Supplementary Figure 1. a. Calibration markers used for gel-filtration chromatography (GFC). b.Four-point (M2-M5) standard curve used for GFC calibration. Molecular weight markers were: M1= thyroglobulin (669 kDa, 18.0 nm HD); M2 = γ-globulin (158 kDa, 11.9 nm HD); M3 = ovalbumin(44 kDa, 6.13 nm HD); M4 = myoglobin (17 kDa, 3.83 nm HD), and M5 = vitamin B12 (1.35 kDa,1.48 nm).

a. b.

M2(11.9 nm)

M3(6.13 nm) M4

(3.83 nm)

M5(1.48 nm)

35 40 45 50 55 60100

101

102

103

Time (min)

20 30 40 50 60 700.0

0.2

0.4

0.6

0.8

1.0

Time (min)

M1(18.0 nm)

Calibration of gel-filtration chromatography

Choi et al.Renal Clearance of Nanoparticles

Supplementary Figure 2. Absorption (a) and fluorescence emission (b; λex = 414 nm) spectraof CdSe/ZnS QDs with cysteine coating. A 525 ± 25 nm filter emission filter was used forQD515 and QD534, and a 560 ± 20 nm emission filter was used for QD554, QD564, andQD574.

a. b.

300 400 500 600 700 8000.00

0.25

0.50

0.75

1.00 QD515

QD534

QD554

QD564

QD574

Wavelength (nm)

450 500 550 600 6500

200

400

600

800

1000

QD534

QD515

QD554

QD564

QD574

Wavelength (nm)

Choi et al.Renal Clearance of Nanoparticles

Absorption and fluorescence emission spectra of CdSe/ZnS QDs with cysteine coating

Supplementary Figure 3: a. Characteristic DMPO-OH adduct signal, averaged over 10scans (aN = aH ≈ 15 G). b. EPR spectrum of QD-Cys (3 μM) in the presence of DMPO (0.1 M)after irradiation with UV light for 15 min. Obtained using same sample geometry andinstrument parameters as in (a), and averaged over 200 scans.

a.

b.

Choi et al.Renal Clearance of Nanoparticles

Electron paramagnetic resonance spectroscopy of QD-Cys

Choi et al.Renal Clearance of Nanoparticles

HD Standards

0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0

6.13 nm

18.0 nm

11.9 nm

3.83 nm

1.48 nm

Time (min)

Non-Radioactive QD515

0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0 QD5159.9 min

Cys13.6 min

0

25

50

75

100

DTT14.8 min

Time (min)

99mTc-Labeled QD515 (Pre-Injection)

0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0Tc-QD515 9.7 min

0

20

40

60

80

100

Tc-MAS3

13.5 min

Time (min)

99mTc-Labeled QD515 (Urine)

0 2 4 6 8 10 12 14 160

1

2

3

4

0

20

40

60

80

100Tc-QD515 9.7 min

Time (min)

Supplementary Figure 4: Molecular weight standards (top left) were used to identify QD515, cysteine (Cys),and dithiothreitol (DTT) in the non-radioactive QD515 preparation (top right). After covalent conjugation usingpre-loaded 99mTc-MAS3-NHS and purification, only trace amounts of 99mTc-MAS3 remain in the preparation(bottom left). After intravenous injection, radiolabeled Tc-QD515 can be identified intact in urine (bottomright). Tracings include absorbance at 280 nm (solid black curve), fluorescence at 515 nm (dotted blackcurve), and gamma-ray detector (red curve).

HPLC GFC analysis of 99mTc-labeled QD515 in vitro and in vivo

350 400 450 500 550 600 650 700 750 800 850

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Abs

Wavelength (nm)

Rhodamine 590

QD565

dye

dye

QD

dye

QD

QD

dyeQY

S

S

n

n

Abs

AbsQY !

""

#

$

%%

&

'!""

#

$

%%

&

'!""

