug loading capacity and drug release study.
Further, in vitro cytotoxic study and internalisation effi-
ciency of the selected formulation were investigated in
both MCF-7 (human breast adenocarcinoma) and MDA-
MB-231 (epithelial human breast cancer) cell lines. The
MCF-7 cells were preferentially selected as HER þve
model cell line of human breast cancer, whereas MDA-
MB-231 were taken as an HER ve cell line model. The
objective of taking both MCF-7 and MDA-MB-231 cell
lines was to test the efficacy of the developed formula-
tion in both HER þve and HER ve cell line models to
get a concrete idea of the potency of the formulation
for breast cancer therapy (Shirshahi et al. 2013,Moses
et al. 2016, Azizi et al. 2017). In addition, the safety of
the developed nanodrug formulation for the intravenous
application was also studied, which is again very crucial
for its clinical use.
2. Materials and methods
2.1. Materials
Ferric chloride (FeCl
3
), cobalt chloride(CoCl
2
), ammonia
aqueous (25% v/v), oleic acid, poly lactic-co-glycolic
acid (PLGA, ratio 85:15), polyethylene glycol (PEG;
JOURNAL OF MICROENCAPSULATION 37
M.W.¼6000), ethanol, dichloromethane (DCM), fluores-
cein isothiocyanate (FITC), Dulbecco’s Modified Eagle’s
Medium (DMEM), foetal bovine serum, Dimethyl
sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium bromide (MTT), etc. were
obtained from Sigma-Aldrich Co. (Bangalore, India).
Docetaxel (DTX) was received as a gift sample from
Cipla laboratories, Pvt. Ltd., Goa, India. MCF-7 and
MDA-MB-231 cell lines were obtained from the
National Centre for Cell Sciences, Pune, India.
2.2. Synthesis of oleic acid stabilised cobalt ferrite
nanoparticles (CF-NP)
For the preparation of oleic acid stabilised cobalt fer-
rite nanoparticles (CF-NP), the technique reported by
Fan et al. has been followed with some modifications
(Fan et al.2018). In brief, CoCl
2
and FeCl
3
with molar
ratio (1:2) were mixed in a solution of ethanol and
double distilled water (1:1 v/v) and then stirred for
30 min. To this mixture, 5 ml of dilute ammonia solu-
tion (25% v/v) was mixed in a dropwise manner with
constant stirring. After that, 0.2 M oleic acid was added
to the above solution mixture as a capping agent.
With continuous stirring for another 1 h, the mixture
was transferred to a 100 ml Teflon-lined stainless-steel
autoclave and heated at 180 C for 16 h in an electric
oven. After attaining the normal room temperature,
the obtained particles were repeatedly washed with
ethanol and hexane. Finally, the resultant particles
were obtained by the magnetic separation technique.
The sample was then dried at 60 C for 10 h for its fur-
ther characterisation.
2.3. Development of docetaxel-loaded cobalt
ferrite nanoparticles
The formulation of docetaxel-loaded CF-NP (DCF-NP)
was developed using multiple emulsion solvent evap-
oration technique following our previously reported
work with minor modification in incubation period,
centrifugation time and concentration of stock solu-
tion (Ling et al.2011, Panda et al.2019). Briefly,
100 mg of PLGA was dissolved in 10 ml of DCM. Then
30 mg of CF-NP was added followed by incubation for
2 h. A weighed amount of DTX was then dissolved in
2 ml DCM and added to the above solution followed
by incubation of 1 h. The drug-loaded PLGA-CF-NP
formed after secondary emulsion was collected by
centrifugation for 2 h. To conjugate PEG over the sur-
face of the drug-loaded nanoparticles, 2 ml of PLGA-
CF-NP was mixed with 10 ml PEG (2% w/v) and incu-
bated for 4 h with constant stirring. After that, the
DCF-NP was obtained by ultrafiltration followed by
lyophilisation at 40 C for 12 h. Here, five different
formulations of DCF-NP were prepared keeping a fixed
amount of polymer (100 mg) and varying the drug
amount. These formulations were then optimised
based on their drug loading efficiency (details in sec-
tion 2.5). A comparison of the formulation compos-
ition, % drug loading and loading efficiency of these
formulations are shown in Table 1. The formulation
having a 1:2 mixture of drug and polymer (DCF-NP
1
)
exhibited excellent drug loading performance with a
drug loading of 8.4% (w/w) and a drug loading effi-
ciency of 81.8%. Thus, the optimised formulation DCF-
NP
1
has been selected for further characterisation and
evaluation of its drug delivery potential, which is
denoted as DCF-NP hereafter. For the development of
fluorescence-labelled DCF-NP, the method adopted in
our previous work was followed (Shaw et al.2017,
Panda et al.2019).
