Materials Research Bulletin 46 (2011) 901904

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Materials Research Bulletin

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Preparation and characterization of nanobiocomposites containing iron

nanoparticles prepared from blood and coated with chitosan and gelatin
M. Chamundeeswari a, V. Senthil b, M. Kanagavel c, S.M. Chandramohan d, T.P. Sastry e,*
a
St.Josephs College of Engineering, Sholinganallur, Chennai 600 119, India
b
Gemini Scans, Chennai 600 029, India
c
St.Isabel Hospital, Mylapore, Chennai 600 004, India
d
Department of Gastroenterology, Madras Medical College, Chennai 600 003, India
e
Bio-products Lab, Central Leather Research Institute, Adyar, Chennai 600 020, India

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

Article history: In this study, we report the preparation of magnetic iron nanoparticles (INPs) from goat blood using
Received 13 July 2010 incineration method. FT-IR and XRD studies have conrmed that the prepared nanoparticles were INPs.
Received in revised form 30 November 2010 These INPs were coated with a mixture of chitosan and gelatin to prepare INPCG nanobiocomposite and
Accepted 14 February 2011
the TEM picture of these composite particles has shown an average particle size of 80300 nm. MRI scan
Available online 18 February 2011
exhibited magnetic property and VSM studies revealed a magnetic saturation of 18.97 emu/g. This may
be used as a MRI contrast agent to enhance cellular imaging and as magnetic nanocarrier for targeted
Keywords:
delivery of drugs in the diagnosis and treatment of cancer.
A. Composites
A. Nanostructures
2011 Elsevier Ltd. All rights reserved.
C. Electron microscopy
C. X-ray diffraction
D. Magnetic properties

1. Introduction charge density, non-toxicity and mucoadhesion [27]. It also

Nanoparticles demonstrate unique properties and are useful in
various elds especially in therapeutic applications [1]. For the past
two decades magnetic nanoparticles (MNPs) such as hematite (a-
Fe2O3), maghemite (g-Fe2O3) and magnetite (Fe3O4) nd wide
biomedical applications [2,3] such as MRI contrast enhancement,
cellular imaging and cancer diagnosis [47]. These MNPs are used as
nanosensors for the assessment of antimicrobial susceptibility
through magnetic relaxation [8], for targeted gene delivery [9], as
drug delivery matrices [10] and as virus magnet hybrid nanoparticle
for targeted infection [11]. Super paramagnetic iron oxide nanopar-
ticles are biocompatible and selectively taken up by living cells [12].
Iron nanoparticles particularly maghemite (g-Fe2O3) is syn-
thesized using electrochemical deposition [13], hydrothermal
synthesis [14], co-precipitation [15], emulsication [16]/micro-
emulsion [17,18] and thermal decomposition methods [19]. An
extensive literature survey on preparation of iron oxide nano-
particles clearly indicates that the precursors are mainly inorganic
sources such as ferric chloride (FeCl2 and FeCl3) [3,4,810,2024],
ferric nitrate [25] and ferrous sulfate [26]. Chitosan is a
biodegradable natural polymer with a great potential for
pharmaceutical applications due to its biocompatibility, high
possesses special properties such as crossing the bloodbrain
barrier [28], haemostatic agent, fat attractor and further
supporting its use in eld bandages due to its natural antibacterial
activity [29]. It nds extensive applications due to its low cost and
large-scale availability [30].
Several authors have used inorganic sources as the starting
materials for the preparation of iron nanoparticles. In this article
we report for the rst time about the usage of animal blood as the
source for synthesizing the iron nanoparticles. Thus prepared
nanoparticles were coated with a mixture of chitosan and gelatin
as these two natural materials contain different functional groups;
chitosan contains amino groups (NH2) and gelatin contains amino
groups (NH2), carboxylic groups (COOH) and hydroxyl groups (
OH). The purpose of adding gelatin to iron nanoparticles is to
investigate whether peptides or/and antibodies could also be
coupled to chitosan coated iron nanoparticles. Further the chitosan
coated iron nanoparticles may also act as potential drug carrier for
therapeutics that contains COOH as their functional groups.
2. Materials and methods
2.1. Materials

* Corresponding author. Tel.: +91 44 24911386; fax: +91 44 24911589. Chitosan was purchased from SigmaAldrich, St. Louis, MO,
E-mail addresses: sastrytp@hotmail.com, sastry56@gmail.com (T.P. Sastry). USA; gelatin was supplied by Maruthi Gel Company, Tamil Nadu,

