Electronic Supplementary Information (ESI) · Brunton,e Luis Yate a and Juan C. Mareque-Rivas*a,b...

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Electronic Supplementary Information (ESI)

QD-filled micelles which combine SPECT and optical imaging

with light-induced activation of a platinum(IV) prodrug for

anticancer applications

Carmen R. Maldonado,c Nina Gómez-Blanco,a Maite Jauregui-Osoro,d Valerie G.

Brunton,e Luis Yatea and Juan C. Mareque-Rivas*a,b

aCooperative Centre for Research in Biomaterials (CIC biomaGUNE), 20009 San

Sebastián, Spain; Fax: (+34) 943 005301; Tel: (+34) 943 005313;E-mail:

[email protected] b Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Spain

cSchool of Chemistry, University of Edinburgh, Edinburgh, U.K. d Division of Imaging Sciences & Biomedical Engineering, King’s College London, St.

Thomas’ Hospital, London, U.K.

eEdinburgh Cancer Research Centre, Institute for Genetics & Molecular

Medicine, University of Edinburgh, Edinburgh, U.K.

Materials. All chemicals were obtained from commercial sources and used as received. For the

synthesis of the hydrophobic QDs, cadmium oxide (CdO, 99.5%), tri-n-octylphosphine oxide

(TOPO, 99%), tri-n-octylphosphine (TOP, 90%), tri-n-butylphosphine (TBP, 97%),

hexadecylamine (HDA, 98%), diethylzinc (ZnEt2, 1M in hexane) and hexametyldisilathiane

[(TMS)2S, 98%] were purchased from Sigma Aldrich. Stearic acid (≥98.5%) was purchased

from Fluka and selenium powder (Se, 99.999%) was obtained from Alfa Aesar.

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

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For the preparation of the water soluble micelles (MQDs), 1,2-dipalmitoyl-sn-glycero-3-

phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG-OMe)

and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]

(ammonium salt) (PEG-NH2) were purchased from Avanti Polar Lipids.

For the synthesis of c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2], potassium tetrachloroplatinate(II)

(99.9%) was purchased from Alfa Aesar and succinic anhydride (99%), potassium iodide

(≥99.5%), potassium chloride (≥99.0%), silver nitrate (≥99.0%), and hydrogen peroxide

solution 30% (w/w) in H2O were purchased from Sigma Aldrich.

QD Synthesis. Hydrophobic core-shell CdSe@ZnS QD was synthesized and purified adapting a

previously reported procedure [1] with some modifications.

CdO (26 mg) and stearic acid (500 mg) were loaded into a three-neck flask and heated to 250°C

under N2 flow and stirring. Once the mixture was completely dissolved, it was allowed to cool

to room temperature. Then, TOPO (6 g) and HDA (2 g) were added and the mixture was heated

to 225°C under N2 flow and vigorous stirring. At this temperature, 2 mL of freshly prepared

TPB-Se solution (see below) was quickly injected into the flask. Following injection the

temperature was adjusted to 220-225°C for 15 min to promote nanocrystal growth.

On reaching the desired nanoparticle core size, determined by UV/vis, the temperature was

lowered to 100°C to stop further nanoparticle growth. Afterwards, the solution was heated to

210°C. At this temperature, 1.2 mL of the ZnS stock solution (see below) was slowly injected in

(5 min). After injection, the temperature of the mixture was set to 100°C and stirred for 3 hours.

After cooling down to 40°C, the nanocrystals were dispersed in chloroform and precipitated by

addition of methanol. After centrifugation the supernatant liquid phase was removed. This

procedure was repeated twice. The precipitate was dried under a stream of N2 at room

temperature.

QDs were characterized by TEM, XPS, UV-vis and fluorescence spectroscopy, NMR and ICP-

AES. The size of the CdSe@ZnS QDs was determined based on TEM and the first exciton peak

(629 nm).

Tri-n-butylphosphine selenide (TBP-Se) was prepared in a N2-filled glove box by shaking 160

mg of selenium powder in 2 ml of TBP. The ZnS stock solution was also prepared in a N2-filled

glove box by reacting (TMS)2S (0.25 mL) with ZnEt2 (1.75 mL) in TOP (3 mL).

Micelle formation (MQD). Hydrophobic CdSe@ZnS QD (1.6 mg, 1 nmol) was combined with

PEG-phospholipid(s) (1 mg of PEG-OMe and 1 mg of PEG-NH2) in chloroform (500 µL). The

solvent was allowed to evaporate overnight in a 3 mL vial at RT. Any remaining solvent was

removed under vacuum for 1 h. Then, the vial was placed in a water bath at 80°C for 30 s, after

which 1 mL of nanopure water was added. The solution was passed through a 0.45 µm filter


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(Iso-DiscTM, Supelco) and then was ultracentrifuged (40 min, 3 cycles, 369000 g) to remove the

empty micelles. Finally the pellet was dissolved in 250 µL of the appropriate solvent (nanopure

water, deuterium oxide or 10 mM PBS). Concentration of the MQDs solutions was estimated by

the method of Peng et al. [2] and confirmed/corrected by ICP-AES. The MQDs were

characterised by XPS, TEM and DLS.