2.4. Characterisation
The crystallinity and phase purity of the experimentally
synthesised CF-NP were investigated by a powder XRD
(Bruker D8 Advanced Diffractometer) using Cu K
a
(k¼1.54059 Å) radiation in the range of 2h¼20–80
with a step size of 0.0199. The surface morphology,
internal structure, and particle size of the CF-NP were
analysed by FESEM (FEI, INSPECT F50, 10 kV) and TEM
Table 1. The composition, % drug loading, and % drug loading efficiency of different formulations of drug-loaded CF-NP; and
the average particle size, polydispersity index (PDI), and Zeta potential of the optimised formulation.
Formulation name
Amount of
drug (mg)
Drug: Polymer
ratio (w/w)
% (w/w)
drug loading
a
% drug loading
efficiency
a
Optimised formulation: DCF-NP
1
Average Particle
diameter
(d nm)
a
PDI
a
Zeta
potential
a
(mV)
DCF-NP
0
100 1:1 7.65 ± 0.05 74.4 ± 0.05 156.8 ± 18.8 0.283 ± 0.07 20.5 ± 4.12
DCF-NP
1
50 1:2 8.40 ± 0.03 81.8 ± 0.04
DCF-NP
2
20 1:5 6.24 ± 0.05 66.45 ± 0.06
DCF-NP
3
14.3 1:7 5.1 ± 0.06 58.27 ± 0.08
DCF-NP
4
10 1:10 4.3 ± 1.1 45.39 ± 1.2
The optimised formulation has been denoted as DCF-NP.
a
Data presented as mean± standard deviation (SD) (n¼3).
38 J. PANDA ET AL.
(JEOL-2010, 120 kV). The TGA study was conducted by a
Mettler Toledo TG-DTA 85 thermal analyser in an N
2
atmosphere. For this, the CF-NP and DCF-NP were
heated up to a temperature of 800 C (10 C/min) in an
alumina crucible. Mean particle diameter (Z-average),
surface charge, and polydispersity index (PDI) of DCF-
NP were investigated by Zetasizer (DLS-nano ZS,
Zetasizer, Malvern Instrument Ltd, Malvern, UK). Fourier
transform infra-red spectroscopy (FTIR) of the free drug,
CF-NP and DCF-NP were carried out in the fingerprint
region (400 cm
1
–4000 cm
1
) on an FTIR spectrometer
(Magna-IR 750, Series II, Nicolet Instruments, Madison,
WI). The magnetic measurements for both CF-NP and
DCF-NP were investigated using a SQUID magnetom-
eter (Quantum Design, MPMS XL-7).
2.5. Drug loading and in-vitro drug release study
The percent loading of DTX drug and its release from
the CF-NP formulations were determined according to
the methods reported in previous works (Satapathy
et al.2016, Panda et al.2019). For the determination
of % DTX loading and loading efficiency, the formula-
tions were dispersed in 1 ml of DCM, sonicated (0.5 h),
magnetically separated, vortexed, and followed by
centrifugation at 15,000 rpm. The % drug loading and
loading capacity were determined in triplicate using a
high-performance liquid chromatography system
(HPLC, Prominence LC-20A, Shimadzu, Japan).
