Preparation of magnetic β - glucan microspheres by microemulsion method for targeting drug delivery system Jun Hee Cho 1*, Sang Gil Ko1, Yangkyu Ahn1, Ki-Chang Song2, Eun Jung Choi3 1Department of Nanochemistry & Biochemistry, Konyang University Nonsan, Chungnam, Korea 320-711 2Department of Chemical Engineering, Konyang University 3Department of Opthalmic Optics, Konyang University Daejeon 302-718, Korea www.themegallery.com

Abstract Magnetic polymer particles can be used for several clinical applications. They were recently introduced as a suitable material for drug targeting, hyperthermia, embolotherapy and magnetic resonance imaging agent. We have synthesized uniform nanometer sized magnetite particles using chemical coprecipitation technique through a sonochemical method with surfactant such as oleic acid. The average size of the magnetite particles can be controlled by the ratio R = [H2O]/[surfactant] in the range of 2 to 8nm. The size of the magnetite nanoparticles prepared by this method show narrow distribution. Powder X-ray diffraction and electronic diffraction measurements show the spinel structure for the magnetite nanoparticles. Morphology of magnetite nanoparticlewas investigated using atomic force microscope(AFM). Magnetic β-glucan microspheres were prepared by a water-in-oil suspension-crosslinking technique. Magnetite nanoparticles were dispersed in a β-glucan solution in alkali solution. The dispersion was added to iso-octane containing Span 80 as a surfactant with stirring. An anticancer drug, 5-Fluorouracil(5-Fu), was immobilized into the magnetic β-glucan microspheres by a swelling method. The magnetic β-glucan microspheres were crosslinked with glutaraldehyde. The surface, and the morphology of the magnetic β-glucan microsphere particles were characterized using optical microscope and scanning electron microscope(SEM). Magnetic hysteresis measurement were performed using a superconducting quantum interference device (SQUID) magnetometer at room temperature to investigate the magnetic properties of the magnetic β-glucan microspheres. The amount of 5-Fluorouracil(5-Fu) released into the aqueous media was measured by UV spectroscopy at 265 nm. DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Requirements of magnetite nanoparticles for medical application Objective of this study Synthesis of uniform magnetite nanoparticles using sonochemical method Control of the size of the magnetite particles Preparation of magnetic β-glucan microspheres capable of targeted drug to minimize burst Control of drug-release rate by concentration of crosslingking agent • Superparamagnetic • Size < 20 nm • Narrow size distribution • Spherical Shape • Low toxicity uniform magnetite nanoparticles Requirements of magnetite nanoparticles for medical application DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Sonochemistry ? Acoustic Cavitation - the formation, growth, and implosive collapse of bubbles in a liquid Cavitational Collapse - intense local heating(~5000 K) - high pressures(~1000 atm) - enormous heating and cooling rates(109 K/sec) Advantages - have a narrow size distribution & control of particle size DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Non toxic, Biocompatible, Structures and properties of β-glucan β-glucan Extraction is extracted from mushroom Molecular form : (-C6H10O5-)n Dissolve in alkali solution Gelate in acid solution Reducing serum cholesterol levels Anticancer effect Diet food Non toxic, Biocompatible, Non carcinogenic DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Targeting drug delivery system ? Drug loaded magnetic biopolymer microspheres Magnet Blood vessel Biopolymer Magnetite nanoparticles Drug Magnetic drug carrier Time Drug Level Maximum Designed Level Minimum Effective Level Toxic Level Dose Controlled Release DDS ODDS Conventional Administration Discontinuous drug-release Side effect Frequent dose Biocompatible Controlled Release Drug delivery system (DDS) DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Synthesis of magnetite nanoparticles FeCl2·4H2O (0.15M, 30ml) add oleic acid (sodium form) heating to 70℃ + sieve washing by centrifuge metal chloride mixture aqueous solution FeCl3·6H2O (0.3M, 30ml) optimum conditions:  add Precipitator ( TMAOH, 72mmol )  irradiation power : 250 W  irradiation time : 30min  irradiation temperature : keeping 70 ~ 80℃ uniform magnetite nanoparticles Biomedical application DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Size distribution of magnetite nanoparticles (1) (3) (4) (2) R=[water] / [surfactant] Particles size (nm) Distribution (%) S.Dev (nm/%) Magnetite nano particles (1) 95 2.2 100.0 0.3/13.3 (2) 104 4.1 99.5 0.4/11.2 (3) 114 5.6 97.9 0.5/9.8 (4) 133 9.0 94.1 0.8/10.8 DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Characterizations of magnetite nanoparticles AFM images R=[water]/[sulfactant]=95 Mean size = 3.5 nm R=[water]/[sulfactant]=133 Mean size = 6.4 nm 220 311 400 422 511 440 X-ray diffraction pattern -- R=[water]/[sulfactant]=133 Mean size= 9.0 nm -- R=[water]/[sulfactant]=95 Mean size= 4.1 nm Magnetic hysteresis curve • Superparamagnetic • Size < 20 nm • Spherical Shape Biomedical application DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Preparation of magnetic β-glucan microspheres magnetite colloid & β-glucan mixture add EtOH vacuum dry washing iso-octane Span 80 at 40℃ ((( ))) H S magnetic β-glucan microspheres optimum conditions:  stirring time : 90 min  stirring temperature : room temperature β-glucan Magnetite nanoparticles DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Preparation of drug loaded magnetic β-glucan microspheres Model drug 5-Fluorouracil(5-Fu) Crosslinking agent Glutaraldehyde Magnetite nanoparticles ` ` ((( ))) ` add EtOH D.I water freeze dry β-glucan 30min room temperature washing ((( ))) H S Drug Sonicator (water bath type) In vitro release studies optimum conditions:  stirring time : 90 min  stirring temperature : room temperature measurement : using UV spectrophotometer  absorption of UV spectrophotometer : 265nm  incubate temperature : keeping 37.0±0.5℃ ` Water bath DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Optical microscope images of magnetic β-glucan microspheres (1) (2) Span 80 (ml) Iso-octane(ml) 5% (w/v) β-glucan(ml) Glutaraldehyde (mol) Size(㎛) Microsphere 1 6 140 20 0.015 31.58 Microsphere 2 10 25.53 DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

SEM images of magnetic β-glucan microspheres Crosslinked with glutaraldehyde DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Characterization of drug release (1) (2) (3) Non crosslinking 0.0005 mol glutaraldehyde 0.0015 mol glutaraldehyde Loading efficiency : ( Calculated drug concentration ) ( Theoretical drug concentration ) ×100 Crosslinking agent (mol) Loading efficiency (%) Released (%) Magnetic β-glucan microspheres (1) non crosslinking 91.0 96.2 (2) 0.0005 25.9 75.5 (3) 0.0015 24.9 65.8 DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Conclusion (1) Magnetite nanoparticles were synthesized by sonochemical method (2) The particle size of magnetite and microsphere can be controlled by concentration of surfactant (3) The synthesized particles show narrow size distribution under S.D 10% (4) Magnetic β-glucan microspheres were prepared by a water-in-oil suspension-crosslinking technique (5) The in vitro release profile revealed the ability of microspheres to prolong the drug release for more than 24 hrs (6) The water soluble magnetic β-glucan microspheres showed more sustained drug release profiles by increasing crosslinking agent. DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY