Supporting Information

© Wiley-VCH 2007

69451 Weinheim, Germany

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Development of a T1 Contrast Agent for Magnetic Resonance Imaging Using MnO

Nanoparticles

Hyon Bin Na, Jung Hee Lee,* Kwangjin An, Yong Il Park, Mihyun Park, In Su Lee, Do-

Hyun Nam, Sung Tae Kim, Seung-Hoon Kim, Sang-Wook Kim, Keun-Ho Lim, Ki-Soo

Kim, Sun-Ok Kim, Taeghwan Hyeon*

Synthesis of water-dispersed MnO nanoparticles

Water-dispersible and biocompatible MnO nanoparticles were prepared by the method described previously with some modifications[1,2]. Uniform-sized MnO nanoparticles (XRD pattern shown in figure S1) dispersed in nonpolar organic solvent were synthesized by the thermal decomposition of Mn-oleate complex. 7.92 g of manganese chloride tetrahydrate (MnCl2•4H2O, 40 mmol, Aldrich Chemical Co., 98 %) and 24.36 g of sodium oleate (80 mmol, TCI Co., 95 %) were added to a mixture composed of 30 ml of ethanol, 40 ml of distilled water, and 70 ml of n-hexane. The resulting mixture solution was heated to 70 °C and maintained overnight at this temperature. The solution was then transferred to a separatory funnel and the upper organic layer containing the Mn-oleate complex was washed several times using distilled water. The evaporation of the hexane solvent produced a pink coloured Mn-oleate powder.

Synthesis of MnO nanoparticles is as follows: 1.24 g of the Mn-oleate complex (2 mmol) was dissolved in 10 g of 1-octadecene (Aldrich Chemical Co., 90%). The mixture solution was degassed at 70 °C for 1 hr under a vacuum to remove the water and oxygen. The solution was then heated to 300 °C at a heating rate of 1.9 °C min-1 with vigorous stirring. As the reaction temperature reached 300 °C, the initially pink coloured solution became transparent. Around 300 °C, the colour of the reaction mixture turned to pale green. This colour change indicated that the Mn-oleate complex was thermally decomposed to generate MnO nanoparticles, whereupon nucleation occurred. The reaction mixture was maintained at this temperature for 1 hr to induce sufficient growth. The solution was then cooled to room temperature, and 20 ml of hexane was added to improve the dispersibility of the nanoparticles, followed by adding 80 ml of acetone to precipitate the nanoparticles. The waxy precipitate was retrieved by

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centrifugation. The above purification procedure was repeated two more times to remove excess surfactant and solvent. The purified MnO nanoparticles were dispersible in many organic solvents such as n-hexane and chloroform.

The size of the MnO nanoparticles could be controlled by varying the synthetic parameters such as solvents and aging time. As the reaction time increased from 1 hr to 2 hr, the particle size of the MnO nanoparticles increased from 25 nm to 35 nm. When 1-hexadecene was used as a solvent instead of 1-octadecene and the aging was performed at 280 °C (300 °C for 1-octadecene), smaller sized particles were obtained. For example, the MnO nanoparticles with particle sizes of 7 nm, 15 nm, and 20 nm were obtained when the synthesis was performed in 1-hexadecene at 280 °C with the aging times of 10 min, 30 min, and 60 min, respectively. The current synthetic procedure is highly reproducible and multi-gram scale products were readily produced.

The resulting MnO nanoparticles dispersed in chloroform were then encapsulated by PEG-phospholipid shell to endow them with biocompatibility. Typically, 2 ml of the organic dispersible 25 nm sized MnO nanoparticles in CHCl3 (5 mg/ml) was mixed with 1 ml of CHCl3 containing 10 mg of 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000 PE, Avanti Polar Lipids, Inc.) in the ratio of 5:1. After evaporating solvent, it was incubated at 80 ºC in vacuum for 1 hr. The addition of 5 ml water resulted in a clear and dark-brown suspension. After filtration, excess mPEG-2000 PE was removed by centrifugation. Herceptin conjugated MnO nanoparticles

Maleimido-MnO nanoparticles was prepared by the similar procedure with water dispersible MnO. To endow maleimide group we used the mixture of 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000 PE, Avanti Polar Lipids, Inc.) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG(2000)Maleimide, Avanti Polar Lipids, Inc.).