For the drug release study phosphate buffer saline
(PBS), pH 7.4 was used as a release medium to create
a simulated physiological condition (Paul et al.2018,
Panda et al.2019). Briefly, 5 mg of lyophilised DCF-NP
(lyophilised at 40 C, 12 h) was dispersed in 1 ml PBS
and was taken inside a cellophane membrane (0.45
micron, Whatmann, UK) based dialysis pouch. The
whole assembly was suspended in PBS and placed on
an orbital shaker bath. About 1 ml of sample solution
was withdrawn at regular intervals, centrifuged and
analysed by the HPLC system. For the HPLC analysis, a
reverse phase C-18 column (150 4.6 mm i.d. pore
size 5 lm, GL Science Inc, Tokyo, Japan) was used.
Acetonitrile-HPLC water-formic acid mixture in the
ratio 60:40:0.05 (v/v/v) was used as a mobile phase.
The flow rate of the mobile phase was kept at 0.4 ml
per minute. About 20 ml of sample solution was
injected into the HPLC column for the study.
2.6. Assessment of in vitro anticancer potential &
internalisation efficiency
The in vitro cytotoxic action of the optimised formula-
tion (DCF-NP) was evaluated in triplicate on both
MCF-7 and MDA-MB-231 cell lines by MTT assay. The
result was compared with that of free DTX. Briefly, the
cells were cultured in DMEM culture medium and
were seeded in a 96-well culture plate (104 cells per
well). The seed culture of cells was incubated over-
night in a bio oxygen demand incubator, followed by
treatment with free DTX suspension, CF-NP, and DCF-
NP (at varying concentrations) for 24 h. After incuba-
tion, the supernatant of each cell was discarded. Cells
were incubated with MTT solution for 4 h, followed by
the addition of dimethyl sulfoxide. A microplate reader
(multimode plate reader, SpectraMax M5; Molecular
Devices, San Diego, CA) was used to measure the %
cell viability at 540 nm (Ansari et al.2016, Pourjavadi
et al.2018).
The internalisation efficiency of the optimised DCF-
NP in MCF-7 and MDA-MB-231 cells was investigated
using confocal laser scanning microscopy system as
well as by flow cytometry. For the qualitative estima-
tion of internalisation efficiency, FITC was incorporated
into the formulation of DCF-NP. For fluorescence
imaging, a similar procedure was followed as reported
in our previous work (Panda et al.2019).
For quantitative analysis, cellular uptake of FITC-
labelled DCF-NP in both MCF-7 and MDA-MB-231 cells
was investigated by flow cytometry (FACS Canto II
TM
cell sorter, BD Biosciences) using FACS Diva software
(BD Biosciences) (Gaonkar et al.2017, Kazi et al.2019).
Both the cells were cultured in the above-mentioned
culture medium in a 35 mm dish. After 24 h incuba-
tion, cells were incubated with fresh medium contai-
ning1 ml of FITC-DCF-NP (50 lg/ml) for 15 and 30 min,
respectively, followed by washing with cold PBS, tryp-
sinized, centrifugation at 1500 rpm. Finally, the cells
were suspended in PBS and analysed by flow cytome-
try to find out the increase in fluorescence intensity as
compared to untreated control cells.
2.7. Haemolysis assay
Haemolysis assay has been taken as a measure of
membrane disruption and safety profile for the nano-
formulation which was intended to be administered
via i.v. route. Here, male Sprague-Dawley rats were
used for the collection of blood samples. The blood
samples were collected in pre-heparinized tubes, cen-
trifuged at 1000 rpm for 5 min followed by washing
the sedimented red blood cells (RBC) with PBS (pH
7.4). Following this, the RBC suspension (190 ll) was
taken in 96 well plates, followed by treatment with
different concentrations of DCF-NP and CF-NP (Lin and
Haynes 2009). After the experimental time period, the
JOURNAL OF MICROENCAPSULATION 39
unlysed RBCs were centrifuged. After centrifugation,
the pellets were discarded and the supernatant was
taken for measuring the absorbance at 570 nm. Finally,
the haemolysis percentage was calculated as per the
previously reported experiment (Lin and Haynes 2009,
Zamani et al.2019).
