Artificial Cells, Nanomedicine, and Biotechnology

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Green synthesis of magnetic Fe3O4 nanoparticles

using Couroupita guianensis Aubl. fruit extract for
their antibacterial and cytotoxicity activities

G. Sathishkumar, V. Logeshwaran, S. Sarathbabu, Pradeep K. Jha, M. Jeyaraj,

C. Rajkuberan, N. Senthilkumar & S. Sivaramakrishnan

To cite this article: G. Sathishkumar, V. Logeshwaran, S. Sarathbabu, Pradeep K. Jha, M. Jeyaraj,

C. Rajkuberan, N. Senthilkumar & S. Sivaramakrishnan (2018) Green synthesis of magnetic
Fe3O4 nanoparticles using Couroupita�guianensis Aubl. fruit extract for their antibacterial and
cytotoxicity activities, Artificial Cells, Nanomedicine, and Biotechnology, 46:3, 589-598, DOI:
10.1080/21691401.2017.1332635

To link to this article: https://doi.org/10.1080/21691401.2017.1332635

Published online: 29 May 2017.

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2018
VOL. 46, NO. 3, 589–598
https://doi.org/10.1080/21691401.2017.1332635

Green synthesis of magnetic Fe3O4 nanoparticles using Couroupita guianensis

Aubl. fruit extract for their antibacterial and cytotoxicity activities
Sathishkumar G.a, Logeshwaran V.a, Sarathbabu S.b, Pradeep K. Jhac, Jeyaraj M.d, Rajkuberan C.a,
Senthilkumar N.b and Sivaramakrishnan S.a
a
Department of Biotechnology, Bharathidasan University, Tiruchirappalli, India; bDepartment of Biotechnology, Mizoram University, Aizawl,
India; cSchool of Medical Science and Technology, Indian Institute of Technology, Kharagpur, India; dNational Centre for Nanosciences and
Nanotechnology, University of Madras, Chennai, India

ABSTRACT ARTICLE HISTORY

In the present study, a sustainable green chemistry approach was established to fabricate magnetic Received 17 February 2017
Fe3O4 nanoparticles (Fe3O4NPs) using the aqueous fruit extract of edible C. guianensis (CGFE). Revised 11 May 2017
Synthesized NPs were further confirmed with different high-throughput characterization techniques Accepted 16 May 2017
such as UV–visible spectroscopy, FT-IR, XPS, DLS and zeta potential analysis. Additionally, XRD, AFM,
HRTEM and SQUID VSM demonstrate the generation of crystalline CGFe3O4NPs with mean diameter of KEYWORDS
17 ± 10 nm. Interestingly, CGFe3O4NPs exhibit a stupendous bactericidal action against different human Phytosynthesis; C.
pathogens which depicts its antimicrobial value. A significant dose-dependent cytotoxic effect of guianensis; magnetic Fe3O4;
CGFe3O4NPs was noticed against treated human hepatocellular carcinoma cells (HepG2). SQUID VSM; bactericidal;
liver cancer

Introduction A very wide range of biological resources from both prokar-

Magnetic nanoparticles (MNPs) are the foremost nanoscale
materials with many practical applications such as ferrofluids,
biosensors, catalysts, separation processes and environmental
remediation [1]. It plays a central role in wide range of bio-
medical applications such as cell therapy [2], drug delivery [3],
photothermal effect [4], tissue engineering [5], regenerative
medicine [6], hyperthermia [7], diagnosis [8] and imaging [9].
Especially, Fe3O4 (ferric oxide) belonging to the class of metal
oxides was very well known for its unanimous photo catalytic
and photo-oxidizing capacities. Due to their unique physio-
chemical properties and capability to facilitate the cellular
and molecular level of biological interactions, MNPs are being
considered as the successful contrast agents of magnetic res-
onance imaging (MRI) in theranostics nanomedicine [10].
Conventional physical and chemical methodologies for
Fe3O4 fabrication were co-precipitation, thermal decompos-
ition, microemulsion, sonochemical synthesis, sol–gel reac-
tions, hydrothermal, decomposition of organometallic
precursors and polyol methods [11]. With the concern of
environmental contaminations and energy consumption, a
sustainable biosynthesis involving either unicellular or multi-
cellular organisms has been deliberated as an eco-friendly
and safe route for nanomaterial preparation [12].
Nature has devised with various processes for the synthe-
sis of nano, micro-length scaled inorganic materials that were
considered as a new and largely unexplored area of research.
yotes and eukaryotes have been used for the synthesis of dif-
ferent nanoparticles such as gold (Au), silver (Ag), copper
(Cu), platinum (Pt), palladium (Pd), iron (Fe), zinc (Zn) and
titanium (Ti) [13,14]. While microbial based protocols suffered
with microbial culture maintenance, tedious procedure, mul-
tiple purification and elongated down-stream processing [15].
Different plant entities were successfully employed to synthe-
size nanostructures of various metals and metal oxides.
Owing to its availability, rapid synthesis, and easy scaling-up
process, phyto-mediated nanoparticles generation was con-
sidered as the most appropriate route. The bioreduction of
metal was achieved by a combination of biomolecules exist-
ing in plant extract, resulting in the formation of metal NPs
has also been extensively reviewed [16].
The main rationale for nano-based therapeutics develop-
ment was to find an effective drug candidate in order to over-
come the persistence of deadliest diseases and prevalence of
multiple drug resistance (MDR) [17]. In particular, cancer con-
tinues to be the worldwide killer resulting in major mortality
and morbidity, as per 2014 statistics reported by World Health
Organization (WHO), more than 10 million new cancer cases
arise, results in six million deaths per year and incidence rate
will get two-fold increase by the end of 2020 [18].
Multifunctional nanomaterials deliver several imaging
options to localize the diseased tissues, also they help in the
delivery of therapeutic agents at desired sites without

