Journal of Biomaterials and Nanobiotechnology, 2011, 2, 194-200
doi:10.4236/jbnb.2011.22024 Published Online April 2011 (http://www.scirp.org/journal/jbnb)
Preparation of Biocompatible Magnetic
Microspheres with Chitosan
Mohamed K. Nasra, Moustafa M. Mohamed, Mohamed A. Elblbesy, Bothaina A. Hefney
Medical Biophysics Departement, Medical Research Institute, Alexandria University, Alexandria, Egypt.
Received January 20th, 2011; revised February 22nd, 2011; accepted March 1st, 2011.
ABSTRACT
Microsphere is a term used for small spherical particles, with diameters in the micrometer range (typically 1 μm to
1000 μm (1 mm)). Microspheres are sometimes referred to as microparticles. Microspheres can be manufactured from
various natural and synthetic materials. The present work we prepared chitosan magnetic microspheres (CMMS) with
simple crosslinking method. The obtained CMMS were in size range of 1000 - 2600 nm with average particle size of
1800 nm. All the essential characterizations of prepared CMMS were done and the results were in a good agreement
with other magnetic microspheres prepared with different method. To test the biocompatibility of CMMS with blood, the
effect of them on erythrocytes aggregation and blood hemolysis were studied. Our results showed that CMMS work as
good compatible materials with blood.
Keywords: Magnetic, Microspheres, Chitosan
1. Introduction
Monodispersed nano/micrometer scale magnetic particles
have drawn considerable attention because of both fun-
damental physics involved and technical applications in
high-density magnetic storage media, ferrofluids and
catalysts. The nano/micrometer spinel ferrite particles
have great potential applications since they are relatively
inert and their magnetic properties can be finely tuned by
chemical manipulations. Especially, in recent years, the
interests in monodispersed spinel ferrite nano/micrometer
particles are growing based on their potential applica-
tions in biomedicine and biotechnology for contrast en-
hancement of magnetic resonance imaging (MRI) and as
drug carriers for magnetically guided site-specific drug
delivery [1].
Magnetic nanoparticles offer some attractive possibili-
ties in biomedicine. First, they have controllable sizes
ranging from a few nanometers, which places them at
dimensions that are smaller than or comparable to those
of a cell (10 - 100 μm), a virus (20 - 450 nm), a protein
(5 - 50 nm) or a gene (2 nm wide and 10 - 100 nm Iong).
This means that can ‘get close’ to a biological entity of
interest. Indeed, they can be coated with biological mo-
lecules to make them interact with or bind to a biological
entity, thereby providing a controllable means of “tag-
ging” or addressing it. Second, the nanoparticles are
magnetic, which means that they obey Coulomb’s law,
and can be manipulated by an external magnetic field
gradient. This ‘action at a distance’, combined with the
intrinsic penetrability of magnetic fields into human tis-
sue, opens up many applications involving the transport
and/or immobilization of magnetic nanoparticles, or of
magnetically tagged biological entities. In this way they
can be made to deliver a package, such as an anticancer
drug, or a coherent of radionuclide atoms, to a targeted
region of the body, such as a tumor. Third, the magnetic
nanoparticles can be made to resonantly respond to a
time-varying magnetic field, with advantageous results
related to the transfer of energy from the exciting field to
the nanoparticles [2].
In recent years, a large number of different clinical ap-
plications of magnetic microspheres have been proposed,
mainly connected to disease treatments. On the contrary,
only few others have proposed to use nanoparticles for
the reliable detection and localization of tumors. Mag-
netic drug delivery by particulate carriers is a very effi-
cient method of delivering a drug to a localized disease
site. In magnetic targeting, a drug or therapeutic radio-
isotope is bound to a magnetic compound, injected into a
patient’s blood stream, and then stopped with a powerful
magnetic field in the targeted area [3].

Preparation of Biocompatible Magnetic Microspheres with Chitosan 195
There is a considerable interest in preparation of mag-
netite (Fe3O4) due to its strong magnetic properties, wh-
ich were used first in biology and then in medicine for
the magnetic separation of biochemical products and
cells as well as the magnetic guidance of particle systems
for site-specific drug delivery. However, the size, charge,
and surface chemistry of magnetic particles could
strongly influence their magnetic properties and distribu-
tion in biological systems. The most common method for
the synthesis of magnetite is by co-precipitation from a
solution of Fe (III) and Fe (II) salts in presence of base.
