Synthesis and doxycycline release profiles from CDHA microspheres.
Doxycyclin is a broad-spectrum antibiotic, with activity against a wide range of gram positive and gram-negative organisms . It is the drug of choice in the treatment of Lyme disease, brucellosis and several rickettsial infections. It is also extensively used for the treatment of juvenile periodontitis . The formation of periodontal pockets has been the result of a localized pathogenic bacterial infection below the gum line . Periodontal pockets are easily accessible from the oral cavity and are therefore a convenient site for treatment by a localized drug delivery system. The particulate based systems for the delivery of drugs are claimed to have enhanced bioavailability, predictable therapeutic response, greater efficacy and safety and controlled and prolonged drug release profile [3,4]. They are especially important in the case of poor drug distribution at the site of infection due to limited blood circulation to the surrounding skeletal tissue . Various particulate systems containing drugs distributed within a biocompatible matrix are under development . Calcium-deficient hydroxyapatite (CDHA) are of greater biological interests than stoichiometric HA bone mineral essentially has a CDHA structure with a Ca/P ratio of about 1.5 which is a Ca/P ratio similar to that of TCP but structurally chemically and compositionally similar to stoichiometric HA. [7, 8]. In addition, CDHAs are more efficient in inducing the precipitation of bone like apatite . So present day studies are focussed on the synthesis and characterization of CDHA bioceramics. The development of CDHA-drug particulate system for controlled drug release seems to be an interesting problem in antibiotic therapy . The purpose of this study was to formulate doxycyclin drug delivery system based on CDHA microspheres for the treatment of juvenile periodontitis. As the behaviour of particulates in the body depends on their morphology and microstructure, microwave processing, a technique for processing of advanced ceramics with desired properties has been used for CDHA synthesis .
Materials and Methods
High purity calcium hydroxide and diammonium hydrogen ortho phosphate were used for the CDHA synthesis. (Sigma Aldrich Chemicals USA). Pharmaceutical grade, low viscosity paraffin oil (SD fine chemicals, India) and the antibacterial drug doxycyclin hyclate (Periostat -a powder for oral suspension from Ranbaxy Pharmaceuticals, India.) were procured locally.
Doxycyclin hyclate is a broad-spectrum antibiotic synthetically derived from oxytetracycline This drug is a tetracycline antibiotic. As doxycyclin is known to be light sensitive, the drug loading and release experiments were protected against exposure to light as much as possible.
Synthesis of CDHA
The CDHA granules were synthesized by the microwave route. Calcium hydroxide and diammonium hydrogen ortho phosphate (DAP) were used as raw materials. The amount of the reactants was calculated from the Ca/P molar ratio of the starting materials. The Ca/P ratio of 1.61 was taken to obtain CDHA. Weighed amounts of the starting granules were dissolved in water and the DAP solution was added to the calcium hydroxide solution under stirring conditions. The solution is then exposed to microwave irradiation in a microwave oven. The product was then filtered and dried in an oven at 100[degrees]C.
Preparation of microspheres
An appropriate aqueous solution of gelatin content was prepared at 30[degrees]C. Fine CDHA powder was added to the above The gelatin slurry with CDHA powder was dispersed in 500 ml of light paraffin oil in an analytical flask by stirring with a glass paddle stirrer. The rpm of the stirrer and the time of stirring were optimized to form microspheres.
The precipitated microspheres were washed in acetone followed by ethanol and dried in air. The gelatin bound beads were heated for an hour at 550[degrees]C to burn off the gelatin The microspheres were then thoroughly washed in distilled water to remove any unburned gelatin and dried in an oven. The microsphere formed with 4% (4gm in 100 ml), 6% (6gm in 100 ml) and 8% (8gm in 100 ml) gelatin have been coded as 4CDHAMS, 6CDHAMS and 8CDHAMS respectively in the text.
X-ray powder diffraction
The composition of the synthesized CDHA ceramic was analyzed by X-Ray powder diffraction (XRD) method (Shimadzu XDD1 X-Ray diffractometer, reflection mode, Japan) using CuK[micro] radiation. Data were obtained for 2? ranging from 20[degrees] to 60[degrees] with a step size of 0.1[degrees] at 4 seconds per step.
Scanning electron microscopy
The morphology of the CDHA granules and microspheres were observed under a scanning electron microscopy (JOEL JSM 5410 & JSM 5300, Japan) after coating with a thin gold film. A few milligram of dried CDHA sample were deposited on a black adhesive tape area, vacuum coated with gold film for 15 minutes and analyzed directly.
Surface area measurements (BET)
The surface area of the microspheres was determined by the triple-point BET method (Sorptomatic 1990) with nitrogen as the adsorbate gas and helium as an inert non-adsorbable carrier.