CONTACT S. Sivaramakrishnan sivaramakrishnan123@yahoo.com Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu

620024, India
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
590 G. SATHISHKUMAR ET AL.
causing adverse effects to normal tissues [19]. In this prime
time, development of theranostic agents based on the “all-in-
one” approach shows great potential in the emerging field of
personalized nanomedicine. It enables early diagnosis and
monitoring of an individual patient and to deliver anticancer
agents over an extended period for enhanced therapeutic
efficacy [20].
A new paradigm for cancer treatment was established
by means of nanocrystallization of bioactive principles, and
the enhanced permeation and retention effect (EPR) of
tumour vessels, which improve the bioavailability of natural
anticancer drugs. It also minimizes the adverse dose limiting
systemic adverse side effects of the drugs [21]. Magnetic NPs
can serve as an efficient vehicle system to reach the tumour
site as well as the tumour microenvironment, which is also
called as the stromal tumour component. Hence, the tumour
microenvironment controls tumour progression and the
spread of cancer in the body. In addition, magnetic NPs can
augment the contrast in tumour MRI [22].
The plant material exploited to synthesize magnetic
Fe3O4NPs was C. guianensis, commonly known as cannonball
tree belonging to Lecythidaceae family. Different extracts
from the leaf and fruit of this tree are known to possess
numerous medicinal values such as antibiotic, antifungal,
antiseptic, anticancer, antioxidant, antihelmintic, wound heal-
ing and analgesic qualities [23]. Juice made from the leaves
was used to cure skin diseases and malaria [24]. It also holds
antiulcer, antifertility, antibiofilm, antipyretic, immunomodula-
tory, antiarthritic, antistress, antidiarrheal, ovicidal, larvicidal
activities and neuropharmacological action in nature [25].
Hence, in the present study, we have approached for the
green synthesis of magnetic Fe3O4NPs using C. guianensis
aqueous fruit extract towards bactericidal and cancer thera-
nostic applications.
Materials and methods
Materials
Ferric chloride hexahydrate (FeCl3
6H2O) was purchased from
Merck Millipore and 25% ammonia solution was purchased
from Sigma Aldrich (Alwar, India). Foetal bovine serum (FBS),
Dulbecco’s Modified Eagle Medium (DMEM), antibiotics,
trypsin–EDTA, phosphate buffer saline, nutrient broth, agar-
agar, streptomycin sulphate, sterile paper discs and other
chemicals were purchased from Himedia Laboratories
(Mumbai, India). HepG2 cell line was procured from National
Centre for Cell Sciences (NCCS) (Pune, India). Bacterial patho-
gens used in this study: S. aureus MTCC 96, E. coli MTCC
2939, S. typhi MTCC 3917, K. penumoniae MTCC 530 were
obtained from Microbial Type Culture and Collection (MTCC)
(Chandigarh, India). Fresh and healthy cannonball fruits were
collected from Gokarnesvarar Temple (Pudukkottai, India).
Synthesis of CGFe3O4NPs
To synthesize CGFe3O4NPs, the collected cannonball fruit
samples were scraped to remove the outer peel, chopped