0025-5408/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.materresbull.2011.02.021
902 M. Chamundeeswari et al. / Materials Research Bulletin 46 (2011) 901904
India; goat blood was collected from nearby municipal slaughter
house; all other chemicals used were of analytical grade.
2.2. Preparation of INPs from goat blood
1 l of goat blood was collected and mechanically stirred using a
glass rod for 15 min continuously to isolate the brin. The
debrinated blood was centrifuged at 10,000 rpm (7155  g) for
20 min; the supernatant (serum portion) was discarded and the
Red blood cells (RBC) collected at bottom of the tube was removed,
washed with water for 10 times and stored at 4 8C. The RBC was
incinerated in a silica crucible using a mufe furnace at 800 8C for
2 h. After cooling the residue (INPs) was collected, dissolved in 5 N
HCl to get Fe as FeCl2 and the undissolved material is settled at the
bottom was discarded. The FeCl2 solution was treated with
ammonium hydroxide and heated at 200 8C for 2 h to get Fe2O3
INPs (150 g of wet weight of RBC gives 50 mg of ash which contains
500 mg of Fe, this is quantied using energy dispersive X-ray
spectroscopy (EDX)). These INPs were stored in a glass container
till further use.
2.3. Preparation of chitosan solution
0.5 g of chitosan was dissolved in 100 ml of 0.3 N acetic acid and
denoted as C.
2.4. Preparation of gelatin solution
1 g of gelatin was dissolved in 100 ml of distilled water at 55 8C
until it dissolves and denoted as G
2.5. Preparation of CG
10 ml of C was mixed with 10 ml of G and stirred continuously
for 15 min and the solution was denoted as CG.
2.6. Preparation of nanobiocomposites using INPCG
50 mg (500 mg Fe) INPs was treated with 500 ml of CG solution
and mixed using a vortex mixer for about 15 min and the pH of
solution was raised to 7 in order to allow the CG to precipitate on to
INPs and allowed to settle for 30 min. The precipitate was then
separated by centrifuging at 10,000 rpm for 10 min. The superna-
tant was discarded and the precipitate was washed thrice with
500 ml of distilled water and dried at 37 8C (INPCG). The INPCG
was dissolved in 1 ml of mild acidic solution (0.5 N HCl) to form
INPCG suspension.
2.7. Characterization
The FT-IR spectra of the prepared samples were recorded on a
Nicolet 360 Fourier transform infrared (FT-IR) spectroscope using
KBr pellet containing 26 mg of sample to conrm the presence of
maghemite and coating of CG on INPs. The XRD of the samples
were done using X-ray diffractor GE model 3003TT German, as a
further conrmatory study for INPs to prove that they were
specically maghemite rather than other forms of iron. The TEM
analysis for the prepared samples was carried out using Tecnai 10,
Philips Transmission electron microscope to determine the size
and shape of the prepared INPCG. The samples were sonicated
using Vibronics, Ultrasonicator processor 2 at 180 W for 20 cycles,
each cycle for 1 min with a gap of 30 s after each cycle. These
particles were then mixed with 2% phosphotungstic acid with the
ratio of 1:2 and 20 ml of the mixture was placed in copper grid
260# and dried at room temperature (RT) for analysis. The
magnetization measurement for the INPs was carried out using
Vibration sample magnetometer (VSM 7300 magnetometer) to
conrm the super paramagnetic effect using the hystereis loop
obtained by applying a maximum magnetic eld of 7500 Oe. The
dried form of sample (INPCG) was weighed in an aluminum foil;
hand pressed as a cylindrical pellet and kept in the sample holder
for analysis. The MRI images for the INPCG were taken using 1.5 T
Avanto high eld Magnetic resonance image analyzer Siemens
Erlanger, Germany to further conrm the super paramagnetic
effect, an ideal characteristic property to be possessed by the
particles acting as MRI contrast agent. The INPCG suspension
containing varying concentration of Fe 50 ml (25 mg), 100 ml
(50 mg), 150 ml (75 mg), 200 ml (100 mg) and 250 ml (125 mg) were
suspended in 500 ml of 1% agarose solution and the imaging
parameters adopted for T2*-weighted MR imaging were
TR = 800 m/s; TE = 26 m/s; FOV = 230  230; ip angle 208; slice
thickness = 5 mm and slice 0.5 mm. The drop in signal with respect
to varying concentration of Fe was determined.
3. Results and discussion
3.1. FT-IR analysis
The FT-IR spectra of INP and INPCG are presented in Fig. 1(A)
and (B). The FT-IR spectrum of INPs exhibited strong bands in the
lower frequency region, 1000500 cm 1 due to the iron oxide
skeleton. This spectrum is comparable with the maghemite [g-
Fe2O3] spectrum reported in earlier study (broad band at
520610 cm 1) [9]. Both INP and INPCG showed the characteris-
tic absorption peak at 564 cm 1 which conrmed the FeO
stretching vibration band and indicated the prepared INPs were
maghemite. The FT-IR spectrum of INPCG shows the OH
stretching absorption bands at around 30383200 cm 1. Peak at
