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FABAD J. Pharm. Sci., 31, 15-21, 2006

RESEARCH ARTICLE

Efficiency of Chitosan Film Formulation Following Experimental Brain EdemaSummary

INTRODUCTION

Chitosan is a well-known natural polymer having

polysaccharide structure and possessing many ad-

vantageous properties, like biodegradability, biocom-

patibility, and anti-infectional and hemostatic

properties1,2. It shows a gel matrix property with

counter ions such as sodium tripolyphosphate (TPP).

Interactions between the positively charged amino

groups of chitosan and negatively charged ion of

sodium TPP creates a matrix structure for delivery

systems3. In many studies, it has been shown that

drug release from this matrix structure may be con-

trolled by the matrix density and amount of crosslink-

ing agent, such as sodium TPP, glutaraldehyde and

NaOH4, 5, 6.

In our previous studies, the efficiency of dexametha-

Efficiency of Chitosan Film FormulationFollowing Experimental Brain Edema

In this study, film formulations containing dexamethasone sodium phosphate were prepared by using a natural polymer chitosan. The in vivo efficiency of film formulations were evaluated by the animal model in which the brain edema was formed on Sprague Dawley rats. The evaluation of the efficiency for film formulations were investigated through the parameters such as wet-dry weight method, determination of the lipid peroxidation ratio, ultrastructural grading system and Transmission Electron Microscopy. The statistical evaluation of te experimental results revealed no significant difference between the experimental groups (p>0.05) which is probably due to the insufficient release medium in the craniectomy area, the movements of the film formulation as a result of the natural movements of the animals after surgery.Keywords: Chitosan, Dexamethasone Sodium Phosphate, Film, cold Injury, Brain EdemaReceived : 01.03.2007Revised : 20.06.2007Accepted : 27.06.2007

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Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100, S›hhiye/Ankara, TurkeyAnkara Atatürk Research and Education Hospital, Department of Neurosurgery, Bilkent/Ankara, TurkeyAnkara Numune Research and Education Hospital, Department of Neurosurgery, 06100, S›hhiye/Ankara, TurkeyHacettepe University, Faculty of Medicine, Department of Biochemistry, 06100, S›hhiye/Ankara, TurkeyHacettepe University, Faculty of Medicine, Department of Anatomy, 06100, S›hhiye/Ankara, TurkeyCorresponding Author e-mail: loner@hacettepe.edu.tr

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sone sodium phosphate (DSP), the most commonly

used steroid in the treatment of brain edema, has

been investigated using different dosage forms, such

as microspheres prepared by using natural (chitosan)

and synthetic (PLGA, L-PLA) polymers7,8. The proven

efficacy of DSP led us to investigate whether the same

efficiency would be observed with a new formulation

prepared by chitosan containing DSP. In this study,

the in vivo efficiency of chitosan film formulations

on an experimental vasogenic brain edema model is

investigated for potential use in treatment.

MATERIALS and METHODS

Materials

The drug substance used in this research, dexametha-

sone sodium phosphate (DSP), was kindly donated

by Deva Drug Company, ‹stanbul. For the control

group, DSP in the sterile parenteral dosage form,

Dekort®, was purchased from Deva Drug Company.

The chitosan used in the formulation of film dosage

forms was low molecular weight from Sigma Aldrich,

Milwaukee. Sodium TPP for the crosslinking process

was also obtained from Sigma Aldrich, Milwaukee.

All other chemicals used were of analytical grade and

were used without further purification. Sprague-

Dawley rats weighing 250 g were used for the animal

experiments.

Animal Model

The protocol of this experimental research was ap-

proved by Hacettepe University’s Experimental An-

imal Ethics Committee (protocol number 2004/3).

The animals used, female Sprague-Dawley rats weigh-

ing approximately 250 g, were divided into five ex-

perimental groups as shown in Table 1. Animals were

anesthetized by the administration of xyla-

zine/ketamine HCl mixture at a dose of 10/90 mg/kg

by the intraperitoneal route. All animals in each group

were stabilized using a stereotaxic frame before

craniectomy. The scalp was incised on the midline

and the skull was exposed. A craniectomy of 5x5 mm

was performed in the left parietal region between

coronary and lambdoid sutures and dura was kept

intact. A metal sterile rod with a diameter of 4 mm,

previously cooled to -80ºC, was applied to the surface

of the parietal cortex9,10,11. The degree of edema is

known to correlate with the period of contact with

the tissue and force of application12. Therefore, in

order to form a standard vasogenic edema, contact

of the metal probes (weighing 10 g) with the parietal

cortex was maintained for 1 min. The chitosan film

formulations containing DSP (0.1 mg/kg) were ad-

ministered to the craniectomy area. DSP at a dose of

0.2 mg/kg was administered intraperitoneally to one

of the control groups (Group B) for systemic applica-

tion. The skin incision was sutured and the animals

were placed in a conditioned room after surgery.

