Jun Hee Cho 1*, Sang Gil Ko 1, Yangkyu Ahn 1, Ki-Chang Song 2, Eun Jung Choi 3 1 Department of Nanochemistry & Biochemistry, Konyang University Nonsan, Chungnam, Korea Department of Chemical Engineering, Konyang University Nonsan, Chungnam, Korea Department of Opthalmic Optics, Konyang University Daejeon , Korea Synthesis and Characterization of Biopolymer-coated Magnetite Nanoparticles

Abstract Magnetic nanoparticles can be used for a variety of biomedical applications. They can be used in the targeted delivery of therapeutic agents in vivo, in the hyperthermic treatment of cancers, in magnetic resonance(MR) imaging as contrast agents and in the biomagnetic separations of biomolecules. We have synthesized magnetite nanoparticles using sonochemical technique 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. Magnetic colloid suspensions containing particles with sodium oleate have been prepared. To prepare a biopolymer-coated magnetite nanoparticles, chitosan or glucan solution was added to the magnetic colloid suspensions under the ultrasonication at room temperature for 30min. Then, tween 20 was added to stabilize the bioplymer- coated magnetite colloid. Nanoparticles, both oleate-coated and bioplymer- coated, have been characterized by several techniques. Atomic force microscope (AFM) was used to image the coated nanoparticles. Magnetic hysteresis measurement were performed using a superconducting quantum interference device (SQUID) magnetometer at room temperature. DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Synthesis of uniform magnetite nanoparticles using sonochemical method Control of the size of the magnetite particles Synthesis of biopolymer-coated magnetite nanoparticles for medical applications One of majors applications is a magnetic resonance imaging (MRI) reagent as a contrast agent Synthesis of uniform magnetite nanoparticles using sonochemical method Control of the size of the magnetite particles Synthesis of biopolymer-coated magnetite nanoparticles for medical applications One of majors applications is a magnetic resonance imaging (MRI) reagent as a contrast agent Superparamagnetic Size < 20 nm Narrow size distribution Spherical Shape Low toxicity Superparamagnetic Size < 20 nm Narrow size distribution Spherical Shape Low toxicity Requirements of magnetite nanoparticles for medical applications Objectives of this study DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

 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(10 9 K/sec)  Advantages - have a narrow size distribution & control of particle size Sonochemistry ? DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Structures and properties of chitosan Chitosan Chitin Deacetylation Chitosan is extracted from shellfish waste Molecular form : (-C 6 H 11 NO 4 -)n Chitosan has an average molecular weight ranging between 3.8X10 3 ~ 2.0X10 6 Dissolve in acid solution (pH<6.5) Gelate in alkali solution Anticancer effect Antacid Non toxic, Biocompatible, Non carcinogenic DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Structures and properties of β-glucan Extraction is extracted from mushroom Molecular form : (-C 6 H 10 O 5 -)n Dissolve in alkali solution Gelate in acid solution Reducing serum cholesterol levels Anticancer effect Diet food Non toxic, Biocompatible, Non carcinogenic β-glucan DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

