Supporting Information

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1 Supporting Information Detailed Experimental Procedure Materials Water-soluble chitosan oligosaccharide (M w =3-5 kda, deacetylation degree=95.2%) was purchased from Kittolife Co. (Pyeongtaek, Korea). Iron (III) acetylacetonate (Fe(acac) 3 ), 4- biphenylcarboxylic acid were purchased from Acros Organics (Geel, Belgium). Oleic acid, benzyl ether, hydrocaffeic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), 4,6- diamidino-2-phenylindole (DAPI), and Nuclear Fast Red solution were purchased from Sigma-Aldrich (Minnesota, USA). Ferumoxide suspension (Feridex) was supplied by Berlex Laboratories (Wayne, NJ). Magic Red caspase detection kit (Immunochemistry Tech.), CCK-8 cell viability assay kit (Dojindo Lab.), and in situ cell death detection kit (Roche) were used according to the manufacturer s instructions. All other chemicals were of analytical grade. Synthesis of DOPA-conjugated chitosan oligosaccharide (chitosan-dopa) Chitosan-DOPA was synthesized by conjugating a carboxylic acid group of hydrocaffeic acid to amino groups of chitosan using carbodiimide chemistry. Briefly, hydrocaffeic acid ( mg) and EDC ( mg) were co-dissolved in 50 ml of deionized water/ethanol mixture (1:1, v/v). This solution was slowly added to a stirred solution (100 ml, ph 5.5) containing 1 g of chitosan oligosaccharide, and reacted for 12 h. The solution ph was maintained at 5.5 to avoid irreversible oxidation of catechol group of hydrocaffeic acid. The resultant chitosan-dopa conjugate was purified by extensive dialysis against ph 5.0 HCl solution for 2 days (M w cutoff of 1 kda) and deionized water for 4 h, and then lyophilized. The chemical structure of chitosan-dopa was examined by Bruker Avance 1 H NMR spectrometer operating at 400 MHz. The degree of substitution was estimated to be ca. 30.9% by comparing the relative peak area of three protons on a DOPA phenyl ring (δ= ppm) and an acetyl group ( COCH 3, δ=1.95 ppm) of the monosaccharide residue in chitosan. Nanoparticle characterization 1

2 The synthesized nanocubes were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), and superconducting quantum interference device (SQUID). The TEM images were taken on a JEOL JEM-2010 electron microscope at 200 kv. The XRD patterns of nanocubes were obtained using a Rigaku D/Max-3C diffractometer equipped with a rotation anode and a Cu Kα radiation source ( = nm). The magnetic data were acquired using a magnetic property measurement system (MPMS) 5XL Quantum design SQUID magnetometer at room temperature. The hydrodynamic diameter and zeta potential value of Chito-FIONs were measured in 0.1 M phosphatebuffered saline (PBS, ph 7.4) solution at 37 C using a dynamic light scattering instrument (Zeta-Plus, Brookhaven, NY) equipped with a He-Ne laser at a wavelength of 632 nm. The metal concentration of the nanocubes was quantified using inductively coupled plasma absorption emission spectroscopy (ICP- AES). Evaluation of magnetic targeting capability of Chito-FIONs Human lung carcinoma A549 cells were seeded on a 4-well chamber slide at a density of cells per well and incubated for 24 h at 37 C. The cells were treated with 50 μl of Chito-FIONs or Feridex (150 μg Fe/mL) and then incubated for 12 h at 37 C either with or without exposing to a cylindrical neodymium magnet (10 mm length, 1.5 mm radius, 3759 G). The treated cells were washed with PBS solution three times, and then fixed with 1% (w/v) formaldehyde solution. The extent of cellular targeting was evaluated by Prussian Blue staining. In brief, the cells were incubated with a 1:1 (v/v) mixture of 2% potassium ferrocyanide solution and 2% HCl solution. After incubation for 30 min at 37 C, the cells were observed using a Nikon TE300 inverted microscope equipped with a digital microscope camera (Polaroid DMC2, USA) and a LSM510 confocal laser scanning microscope (Carl Zeiss, Germany). The cellular Fe content was quantified using inductively coupled plasma absorption emission spectroscopy (ICP-AES). Briefly, the treated cells were collected by scraping and counted by using a hemacytometer. After the cells were lysed with 10 μl of nitric acid, the lysate was resuspended in 5 ml of deionized water and then analyzed by using a Polyscan 60E ICP-AES equipment (Thermo Jarrel Ash, USA). 2

3 Cell viability assay A549 cells were seeded in a 96-well plate at a density of cells per well and grown for 24 h at 37 C. The cells were then incubated with the culture medium containing Chito-FIONs or Feridex at varying concentrations up to 100 μg/ml for 12 h at 37 C. The number of viable cells was determined by the CCK-8 cell viability assay. In brief,10 μl of CCK-8 solution was added to 100 μl of serum-free RPMI medium in each well of the plate. After incubating the plate for 1 h at 37 C, the absorbance at 450 nm was measured using a Bio-Rad microplate reader. In vivo magnetic hyperthermia and anti-tumor effect of Chito-FIONs Male BALB/C nude mice (7-8 weeks of age, about 20 g) were housed in a pathogen-free environment, and provided with sterilized food and water. All animal experiments were conducted in accordance with the guidelines provided by Institutional Animal Care and Use committee of KAIST. The mouse tumor model was generated by injecting 200 μl ( cells) of A549 cell suspension into the subcutaneous region of the mice. When the volume of the A549 tumor xenograft reached 50 mm 3, Chito-FIONs or Feridex (375 μg Fe/kg body weight) was intratumorally administered. Saline was used for the control group of mice. Magnetic hyperthermia was carried out for 20 min using a RF generator (T&C Power Conversion, Inc., Rochester, NY) on the same day of injection. Tumor growth was monitored daily by measuring perpendicular diameters of the tumors using a digital caliper. The respective tumor volume was calculated from the following formula: tumor volume = 0.5 (major axis) 2 (minor axis). Histological examination For histological analysis, the mice bearing the A549 tumor xenografts were sacrificed and dissected at 6 days post-injection. Excised tumor tissues were fixed with 4% (v/v) formaldehyde in PBS solution (ph 7.4), embedded in paraffin blocks, and then sectioned into 10-μm-thick slices. The sections were stained with Prussian Blue and counterstained with Nuclear Fast Red to examine the distribution of Chito-FIONs or Feridex inside the tumor tissues. The Prussian Blue-stained tumor area was quantitatively assessed by using Image J software (NIH Image) to examine the intratumoral amount of Chito-FIONs and Feridex after the hyperthermia treatment. Apoptotic area in the tumor was observed 3