#

$

%%

&

'=

2

2

Supplementary Figure 5: Absorbance spectra (left) and fluorescence emission spectra(right) of the QY standard Rhodamine 590 and QD565.

where Abs is the absorbance at 490 nm, n is the index ofrefraction of the solvent (water for QD and ethanol for dye),S is the integrated area under the fluorescence spectrum,and QYdye is the absolute QY reported for rhodamine 590(99% in ethanol).

Choi et al.Renal Clearance of Nanoparticles

Quantum yield measurements using rhodamine 590

500 550 600 650 700 750-1x10

6

0

1x106

2x106

3x106

4x106

5x106

6x106

7x106

8x106

9x106

1x107

Counts

Wavelength (nm)

Rhodamine 590

QD565QY ~10%

99mTc-MAS3-NHS

N

TcN N

S

O

O

O

OHN

O

O O

HO

O OH

O

MW = 561.3 Da

a. b.Abs-FL QD515

350 450 550 650 7500.0

0.2

0.4

0.6

0.8

1.0

0

250

500

750

1000

Wavelength (nm)

Supplementary Figure 6: a. Chemical structure of 99mTc-MAS3-NHS. b. Absorption (solidcurve) and fluorescence emission (dotted curve; λex = 414 nm) of QD515.

Choi et al.Renal Clearance of Nanoparticles

Radiolabeling of QD515 with MAS3-chelated 99mTc

- 1 -

SUPPLEMENTARY METHODS

Renal Clearance of Nanoparticles

Hak Soo Choi 1, Wenhao Liu 2, Preeti Misra 1, Eiichi Tanaka 1, John P. Zimmer 2, Binil Itty Ipe 2,

Moungi G. Bawendi 2,*, and John V. Frangioni 1,3,*

1 Division of Hematology/Oncology, Department of Medicine and 3 Department of Radiology,

Beth Israel Deaconess Medical Center, Boston, MA 02215 2 Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139

Quantum dot (QD) chemicals

Trioctylphosphine oxide (TOPO), hexadecylamine (HDA), hexamethyldisilathiane

((TMS)2S), DL-cysteine, and dithiothreitol (DTT) were purchased from Sigma Aldrich (St.

Louis, MO) and used as received. Selenium shot, n-hexylphosphonic acid (HPA), n-

tetradecylphosphonic acid (TDPA), cadmium 2,4-pentanedionate (Cd(acac)2), and cadmium

oxide were purchased from Alfa Aesar (Ward Hill, MA). Diethylzinc (ZnEt2) was purchased

from Fluka and stored at -30°C under nitrogen atmosphere after being filtered through a 0.2 µm

filter. Trioctylphosphine (TOP) and tributylphospine (TBP) were purchased from Strem

Chemicals (Newburyport, MA). Tributylphosphine selenide (TBP-Se) was prepared by

dissolving 0.15 mmol of selenium shot in 100 mL of TBP under inert atmosphere and stirring

vigorously overnight, forming a 1.5 M TBP-Se solution. All air sensitive materials were handled

in an Omni-Lab VAC (Newburyport, MA) glove box under dry nitrogen atmosphere with

oxygen levels < 0.2 ppm. All solvents were spectrophotometric grade and purchased from EMD

Biosciences (San Diego, CA).

Synthesis of CdSe/ZnS QDs

Core/shell CdSe/ZnS nanocrystals with emission wavelengths of 534 nm and higher were

synthesized via modification of previously reported procedures.1, 2 Briefly, a precursor solution

composed of Cd(acac)2 (155 mg, 0.5 mmol) and HDA (0.25 mL) in TOP (2.8 mL) was degassed

at 100˚C for 1 hr, followed by the addition of TBP-Se (5 mmol) after cooling to room

- 2 -

temperature. This mixture was loaded into a syringe under dry N2 atmosphere. In a separate 3-

neck round bottom flask, 90% TOPO (6.25 g) and 90% HDA (5.75 g) were degassed at 135˚C

for 2 hr and backfilled with N2. The temperature was increased to 360˚C before rapidly injecting

the precursor solution. The cores were grown at 280˚C until the desired wavelength was reached.

