5. PREPARATION OF GELATIN MAGNETIC MICROSPHERES. Targeting the drug with magnetic microspheres was first described by Widder et

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1 5. PREPARATION OF GELATIN MAGNETIC MICROSPHERES 79 Targeting the drug with magnetic microspheres was first described by Widder et al. (1979a), who used magnetically responsive biodegradable drug carrier with the capacity to localize both carrier and therapeutic agent, by magnetic means to a specified in vivo target site. These magnetic microspheres consist of magnetite (Fe 3 O 4) particles, which are responsible for magnetic property, and a therapeutic agent entrapped in the biodegradable polymer matrix. Magnetite are biocompatible iron (II, III) oxide particles with no toxicity (Lübbe et al., 1996), hence many authors utilized this material in the preparation of magnetic microspheres, to target toxic drugs particularly for anticancer therapy (Kato, 1983; Gupta and Hung, 1989). Very few literature are available in the development of magnetic microspheres with anti-inflammatory drugs. Lalla and Ahuja (1991) attempted to develop magnetic microspheres with anti-inflammatory drug in order to target it into inflammatory site. In general magnetic microspheres are infused into an artery supplying a given in vivo target site. A magnet of sufficient field strength to retard the microspheres solely at the capillary level vasculature is placed externally over the target area. These procedures result in a localized depot of the drug at the site of action which is the desired property of this target system. Because of arterial administration with subsequent magnetic localization, the majority of infused microspheres does not circulate systemically. Hence the infused microspheres are not cleared by the macrophages/reticuloendothelial system. This targeting system allows therapeutic levels of drugs to be attained at a desired target with smaller doses and thus avoiding side effects due to accumulation of the drug at non-target area.

2 80 Albumin magnetic microspheres are usually prepared by dispersing the magnetite and drug in albumin solution, then emulsified in suitable dispersion medium and crosslinked with formaldehyde or denatured by heating to form microspheres (Widder et al., 1979a; Gupta et al., 1988). Two types of ferromagnetic ethylcellulose microcapsules containing mitomycin C as core material were reported by Kato (1983). In the first type the ferrite particles responsible for magnetic property are attached to the microcapsule surface and in the second type ferrite particles were encapsulated inside the microcapsules. Microcapsules of first type were prepared by coacervation technique and the second type were prepared by solvent evaporation technique. The microspheres prepared by the later technique had high magnetic response because of high ferrite content. As reported in the literature, the following parameters are to be considered in the preparation of magnetic microspheres (Kato, 1983; Langer et al., 1985; Papisov et al., 1987; Gupta et al., 1988; Gupta and Hung, 1989). Particle size: The size of microspheres to be prepared mainly depends on the route of administration. A size range of μm was suggested for injection into aorta and artery. For intravenous administration size range of below 5 μm was suggested. The drug release from the microspheres is also influenced by the particle size. Magnetite content: High magnetic response could be obtained by preparing microspheres with high magnetite content. The magnetite content of the magnetic microspheres plays a crucial role in the targeting efficiency and the microspheres prepared with high magnetite content can be retained in the target site with help of suitable magnet (Gupta and Hung, 1990b).

3 81 Drug loading and release kinetics: The magnetic microspheres should be formulated with optimum drug loading, in order to deliver effective dose at the site of action, with single administration for prolonged period. The drug release characteristics of the magnetic microspheres should be optimized to get required release kinetics. In the present study the magnetic microspheres loaded with diclofenac sodium is prepared by using gelatin as a carrier. The development of magnetic microspheres by using gelatin as carrier material is a new area and however a meager amount of research work has been carried out (Wu et al., 1993) in this area. The gelatin magnetic microspheres were prepared by emulsification and cross-linking technique by using glutaraldehyde as cross-linking agent Materials and Methods Gelatin, type-b, 300 bloom strength was purchased from Sigma Chemicals, USA. Magnetite powder, less than 5 μm was purchased from Sigma-Aldrich Chemie GmbH, Germany. Magnetite powder, less than 1 μm was supplied by Liquids Research Limited, United Kingdom. Diclofenac sodium, gifted by MARAL, Chennai, India. Anhydrous ether, isopropyl alcohol, toluene, span 80 and glutaraldehyde were purchased from S.D. Fine Chemicals Ltd, Boisar, India and Sesame oil (Idhayam) was purchased from Food World, Chennai, India. Neodymium magnet, 8000 Gauss field strength and 400 Gauss/cm field gradient, 30 mm diameter and 4 mm thickness was purchased from ABY systems (PVT) Ltd., Red Hills, Chennai, India. All other reagents used were of analytical grade. Glutaraldehyde-saturated toluene solution was prepared as described in the Section 4.

4 Preparation of gelatin magnetic microspheres loaded with diclofenac sodium for intra-arterial administration Gelatin magnetic microspheres MG 1, MG 2 and MG 3 loaded with diclofenac sodium for intra-arterial administration were prepared as follows. As given in the Table 5.1, required quantity of gelatin was dissolved in 3 ml of phosphate buffer (ph 7.4) by heating at 60ºC. Specified quantity of diclofenac sodium was dissolved separately in 3 ml of phosphate buffer (ph 7.4) by heating at 60ºC and added to gelatin solution. Required quantity of magnetite (less than 5 μm) was wetted with 1 ml of 50% v/v alcohol and added to gelatin-diclofenac solution. The mixer was homogenized (Remi, India, 5000 rpm) to make uniform dispersion of magnetite. Then the mixture was added drop wise to 100 ml of sesame oil with 1% w/v span 80 preheated to 60ºC and emulsified by stirring with help of hand blender (10,000 rpm/5 min). After getting the required globule size, the stabilized emulsion was stirred with a stirrer (Remi, India) attached to a motor (approx.1000 rpm). Table 5.1. Formula for the preparation of gelatin magnetic microspheres loaded with diclofenac sodium. Batch No. Amount of gelatin used Amount of diclofenac sodium used Amount of magnetite used Yield a Theoretical drug loading (%) Theoretical magnetite content (%) MG ! MG ! MG ! MG ! MG ! MG ! a Values are mean! SE (n=3).