Preparation of size-controllable magnetite nano-particles
was performed in presence of different water-soluble
polymers such as lignosulfonate, mesoporous sulfonated
styrene-divinylbenzene, polypeptide [4,5] etc.
Magnetite nano-particles have been also incorporated
into more complicated architectures, such as polymeric
gels providing formation of magnetic field sensitive gels
[6]. These gels contain magnetic particles dispersed ho-
mogeneously and confined in a polymer network. Under
a nonuniform magnetic field, the particles undergo mo-
tion; which in turn induces elongation, contraction, or
bending of the gels with short response time. Incorpora-
tion of magnetite into spherical polymeric particles has
been achieved by following two approaches: 1) hetero-
coagulation of magnetite on the surface of pre-formed
polymeric particles [7], 2) encapsulation of magnetite
particles during emulsion polymerization process [8] or
by using the microemulsion approach [9], or 3) by
layer-by-layer deposition method [10,11].
As the pharmaceutical industry continues to develop
new effective medications, the need to develop efficient,
minimally invasive delivery techniques is paramount. A
successful targeted drug delivery system will allow clin-
ical usage of drug not currently accessible to physicians,
as well as a more efficient means for delivering those
already available [12].
Chitosan is a heteropolysaccharide composed of β-
(1–4)-2-deoxy-2-amino-d-glucopyranose units and of β-
(1–4)-2-deoxy-2-acetamino-d-glucopyranose units. Chi-
tosan occurs in nature, particularly in the cell wall of
some fungi such as Mucor rouxii [13] and is mainly pro-
duced by the deacetylation of chitin, which is a major
component of the exoskeleton of insects, the cuticles of
annelids and mollusks, and the shell of crustaceans such
as shrimp, crab, and lobster. Chitosan has been exten-
sively examined in the pharmaceutical industry for its
potential in the development of controlled release of drug
delivery due to its excellent biocompatibility, biode-
gradability, bioactivity and nontoxicity [14]. Various
sustained release drug carriers have been made from chi-
tosan such as microparticles [15], tablets [16], gel [17]
and beads [18]. Chitosan microspheres have been widely
investigated for use as controlled release delivery sys-
tems for hormones [19], vitamins [20], proteins [21] and
enzymes [22].
This work aims to prepare the chitosan magnetic mi-
crospheres and to study its effect on dynamic properties
(blood viscosity, erythrocytes aggregation, osmotic fra-
gility and hemolysis) (in vitro) under the influence on
external magnetic field.
2. Materials and Methods
Chitosan polymer, glutraldhyde, Sodium hydroxide, fer-
rous sulfate, ferric chloride, sodium hydroxide and poly-
ethylene glycol-10000 (PEG-10000) were obtained from
Sigma (UAS). All chemicals were of analytical grade and
no further purification was required.
Preparation of Chitosan Magnetic Microsphe r es
Acetate buffer was used as solvent for the chitosan po-
lymer and glutraldhyde was used as the cross-linker. So-
dium hydroxide solution was used as medium. Magnetic
fluid was synthesized as follows: a 35% (w/v) ferrous
sulfate solution, 54% (w/v) ferric chloride solution and
36% (w/v) sodium hydroxide solution were prepared
using distilled water. Then the ferric salt and ferrous salt
were mixed, stirred and heated. When the temperature
reached 55˚C, the alkaline solution was added. The mix-
ture was stirred for 30 min, and then 5 g of polyethylene
glycol-10000 (PEG-10000) was added. The temperature
was raised to 80˚C and maintained for 30 min. The mix-
ture was then neutralized while cooling, and the magnetic
fluid was prepared. 1% (w/w) chitosan was dissolved in
acetate buffer at pH 4.5. The dissolved chitosan was
added drop wise on the magnetic fluid using Syringe
Pump. Formed CMMS were washed with deionized wa-
ter and soaked in 1, 3, and 5 mol % glutraldhyde solution
for 2 hr, and then washed with deionized water.
Scanning electron microscopy (SEM) (Joel, JSM-6360
LA-Japan) images were taken after sputter coating the
CMMS with gold (SPI-module TM Sputter coater, Ja-
pan).the size of CMMS was analyzed by Beckman Coul-
ter Particle Size Analyzer (N5 submicron particle size
analyzer, Japan). The magnetic properties of air dried
MMS were determined by a vibrating-sample magne-
tometer (VSM-9600-1DSM-LDG-USA). Thermo gra-
vimetric analyses (TGA) were measured by (Shimadzu,
TGA-50, Japan) with a heating rate of 10˚C/min in ni-
trogen flow. Fourier transforms infrared spectropho-
tometer (FT-IR) spectra were recorded before and after
processing of CMMS. The FTIR spectra were obtained
using a FT-IR spectrophotometer (Alpha-centauri) (Shi-
madzu, Japan, FT-IR-8400S). Around 4 - 8 mg of MMS
were thoroughly mixed with IR-grade potassium bromide
(KBr) (200 mg) and grinding together, then compressed
into tablet form with the compressor (Shimadzu-Com-
pressor-Japan) in order to record the spectrum.
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196 Preparation of Biocompatible Magnetic Microspheres with Chitosan
Microscopic S tudy of RBC’s Aggreg ation
For examine the effect of CMMS on erythrocytes aggre-
gation. 2 ml of erythrocytes suspension with 1% concen-
tration in PBS was incubated with 1 gm of CMMS for
two hours. The incubated erythrocytes were then allowed
to immigrate freely on an inclined slide at angle of 45˚ in
order to make erythrocytes film [23]. Erythrocytes film
was left to dry for two minutes. The erythrocytes film
was examined using light microscope connected to CCD
camera. The image of erythrocytes film was transferred
to computer through an interface connected to CCD
camera. The images of erythrocytes film were analyzed
by imaging processing software in order to calculate ag-
gregation shape parameter (ASP) which is used to quan-
tify erythrocytes aggregation. The following equation
was used to calculate ASP [24]:
2
ASP = 4πAP (1)
where A is the projected area of the aggregate, P is the
perimeter of the projected area. The ASP of ten images
of incubated erythrocytes was computed in order to cal-
culate the mean value of ASP and then compared to the
mean value of unincubated erythrocytes.
Hemolysis
Hemolysis has been determined to confirm that the cho-
sen magnetic field intensity for the chosen time period
affects the cellular membrane. Erythrocyte suspensions
(Ht = 5%) was incubated with CMMS, for two hours.
After centrifugation the degree of hemolysis (%) was
determined by measuring the hemoglobin content in the
supernatant at 540 nm. Hemolysis was expressed as a
percentage of the absorbance in distilled water [25].
3. Results and Discusssion
The observation of size,shape and surface topography of
the dried CMMS are shown in Figure 1.The shapes of
the dries microspheres were soherical, and the surface
was rough, porous and unfolded. Furthermore the mi-
crosphere was not hollow.
The particle size distribution curve (Figure 2.) showed
sharp distribution range of microspheres, with 90% of
spheres in size range of 1000-2600 nm with average par-
ticle size of 1800 nm and only 10% were oversized. The
formed microspheres have uniform surface structure in
which the microspheres lose their shape upon drying but
regain them upon re-swelling. Which agreement with the
results obtained by Erika Aranas et al. [26]. Erika Aranas
et al. [26] prepared the spheres with technique similar to
emulsion polymerization. Zhanga Ji et al. prepared
composite magnetic microspheres based on artemisia
seed gum and chitosan using the suspension cross-linking
technique for use in the application of magnetic carrier
technology [27]. Their results showed that composite
(a)
(b)
(c)
Figure 1. SEM images of CMMS (a) CMMS with different
size; (b) Rough surface of CMMS; (c) None hollow CMMS.
magnetic microspheres can be produced in the size range
230 - 460 μm.
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Preparation of Biocompatible Magnetic Microspheres with Chitosan 197
Figure 2. Particles size distribution of CMMS with size
range 1000 - 2600 nm.
From the hysteresis loops of CMMS (Figure 3), the
saturation magnetization was 8.065 emu/g, coercivity was
76.05 Oe and retentivity was 1.057 emu/g. These results
are in agreement with Zhanga Ji et al. the magnetic
properties of the composite magnetic microspheres were
evaluated using a VSM [27].
The most effective parameters determining the mag-
netic properties were the stirring rate of the suspension
medium and the Fe3O4/chitosan ratio, as in the case of
the size/size distribution evaluation.The stirring rate of
the suspension medium was varied between 500 and
1000 rpm for investigation of the effects of stirring rate
on the magnetic properties of the composite magnetic
microspheres. Furthermore, similar behavior for this pa-
rameter had been reported previously. Chiriac H. et al.,
showed that the variation of the saturation magnetization
and the coercive field as a function of the particle sizes
for the atomized magnetic microspheres obtained for a
diameter nozzle of 91 mm [28]. Their result showed that
the saturation magnetization and the coercive field of the
atomized microspheres with sizes between 26 and 300
mm range between 79.8 and 42 emu/g and 305 and 384
Oe, respectively.