3. Results and discussion
For exploiting nanoparticles in therapeutic applica-
tions, the size, and proper surface coating with
organic or inorganic material are very much essential
to enhance their blood pharmacokinetic profile and
bioavailability (Gupta and Wells 2004, Tietze et al.
2015). To achieve effective cancer cell penetration and
enhanced tumour inhibition, particle size should be in
the range 10–200 nm (Gupta and Wells 2004). In add-
ition, good drug loading efficiency along with a pro-
longed drug release profile, significant cytotoxicity,
cell-internalisation and excellent bio-compatibility of
the drug-loaded CF-NP (DCF-NP) is very important for
its possible clinical application. Thus, determination of
the size, structural, morphological, chemical and mag-
netic property of CF-NP and evaluation of the drug
loading efficiency, cytotoxicity and haemolysis of the
DCF-NP has been carried out, the results of which are
discussed below.
3.1. XRD analysis
The XRD pattern of the synthesised nanoparticles is
presented in Figure 1(a), which confirms the formation
of CF-NP. All peaks of the CF-NP are well matched with
those of the pure cubic spinel ferrite phase of CoFe
2
O
4
(JCPDS card no. 22–1086) (Ansari et al. 2016,Wuet al.
2016). The observed characteristic peaks at 2hvalues of
30.4,35.6
,43.3
, 53.8,57.3
,62.7
, and 74.7corres-
pond to the reflections from (220), (311), (400), (422),
(511), (440), and (533) crystal planes of CoFe
2
O
4
(Ansari
et al. 2016,Wuet al. 2016). The sharp and intense
peaks observed define the crystalline nature of the
nanoparticles. The average crystallite size of the CF-NP
as calculated from the Debye-Scherer formula consider-
ing the strongest XRD peak of (311) is about 18 nm
(Patil et al. 2014,Ansariet al. 2016).
3.2. Structural and morphological analysis
The TEM image shown in Figure 1(b) indicates that
the nanoparticles formed were of spherical shape with
uniform size distribution and negligible agglomeration.
The particle size distribution of the CF-NP as obtained
from TEM analysis as shown in Figure 2(a) depicts an
average particle size of 21 nm which is consistent with
the crystallite size calculated from the XRD pattern
and with the FE-SEM analysis of the nanoparticles
(inset of Figure 1(b)).
3.3. Size distribution and f-potential
The surface charge of the drug-loaded nanoparticles is
an important parameter that determines the colloidal
stability, recognition by the reticuloendothelial system,
and cell internalisation of the nanocarriers (Pourjavadi
et al. 2018). The particle size distribution and polydis-
persity index (PDI) of DCF-NP as measured by DLS and
Figure 1. (a) XRD pattern of the synthesised cobalt ferrite nanoparticles (CF-NP) showing characteristic peaks of cubic spinel
cobalt ferrite. (b) TEM image of the CF-NP. Inset shows the SEM image of the same.
40 J. PANDA ET AL.
its corresponding Zeta potential as measured by zeta-
sizer are presented in Table 1. The average hydro-
dynamic diameter of the DCF-NP formulation was
obtained to be 156.8 ± 18.8 nm with a polydispersity
index (PDI) of 0.283 ± 0.07. The polymer coating which
was done on the surface of CF-NP cannot be differen-
tiated by DLS and hence it measures the solvated par-
ticles dispersed in water. The larger particle size of
DCF-NP may be due to the contribution of PEG and
PLGA on the surface of CF-NP. The zeta potential of
DCF-NP was obtained to be 20.5 ± 4.12 mV. The zeta
potential of CF-NP could not be measured as they are
only dispersible in an organic solvent. A smooth sur-
face morphology, spherical shape, uniform size distri-
bution, optimum surface charge, etc. are considered
ideal for nanoparticle formulations for in vivo applica-
tions, which has been successfully achieved in our
case and can be attributed to our selected specific
fabrication parameters.