5 83 Ten ml of glutaraldehyde-saturated toluene solution was added slowly and stirring was continued for 6 h at room temperature. The cross-linked microspheres were allowed to sediment by placing a 8000 G magnet at the bottom of the beaker. Then the microspheres were washed with anhydrous ether to remove sesame oil. Then it was washed with 3x10 ml of 5% w/v sodium metabisulphite, 2x10 ml water and 2x10 ml of isopropyl alcohol. During each washing, the microspheres were collected by placing a magnet of 8000 G strength at the bottom of the beaker. After washing, the microspheres were dried at 45ºC, transferred to glass vials and stored in a desiccator. Batches MG (without both drug and magnetite) and MG 0 (without drug but with magnetite) were prepared similarly in order to study the effect of drug loading and magnetite content on the particle size of the microspheres Preparation of gelatin magnetic microspheres loaded with diclofenac sodium for intravenous administration Gelatin magnetic microspheres loaded with diclofenac sodium MG 4, for intravenous administration, was prepared by the slight modification of the above procedure. Magnetite of lesser particle size (less than 1 μm) was employed in the preparation. Gelatin (750 mg) was dissolved in 3 ml of phosphate buffer (ph 7.4) heated to 60ºC. Diclofenac sodium (125 mg) was dissolved separately in 3 ml of phosphate buffer (ph 7.4) by heating and added to gelatin solution. Required quantity of magnetite was wetted with 1 ml of 50% v/v ethanol, added to gelatin-diclofenac sodium solution and the mixture was homogenized (Remi, India, 5000 rpm). Then the resulting mixture was added drop wise to 100 ml of oil phase (75 ml of sesame oil and 25 ml of anhydrous ether containing 1% v/v span 80) preheated to 45ºC and emulsified by stirring with help of hand blender (10,000 rpm/10 min). After getting the required

6 84 globule size, the stabilized emulsion was stirred with help of a stirrer attached to a motor (Remi, India, approx.1000 rpm). Then the magnetic microspheres were crosslinked and recovered as described in the preparation of magnetic microspheres for intraarterial administration Results and Discussion The procedure followed to prepare the magnetic microspheres produced good yield of microspheres with free flowing nature. When the suspension of microspheres was placed near a magnet of 8000 G, their magnetic response was good and they were readily attracted by the magnet. This property was exploited in the recovery of the microspheres as described in the preparation procedure. The sesame oil with 1% v/v span 80 was found to produce spherical microsphere without aggregation. Glutaraldehyde-saturated toluene solution was used to cross-link and stabilize the magnetic microspheres. After stabilization the unreacted glutaraldehyde was neutralized by using sodium metabisulphite. Gelatin magnetic microspheres MG 1, MG 2 and MG 3, loaded with diclofenac sodium were prepared for intra-arterial administration. Magnetite particles of less than 5 μm was used in the preparation of magnetic microspheres. Since particles of μm can be administered conveniently (Kato, 1983; Gupta and Hung, 1989) by intra-arterial route, the preparation conditions were standardized (Saravanan et al., 2003ab) to produce microspheres of this range. Since gelatin magnetic microspheres MG 4, was prepared for intravenous injection, a size range of less than 5 μm was prepared by slightly modifying the preparation conditions. Magnetite particles of less than 1 μm were used in this formulation. The stirring speed and gelatin/polymer content were optimized by observing the particle size under microscope attached with micrometer.

7 85 The formula given in Table 5.1 and the preparation conditions described in the procedure produced the particles of desired size. In the preparation of MG4 the viscosity of external phase was reduced by diluting the oil with ether. The viscosity of internal phase was reduced by using less concentration of gelatin and drug. Thus reduction in viscosity of the phases in combination with high stirring conditions resulted in reduced particle size. The process of drug localization by magnetic microspheres is based on the competition between forces exerted on the blood compartment, and magnetic forces exerted between the microspheres and applied magnetic field. When the magnetite forces exceed the linear blood flow rates in arteries (10 cm/sec) or capillaries (0.05 cm/sec), the microspheres are retained at the target site. It has been suggested that at the arterio-capillary blood flow rate of to 0.1 cm/sec, 20% w/w magnetite is sufficient to achieve 100% retention of the magnetic carrier using 8000 Gauss magnet (Gupta and Hung, 1989). In an in vitro experiment it was demonstrated that 28% w/w of magnetite in nanoparticles is necessary for their effective targeting. Many authors were formulated magnetic microspheres with magnetite content of 15-22% w/w and reported their targeting efficiency (Gupta and Hung, 1989 ) in various tissues. After reviewing all above factors available in the literature and since the magnetic microspheres are going to be localized in the femoral artery, in the present study, they were prepared with higher amount of magnetite (30% w/w) to withstand arterial pressure under a magnetic field produced by 8000 Gauss magnet. Magnetic microspheres formulated as per the formula shown in Table 5.1 were used for further physiochemical characterization.