6 mg of Herceptin (Roche Parma Ltd.) was dissolved in 0.5 ml phosphate buffered saline (PBS, pH 7.2) and mixed with 60 µl of N-succinimidyl S-acetylthioacetate (SATA) in dimethyl sulfoxide (1.5 mg/ml). After 30 min, 120 µl of 0.5M hydroxylamine in PBS was added and the solution was incubated for 2hr at RT. Thiolated Herceptin was purified with desalting column (Sephadex G-25) and added to 0.3 ml of maleimido-MnO (10 mg/ml). It was incubated overnight at 4 ºC and Herceptin conjugated MnO nanoparticles were isolated by gel filtration with Sephacryl S-200.

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Measuring MRI relaxation properties of MnO nanoparticles. We measured T1 and T2 relaxation times of the size-tuned water dispersed MnO

nanoparticles that were prepared in test tubes with varying concentrations on a 3.0T clinical MRI scanner (Philips, Achieva ver. 1.2, Philips Medical Systems, Best, The Netherlands) equipped with 80 mT/m gradient amplitude and 200 ms/m slew rate. A Look-Locker sequence (TR/TE = 10/1 ms and flip angle = 5) was used to measure T1 by acquiring 17 gradient echo images at different inversion delay times using minimum inversion time of 87 ms with a phase interval of 264 ms with in-plane image resolution

= 625 × 625 µm2 and slice thickness = 500 µm. The images were fitted into a 3-parameter function to calculate T1 values using Matlap program. T2 measurements were performed by using 10 different echo times in a multislice turbo spin echo sequence (TR/TE = 5000/20, 40, 60, 80, 100, 120, 140, 160, 180, 200 ms, in-plane resolution =

200 × 200 µm2, slice thickness = 500 µm). The images were fitted into Levenberg-Margardt method to calculate T2 values using Matlap program.

We measured the specific relaxivities (r1 and r2) of the MnO nanoparticles. The signal intensities of each ROIs in the T1 map (60 - 80 pixels) and the T2 map (200 - 300 pixels) were measured for each concentration, which were then used for r1 and r2 calculations, respectively. We derived the following three specific relaxivities based on different expression of Mn concentration: r1 (mM-1s-1) based on the molar concentration of manganese atom ( MnC ) measured by ICP-AES, rN1 (µM-1s-1) based on the number of MnO nanoparticles ( NPC ), and rS1 (m·s-1) based on the surface area of MnO nanoparticles ( NPS ) (see Supplementary Method online).

Cytotoxic evaluation of the human cell lines in vitro.

The human cancer cell lines PC-3, U-87MG, MCF7, Huh7 and MRC5 were obtained from Korea Cell Line Bank (KCLB, Seoul, Korea) and NCI H460, HEK 293, HL-60 were purchased from American Type Culture Collection (ATCC, Manassas, USA). Cells were grown as monolayer cultures in 100 mm dish and subcultured 3 times

for a week at 37 °C in atmosphere containing 5% CO2 in air and 100% relative humidity, which were maintained at low passage number of 3 to 15. For in vitro cytotoxic assay,

cells at a logarithmic growth phase were detached and plated (180 µl per well) in 96- well flat-bottom microplates at densities of 1,000-25,000 cells per well, which were

then left for 20-24 h at 37°C to resume exponential growth. After 24 h recovery, 20 µl distilled water (8 control wells per plate) and various concentration of the MnO nanoparticles (final concentrations are 450, 45, 4.5, 0.45, and 0.045 µg/ml) were added to the wells in triplicate. For control wells, the same volume of complete culture

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medium was included in each experiment. Following 72 h of continuous MnO

nanoparticle exposure to the cells under 5% CO2 atmosphere at 37 °C, cell survival was assessed by SRB (sulforhodamine B)[3] or WST-8 assay.[4] The IC50 (the concentration of MnO nanoparticles that inhibited 50% of cell growth) was obtained for each cell type by plotting a concentration-effect curve. Animal preparation.