3.4. FTIR analysis
A comparison of the FTIR spectra of the bare CF-NP,
free DTX drug, and DCF-NP formulation is presented
in Figure 2(b). FTIR spectra for CF-NP clearly show the
presence of oleic acid on the CF-NP surface which is
in good agreement with other studies (Ling et al.
2011, Patil et al. 2014). The vibration due to the
stretching of Fe–O at 574 cm
1
can be assigned to the
characteristic band for CF-NP (Patil et al. 2014). The
presence of carboxylate symmetric stretching and
asymmetric stretching band at 1435 cm
1
and
1570 cm
1
affirmed the conjugation of oleic acid on
the surface of CF-NP (Salunkhe et al. 2016). The bands
near 3480 cm
1
can be attributed to the O-H stretch-
ing of the carboxyl group or H
2
O adsorbed on the sur-
face of DCF-NP. The sharp peak corresponding to
carbonyl stretching was observed near 1721 cm
1
, jus-
tifying the presence of PLGA in the DCF-NP
Figure 2. (a) Histogram showing the particle size distribution of CF-NP as obtained from the TEM image. (b) FTIR spectra of DTX,
CF-NP, and DCF-NP. (c) TGA curves for CF-NP and DCF-NP. (d) In vitro drug release profile of DTX from DCF-NP in PBS (pH 7.4).
Data shown as the mean ± SD (n¼3).
JOURNAL OF MICROENCAPSULATION 41
formulation (Ling et al. 2011, Panda et al. 2019). The
N–H stretching vibration for DCF-NP was noticed at
3420 cm
1
. The presence of bands at 2960 cm
1
and
1450 cm
1
are attributed to the methane stretching
vibration of DCF-NP (Mandal et al. 2018). It is also
observed that the characteristic peaks of the DTX drug
at 780 cm
1
(aromatic mono-substituted benzene) and
1245 cm
1
(for esters) are present in the DCF-NP for-
mulation, suggesting successful incorporation of the
drug into the formulation (Paul et al. 2018).
3.5. TGA analysis
TGA analysis was done for the confirmation of an
organic coating on the surface of CF-NP and DCF-NP
which is shown in Figure 2(c). It can be seen that the
weight loss took place slightly up to 150 C for both
the CF-NP and DCF-NP. This can be ascribed to the
evaporation of volatile solvents and adsorbed water
present on the nanoparticle surface. The weight loss
of CF-NP at about 240 C can be attributed to the deg-
radation of oleic acid from the surface of CF-NP.
Above 400 C, the curve becomes nearly flat which
shows good agreement with patterns reported earlier
for oleic acid-coated CF-NP (Jain et al. 2005, Salunkhe
et al. 2016). This result suggests that around 16.5 (wt
%) of the oleic acid was adsorbed on the surface of
CF-NP. The thermal degradation pattern of DCF-NP is
unidentical to that of CF-NP which demonstrates the
presence of other polymeric shells on its surface. The
weight loss observed within the temperature range of
230–600 C can be attributed to the decomposition of
PLGA and PEG coating along with the docetaxel drug.
The thermogravimetric curve of DCF-NP above 600 C
demonstrates that the residual weight of this formula-
tion was around 69.24% of CF-NP which corroborates
well with earlier reports (Ling et al. 2011). The TGA
analysis indicates that most of the CF-NP have been
encapsulated into the PLGA polymeric matrix, and
hence are suitable for applying these formulations as
drug carriers.
3.6. Magnetic property analysis
The field-dependent magnetisation (M–H) curves of
the synthesised CF-NP and DCF-NP formulation were
recorded at different temperatures in the field range
of 5toþ5 T which is shown in Figure 3(a,b), respect-
ively. The saturation magnetisation (M
s
)values of the
CF-NP were found to be 43.58 and 47.78 emu/g at
300 K and 200 K respectively. This M
s
value of CF-NP
obtained at room temperature is less than the bulk
CoFe
2
O
4
(80 emu/g) suggesting the nano-size effect
of these particles (Ansari et al. 2016). A significantly
smaller value of saturation magnetisation was noticed
for DCF-NP with a value of 13.74 and 15.13 emu/g at
300 K and 150 K, respectively, which is due to the
existence of the polymeric shell on the surface of
DCF-NP. Also, both CF-NP and DCF-NP exhibit small
hysteresis loops at 300 K, which indicates the ferro-
magnetic nature of the nanoparticles. At lower tem-
peratures, clear hysteresis loops with increased values
of magnetisation and coercive field were observed for
both CF-NP and DCF-NP. This increment can be attrib-
uted to the absence of thermal effects which results in
the growth of magnetic anisotropy hindering the
orientation of the magnetic moments (Dey
et al. 2017).
3.7. Percent drug loading and in vitro drug
release study
A suitable amount of drug loading efficiency along
with a prolonged drug release profile plays a vital role
Figure 3. M–H curves of CF-NP and DCF-NP measured at (a) 300 K and (b) 200 K and 150 K, respectively.
42 J. PANDA ET AL.
in in vivo applications of nano-drug formulations. The
DCF-NP showed a significantly good amount of drug
loading of 8.4% (w/w). The drug loading efficiency
was found to be 81.8%, which again can be consid-
ered admissible for magnetic nano-drug formulations.
A sustained release of DTX from the nanoparticle for-
mulation is regarded as a crucial factor, which has a
direct influence on the dosing frequency and related
pharmacodynamic effect of the drug inside the body.
In our case, the in vitro drug release study presented
in Figure 2(d) showed a sustained drug release pattern
of DTX for the DCF-NP in PBS (pH 7.4) over a 17 days
experimental drug release period. Though initially a
burst release was noticed for the first few hours after
that DTX was found to be released in a sustained
manner from the DCF-NP over the rest of the experi-
mental time period.
3.8. Studies on in vitro breast cancer cell
line system
3.8.1. In vitro cytotoxicity study
Evaluation of the cytotoxicity of the as-synthesised NP
to cancer cells is very crucial for its drug delivery
applications. The cytotoxicity of CF-NP and DCF-NP
were compared with that of free DTX drug using MTT
assay against both MCF-7 and MDA-MB-231 breast
cancer cell lines which is shown in Figure 4(a,b). CF-
NP without DTX showed less toxicity than DCF-NP
towards MCF-7 cells. More than 70% cell viability was
observed in the case of CF-NP, whereas DCF-NP
showed very high toxicity than both free DTX and CF-
NP at the same equivalent concentration. The IC
50
value was found to be 14.05 ± 1.2 mg/ml for DCF-NP
for MCF-7 cells. CF-NP had negligible toxicity on the
MDA-MB-231 cells as compared to its toxicity on MCF-
7 cells. However, higher toxicity was noticed when
MDA-MB-231 cells were treated with DCF-NP and DTX.
The toxicity of DCF-NP was higher than free DTX for
the MDA-MB-231 cells, alike MCF-7 cells. The IC
50
value was found to be 15.58 ± 1.2 mg/ml for DCF-NP.
The overall results for both the HER þve and HER –ve
cell lines suggest that DCF-NP could be potentially
applicable for breast cancer treatment.
3.8.2. In vitro cellular uptake study:
The DCF-NP was also investigated for its internalisa-
tion efficiency in the tested cell lines. Confocal laser
scanning microscopy images are shown in Figure
5(a,b) showed that FITC labelled DCF-NP (100 mg/ml
concentration) were preferentially taken up by both
MCF-7 and MDA-MB-231 cells over a 0.5 h incubation
period. The fluorescent DCF-NP was found to be accu-
mulated mostly throughout the cytoplasm but were
unable to penetrate the nucleus as clearly seen in con-
focal images of MCF-7 cells. The nanosize of the DCF-
NP might be responsible for such prominent
internalisation.
