Synthesis, characterization and preliminary analysis of in vivo biological activity of chitosan/celecoxib microcapsules

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 14, 2010, Pages 4147-4151

  Cheng, Shuk Yan ^a   Yuen, Marcus Chun Wah ^a   Lam, Pik Ling ^a   Gambari, Roberto ^b   Wong, Raymond Siu Ming ^c   Cheng, Gregory Yin Ming ^c   Lai, Paul Bo San ^c   Tong, See Wai ^c   Chan, Kit Wah ^c   Lau, Fung Yi ^c   Kok, Stanton Hon Lung ^c   Lam, Kim Hung ^a   Chui, Chung Hin ^a,c  

^a HONG KONG POLYTECHNIC UNIVERSITY   (Hong Kong)

^b UNIVERSITY OF FERRARA   (Italy)

^c CHINESE UNIVERSITY OF HONG KONG   (Hong Kong)

Author keywords

Celecoxib; Chitosan; Cyclooxygenase 2; Oral administration

Indexed keywords

CELECOXIB; CHITOSAN; CYCLOOXYGENASE 2;

ANIMAL CELL; ANIMAL EXPERIMENT; ARTICLE; BIOLOGICAL ACTIVITY; CONTROLLED STUDY; DRUG ABSORPTION; DRUG ANALYSIS; DRUG BLOOD LEVEL; DRUG DELIVERY SYSTEM; DRUG SYNTHESIS; ENCAPSULATION; ENZYME INHIBITION; GAS CHROMATOGRAPHY; IN VIVO STUDY; LIVER CELL; MASS SPECTROMETRY; MICROCAPSULE; MOUSE; NONHUMAN; PROTEIN EXPRESSION;

CHITOSAN; CYCLOOXYGENASE 2 INHIBITORS; GAS CHROMATOGRAPHY-MASS SPECTROMETRY; MICROSCOPY, ELECTRON, SCANNING; MICROSPHERES; PYRAZOLES; SULFONAMIDES;

ANIMALIA; MUS;

EID: 77953873272     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.05.054     Document Type: Article

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13. Chemicals and reagents. Unless specified, all the chemicals and reagents were purchased from Sigma-Aldrich. 1% Chitosan (CTS, practical grade, >85% deacetylation) was dissolved in 1% acetic acid followed by filtration to remove insoluble impurities. Celecoxib, as used in the treatment of osteoarthritis, rheumatoid arthritis, acute pain, painful menstruation and menstrual symptoms, and to reduce numbers of colon and rectum polyps in patients with familial adenomatous polyposis, was obtained from Pzifer pharmaceutical company. Jojoba oil (obtained from Simmondsia chinensis) was obtained from the Easy Creation Asia Limited.
14. Synthesis of chitosan/celecoxib microcapsules. Fifty micrograms of celecoxib was firstly dissolved in 5 ml of jojoba oil. The mixture of celecoxib/jojoba oil was poured into 50 ml of acidic chitosan solution followed by intermixing for 10 min using a magnetic stir plate, Heidolph MR3001 (Heidolph, Germany), at a speed of 1200 rpm to form o/w emulsion. The emulsion was then further mixed using an ultrasonic processor (Vibra Cell™ VCX 750, Sonics and Materials Inc., USA) with the ultrasonic amplitude being 85% for 1 min to break down the emulsion into smaller droplets. Fine emulsion was produced without coalescence. 1% sodium hydroxide solution was slowly dipped into the emulsion for precipitation; and the precipitates were formed when the pH value reached around 7.5. Afterwards, pH value was further adjusted to pH 10.5 by sodium hydroxide solution to ensure complete precipitation. The precipitates, that is, chitosan/celecoxib microcapsules were rinsed with hexane followed by deionised water and spun down using a centrifuge in order to remove the unwanted materials including excess chitosan and uncapsulated oil as well as celecoxib. Analytical work for chitosan/celecoxib microcapsules. The morphology of novel synthesized chitosan/celecoxib microcapsules was recorded by scanning electron microscopy. The range and mean of particle size of microcapsules was evaluated with a zetasizer (Malvern Instruments, Zetasizer 3000 HSA). The microcapsules were firstly diluted with deionised water at the ration of 1:20 and 4 ml of the solution was taken for evaluation. Encapsulated celecoxib was extracted with ethanol and purified from known amount of microcapsules. The concentration of isolated celecoxib was further determined by absorption spectrophotometry and compared with the celecoxib positive reference curve.
15. Animal care and mice treatment. Eight weeks old C57BLC mice, weighing approximately 20-25 g, were purchased from the animal unit of The Chinese University of Hong Kong and maintained in a conventional sanitary facility, in accordance with the institutional guidelines on animal care, with the required consistent temperature and relative humidity. All the procedures were approved by the Animal Research Ethics Committee. A total of nine mice were included in our preliminary study and they were divided into three groups, respectively. The free celecoxib was mixed directly with a physiological saline solution containing 0.5% methylcellulose and 0.025% Tween 20 while the chitosan/celecoxib microcapsules were also mixed with the physiological saline solution. From day one to day four, treatment groups received celecoxib or chitosan/celebrex microcapsules once daily at a concentration of 4 μg/g of body weight. The concentration of free celecoxib solution was adjusted to the same as the celecoxib in the chitosan/celecoxib. Control group received the same volume of physiological saline orally. The animals were monitored and recorded if there had any abnormal behaviors. On day 5, all the mice were sacrificed and autopsies was performed to collect peripheral blood and livers for analytical chemistry and pathological analysis.
16. Immunohistochemistry analysis of cyclo-oxygenase 2 protein expression. Sections of mouse liver from autopsy samples were dewaxed with xylene and gradient concentrations of ethanol. Possible endogenous peroxidase was blocked and slides were washed with PBS. Slides were then blocked again and treated with diluted primary antibody (rabbit anti-rat cyclo-oxygenase 2) in PBS. Slides were washed with PBS and then treated with the secondary antibody CSA II rabbit link. After washing, slides were further treated with amplification reagent and anti-fluorescein-HRP. Afterwards, slides were incubated with DAB substrate. Nuclei were stained with haematoxylin and finally slides were inspected under a light microscope.
17. Whole blood was collected after mice were sacrificed and plasma was isolated after centrifugation. Afterwards, plasma liver enzymes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by the Vet biochemistry assay kits for the IDEXX laboratories machine in order to determine whether there are any liver failure phenomena from chitosan/celecoxib microcapsules treated groups of mice by comparing with the free celecoxib group and vehicle treatment group as well as the normal control ranges.
18. Plasma concentration of celecoxib. Plasma concentration of celecoxib from mice after the four day treatment protocol was determined semi-quantitatively by gas-chromatography-mass-spectrometry (GC/MS) and compared with the celecoxib positive reference curve.
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