Preparation of biocompatible magnetic microspheres with chitosan

doi:10.4236/jbnb.2011.22024

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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.

Email: [email protected]

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.

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]:

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.

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-

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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