into small pieces and dried under shade condition for
8–10 days. Then, the dried fruit samples were ground into
fine powder using commercial electrical stainless steel kitchen
blender and the extract was prepared by simple decoction
method. Briefly, 20 g of autoclaved fruit powder was mixed in
400 ml of sterile distilled water and boiled in 60  C for 20 min
to get maximum extraction. Subsequently, the extracts were
filtered through Whatman no. 1 filter paper to remove
the debris and stored at 4  C for the further studies. The
CGFe3O4NPs were prepared via co-precipitation method, for
that 0.1 M FeCl3
6H2O was continuously mixed with CGFE
extract in a 1:1 ratio at room temperature for 30 min. Then,
the pH was adjusted to 10.5 with 25% ammonia solution,
and heated at 80  C for 30 min under constant stirring with a
magnetic stirrer in N2 atmosphere. Finally, the precipitate was
centrifuged at 20,000 rpm for 20 min at 10  C (Eppendorf)
then the pellet was re-suspended in N2 purged water and
washed repeatedly for three times. The final pellet was
dispersed in 5 ml of MilliQ water, frozen at 80  C and lyophi-
lized for two days to get fine magnetic powder for further
studies.
Instrumentation
Generation of CGFe3O4NPs by reducing the respective metal
ion Fe3þsolution with aqueous CGFE was easily confirmed
using the Perkin-Elmer Lamda-45 UV–Vis spectroscopy. The
absorption spectra of metal ion precursor FeCl3
6H2O and
CGFe3O4NPs were measured using a UV–Vis spectrophotom-
eter in 300–700 nm range. FTIR measurement was carried out
to identify the potential biomolecules in CGFE responsible for
reduction and stabilization of CGFe3O4NPs. The samples were
pelletized using KBr powder and dried, then the transmit-
tance was recorded using JASCO 460 plus FTIR spectrometer
in wavelength range between 4000 and 400 cm1. Further,
the X-ray diffraction pattern of CGFe3O4NPs was acquired
using PAN analytical X pert PRO Model in conditions at 40 kV
and 30 mA in Cu, Ka radiation and mean particles size (L) of
CGFe3O4NPs was calculated using following Debye–Scherrer’s
equation.
L ¼ 0 : 9k=b cosh
where k is the wavelength of the X-ray, b is the full width and
half maximum and h is Bragg's angle. The surface oxidation
state and presence of elemental composition were studied
with XPS analysis using an omicron ESCA spectrometer with
monochromatized Al Ka radiation. Dynamic light-scattering
measurement was obtained to analyse the size dispersity of
synthesized CGFe3O4NPs using Nano-ZS90 analyser apparatus
at 25  C and started 2 min after the cuvette was placed in the
DLS apparatus to allow the temperature to equilibrate. In add-
ition, the zeta potential analysis of CGFe3O4NPs was carried
out using a Malvern Zetasizer (Malvern, UK). Further, the pre-
cise size, shape and distribution pattern of CGFe3O4NPs and
EDAX were obtained using a (TECNAI-10) TEM operated at an
accelerating voltage of 100 kVe. A VSM-BS2–11 Tesla was used
to study the magnetic properties of synthesized CGFe3O4NPs.
The field dependence of magnetization was recorded at 10 K
and 300 K under circulate magnetic field ranging between
2 and –2 T.

