FABAD J. Pharm. Sci., 31, 15-21, 2006 RESEARCH ARTICLE
Efficiency of Chitosan Film Formulation Following Experimental Brain Edema
Summary
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 Formulation Following 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 Edema
Received : 01.03.2007 Revised : 20.06.2007 Accepted : 27.06.2007
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Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100, S›hhiye/Ankara, Turkey Ankara Atatürk Research and Education Hospital, Department of Neurosurgery, Bilkent/Ankara, Turkey
Ankara Numune Research and Education Hospital, Department of Neurosurgery, 06100, S›hhiye/Ankara, Turkey Hacettepe University, Faculty of Medicine, Department of Biochemistry, 06100, S›hhiye/Ankara, Turkey
Hacettepe University, Faculty of Medicine, Department of Anatomy, 06100, S›hhiye/Ankara, Turkey Corresponding Author e-mail: [email protected]
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
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.
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 Codes A B C D E F
Edema Formation + + + + + -
Treatment None i.p. Dekort®
i.p. saline Chitosan film
Chitosan film containing DSP None
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
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.
A- Intraneuronal vacuoles No vacuoles: 0
Small vacuoles: 1 Large vacuoles: 2
B- Mitochondria in neurons Normal: 0
Mild swelling: 1 Severe edema: 2
C- Perineuronal edema No edema: 0
Mild edema (+): 1 Severe edema (++): 2
D- Large sized myelinated axons Normal: 0
Separation in myelin configuration: 1 Interruption in myelin configuration: 2 Honeycomb appearance: 3
E- Medium sized myelinated axons Normal: 0
Separation in myelin configuration: 1 Interruption in myelin configuration: 2 Honeycomb appearance: 3
F- Small sized myelinated axons Normal: 0
Separation in myelin configuration: 1 Interruption in myelin configuration: 2 Honeycomb appearance: 3
b.
Ultrastructural Scoring Parameters Small myelinated axons Medium myelinated axons Large myelinated axons Perineuronal edema Intraneuronal vacuoles Mitochondria in neurons TOTAL
Group A 7 35 44 61 61 56 264
Group B 2 26 38 46 42 42 196
Group C 5 34 44 61 58 53 255
Group D
3 37 49 56 56 49 250
Group E 3 30 41 62 54 46 236
Group F 0 1 2 0 0 2 5
Figure 2. Water contents of the experimental groups.
Figure 3. Lipid peroxidation ratios of the experimental groups.
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).
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.
san: a biocompatible biomaterial for oral and intravenous administration, Gebelin CG, Dunn RL (eds.), Progress in Biomedical Polymers, Ple- num Press, New York, 1990, pp. 283-290.
Malette WG, Quigley HJ, Gaines RD, Johnson ND, Rainer WG. Chitosan: a new haemostatic, Ann Thorac Surg, 36, 55-58, 1983.
Shu XZ, Zhu KJ. A novel approach to prepare tripolyphosphate/chitosan complex beads for controlled drug delivery, Int J Pharm, 201, 51-58, 2000.
Puttipipatkhachorn S, Nunthanid J, Yamamoto K, Pech GE. Drug physical state and drug poly- mer interaction on drug release from chitosan matrix films, J Controlled Rel, 75, 143-153, 2001.
Remunan-Lopez C, Bodmeier R. Mechanical, water uptake permeability properties of crosslinked chitosan glutamate and alginate films, J Controlled Rel, 44, 215-225, 1997.
Sawayanagi Y, Nambu N, Nagai T. Permeation of drugs through chitosan membranes, Chem.
Pharm. Bull., 30(9), 3297-3301, 1982.
Ero¤lu H, Kafl HS, Öner L, Turkoglu OF, Akalan N, Sargon MF, Özer N. The in vitro and in vivo characterization PLGA:L-PLA microspheres con- taining dexamethasone sodium phosphate, J Microencapsulation, 18(5), 603-612, 2001.
Turkoglu OF, Ero¤lu H, Okutan O, Bodur E, Sargon MF, Özer N, Öner L, Beskonakli E. The efficiency of dexamethasone sodium phosphate- encapsulated chitosan microspheres after cold injury, Surgical Neurology, 64(S2), 11-16, 2005.
Chan PH, Yang GY, Chen SF, et al. Cold induced brain edema infarction are reduced in transgenic mice over expressing CuZn-superoxide dismu- tase, Ann. Neurol., 29, 482-486, 1991.
Klatzo I. The relationship between edema, blood brain barrier and tissue elements in a local brain injury, J. Neuropathol. Exp. Neurol., 17, 548-564, 1958.
Murakami K, Kondo T , Sato S, et al. Occurrence of DNA fragmentation following cold injury induced brain edema, Neuroscience, 87, 231-237, 1997.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
FABAD J. Pharm. Sci., 31, 15-21, 2006
Murakami K, Kondo T, Yang G, et al. Cold injury in mice: a model to study mechanisms of brain edema and neuronal apoptosis, Prog Neurobiol., 57, 289-299, 1999.
Wallace S. A biochemical study of cerebral tissue and of the changes in cerebral oedema, Brain, 62, 426-438, 1939.
Sahin S, Kaynak MS, Hincal AA. Potential use of freeze drying technique for estimation of tissue water content, Methods Find Exp Clin Pharmacol, 28(4), 211-215, 2006.
Freeman BA, Crapo JB. Biology of disease: free radicals and tissue injury, Lab. Invest., 47, 412- 426, 1982.
Murokami K, Kondo T, Chan PH. Blood-brain barrier disruption, edema formation, and apop- totic neuronal death following cold injury, Acta Neurochir. Suppl. (Wien), 70, 234-236, 1997.
Orita T, Nishizahi T, Kamiryo T, Harada K, Aoki H. Cerebral microvascular architecture following experimental cold injury, J. Neurosurgery, 68(4), 608-612, 1988.
Ero¤lu H, Sargon MF, Öner L. Chitosan formu- lations for steroid delivery: effect of formulation variables on in vitro characteristics, Drug Dev.
Ind. Pharm., 33, 265-271, 2007.
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13.
14.
15.
16.
17.
18.