Thirty adult C3H mice weighting 25-35 g were employed under the institutional guideline of Samsung Biomedical Research Institute and Asan Institutes for Life Sciences for animal handling. The MnO nanoparticles with the core size of 25 nm were injected bolus through a tail vein line with a dose of 35 mg of Mn (measured by ICP-AES) per kg of mouse body weight for all experiments. For MRI, the animals were anesthetized and set into an MR-compatible cradle. During MRI, the animals were anesthetized by breathing 2% isoflurane into oxygen-enriched air with a facemask. The

rectal temperature was carefully monitored and maintained at 36 ± 1°C. To investigate the time course of distribution of the MnO nanoparticles in the brain, MRI was performed before, and 1-hr, 2-hr, 24-hr, 48-hr, 72-hr, 96-hr, 120-hr, 7-day, 11-day, 14-day, and 21-day after administration of the MnO nanoparticles for 6 animals. To investigate the time course of distribution of MnO particles in the abdomen, MRI was performed before, and 30-min, 1-hr, 6-hr, 24-hr, 48-hr, 72-hr, 96-hr, and 120-hr after administration of the MnO nanoparticles for 3 animals. Three animals were separately injected with the MnO nanoparticles and MRI was performed at 72 hours, which were then sacrificed for extracting tissues from cerebral cortex, hippocampus, kidney cortex, and liver parenchyma for transmission electron microscopic (TEM) measurements. Eighteen animals were injected with the MnO nanoparticles for T1 measurements of the blood. Blood samples were drawn from 2 mice each at 30-min, 1-hr, 3-hr, 6-hr, 1-day, 3-day, 7-day, 14-day and 24-day after the MnO injection. The T1 values of the blood samples at each time points were measured and the corresponding Mn contents were measured by ICP-AES analysis.

The breast cancer brain metastatic tumor model was made by inoculating the MDA-MB-435 human breast cancer cells (2 x 105 cells/5 µl of HBSS) purchased from Korea Cell Line Bank (KCLB, Seoul, Korea) into the specific pathogen-free male balb/c-nu mice (6 weeks old) brain. The tumor was grown for 12-17 days until MRI examination, which was performed before, and 1-hr, 3-hr, 9-hr, and 24-hr after administration of the Herceptine functionalized (20mg of Mn /kg of body weight) or non-functionalized MnO nanoparticles (20mg of Mn /kg of body weight) for 4 animals.

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Calculation specific relaxivities, the number and the surface area of MnO nanoparticles.

The specific relaxivities of MnO nanoparticles, which means the contrasting effect of nanoparticles per unit concentration, were calculated by

CriTT ii

⋅+=0,

11,

where i = 1 (longitudinal relaxivity) and i = 2 (transverse relaxivity).

The number of manganese atoms per a nanoparticle was derived from an equation

3

3

32

4ad

VV

Nunit

NPMn

π== , where

3aVunit = is the volume of a unit cell of fcc structure and 3

234

=

dVNP π is the

volume of a spherical nanoparticle with a given diameter ( d ). The surface area of MnO nanoparticles ( NPS ) was calculated by

daNC

NCAS AMnANPNPNP 2

3 3

=××= ,

where the number of MnO nanoparticles ( NPC ) is

3

3

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daC

NC

C Mn

Mn

MnNP π

== ,

the surface area of a spherical nanoparticle is

2

24

=

dANP π ,

and 12310022.6 −×= molN A is the Avogadro constant, and the molar concentration of manganese ( MnC ) was measured by ICP-AES.