In vitro anticancer activity
Cell lines and culture medium
The HepG2 cells were grown in DMEM supplemented with 10%
inactivated FBS, penicillin (100 IU/ml), streptomycin (100 lg/ml)
and amphotericin B (5 lg/ml) in humidified atmosphere of 5%
CO2 at 37  C until confluent. The cells were dissociated with
trypsin–EDTA solution (0.2% trypsin, 0.02% EDTA, 0.05% glu-
cose in PBS). The stock cultures were grown in 25 cm2 culture
flasks, and all experiments were carried out in 96 microtiter
plates (Tarsons India Pvt. Ltd., Kolkata, India).
Cell viability assay
The cytotoxic effect of CGFe3O4NPs and crude CGFE was
tested by MTT (Sigma, St. Louis, MO) assay on HepG2 cells
[26]. For that, the cells were seeded in 96-well plates at a
density of 1  104 cells per well. After incubation for 20–24 h,
the cells with 70–80% confluency were treated with
CGFe3O4NPs and crude CGFE at different concentrations (1, 5,
10, 25, 50, 75, 100, 150, 200 and 250 lg ml–1) and incubated
for 24 h. Then, 20 ll of MTT (5 mg/ml) solution was added to
cells per well, and the plate was further kept in 5% CO2 at
37  C for another 4 h. After that, the medium was removed,
and 150 ml of DMSO was added to the cells. The plate was
gently shaken for 15 min to dissolve the formazan crystals
generated by proliferating cells, and the measurement was
performed using a Spectramax M2 Microplate Reader
(Molecular Diagnostic, Inc., Tucson, AZ) at 570 nm with refer-
ence filter as 655 nm. Relative viability was calculated with
non-treated cells as 100% control. The results were expressed
as mean values (±SD) of six repeats.
Measurement of cytomorphological changes in MCF-7
HepG2 cells were pre-treated with different concentration of
synthesized CGFe3O4NPs and crude CGFE for 24 h at 37  C in
5% CO2 atmosphere. After the incubation of cells, the gross
morphological changes in the cells were observed under an
inverted phase contrast microscope (Nikon, Tokyo, Japan).
Fluorescent imaging
Acridine orange (AO) and ethidium bromide (EtBr) staining
was used to detect the morphological features of apoptotic
and necrotic cell death. The IC50 concentration of CGFe3O4NPs
and crude CGFE was treated with HepG2 cells (1  105 cells/
ml). After 24 h, the cells were washed with ice-cold 1 PBS
and stained with a mixture of AO (100 mg/ml) and EtBr
(100 mg/ml) at room temperature for 5 min. After washing
with PBS, the stained cells were observed under a fluorescence
microscope (Model: OLYMPUS IX70, Olympus Optical Co., Ltd.,
Tokyo, Japan) at 100 magnification via a particular filter at
510–590 nm and photographs were acquired.
Haemocompatibility assay
To assess the haemocompatibility of synthesized
CGFe3O4NPs, fresh blood was collected from healthy volun-
teers in sterile lithium heparin vacutainers. The samples were
individually suspended in 10 mM HEPES buffered saline and
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 591
red blood cells (RBCs) were separated by Ficoll density gradi-
ent centrifugation (1500 rpm for 10 min at 4  C). Collected
RBCs were further diluted in 20 mM HEPES buffered saline
(pH 7.4) to form a 5% v/v solution. Then, the RBC suspension
was added to HEPES saline, 1% Triton X-100 and samples
with different concentrations and incubated at 37  C for
60 min. All the samples were prepared in triplicate, after
being slightly vortexed the suspension was incubated under
static conditions for 4 h at 37  C. Then, all the samples were
centrifuged (Heraeus table top centrifuge 5805R, Hanau,
Germany) at 12,000 rpm at 4  C and the supernatants were
transferred to a 96-well plate. The haemolytic activity was
determined by measuring the absorbance at 570 nm (Biorad
microplate reader model 550, Tokyo, Japan). Control samples
of 0% lysis (in HEPES buffer) and 100% lysis (in 1% Triton
X-100) were employed in the experiment. The percentage of
haemolysis was calculated as follows:
Hemolysis % ¼ ½ðsample absorbance  negative controlÞ
=ðpositive control  negative controlÞ  100:
This study was approved by the Institutional Ethics
Committee (IEC) of Bharathidasan University (Ref no.
DM/2014/ 101/54). A consent form with all the mandatory
information was collected from the healthy volunteers for
their participation in this study, and the form was submitted
to IEC, Bharathidasan University.
Antibacterial activity
The antimicrobial effect of synthesized CGFe3O4NPs and crude
CGFE was evaluated against different bacterial human patho-
gens such as S. aureus MTCC 96, E. coli MTCC 2939, S. typhi
MTCC 3917 and K. penumoniae MTCC 530 by disc
diffusion method [27]. Briefly, the bacterial strains were sub-
cultured in nutrient broth for 24 h at 30  C. Each strain was
swabbed into the separate nutrient agar plates using sterile
cotton swabs. Using sterile micropipette, different volumes (25,
50 and 75 lg/ml) of CGFe3O4NPs and crude CGFE were loaded
onto sterile discs and allowed to dry. The sample loaded discs
and standard antibiotic discs (streptomycin 1 mg/ml) were
impregnated in the nutrient agar medium. In this study, the
doses were fixed based on the preliminary data obtained from
