1. Synthesis and Characterization of Magnetic ` Chitosan Microspheres for Medical Applications ` Sang Gil Ko 1 *, Jun Hee Cho 1, Yang kyu Ahn 1, Ki Chang Song 2, Eun Jung Choi 3 1 Department of Nanochemistry & Biochemistry, Konyang University Nonsan, Chungnam, Korea 320-711 2 Department of Chemical Engineering, Konyang University Nonsan, Chungnam, Korea 320-711 3 Department of opthalmic Optics, Konyang University Daejeon 302-728, Korea

2. Microspheres composed of superparamagnetic magnetite nanoparticles and chitosan were developed as a novel MRI-detectable embolic material. We have synthesized magnetite nanoparticles using sonochemical method with sodium oleate as surfactant. Nanoparticle size can be varied from 2 to 8 nm by controlling the sodium oleate concentration. Magnetite phase nanoparticles could be observed from X-ray diffraction. The synthesized magnetite nanoparticles were embedded in a polyglucosamine (chitosan) by sonochemical method. The ferrofluid, solution of magnetite-embedded chitosan, was sprayed on the surface of an alkali solution with a nozzle to produce magnetic chitosan microspheres. The morphology of the magnetic chitosan microsphere was characterized using optical microscope and scanning electron microscope. Magnetic hysteresis measurement were performed using a superconducting quantum interference device (SQUID) magnetometer at room temperature to investigate the magnetic properties of the chitosan microspheres including magnetite nanoparticles Abstract

3. Magnetic inks Magnetic memory device Magnetic fluids Clinical applications (NT+BT) - drug delivery - cell separation - hyperthermia - MRI imaging - embolotherapy Applications of magnetite nanoparticles

4. Embolization - is a process in which a blood vessel or organ is obstructed by the lodgment of a material mass (as an embolus): used in cancer treatment Diseases treated by embolotherapy - Blood vessel trouble - Bleeding - Tumor treatment Requirements for Embolization - Biocompatible and non-toxic - No carcinogenic - Relatively benign inflammatory response - No aggregation - Spherical shape Embolotherapy

5. MRI detectable embolus A problem in embolotherapy - Most embolic materials can not be detected by an MRI. Ex) Gel form, PVA (polyvinyl alcohol), coil, micro balloon, silicon bead, glass bead, dehydrogenated ethanol, NBCA(N-butyl 2-cyano acrylate) - MRI-detectable embolus can be effectively used for embolotherapy The objective is to synthesize an MRI-detectable embolus - narrow size distribution of SPIO nanoparticles - chemically stable SPIO ferrofluids - spherical and homogeneous SPIO-chitosan microspheres - contrast enhanced T2-weighted MR images of the microspheres medical application Microcapsule Microsphere magnetite biopolymer

6. FeCl 3 ·6H 2 O (0.3M, 30ml) FeCl 2 ·4H 2 O (0.15M, 30ml) optimum conditions:  add Precipitator ( NH 4 OH, TMAOH or NaOH ) 180mmol, 72mmol, 51mmol  irradiation power : 250 W  irradiation time : 30min  irradiation temperature : keeping 70 ~ 80 ℃ optimum conditions:  add Precipitator ( NH 4 OH, TMAOH or NaOH ) 180mmol, 72mmol, 51mmol  irradiation power : 250 W  irradiation time : 30min  irradiation temperature : keeping 70 ~ 80 ℃ + metal chloride mixture aqueous solution add oleic acid (sodium form) 8~12ml heating to 70 ℃ sieve washing by centrifuge uniform magnetite nanoparticles Synthesis of magnetite nanoparticles