Thermal gravimetric Analysis (Figure 4.) showed that
the chitosan powder a small peak at 26˚C - 106˚C due to
the volatization of the solvent. Other strong transitions
were found at 253˚C - 337˚C due to faster decomposition
of chitosan powder. The difference in the thermal analy-
sis between the two figures of chitosan powder and
CMMS may be due to increase of thermal stability of
magnetic microspheres than chitosan. This results in
agreement with results obtained by Pich A. et al. [29].
Liu ZL et al. indicated that in their Thermal gravimetric
Analysis of magnetic microspheres that the microspheres
start degrading at about 260˚C. The first mass loss is
about 63% at the temperature between 260 and 280 1C,
Figure 3. Hysteresis loop of CMMS.
(a)
(b)
Figure 4. Thermal gravimetric analysis of chitosan (a) and
CMMS (b).
and the second mass loss is about 10% at the temperature
between 360 and 400 1C. The Tg is about 280 and 380
1C, respectively.
For the IR spectrum of chitosan (Figure 5) the char-
acteristic absorption bands appeared at 3433 cm−1 indi-
cate to hydroxyl group, 1641 cm−1 indicate to amide I,
1573 cm−1 indicate amide II, 1382 cm-1 indicate to am-
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198 Preparation of Biocompatible Magnetic Microspheres with Chitosan
(a)
(b)
Figure 5. the IR spectrum of chitosan (a) and CMMS (b).
ide III, band at 2920 cm−1 indicate to C-H stretching of
coplymer, band at 1029& 1080 cm−1 indicate to C-O
stretching and band at 1622 cm−1 indicate to NH3 ab-
sorption of chitosan. Compared with the spectrum of
chitosan magnetic microsphere (Figure 5) the 3433 cm-1
peak of hydroxyl group shifted to 3409 cm−1 and a de-
crease in peak intensity of amid I (–NH2) group at 1566
cm−1 which indicated the ionic cross-linking between
amid I (–NH2) group of chitosan and –C=O groups of
glutraldhyde and new characteristic band appear at 1650
cm−1 indicate to C=N group which indicate to cross-
linking. The functional groups of materials are very im-
portant for diverse applications, especially for biotech-
nological purposes. Therefore, the present functional
groups should be kept even if the shape (or geometry) is
changed into a new form (i.e. microspheres or mem-
brane). In this study the FT-IR of the spectra results ob-
tained the peaks expected due to the geometry. In which
the spectra of chitosan powder indicated the presence of
the characteristic peaks such as hydroxyl group, amid-І,
and amid-Π, amid-ІІІ, and NH3 group. On the other hand
the spectra of chitosan magnetic microspheres obtained
shift in the characteristic peak of hydroxyl group and
decrease in the peak intensity of amid-І due to the
cross-linking between amid- and glutraldhyde which
Figure 6. ASP of erythrocytes incubated with CMMS com-
pared to control.
Figure 7. Hemolysis of blood incubated with CMMS.
obtained by the presence of characteristic peaks of C=N
group. In the FTIR spectra of magnetic chitosan micro-
spheres obtained by De-Sheng J et al. the (C), the peak
was at 3427 cm−1 corresponds to stretching vibrations of
hydroxyl. The C-H stretching vibration of the polymer
backbone is manifested through strong peak at 2925 cm−1
and 2855 cm−1 [30]. The stretch vibrations of C-O are
found at 1084 cm−1 and 1032 cm−1. For cross-linked chi-
tosan microspheres, an additional peak at 1656 cm−1
which corresponds to stretching vibrations of C-N bond.
This peak indicates the formation of Schiff’s base as a
result of the reaction between carbonyl group of glu-
taraldehyde and amine group of chitosan chains. The
little peak at 1721 cm−1 shows the existence of impend-
ent aldehydic group in magnetic chitosan microspheres.
The erythrocytes incubated with CMMS showed lower
values of ASP than control (unicubated erythrocytes)
Figure 6. But the decrease in ASP was slightly small.
This indicated that there was no harmful effect of CMMS
on erythrocytes aggregation.
There was no difference in hemolysis curve between
control and blood incubated with CMMS Figure 7. This
make sure that CMMS have no harmful effect on blood.
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