the earlier studies in our laboratory. After 24 h incubation at
37  C, the different levels of zone of inhibition were measured.
Results and discussion
The essential phytochemicals such as terpenoids, flavones,
ketones, aldehydes, amides and carboxylic acids are mainly
responsible for the instant direct reduction of metallic ions
and produce nano-sized particles [28]. In this study, we
have developed a new facile route to fabricate magnetic
CGFe3O4NPs functionalized with active constituents of
C. guianensis for therapeutic applications.
Synthesis of CGFe3O4NPs
After the addition of CGFE into precursor FeCl3 solution,
formation of CGFe3O4NPs was noticed with a change in the
592 G. SATHISHKUMAR ET AL.
colour of reaction medium (Figure 1(a)). Synthesized NPs
exhibit light red to dark brown colour whereas the precursor
FeCl3 was golden yellow in colour. Water-soluble fractions
such as phenols and phenolic acids of CGFE mainly act as
reducing and surface functionalization agents. The possible
reduction mechanism of CGFe3O4NPs formation can be eluci-
dated by comparison with the related mechanism through
reduction of FeCl3 using bioactive phyto-molecules [29].
Based on this mechanism, the hydroxyl and amine groups of
bioactive molecules in plant extracts tend to bind with Fe3þ
and form ferric hydroxide, which is partially reduced by other
bioactive materials to produce Fe3O4 particles.
Characterization of CGFe3O4NPs
UV–visible spectrum of synthesized CGFe3O4NPs produces a
strong absorbance in the visible region at around
250–350 nm (Figure 1(b)), confirms that the particles were sta-
ble and well dispersed in the solution. This result also clearly
depicts that the hydroxyl group containing plant biomaterials
are in strong coordination with transition metals. Our results
highly corroborate with Zhang et al. [30] where folate-conju-
gated polymer coated supermagnetic iron oxide nanopar-
ticles have shown the absorbance values in visible region.
FTIR spectroscopic analysis was used to study the
interactions metal ions (Fe) with major functional groups of
CGFE biocompounds as well as the functionalization of
CGFe3O4NPs. As shown in Figure 1(c), for CGFE, a broad char-
acteristic peak from 3600 to 3100 cm–1 could be assigned to
Figure 1. Synthesis of CGFe3O4NPs (a). Formation of dark brown color in tube 3
indicates the generation of CGFe3O4NPs (b). UV–visible spectroscopic analysis of
CGFe3O4NPs produces the absorbance spectra around 250–300 nm whereas
FeCl3 generates no absorbance spectra (c). FTIR spectrum of CGFE (bottom) and
CGFe3O4NPs (top) specifies the functional groups active bio constituents respon-
sible for reduction and stabilization.
the O–H or N–H stretching vibration arising from hydroxyl
groups of polyphenols and some aromatic heterocyclic
organic compounds [31]. A band at 1640 cm–1 attributes the
presence of carbonyl groups C¼O (1610–1780 cm–1) and
existence of C–H was confirmed by the stretching vibrations
at 2925 cm–1. FTIR spectrum of synthesized CGFe3O4NPs
shows variation in the intensities of transmittance due to the
interaction of metal ions with CGFE biocompounds, which
leads to the reduction and stabilization of the Fe3O4 nanopar-
ticles [32]. The intensities of transmittance at 3421, 2925,
1626, 1407, 1234 and 1067 cm–1 were increased dramatically
for Fe3O4 nanoparticles. Interestingly, a new band at 590 and
622 cm–1 attributes the intrinsic stretching vibrations of the
metal at a tetrahedral site [33]. In addition, the transmittance
located at 1067 cm–1 represents the presence of C–O stretch-
ing in the region of 1300 and 1000 cm–1 which might be due
to the existence of covalent linking of ether or ester groups
with NPs [34]. Consequently, the FTIR results indicate the vital
role of plant bioactive metabolites such as polyphenols, pro-
teins and reducing sugars in reduction and stabilization of
CGFe3O4NPs.
XPS spectra of CGFe3O4NPs prepared via bioreduction
method show distinct spectra for O1s, C1s and N1s region at
529.6, 283.8 and 398.5 eV, respectively (Figure 2(d)), which
represents the existence of different biomoieties onto the sur-
face of Fe3O4 modified with CGFE. The binding energy values
for the main peak maxima Fe 2p3/2 and 2p1/2 were found at
711.1 and 724.5 eV, respectively (Figure 2(a)). Our results are
in good agreement with the values reported for Fe3O4 in the
literature [35]. The main C1s peak at 283.8 eV (Figure 2(b))
can be assigned to the carbon atoms within phenyl and car-
bonyl groups residues of the polyphenolic constituents. The
O1s core level (Figure 2(c)) decomposed into three chemically
distinct components at 529.8 eV denotes the existence of
carboxylate groups. The N1s core level centred at 398.5
attributes the amine groups present in the indole derivatives
and protein moieties. From these results, we speculate that
the Fe3þ ions are in strong coordination with water-soluble
heterocyclic metabolites of CGFE. During nucleation, these
active bioconstituents could be preferentially adsorbed onto
Figure 2. XPS analysis of CGFe3O4NPs (a) Fe 2p, (b) O1s, (c) C1s and (d) survey
scan.