After cooling to 80˚C, butanol (4 mL) was added to prevent solidification of the product.

Core/shell CdSe/ZnS nanocrystals with emission wavelengths less than 534 nm were

synthesized via a previously reported procedure by Peng et al.3 Briefly, CdO (64.5 mg, 0.5

mmol), TDPA (176 mg), and 99% TOPO (3.02 g) were degassed at 155˚C for 1 hr. The flask

was backfilled with N2 and heated to 320˚C with stirring until a clear homogenous solution was

formed. The solution was cooled to 270˚C before injecting a precursor solution consisting of

TOP (1.5 mL) and 3 mmol of TBP-Se. The cores were grown at 210˚C until the desired first

absorbance maximum was reached (typically 470 – 480 nm). Butanol (2 mL) was added to the

solution before cooling to room temperature to prevent solidification of the product.

In all cases, the core stock solutions were allowed to settle for 1 hr and then centrifuged

at 3000 g for 4 min. The pellet was discarded and the supernatant was precipitated twice with

the addition of acetone followed by centrifugation. The pellet was redispersed in hexane, filtered

through a 0.2 µm filter, and injected into a degassed solution of 99% TOPO (10 g) and n-

hexylphosphonic acid (0.4 g). After removal of the hexane under reduced pressure at 80˚C, the

flask was back-filled with dry N2 and the temperature increased to 130˚C before adding 0.25 mL

of decylamine and stirring for 30 min. Precursor solutions of ZnEt2 and (TMS)2S were prepared

by dissolving the appropriate amounts of each in 4 mL of TOP and loading them into two

separate syringes under inert atmosphere. The amount of ZnEt2 was calculated by assuming a 3

monolayer overcoat according to the methods of Dabbousi.1 A twofold molar excess of (TMS)2S

was used. The precursor solutions were injected simultaneously into the 130˚C bath at a rate of 4

mL/hr and annealed overnight at 80˚C. QD stock solutions were stored under ambient conditions

in a solution of butanol (4 mL) and centrifuged once more before use.

Gel-Filtration Calibration Standards and QD Spectral Analysis

The gel-filtration chromatography calibration standards used in these experiments are

shown in Supplementary Figure 1 online. Absorption and fluorescence emission spectra of QD-

Cys used in these experiments are shown in Supplementary Figure 2 online.

- 3 -

Electron Paramagnetic Resonance (EPR) Spectroscopy

To investigate potential radical generation by QD-Cys, we performed electron

paramagnetic resonance (EPR) spectroscopy on QD samples incubated with spin trapping 5,5-

dimethyl-1-pyrroline-N-oxide (DMPO). EPR spectra were taken on a Bruker EMX EPR

instrument equipped with a Gunn diode operating at 9.5 GHz with 100 kHz modulation

frequency, 2 G modulation amplitude, and a microwave power of 2 mW. QD-Cys in DMPO

solutions were irradiated in a quartz cuvette with a 365 nm UV lamp, and immediately loaded

into a quartz flat cell for measurement. Hydroxyl radicals react rapidly with DMPO to form the

DMPO-OH adduct, which produces a characteristic EPR signal. We validated our experimental

setup by mixing DMPO (0.1 M, 200 μL) with H2O2 (1 equiv) and a catalytic amount of FeCl2 in

water to generate the DMPO-OH adduct and measured the resulting EPR spectra. The

characteristic DMPO-OH signal clearly seen in Supplementary Figure 3a online (n = 10 scans).