MRI contrast agent Magnetite nanoparticles Magnetite nanoparticles Biopolymer(chitosan or beta glucan) Gadopentetate type Magnetite nanoparticle type In liver  T1WI(T1-weighted image): spin-lattice relaxation time → increased signal intensity of normal liver parenchyma → cancer cell was presented darker than normal cell  T2WI(T1-weighted image): spin-spin relaxation time → decreased signal intensity of normal liver parenchyma → cancer cell was presented brighter than normal cell Not change of contrast of the cancer cell by MRI contrast agent. The contrast of cancer cell does not change by MRI contrast agent Magnetite nanoparticles are useful for T2-weighted MRI Magnetite nanoparticles are easily taken up by Kupper cells in the liver via reticuloendothelial system (RES), because the SPIO are smaller than erythrocytes. The SPIO enhanced MRI contrast for liver and spleen DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Synthesis of magnetite nanoparticles FeCl 3 ·6H 2 O (0.3M, 30ml) FeCl 2 ·4H 2 O (0.15M, 30ml) optimum conditions:  add Precipitator ( TMAOH, 72mmol )  irradiation power : 250 W  irradiation time : 30min  irradiation temperature : keeping 70 ~ 80 ℃ optimum conditions:  add Precipitator ( TMAOH, 72mmol )  irradiation power : 250 W  irradiation time : 30min  irradiation temperature : keeping 70 ~ 80 ℃ + metal chloride mixture aqueous solution add oleic acid (sodium form) heating to 70 ℃ sieve washing by centrifuge uniform magnetite nanoparticles Biomedical application DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Size distribution of magnetite nanoparticles R=[water] / [surfactant] Particles size (nm) Distribution (%) S.Dev (nm/%) Magnetite nano particles (1) /13.3 (2) /11.2 (3) /9.8 (4) /10.8 (1) (3) (4) (2) DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

AFM images R=[water]/[sulfactant]=95 Mean size = 3.5 nm R=[water]/[sulfactant]=133 Mean size = 6.4 nm 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 R=[water]/[sulfactant]=133 Characterizations of magnetite nanoparticles Superparamagnetic Size < 20 nm Spherical Shape Biomedical application DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

10%(W/V) Ferrofluid 10ml Optimum conditions:  irradiation power : 150 W  irradiation time : 20min 0.07% (w/v) Chitosan or 0.1%(w/v) beta glucan solutin Biopolymer coated magnetite Separation of large size particles biopolymer coated magnetite fluid Preparation of biopolymer coated magnetite nanoparticles MRI contrast agent DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

IR spectra of magnetite & biopolymer coated magnetite nanoprticles cm -1 Transmittance (%) cm -1 Transmittance (%) (a) (b) (c) (a) (b) (c) (a) Magnetite nanoparticles (b) chitosan (c) chitosan coated magnetite (a) Magnetite nanoparticles (b) Beta glucan (c) Beta glucan coated magnetite 1643Cm Cm Cm Cm Cm Cm -1 DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

[water]/ [sulfactant] Magnetite nanoparticles size (nm) Chitosan coated magnetite Beta glucan coated magnetite Avr, size (nm) S.Dev (nm/%) Avr, size (nm) S.Dev (nm/%) R=952.2(100%)4.10.4/ /11.2 R=1044.1(99.5%)5.60.6/ /10.5 R=1145.6(97.9%)7.70.8/ /10.5 R=1339.0(94.1%) / /10.7 Size distribution of biopolymer coated magnetite nanoparticles Beta glucan coated magnetite (R=95) Chitosan coated magnetite (R=95) DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Characterizations of biopolymer coated magnetite nanoparticles Magnetic hysteresis curve Chitosan coated magnetite(R=133) Beta glucan coated magnetite(R=133) AFM image Chitosan coated magnetite(R=133) Mean size = 10.7 nm Beta glucan coated magnetite(R=133) Mean size = 10.1 nm Superparamagnetic Size < 20 nm Spherical Shape MRI contrast agent DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

MR images of biopolymer coated magnetite nanoparticle suspension ※ Fe concentration M M M M M Chitosan coated magnetite nanoparticles Beta glucan coated magnetite nanoparticles T1(left)- and T2(right)-weighted MR Images DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY

Conclusion s (1) Magnetite nanoparticles were synthesized by sonochemical method (2) The size of particles can be controlled by concentration of surfactant (3) The synthesized particles show narrow size distribution under S.D 10% (4) The magnetite nanoparticles were coated by biopolymer as a chitosan and beta glucan (5) Both magnetite nanoparticles and biopolymer coated magnetite nanoparticles were revealed superparamagnetism (6) The biopolymer coated magnetite nanoparticles showed a strong enhancement of MR image contrast in vitro. DEPARTMENT OF CHEMISTRY, GRADUATE SCHOOL, KONYANG UNIVERSITY