the surface, also it controls the nucleation position through
special confinement [36].
The crystalline structure of CGFe3O4NPs was characterized
by XRD. As shown in Figure 3(a), the interplanar distances for
biosynthesized CGFe3O4NPs were noticed at 2h ¼ 30.08 ,
35.54 , 43.07 , 53.4357 , 56.95 and 62.54 , corresponding to
Figure 3. XRD pattern of synthesized CGFe3O4NPs: (a) diffraction planes of
CGFe3O4NPs denote the cubic spinel structure and (b) zeta potential analysis of
CGFe3O4NPs.
Figure 4. AFM images show the topography and size distribution of synthesized CGFe3O4NPs (a) 2D-micrographs, (b) 3D-micrographs, (c) 2D-roughness, (d) 3D-
roughness and (e) size distribution.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 593
each Bragg reflection (220), (311), (400), (422), (511) and (440)
can be well indexed as the inverse cubic spinel structure of
magnetite Fe3O4 in accordance with Joint Committee on
Powder Diffraction Standards (JCPDS File No.: 75-1610). As
per Scherrer’s equation, D¼kk/B cos h, the average crystallite
size was calculated to be 7.2 nm. Similar results were
obtained by Silva et al. [37] for fucan polysaccharides coated
magnetite MNPs.
The stability of CGFE-stabilized Fe3O4 colloidal solution has
been investigated based on the zeta potential (f), which is a
measure of electrostatic potential on the surface of nanopar-
ticles, and it is associated with the electrophoretic mobility of
colloidal suspension. The zeta potential of CGFe3O4NPs was
measured in the growth medium as 26.0 mV and the surface
of CGFe3O4NPs was negatively charged (Figure 3(b)). Due to
the low surface charge, Fe nanoparticles get easily aggregated
and become unstable, the synthesized CGFe3O4NPs exhibit a
large negative zeta value (26.0 mV) mainly because of the
surface coating with plant biomolecules which generates a
repulsive forces between nanoparticles [38].
Surface topography of synthesized CGFe3O4NPs was
studied with AFM images. Unlike the electron microscopic
analysis, which provide a two-dimensional projection or a
two-dimensional image of a sample, AFM provides a three-
dimensional surface profile (Figure 4(a–d)). Although, the lat-
eral dimensions are influenced by probe shape, the height
measurements can provide the height of nanoparticles with a
high degree of accuracy and precision. As shown in the histo-
gram of AFM (Figure 4(e)), the size distribution of magnetic
CGFe3O4NPs was between 7 and 77 nm. Our data highly
match with the previous report by Banerji et al. (2011) [39]
594 G. SATHISHKUMAR ET AL.
where Fe3O4NPs conjugated with 5-fluorouracil show highly
stable, oval shaped Fe3O4 with nearly 10 nm diameter. It was
also observed that the free iron oxide nanostructures are
prone to quick agglomeration whereas the 5-FU coated NPs
are stable to coagulation because of their lack of free reactive
surface for agglomeration [39].
The TEM micrographs show spherical and polydispersed
NPs with sizes ranging between 7 and 80 nm; the mean size
was found to be 17 ± 10 nm. The HRTEM micrograph shows
the fine configuration of the synthesized CGFe3O4NPs. A par-
ticle size distribution histogram based on the size of 50 par-
ticles was measured from the TEM image as shown in Figure
5(a). The mean particle size was 17 nm with the standard
deviation of 10, it was quite close to the calculated crystal-
lite size of 7 nm, suggesting that the majority of the
observed spherical nanoparticles might be single crystals
[40]. Besides, Figure 5(b) exposes the EDX spectrum of
CGFe3O4NPs, a strong signal with the highest percentage at
6.2 keV region was noticed for Fe indicating the purity and
stability of synthesized NPs. It was also revealed that the
existence of iron and oxygen confirmed the formation of
Fe3O4 nanoparticles, and a typical optical absorption peak at
B 0.5 keV due to surface plasmon resonance was observed.
The presence of Cu was attributed to the carbon coated
copper grid used for sample preparation. According to many
[41] earlier studies, our results clearly confirm the reduction
and formation of highly stable, surface modified spherical
Fe3O4NPs, via direct reduction, using C. guianensis fruit
extract.
Figure 5. HRTEM measurements of synthesized CGFe3O4NPs: (a) micrographs display well dispersed spherical CGFe3O4NPs with an average size of (b) EDAX analysis
of CGFe3O4NPs.
Interestingly, the magnetic properties of the NPs were not
damaged by the plant biomolecules, in contrast they protect
them from intraparticle interaction and make the properties
more stable against the environment. Magnetic parameters
like saturation magnetization (MS), coercivity (HC) and
remanence magnetization (MR) have been evaluated. At
room temperature, the synthesized CGFe3O4NPs exhibit
superparamagnetism with negligible hysteresis loop (Figure
6), coercivity (HC) of 48 Oe and MR of 0.1 emu g1, and MS of
0.11 emu g1, respectively. Our result highly corroborates
with the previous report [42] where pristine Fe3O4 exhibits
superparamagnetic behaviour at room temperature, it was
also hypothesized that the low MS value could be due to
crystalline defects and alignment of magnetic moment in
nanocrystals. On the contrary, at low temperature, synthe-
sized CGFe3O4NPs show transitional behaviour from superpar-
amagnetic to ferromagnetic with increased symmetric
hysteresis loop. The increased magnetic behaviour and trans-
lation of superparamagnetic to ferromagnetic was mainly
because of the long-range magnetic dipole–dipole interaction
between the particles at low temperature [10].
In vitro anticancer activity
MTT assay was carried out to assess the cytotoxic ability of
CGFE and CGFe3O4NPs on HepG2 cells and to identify the IC50
values at 24 h. The present findings revealed that both CGFE
and CGFe3O4NPs significantly inhibited the growth of HepG2