4 by terminal deoxynucleotidyl transferase-mediated 2 -deoxyuridine 5 -triphosphate-biotin nick end labeling (TUNEL). TUNEL assay was performed by using in situ cell death detection kit (Roche) which produces a green fluorescence signal within apoptotic area. The cell nuclei were also stained with DAPI (1.5 μg/ml in PBS solution). The resulting images were then acquired using a Nikon TE300 inverted microscope equipped with a digital microscope camera (Polaroid DMC2, USA). Statistical analysis Statistical analysis was performed using a standard Student s t-test. All data are expressed as mean±standard deviation. Statistical significance was determined, with P values less than 0.05 considered statistically significant. 4

5 Figure S1. 1 H NMR spectrum of DOPA-conjugated chitosan oligosaccharide (chitosan-dopa). Figure S2. TEM images of pristine ferrimagnetic iron oxide nanocubes (FIONs) and chitosan-dopastabilized ferrimagnetic iron oxide nanocubes (Chito-FIONs). 5

6 Figure S3. FT-IR spectra of (a) pristine FIONs, (b) Chito-FIONs, and (c) chitosan-dopa. Figure S4. Field-dependent magnetization curve of Chito-FIONs, FIONs, and Feridex at room temperature. 6

7 Figure S5. Temperature versus time graphs of (a) Chito-FIONs and (b) Feridex dispersion with various Fe concentrations (75, 150, and 225 μg Fe/mL) under an AC magnetic field. Figure S6. (a) TEM image and (b) DLS data of polyethylene glycol (PEG)-phospholipid-coated FIONs. (c) Temperature versus time graphs of Chito-FIONs, PEG-phospholipid-coated FIONs, and Feridex dispersion at the same Fe concentration (150 μg Fe/mL) under an AC magnetic field. 7

8 Figure S7. Prussian Blue staining images of A549 cells treated with Chito-FIONs or Feridex (7.5 μg Fe/mL) for 12 h at 37 C either with or without exposing to a cylindrical neodymium magnet (10 mm length, 1.5 mm radius, 3759 G). Figure S8. Cellular Fe content of A549 cells following treatment with Chito-FIONs or Feridex (7.5 μg Fe/mL) for 12 h at 37 C either with or without applying a magnet (3759 G). After the treated cells were harvested and counted, the cellular Fe content was quantified using inductively coupled plasma absorption emission spectroscopy (ICP-AES). 8

9 Figure S9. Confocal Z-stack images of A549 cells treated with (a) Chito-FIONs or (b) Feridex (7.5 μg Fe/mL) for 12 h at 37 C with exposing an exterior magnet (3759 G). The top and right side of the image shows a cross-section of the cells in the x-z and y-z axis, respectively. Red and green arrowheads denote the location of the cell and iron oxide nanoparticles (Chito-FIONs or Feridex), respectively. Figure S10. Viability of A549 cells after treatment with Chito-FIONs or Feridex at various concentrations for 12 h at 37 C. Both Chito-FIONs and Feridex were not toxic to A549 cells at a concentration (7.5 μg Fe/mL) used in this study. 9

10 Figure S11. Confocal microscopic images of A549 cells treated with magnetic targeting of ChitoFIONs or Feridex (7.5 μg Fe/mL) without an AC magnetic field. Fluorescent images were acquired after staining with Live-dead cell staining reagent. Live and dead cells appear green and red, respectively. Figure S12. Confocal microscopic images of A549 cells treated with an AC magnetic field without magnetite nanoparticles. Fluorescent images were acquired after staining with (a) Live-dead cell staining reagent and (b) Magic Red caspase detection kit. This result clearly revealed that application of an AC magnetic field itself did not exert any cytotoxic and apoptotic effect against A549 cells. 10

11 Figure S13. Representative photographs of A549 tumor-bearing mice taken at day 6. The mice were intratumoral injected with Chito-FIONs or Feridex (375 μg Fe/kg body weight) and subsequently treated with an AC magnetic field for 20 min. Saline was used for the control group of mice. 11

12 Figure S14. Histological cross sections of A549 tumors unexposed to an AC magnetic field. (a) Prussian Blue staining images of tumor sections taken at day 6 following intratumoral injection of Chito-FIONs or Feridex. (b) Confocal microscopic images of TUNEL-stained tumor sections. Apoptotic cells emit green fluorescence signal. Cell nuclei were also stained with DAPI (blue fluorescence). 12

13 Figure S15. The Prussian Blue-stained tumor area determined at day 6 following intratumoral injection of Chito-FIONs or Feridex. The histological cross sections of A549 tumors were quantitatively assessed by using Image J software (NIH Image). Statistically significant difference between two groups, ** = P < Figure S16. Time course changes of body weight of mice during the treatment. 13