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The stability of the water-dispersed MnO nanoparticles

The stability of the MnO nanoparticles in water or a possibility of leaching of the manganese ion (Mn2+) from the MnO nanoparticles was tested by measuring the Mn concentrations of the supernatants and the precipitate after centrifugation and the corresponding relaxation rates (R1). We conducted elemental analysis by inductively coupled plasma atomic emission spectroscopic (ICP-AES) and MRI experiments for the supernatants of MnO nanoparticles dispersion incubated for 7 days at room temperature followed by 3 cycles of centrifugation and aspiration. The Mn concentrations and the corresponding relaxation rates (1/T1) of the supernatants (S1, S2, S3) and the precipitate (P) obtained after the third centrifugation were measured (Table S1). Neither T1 contrast enhancement in MRI, nor Mn contents in inductively coupled plasma atomic emission spectrometry (ICP-AES) was observed from all of the supernatants. This indicates that the nanoparticles are highly stable and no appreciable leaching-out occurs from the MnO dispersions. Mouse in vivo MRI.

All in vivo MRI were carried on a 4.7T/30 MRI System (Bruker-Biospin, Fallanden, Switzerland) equipped with a 20 cm gradient set capable of supplying up to

100mT/m in 200 µsec rise-time. A birdcage coil (72 mm i.d.) (Bruker-Biospin, Fallanden, Switzerland) was used for excitation, and actively decoupled from a 20 mm diameter saddle-shaped surface coil (homebuilt), which was used for receiving the signal for brain imaging. High-resolution 3D MnO nanoparticle contrast enhanced MRI was obtained from each mouse brain using a fast spin-echo T1-weighted MRI sequence (repetition time (TR) / echo time (TE) = 300/12.3 ms, number of experiment (NEX) =1,

echo train length = 2, 140 µm 3D isotropic resolution, field of view (FOV) = 2.56 × 1.28 × 1.28 cm3, matrix size = 256 × 128 × 128, slice selection direction = sagittal, and readout direction = head-to-foot) to evaluate the contrast. For imaging abdomen, a birdcage coil (72 mm. i.d) (Bruker-Biospin, Fallanden, Switzerland) was used for excitation and receiving the signal with respiratory gating at expiration point (SA instrument, Stonybrook, NY, USA). Multi-slice MR images were obtained from each mouse using a spin-echo T1-weighted MRI sequence (TR/TE = 400/12 ms, NEX = 16,

slice thickness = 1.5 mm, FOV = 2.78 × 168 cm2, matrix size = 192 × 192, slice selection direction = sagittal, and readout direction=head-to-foot) to evaluate the contrast.

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To plot the time course of the signal enhancement, the signal-to-noise ratio (SNR)

was measured according to the equation, SNR = 0.655 × [signal intensity (SI)/standard deviation (SD) of noise] using Bruker ParaVision Software (Bruker-Biospin, Fallanden, Switzerland). The SIs were measured for various tissues in the organ, and the SD of the noise was measured outside the organ in the air, but avoiding areas that might be affected by motion.

Discussion on the time courses of in vivo mouse MRI

The time courses of the enhancement show the profiles of the intracellular uptake, the duration of the enhancement, and the organ specific wash-out times after the administration of the MnO nanoparticles. The maximum signal enhancement was observed at 48-96 hours in the brain, and the signal half life was about 21 days after the administration of MnO nanoparticles (see Figure S5). The MRI T1 value of the blood drawn from mice at various time points after the injection was maximum at 1 hr and reached the control value within 6 hours after the administration, which was in concordance with the blood level of Mn measured by ICP-AES (see Figure S9). The long signal enhancement after the rapid clearance from the blood indicates the intracellular uptake of the MnO nanoparticles. The maximum enhancement of renal pelvis at 1 hour followed by a rapid clearance and a reach at the baseline value by 72 hours after the administration was observed in kidney (see Figure S6). The spinal cord was continuously enhanced and the liver parenchyma stayed enhanced throughout the entire period investigated (see Figure S6).