Twenty-four hours after the trauma, the animals were

sacrificed and their brains were decapitated intact12.

The Sprague-Dawley rats in stereotaxic frame and

the application of the chitosan film formulations are

shown in Figure 1.

Figure 1. Animals in stereotaxic frame and application of the chitosan film formulations.

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Evaluation of the Brain Edema

Wet-dry tissue weight method

The water content of brain tissues in rats has been

evaluated in the literature using different methods13,14,

one of which is the estimation of water content by

freeze drying. In this method, after the decapitation,

brain tissue was immediately weighed and frozen at

-20°C. After 24 h, the tissues were lyophilized for a

day. Their weights were determined and the results

were expressed as the water volume in gram tissue.

In our experimental research, the water content per-

centages of the brain tissues were evaluated using

the basic method described by Wallace13. Briefly, both

of the cerebral hemispheres were immediately

weighed after decapitation and were kept in an oven

at 70°C for 36 h until they reached a constant weight.

The percentage of tissue water content was calculated

using the formula:

% water content = [(wet-dry weight)/wet weight]*100

Eq. 1

The results of the experimental groups were compared

using the Mann-Whitney U test, and a value of p=0.05

was considered to indicate statistical significance.

Malondialdehyde Amount

Hydroxyl radical, an example for free radicals, may

easily react with the membrane phospholipids result-

ing in the provocation of lipid peroxidation. Malon-

dialdehyde, as the final product of lipid peroxidation,

can be identified via thiobarbituric acid (TBA)

reaction15. Malondialdehyde is a volatile substance

that can rapidly evaporate, causing polymerization

of the membrane structure. As a result of this poly-

merization, the membrane loses some properties,

such as ion transport, regulation of enzyme activity,

stabilization of the membrane surface, and ability to

change its shape. The membrane is destabilized by

lipid peroxidation and loses its property to form a

potential barrier, vascular permeability increases and

as a result of excess accumulation of Ca++, cell death

occurs. The destruction of the blood brain barrier by

increasing vascular permeability results in edema.

The absorptivity of the color formed by malondialde-

hyde with TBA is determined for the lipid peroxida-

tion ratio per gram of wet tissue in nmol unit. The

fresh tissues were homogenized in 150mM KCl con-

taining 25mM Tris-HCl pH7 buffer at 12000 rpm. 0.5

ml of the resulting homogenates were mixed with 3

ml 1% H3PO4 and 1 ml TBA and placed in a water

bath for 45 min. After cooling, 4 ml of n-butanol was

added and vortexed for 2 min. After centrifugation,

the color of the butanol was measured at λ=532 nm.

Ultrastructural Grading System (UGS)

The brain tissues were dissected by the neurosurgeon

just after decapitation in dimensions of 2x2x2 cm and

immediately placed in the fixation solution containing

2.5% glutaraldehyde in water. After 24 h, they are

washed with pH 7.4 phosphate buffer solution. The

samples were post-fixed in 1% osmium tetroxide in

pH 7.4 phosphate buffer and dehydrated in an in-

creasing concentration of alcohol. They were washed

with propylene oxide and embedded in epoxy resin

embedding media. Semi-thin sections, approximately

2 µm in thickness and ultra-thin sections, approxi-

mately 69 nm in thickness, were cut with a glass knife

on a LKB-Nova ultramicrotome. Ultra-thin sections

were collected on copper grids, stained with uranyl

acetate and lead citrate and examined with a Jeol-

JEM 1200 EX transmission electron microscope. The

magnification was x10000 for transmission electron

microscopy (TEM). In order to investigate the tissues,

the Ultrastructural Grading System (UGS) was used

for the evaluation of a more quantitative data (Table

2).

FABAD J. Pharm. Sci., 31, 15-21, 2006

Table 1. Experimental groups

Group CodesABCDEF

Edema Formation+++++-

TreatmentNonei.p. Dekort®i.p. salineChitosan filmChitosan film containing DSPNone

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RESULTS

Wet-Dry Tissue Weight Method

The water content percentages were calculated using

Equation 1 and the total results are given in Figure 2.