7. Preparation of magnetic biopolymer microspheres 1. 3g chitosan powder + 147ml 1% acetic acid solution → 2% chitosan solution 2. 7.5g β-glucan powder + 142.5ml 1% NaOH solution → 5% β-glucan solution 1. 3g chitosan powder + 147ml 1% acetic acid solution → 2% chitosan solution 2. 7.5g β-glucan powder + 142.5ml 1% NaOH solution → 5% β-glucan solution Magnetite colloid & chitosan solution or β-glucan solution microspheres of magnetic nanoparticles –chitosan or magnetic nanoparticles- β-glucan Nozzle Alkali solution NaOH/Ethanol/Water, 4/30/66, W/V/V Acid solution Acetic acid/Ethanol/Water, 4/30/66, W/V/V N 2 gas 10L/min magnetite nanoparticles(0.5g) + 2% chitosan solution(150ml) magnetite nanoparticles(0.5g) + 5% β-glucan solution 150ml) Optimum conditions:  Irradiation Power: 665 W  Irradiation Time: 15min

8. Molecular form : (-C 6 H 11 NO 4 -)n Chitosan has an average molecular weight ranging between 3.8X10 3 and 2.0X10 6 Dissolved in acid solution (acetic acid) Gelation in alkali solution (NaOH solution) Anticancer effect These biopolymers are nontoxic, biocompatible, non carcinogenic material Chitosanβ-Glucan Chitosan Chitin Deactylation Extraction from mushroom Molecular form : (-C 6 H 10 O 5 -)n Dissolved in alkali solution (NaOH solution) Gelation in acid solution (acetic acid) Anticancer effect Diet food Structure and property of chitosan and β-glucan

9. XRD results of synthesized nanoparticles Sample 1 TMAOH 72 mmol Sample 2 72 mmol [Water] / [Oleic acid]` 104.16 133.32 ` 220 311 400 422 511` 440 Size 6.1 nm 7.5 nm

10. (a)(b)(d)(c) Size distributions of magnetite nanoparticles ( a ) ( b ) ( c ) ( d ) [Water]/ [Oleic acid] 95.23 104.16 114.83 133.32` Particle size (nm) 2.0 3.7 5.4 7.4 S.Dev (%) 12.6 10.6 9.8 10.6

11. 6.4 nm13372mmolMagnetite 2 3.5 nm9572mmolMagnetite 1 Mean sizes[Water] / [Oleic acid]TMAOH` AFM images of magnetite nanoparticles Magnetite 1Magnetite 2

12. Optical microscope image of magnetic biopolymer microspheres Magnetic chitosan microspheres Magnetic β-glucan microspheres

13. SEM image of magnetic biopolymer microspheres Magnetic chitosan microspheres Magnetic β-glucan microspheres

14. Magnetic propertion of magnetite nanoparticles and magnetic biopolymer microspheres ( a ) ( b ) ( c ) Magnetite nanoparticle Magnetic-chitosan microsphere Magnetic-β-glucan microsphere

15. β-glucan microspheres chitosan microspheres MR Imaging results of magnetic biopolymer microspheres T1(left)- and T2(right) weighted MR Images 1 2 3 4 Fe concentration 1. 0.02 M 2. 0.002 M 3. 0.0002 M 4. 0.00002 M 1 2 3 4 1 2 3 4

16. Conclusion (1) We synthesize magnetite nanoparticles by sonochemical method (2) The size of particles can be controlled by the ratio of oleic acid vs water (3) The synthesited particles show narrow size distribution under S.D 5% (4) 2% chitosan, 5% β-glucan solution are excellent viscosity, and could know that form of sphere is made because viscosity does not decrease much even if irradiate ultrasonic (5) Fe concentration of 2mM, the MRI contrast was much higher than that of pure water. Therefore, the ferrofluid may potentially be useful in developing an MR detectable embolic material. (1) We synthesize magnetite nanoparticles by sonochemical method (2) The size of particles can be controlled by the ratio of oleic acid vs water (3) The synthesited particles show narrow size distribution under S.D 5% (4) 2% chitosan, 5% β-glucan solution are excellent viscosity, and could know that form of sphere is made because viscosity does not decrease much even if irradiate ultrasonic (5) Fe concentration of 2mM, the MRI contrast was much higher than that of pure water. Therefore, the ferrofluid may potentially be useful in developing an MR detectable embolic material.