Quantitative cellular uptake of fluorescent DCF-NP
for both MCF-7 and MDA-MB-231 cells was also esti-
mated by flow cytometry analysis which is shown in
Figure 6(a,b). A time-dependent enhancement in the
median fluorescence intensity at a post-incubation
time period of 15 and 30 min was observed. As per
earlier reports, uptake of nanoparticles by the cancer
cells could be attributed to the endocytosis mechan-
ism rather than passive diffusion (Gaonkar et al. 2017,
Kazi et al. 2019). Thus, it is presumed that DCF-NP
entered the cells through endocytosis. A higher
uptake intensity was observed at 30 min, which dem-
onstrates the time-dependent uptake of DCF-NP.
Higher intracellular accumulation of the DCF-NP
Figure 4. Comparison of % cell viability upon treatment with DCF-NP, DTX and CF-NP on (a) MCF-7 and (b) MDA-MB-231 cells.
Data shown as the mean ± SD (n¼3).
JOURNAL OF MICROENCAPSULATION 43
Figure 5. Confocal laser microscopy images of (a) MCF-7 (b) MDA-MB-231 cells treated with FITC labelled DCF-NP (100 mg/mL)
over 0.5 h incubation. The same cells were analysed with differential interference contrast (DIC) for comparison. Fluorescent DCF-
NP can be seen accumulated throughout the cytoplasm in MCF-7 cells and throughout the cell in MDA-MB-231 cells (scale
bar: 10 mm).
Figure 6. Cellular uptake of FITC labelled DCF-NP formulation observed by flow cytometric studies and the corresponding flow
cytometric distribution of FITC labelled DCF-NP in KB (DI) for the (a) MCF-7 and (b) MDA-MB-231 cell lines, at a different post-
incubation time period of 15 min and 30 min.
44 J. PANDA ET AL.
formulation in the tested cells signifies its enhanced
therapeutic effect.
3.9. Haemolysis study
Excellent blood compatibility i.e. very low haemolytic
effect is very much essential for in vivo application of
the magnetic nanodrug formulations. Here, the
haemolytic assay analysis was carried out in human
RBCs to estimate the blood compatibility of the as-
synthesised CF-NP and DCF-NP at different concentra-
tions (2.5–40 mg/ml) which is shown in Figure 7. The
absorbance values obtained for RBCs treated with CF-
NP exhibited no impressive haemolysis as compared
to the control. The maximum haemolytic activity range
was observed up to 3.1% for CF-NP and 7.2% for
DCF-NP, respectively. Thus, we may conclude that the
developed DCF-NP is well compatible with blood and
could be a promising choice for future clinical applica-
tions (Ansari et al. 2016, Zamani et al. 2019).
4. Conclusion
A biocompatible cobalt ferrite nanocarrier formulation
has been successfully developed for sustained and
cell-specific delivery of docetaxel to breast cancer cells
which showed extensive intracellular distribution on
both MCF-7 and MDA-MB-231 cell lines. A good
response of the formulation in terms of its antiprolifer-
ative nature against both HER þve and HER –ve
human breast cancer cells signify its potency for clin-
ical application. Further, negligible toxicity towards
blood cells reflects its human-safe nature. All these
results suggest the potential application of DCF-NP for
the delivery of DTX for breast cancer treatment.
However, further in vivo testing on suitable animal
models is warranted for its successful clinical
translation.
Acknowledgement
J. P. acknowledges UGC for providing UGC-BSR fellowship
(Ref. No.- P-1/RS/86/16). Science & Engineering Research
Board (SERB), Govt. of India is thankfully acknowledged for
research funding (project no. ECR/2017/003250 and EEQ/
2016/000617). The UPE and DSA program of UGC and the
PURSE program of DST, Government of India are also
acknowledged. Further Indian Institute of Chemical Biology
(IICB) and UGC-DAE CSR, Kolkata are also being acknowl-
edged for providing instrumentation facilities.
Disclosure statement
No potential conflict of interest was reported by
the author(s).
Funding
This work was supported by the Science and Engineering
Research Board (SERB), DST, Govt. of India.
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