Under the same sample geometry and instrument parameters, we measured radical generation of

QD-Cys. QD-Cys (3 μM, 200 µL) was mixed with DMPO (0.1 M) and irradiated with 365 nm

UV light for 15 min. ESR measurements of the resulting solution showed negligible DMPO

signal (n = 200 scans), indicating that QD-Cys is not efficient at radical generation (see

Supplementary Fig. 3b online). The QD concentration was chosen to be representative of the

concentration used in the in vivo studies. The results are in good agreement with previous studies

showing that radical generation is greatly diminished with CdSe cores over-coated with a ZnS

shell verses CdSe cores alone.4 Theoretical predictions suggest that in the CdSe(ZnS) core(shell)

system, the much higher bandgap of the ZnS shell efficiently confines both the hole and the

electron to the core,1 thus reducing the likelihood of radical generation.

Quantum Yield Measurements

QY was calculated using the method and data shown in Supplementary Figure 5 online.

99mTc-MAS3 Conjugation to QDs

Prior to conjugation, any free ligands were removed by three cycles of

dilution/concentration through Vivaspin 6 (MWCO 10,000) spin concentrators (Vivascience,

Stonehouse, UK). The pre-loaded NHS ester of 99mTc-MAS3 (see Supplementary Fig. 6a online)

was prepared as described in detail by Misra et al.5 Using a solid-phase strategy, it is produced at

- 4 -

high-specific activity (typically 4.1 x 108 MBq/mmol = 1.1 x 104 Ci/mmol), in neat aprotic

solvent, and without contaminating co-ligands. Tc-QD conjugation was performed by the

addition of 80 µL of [99mTc-MAS3]-NHS (~ 2 mCi) in DMSO to 1 mL (1 µM) of QD515 (see

Supplementary Fig. 6b online) in pH 7.8 PBS. After stirring for 1 hr, the radiolabeled conjugates

were purified by washing five times in Vivaspin concentrators (MWCO 10,000) with pH 7.4

PBS and analyzed by RP-HPLC on 8 x 300 mm, 200 Å Diol (YMC, Japan) size-exclusion

chromatography column using PBS, pH 7.4 supplemented with 1 mM cysteine as mobile phase

(see Supplementary Figure 4 online). The HPLC platform is composed of binary pump (model

1525, Waters, Milford, MA), UV detector (model 2487, Waters), fluorescence detector (model

2475, Waters), and high-sensitivity PMT gamma detector (FC-3200, Bioscan, Washington, DC),

and all housed within a Capintec (Ramsey, NJ) hot cell equipped with a model CRC-15R

(Capintec) dose calibrator.6

References

1. Dabbousi, B.O. et al. (CdSe)ZnS Core-Shell Quantum Dots: Synthesis and Characterization

of a Size Series of Highly Luminescent Nanocrystallites. J. Phys. Chem. B 101, 9463-9475

(1997).

2. Fisher, B.R., Eisler, H.-J., Stott, N.E. & Bawendi, M.G. Emission Intensity Dependence and

Single-Exponential Behavior In Single Colloidal Quantum Dot Fluorescence Lifetimes. J.

Phys. Chem. B 108, 143-148 (2004).

3. Peng, Z.A. & Peng, X. Formation of High-Quality CdTe, CdSe, and CdS Nanocrystals

Using CdO as Precursor. J. Am. Chem. Soc. 123, 183-184 (2001).

4. Ipe, B.I., Lehnig, M. & Niemeyer, C.M. On the generation of free radical species from

quantum dots. Small 1, 706-709 (2005).

5. Misra, P., Humblet, V., Pannier, N., Maison, W. & Frangioni, J.V. Production of multimeric

prostate-specific membrane antigen small molecule radiotracers using a solid-phase 99mTc

pre-loading strategy. J Nuc Med In Press (2007).

6. Humblet, V., Misra, P. & Frangioni, J.V. An HPLC/mass spectrometry platform for the

development of multimodality contrast agents and targeted therapeutics: prostate-specific

membrane antigen small molecule derivatives. Contrast Media Mol Imaging 1, 196-211

(2006).