Table S1. Mn concentrations and the corresponding relaxation rates (R1) of the supernatants obtained (S1, S2, S3) from the three cycles of centrifugation and aspiration of MnO nanoparticles, and the precipitate (P) obtained after the third centrifugation.

Initial S1 S2 S3 P

Mn Concentration (mM)

17.07 0.02 0.00 0.00 15.87

R1 (sec-1) 2.70 0.66* 0.66* 0.71* 1.97*

* R1 of water: 0.67 sec-1

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Table S2. Comparative MRI results of MnO and MnO/SiO2 core/shell nanoparticles.

T1 (ms)† r1 (mM-1s-1) T2 (ms)† r2 (mM-1s-1)

MnO 481 0.37 85 1.74

MnO/SiO2

core/shell 1157 0.04 130 1.46

Water 1430 328

†The longitudinal relaxation time (T1) and the transverse relaxation time (T2) were measured at 5 mM

of Mn measured by ICP-AES analysis.

Table S3. The concentration of MnO nanoparticles that inhibited 50% of cell growth

(IC50).

Cell line Characteristics IC50 ([Mn], mM)

MRC-5 Human normal lung fibroblast 4.73

HEK 293 Human embryonic kidney cell 1.33

NCI-H460 Human large cell lung cancer cell 0.36

Huh7 Human hepatoma cell 0.66

U87-MG Human glioblastoma cell 3.57

MCF-7 Human breast adenocarcinoma cell 0.44

PC-3 Human prostate adenocarcinoma cell 0.53

HL-60 Human acute promyelocytic leukemia cell 0.66

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Figure S1. The powder X-ray diffraction (XRD) pattern of the organic dispersible 25 nm

sized MnO nanoparticles.

Figure S2. The magnetization curve (applied magnetic filed (H) vs. magnetization (M)) of the organic dispersible 25 nm sized MnO nanoparticles at 300 K. Magnetic study was carried out on a powder of the organic dispersible MnO nanoparticles using a commercial vibrating sample magnetometer (Lake Shore 9300) at 300 K.

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Figure S3. TEM image of MnO/SiO2 core/shell nanoparticles. Figure S4. A plot of the change in body weight of mice after the injection of the MnO nanoparticles. No appreciable weight loss for 3 weeks was observed although the initial drop was noticed. The initial drop could be associated with the animal preparation, anesthesia used to maintain during MRI, and any others that might have caused stresses to mice for the MRI examinations.

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Figure S5. The time course of the signal enhancement in a mouse brain. a) Axial images of the brain show the temporal evolution of signal at various time points after the injection of the MnO nanoparticles. Enhancement occurs as early as 1 hour in cerebral spinal fluid (CSF) spaces and reaches at a maximum signal enhancement in the entire brain after 20 hours. The maximum signal enhancement in the brain is maintained until 96 hours, which was then gradually decreased reaching at the signal half life at about 21 days. b) A plot of the time courses of the signal enhancements in cortex, basal ganglia, and hippocampus indicates that the maximum enhancement in the brain generally occurs around 48-96 hours after the administration of MnO nanoparticles, and the signal half life is about 21 days.

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Figure S6. a) The time course of the signal enhancement shows a strong signal enhancement in renal pelvis at 30 – 60 minutes followed by a rapid drop and a reach at a baseline value at 120 hours after the administration, b) the entire liver tissue was generally enhanced, and c) the steady increase of the signal in the spinal cord was seen throughout the period investigated. d) A plot of the signal enhancement in time after the administration of the MnO nanoparticles.

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Figure S7. TEM images of the brain cells containing 25 nm sized MnO nanoparticles. The TEM images were taken 72 hours after the administration of the MnO nanoparticles.

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Figure S8. TEM images of Liver (left) and Kidney (right) from the tissues extracted 72 hours after the administration of the 25 nm sized MnO nanoparticles. The MnO nanoparticles were found in the cytoplasmic spaces. Figure S9. Mn Concentration (measured by ICP-AES) and T1 values of the mouse blood measured at various time points after the injection of the MnO nanoparticles.

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