cells in a dose-dependent manner. The IC50 values of CGFE and
CGFe3O4NPs were 120 and 44.51 lg ml1 for a 24 h treatment
period, respectively (Figure 7(a)). Cell viability assay clarifies
that the toxicity of NPs mainly depends on their surface morph-
ology and size. Taken together, polyol adsorbed Fe3O4NPs sur-
face may offer a synergistic cytotoxicity effect against the
treated human hepatocellular carcinoma cells (HepG2).
As described in many earlier studies, nanoscale materials
were easily internalized by cells through either fluid phase
pinocytosis or clathrin-mediated endocytosis [43]. Also, it is
now well established that the enhanced permeation retention
(EPR), leaky vasculature and poor lymphatic drainage of
cancer cells easily let the intravenously injected NP systems
extravasate and preferentially accumulate in the tumour tis-
sue [44]. Isatin (1H-indole-2,3-dione), an endogenous com-
pound isolated from the fruits of C. guianensis Aubl.
possesses a wide range of biological activities such as anxio-
genic, sedative, anticonvulsant activities and also act as a
potent antagonist agent [45]. Derivatives of isatin are known
to have cytotoxicity against human carcinoma cell lines [46].
We found that the active constituents of CGFE were being
functionalized with Fe3O4NPs, which was confirmed by FT-IR
and XPS analysis.
Figure 6. SQUID VSM magnetization curves of CGFe3O4NPs at different tem-
perature (10 K and 300 K).
Figure 7. In vitro anticancer activity of CGFE and CGFe3O4NPs against human hepatocellular carcinoma cells (HepG2) cells: (a) cell viability assay, (b) phase contrast
images show the gross cytomorphological changes and AO-EB staining displays the CGFe3O4NPs induced apoptotic effect in treated HepG2 cells (A-Control, B-CGFE
and C-CGFe3O4NPs) and (c) haemocompatibility assay of CGFE and CGFe3O4NPs.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 595
The first and most readily noticeable effect following
exposure of cells to toxic materials was the alteration in cell
shape or morphology in a monolayer culture. Microscopic
observations of treated cells showed distinct morphological
changes indicating unhealthy cells, whereas the control
appeared normal. Diverse morphological alterations were
observed in CGFE and CGFe3O4NPs treated HepG2 cells, how-
ever no such effects were seen in untreated control cells. It
was also shown that the morphological variations observed
such as loss of membrane integrity, inhibition of cell growth,
cytoplasmic condensation and cell clumping (Figure 7(b))
results indicate that the CGFE and CGFe3O4NPs treated cells
undergo cell death, whereas the non-treated cells were
active.
Initially CGFE and CGFe3O4NPs induced apoptosis was
noticed with the morphological changes in the cell shape
and chromatin condensation. The ability of CGFE and
CGFe3O4NPs to induce apoptosis was determined by AO/EtBr
staining method. Fluorescence microscopic observations
showed viable (light green), early apoptotic (bright green
fluorescence and condensed chromatin), late apoptotic cells
(orange fluorescence) and nonviable cells (red coloured fluor-
escence) (Figure 7(b)). Additionally, we also observed that
CGFE and CGFe3O4NPs treated HepG2 cells showed different
characteristic features like condensed nuclei, membrane bleb-
bing and apoptotic bodies of nuclear morphology whereas
the untreated control cells remain intact. The above results
strongly support that the formulated CGFe3O4NPs trigger
HepG2 cell death via caspase–cascade mediated mitochon-
drial dysfunction and apoptosis [47].
Haemocompatibility assay
Moreover, the haemocompatibility assay of synthesized
CGFe3O4NPs and CGFE was evaluated upon calculating the
damage to the human RBCs. The results showed that the
CGFe3O4NPs exhibit comparatively lower red haemoglobin
release than crude CFGE (Figure 7(c)). As per the result,
596 G. SATHISHKUMAR ET AL.
Figure 8. Bactericidal effect of CGFe3O4NPs: (a) disc diffusion assay shows clear zone of inhibition in dose-dependent manner against gram negative: (1) E. coli
MTCC 1687, (2) S. typhi MTCC 3917, (3) K. pneumonia MTCC 530 and gram positive and (4) S. aureus MTCC 96(A- Positive control, B,C & D-25,50 &75µg/ml of