Statistical comparison of the results was done using

Mann-Whitney U test with a significance level of

p=0.05. The water content of the systemic DSP-

administered group (Group B) was found to be sta-

tistically different from the control group (Group A)

(p<0.05). On the other hand, the chitosan film-

implanted group’s (Group E) water content was lower

than Group A’s value, but no statistically significant

difference was found (p>0.05). The other two control

groups’ results (Groups C and D) showed no statistical

difference from Group A (p>0.05).

Malondialdehyde Amount

Lipid peroxidation results were similar to the wet-

dry weight findings. Although the lipid peroxidation

ratio of Group E had a decreased malondialdehyde

amount with respect to Group A, the statistical eval-

uations showed that the decrease was not statistically

significant (p>0.05). On the other hand, the other

control group, (Group B), in which the animals re-

ceived systemic DSP, had a statistically significant

decreased malondialdehyde amount with respect to

the control Group (Group A) (p>0.05).

Ultrastructural Grading System

After evaluation of the TEM photographs of the

experimental groups according to the UGS scores,

the differences between the groups were investigated

according to the parameters described in Table 2a,b.

The total score of Group E (236), in which dexametha-

sone film formulation is implanted, was decreased

with respect to the control Group A (264), in which

edema was formed after craniectomy but no treatment

was applied.

Table 2. a./b. Ultrastructural grading system

A- Intraneuronal vacuolesNo vacuoles: 0Small vacuoles: 1Large vacuoles: 2

B- Mitochondria in neuronsNormal: 0Mild swelling: 1Severe edema: 2

C- Perineuronal edemaNo edema: 0Mild edema (+): 1Severe edema (++): 2

D- Large sized myelinated axonsNormal: 0Separation in myelin configuration: 1Interruption in myelin configuration: 2Honeycomb appearance: 3

E- Medium sized myelinated axonsNormal: 0Separation in myelin configuration: 1Interruption in myelin configuration: 2Honeycomb appearance: 3

F- Small sized myelinated axonsNormal: 0Separation in myelin configuration: 1Interruption in myelin configuration: 2Honeycomb appearance: 3

a.

b.

Ultrastructural Scoring ParametersSmall myelinated axonsMedium myelinated axonsLarge myelinated axonsPerineuronal edemaIntraneuronal vacuolesMitochondria in neuronsTOTAL

GroupA73544616156264

GroupB22638464242196

GroupC53444615853255

GroupD33749565649250

Group E3

3041625446236

Group F0120025

Figure 2. Water contents of the experimental groups.

Figure 3. Lipid peroxidation ratios of the experimental groups.

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FABAD J. Pharm. Sci., 31, 15-21, 2006

Figure 4b. TEM photograph of Group B (i.p. DSP administered).

Figure 4a. TEM photograph of Group A (no treatment).

Figure 4c. TEM photograph of Group C (i.p. saline administered).

Figure 4d. TEM photograph of Group D (empty chitosan film).

Figure 4e. TEM photograph of Group E (DSP chitosan film).

Figure 4f. TEM photograph of Group F (only craniectomy).

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DISCUSSION

The potential use of chitosan film formulations in an

experimental vasogenic brain edema model was in-

vestigated using the parameters and detailed proce-

dures as stated above. Maximum edema after cold

injury can be observed 24 h after trauma16,17. The

chitosan film formulations were prepared using TPP

as the crosslinking agent. The in vitro characterization

of the film formulations were previously investigated

by Ero¤lu et al.18. The total percentage of the in vitro

release of DSP from film formulations was 15%, which

corresponds to half of the systemic dose of DSP (0.2

mg/kg). With respect to the control groups, it was

observed that the scores for all parameters investigated

were decreased. On the other hand, the statistical

evaluation demonstrated no significant difference

between the experimental groups (p>0.05). The most

probably explanation is the insufficient release medi-

um in the craniectomy area over the brain tissue.

Furthermore, when compared with the microsphere

formulations, the surface area of the film formulations

is less, which might possibly affect the released

amount of the active content in the formulation.

Although the formulations were implanted so as to

be in contact with the brain tissue in the craniectomy

area, movement of the animals might have caused

displacement from the site of action. In an effort to

overcome these problems, the authors plan to perform

additional experiments, including the trial of implan-

tation of chitosan films that are cut into small pieces

despite using them as a single piece. As a second

alternative, before the suturing of the skin incision,

a biodegradable gelatin sponge material with a 3 mm

thickness will be placed over the chitosan film piece

to retain it in the craniectomy area; gelation of this

film piece with a suitable amount of saline will provide

an additional dissolution medium for the release of

DSP from chitosan film formulations.

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