Microparticle drug loading
The 10 mg of CDHA microspheres was immersed in 10 ml of PBS containing 10 mg of doxycyclin for 24 hrs. After 24 h, the microspheres were separated by centrifugation and dried at room temperature for 48 h. The microspheres were then separated and the concentration of the doxycyclin was measured using UV-VIS spectrophotometry (Varian Cary 5E UV-VIS-NIR Spectrophotometer, USA) with a 1-cm path length cuvette. Subsequently the loading was carried out at the pH 7.4 of the buffer.
The loading was optimized by varying the concentration of drug and the CDHA particles. The amount of drug absorbed by the CDHA is calculated by finding the difference in doxycyclin concentration in the loading buffer, before and after loading by spectrophotometrically using the equation:
Percentage drug loading = ((A-B)/A)*100 Where A and B represent the initial and final drug concentrations of the buffer solution, respectively.
The in-vitro studies were carried out soaking the specimens in sodium phosphate buffer solution as the medium for release of drug at pH 7.4 and 37[degrees]C. Triplicate samples of 10 mg of granules were suspended in 20 ml phosphate buffer in tubes. The tubes are placed in a bench top constant temperature water bath. At regular intervals of time samples are withdrawn and analyzed using ultraviolet spectroscopic techniques. The drug release profiles were obtained found out by the UV-Visible spectrophotometry as mentioned above. The doxycyclin release from the specimen to the buffer was determined by measuring the absorbance values at the maximum observed at [lambda]=270 nm. The spectra were recorded for every 30 minutes until there was no change in the subsequent values
XRD Phase Analysis
The XRD pattern of the microspheres looks similar to that of the starting CDHA powder also shown in the figure 1 for comparison. This indicates the stability of the CDHA microspheres formed through heating at 650o C to remove the gelatin. The broad characteristic XRD peaks indicate the microcrystalline nature of the samples.
[FIGURE 1 OMITTED]
The SEM morphology of the CDHA microspheres are shown in Figure 2. All particles were edged, irregular and some are spherical. There is a considerable agglomeration of granules. Distinct difference in the morphology of the microspheres corresponding to a transition from irregular shape to spherical with smooth surfaces as a function of gelatin content has been clearly shown The open pore content in the sintered body is dependent on the gelatin content in starting CDHA/ gelatin mixture.
[FIGURE 2 OMITTED]
In-vitro release profile
A typical UV visible spectrum of the doxycyclin hyclate shows very prominent peaks at 270 nm and 350 nm, which were monitored for the drug release profile.
Intermittent sampling is used for in-vitro dissolution testing. At each scheduled sampling point, the UV absorption spectrum was taken and the amount of drug released was calculated. The doxycyclin release profiles with different CDHA microspheres are shown in Figure 3. The morphological features of the microsphere were found to correlate well with the in-vitro doxycyclin release. All the microspheres exhibit similar release profiles with an initial gradual increase reaching a maximum value and then nearly a constant release profile.
[FIGURE 3 OMITTED]
A maximum amount of about 80% drug release was observed for the 6CDHAMS microspheres while 8CDHAMS shows about 60% drug release. The behaviour of a particulate in the body depends on its morphology and microstructure. Irregular morphology of the particulate is known to cause inflammatory reactions so rounded granules with smooth geometry are preferred as obtained in the present study.
The 6CDHAMS microspheres were relatively found to be uniformly spherical and with a smoother surface. The observed release profile has been attributed to the surface bound drug (11). It also has more surface area as indicated in table 1.
Hence, more amount of drug could have been absorbed on the 6CDHAMS microspheres, which results in the maximum amount of drug released. The 4CDHAMS microspheres were found to be larger which results in lesser amount of drug absorption and release at a shorter interval relatively. The results thus suggest optimum morphology, size and presence of micro pores in 6CDHAMS. In many therapeutic delivery systems the rate of release should be relatively constant or of zero order dependence i.e. rate of release is independent of time (12,13).
The constant release of the drug however can be controlled by appropriate selection of the microspheres by optimizing and fabrication methods.
CDHA microspheres with optimum pore content and morphology were prepared. Drug loading at pH 7.4 leads to higher amount of loading. Release pattern strongly depend on the morphology of CDHA microspheres and on optimum release upto 80% has been recorded.
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Sunita Prem Victor and T S Sampath Kumar * Department of Metallurgical and Materials Engineering Indian Institute of Technology Madras, Chennai 600 036
* corresponding author
Table 1 -Surface area and drug release of microspheres Microspheres BETArea %Drug ([m.sup.2]/gm) release 4CDHAMS 40 53 6CDHAMS 75 80 8CDHAMS 53 65
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|Title Annotation:||calcium deficient hydroxyapatite|
|Author:||Victor, Sunita Prem; Kumar, T.S. Sampath|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jul 1, 2006|
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