CGFe3O4NPs) (b) Zone of inhibition of synthesized CGFe3O4NPs against tested bacterial pathogens.
compared to the positive control (Triton-1X) haemolytic activ-
ity of NPs was less considerable, implying its safe nature in
chemotherapeutic application. The mechanisms of direct
haemolytic activity for different toxic agents were found to
be non-specific. Our results demonstrated that the active bio-
compounds capped on the surface of CGFe3O4NPs largely
strengthen its biocompatibility. Many earlier studies have
proved that the surface passivation of nanomaterials with
active biomolecules will improve their biocompatibility [48].
Eventually, this can be easily used for different pharmaco-
logical applications, including drug release. According to
International Organization for Standardization/Technical
Report 7406, for bio-based materials the haemolytic permis-
sible level was fixed as less than 5% [49]. The exposure of
phenol functionalized nanomaterials shows meager level of
haemolysis, revealing their biocompatibility and suitability for
clinical trials.
Antibacterial activity
The bacterial culture plates were impregnated with
CGFe3O4NPs loaded discs showing different levels of zone of
inhibition in dose-dependent manner (Figure 8(a,b)).
CGFe3O4NPs intrinsically exhibit highest level zone of inhib-
ition against gram negative E. coli MTCC2939, S. typhi
MTCC3917 and K. penumoniae MTCC 530 than gram positive
S. aureus MTCC 96. This difference can be attributed to differ-
ences in cell wall structure inherent in gram-negative and
gram-positive bacteria. Gram-positive and gram-negative
bacteria have similar internal, but very different external
structures. Gram-positive bacterial cells possess thick peptido-
glycan layer that contains teichoic and lipoteichoic acids
whereas gram-negative bacteria have thin peptidoglycan
layer and an outer membrane that contains lipopolysacchar-
ide, phospholipids and proteins [50].
This gives a clear view on the bactericidal effect of
synthesized CGFe3O4NPs which was totally relying on cell
wall thickness, morphology of the cell envelope and resist-
ance of outer membrane to the ROS produced by the metal
ions. However, the antimicrobial activity of nanoparticles
was explained by several mechanisms such as cell membrane
damage, generation of reactive oxygen species, release of
toxic metals and DNA damage. When these nanoparticles
enter the bacterial cell and interact with sulphur containing
proteins of cytoplasm, ribosomes and phosphorus containing
DNA, they disrupt the respiratory chain and cell division [51].
Internalization may result in either cell rupture and
exocytosis or intercalation with DNA/inactivation of key
enzymes, thereby, causing cell damage. Further studies are
required to understand the exact mode of action of these
nanomaterials and the mechanism of growth inhibition in
bacterial cells [52].
Conclusions
In conclusion, we have successfully developed a facile, green
chemistry and an economically viable route to fabricate
multifunctional magnetic Fe3O4 nanoparticles. The fabricated
nanomaterials were well-characterized with different exclusive
instrumentation, which demonstrated the generation of
highly stable, crystalline, spherical-shaped magnetic Fe3O4 in
co-ordination with functional groups of the active biocom-
pounds. Furthermore, in vitro anticancer studies show that
the synthesized CGFe3O4NPs induce significantly enhanced
cytotoxicity effect at a minimal dosage of 44.5 lg ml1 com-
paratively with CGFE 120 lg ml1 against HepG2 cells. In
comparison with crude CGFE, the synthesized CGFe3O4NPs
possess a stupendous bactericidal effect against different bac-
terial human pathogens. Last but not least, the synthesized
CGFe3O4NPs showed excellent magnetic properties and bio-
compatibility which let them to explore for MRI and their
serum stability encourages further in vivo preclinical studies.
Overall, the magnetic CGFe3O4NPs could be a prominent
multifunctional nanomaterial for concurrent theranostics,
drug delivery and image guided real time therapies to treat
human cancer and other infectious diseases.
Disclosure statement
No potential conflict of interest was reported by the author(s).

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