1645 cm 1 represents NH stretching vibration of amide I bond of
gelatin. Broad absorption band around 1003 cm 1 represents free
primary amino group at C2 position of chitosan molecule. Bands at
1404 and 1431 cm 1 represent CO stretching of chitosan [22].
These results indicate the coating of CG onto INPs.
3.2. XRD analysis
Fig. 2 shows the X-ray diffraction pattern of the prepared INPs.
All the detected diffraction peaks could be attributed to the
characteristic peaks of g-Fe2O3 lattice parameters. The main peaks
observed were at 2u = 30.188, 30.288, 35.418, 43.458, 53.808 and
56.778. The results are in line with the observations made by
Faunconnier et al. [31].
3.3. VSM analysis
The magnetic behavior of the prepared INPCG was studied
using the Hysteresis loop measured at a maximum eld of
7500 Oe at room temperature and plotted in Fig. 3. The specic
magnetic saturation Ms was found to be 18.97 emu/g. The
magnetic retentivity and coercivity were found to be 5.36 emu/g
and 233.4 Oe respectively, the Ms value was found to be lower
than that of theoretical bulk g-Fe2O3 (76 emu/g). The lower Ms
value for g-Fe2O3 nanoparticle is due to the decrease in particle
size and their large surface to volume ratio [26,32]. These results
suggest that the nanobiocomposites exhibit ferromagnetic
behavior and this magnetic property is required for the soft
magnetic applications. These INPs can also be tried as MRI
contrast agents to enhance the cellular imaging of cancer lesions
both for in vitro and in vivo applications. Due to the
characteristic super paramagnetic effect exhibited by the INP
CG, it also paves a way to prepare magnetic carriers for targeted
drug delivery [33].
 M. Chamundeeswari et al. / Materials Research Bulletin 46 (2011) 901904
Fig. 1. (A) FT-IR spectrum of INPs and (B) FT-IR spectrum of INPCG.
Fig. 2. XRD spectrum of INPs showing characteristic peaks of maghemite (g-Fe2O3).
3.4. TEM analysis
TEM studies (Fig. 4) revealed the size and shape of the
nanoparticles. INPCG exhibited spherical shaped nanoparticles
with size in the range of 80300 nm. Most of the nanoparticles are
agglomerated forming nanoclusters probably due to the magne-
tism and surface tension of the droplet on the carbon coated TEM
grid as it dries during preparation for imaging [34].
903
Fig. 3. VSM shows the super paramagnetic effect exhibited by INPCG at room
temperature
Fig. 4. TEM micrograph showing the spherical shaped INPCG particles with the
size in the range of 80300 nm.
3.5. MRI studies
The dark circles observed in Fig. 5 denote the loss of magnetic
resonance signal with respect to the concentration of Fe. The
control (a) which has no Fe shows absence of drop in signal. But the
INPCG (bf) shows a drop in signal with respect to increasing
concentration of Fe which is directly proportional to the
concentration of Fe content in 1% agarose. A maximum drop in
signal was observed in 125 mg/ml Fe when compared with
minimum concentration of Fe in 25 mg/ml. This phenomenon
indicates the magnetic property of the INPCG, which might
enable it for its dual purpose as magnetic carrier for targeted drug
904 M. Chamundeeswari et al. / Materials Research Bulletin 46 (2011) 901904
Fig. 5. MRI images of INPCG containing varying concentration of Fe. (a) control (1% agarose gel without INPCG), (b) 25 mg, (c) 50 mg, (d) 75 mg, (e) 100 mg and (f) 125 mg of
Fe concentrations in 1% agarose gel.
delivery to the cancer lesion via magnetic guiding through the
application of strong external magnetic eld and also as MRI
contrast agent to enhance the cellular imaging to provide high
resolution images. Our preliminary studies showed encouraging
results, in future we are planning to prepare nanobiocomposites
using chemotherapeutic agent and monoclonal antibody to
produce target specic magnetic carriers with less toxic effect
and high therapeutic efciency.
4. Conclusion
In summary, we have prepared INPs from the natural resource,
goat blood by incineration method. Using these INPs, INPCG
nanobiocomposite was prepared which may be used as a
substitute for the commercially available MRI contrast agents,
after comparing their toxicity, biocompatibility and economic
benets. It has also shown super paramagnetic property with a
particle size in nanometer which is an additional benet since it
can promote cellular uptake by the micrometer sized mammalian
cells through receptor mediated phagocytosis when coupled with
the chemotherapeutic agent and monoclonal antibodies. Prelimi-
nary studies indicated that the INPCG composites may be tried as
MRI contrasting agents and further studies are in progress to show
whether these nanobiocomposites can be used as a targeted
magnetic carrier particularly for cancerous cells.
Acknowledgements
The authors would like to thank Khemlal Adikarai, Technical
assistant St.Josephs College of Engineering, Chennai for his help in
collecting the blood.
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