Labeling of micelles with fac-[99mTc(OH2)3(CO)3]+. 99mTc pertechnetate (Na[99mTcO4]) was

eluted with saline from a Drytec generator (GE Healthcare, Amersham, UK) at the

Radiopharmacy at Guy’s and St Thomas’ Hospital NHS Trust (London, UK) and converted to

fac-[99mTc(OH2)3(CO)3]+ using the lyophilized kit “Isolink” (Covidien, Petten, The

Netherlands). The synthesis and quality control of tricarbonyltechnetium (99mTc-TC) were

carried out following the manufacturer’s instructions. Briefly, 1 mL of Na[99mTcO4] (1.0-1.7

GBq) was injected into the kit and the mixture was heated at 100°C for 30 min. The vial was

allowed to cool to room temperature, and its content was neutralised with 1M HCl. The reaction

was monitored by thin layer chromatography (TLC) using silica gel TLC strips (Merck) with

methanol/HCl 99/1 as mobile phase. 99mTc-TC has a Rf of about 0.3-0.4, while [99mTcO4]-

moves with the solvent front. TLC strips were scanned using a Mini-scan radio TLC scanner

with a FC3600 FlowCount detector of γ photons (LabLogic, Sheffield, UK).

400 µL of fac-[99mTc(OH2)3(CO)3]+ was added to 100 µL of an aqueous micelle solution (at

concentrations ranging from 500 to 1 nM) in an Eppendorf tube and the mixture was heated at

90°C for 25 min. The Eppendorf was then allowed to cool down to room temperature and the

contents were transferred to a Nanosep 100k molecular weight cutoff ultrafiltration centrifugal

device (Pall Life Sciences), which was centrifuged at 12500 rpm for 10 min. This allowed

separating the radiolabelled micelles, which remained in the retentate, from the unbound 99mTc-

TC, which was in the filtrate. The filtrate was discarded, and deionized water of injection (500

µL) was added to the retentate. Once more, the filter was centrifuged at 12000 rpm for 10 min.

This process was repeated twice. The total radioactivity in the filtrates and retentates was

measured in a CRC-25R dose calibrator (Capintec, USA) in order to determine the radiolabeling

yield (%). The radiolabelled micelles were recovered from the filter by the addition of water

(200 µL) to the retentate. After mixing, 100 µL was aliquoted into Eppendorfs and their activity

was measured before placing the tubes in the scanner. SPECT scans were acquired using a

NanoSPECT/CT scanner (Bioscan, Paris, France) with SPECT acquisition time 3300 s,

obtained in 55 projections using a 4-head scanner with 4 x 9 (1 mm) pinhole collimators in

helical scanning mode, acquiring a transaxial field of view of 206.6 mm and an axial field of

view of 24 mm. Images were reconstructed using the HiSPECT (Scivis GmbH) reconstruction

software package, and fused using proprietary Bioscan InVivoScope (IVS) software.


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Interaction of fac-[99mTc(OH2)3(CO)3]+ with micellar quantum dots (MQDs):

Stability studies in vitro. Three MQDs samples were labelled with 235 MBq of 99mTc-TC

following the above procedure. Once labelled and washed, the samples were diluted in 200 µL

of either saline, PBS or human serum and incubated at room temperature or 37 °C (in the case

of human serum) for 24 h. The stability of the radiolabelled micelles was monitored by thin

chromatography (TLC) using silica gel TLC strips (Merck) with methanol/HCl 99/1 as mobile

phase at 1 h and 24 h. The results revealed no change in the TLC patterns for the different time-

points in each system, indicating the interaction between the micelles and the tricarbonyl moiety

is stable.

Stability studies in vivo: The stability of the 99mTc-TC-MQD interaction was also investigated

in vivo. These studies were carried out in accordance with UK Research Councils’ and Medical

Research Charities’ guidelines on Responsibility in the Use of Animals in Bioscience Research,

under a UK Home Office licence. The female BALB/c mouse used in this study (aged 8 weeks,

20.8 g) was purchased from Harlan Laboratories, UK.

Mice received an intravenous (i.v.) tail vein injection of 70 MBq of radiolabelled micelles in

100 uL of saline (FreseniusKabi) (nanoparticle concentration: 250 nM). With the mouse under

isofluorane anaesthesia in a Minerve imaging chamber, SPECT/CT scans were acquired either

from 0 min to 1 h post-injection (p.i.) and at 24 h p.i. respectively, using a NanoSPECT/CT

scanner (Bioscan, Paris, France) with SPECT acquisition time 1800 s, obtained in 30

projections using a 4-head scanner with 4 x 9 (1 mm) pinhole collimators in helical scanning

mode and CT images with a 45 kVP X-ray source, 500 ms exposure time in 180 projections

over 9 min. Images were reconstructed in a 256 _ 256 matrix using the HiSPECT (Scivis

GmbH) reconstruction software package, and fused using proprietary Bioscan InVivoScope

(IVS) software. Quantification was performed by selecting the desired organs as regions of

interest (ROI) using the quantification tool of the IVS software.

Early SPECT images clearly show that the radiolabelled micelles accumulate quickly in the

liver and spleen, with some activity seen in the bladder and some still circulating in the blood at

the early time points. Late SPECT scans carried out at 24 h p.i. show that most of radioactivity

remains in the liver and spleen. No radioactivity can be seeing circulating in the blood, and

although there is some activity in the bladder, the kidneys are not lit up in the image. In contrast, 99mTc-TC quickly accumulates in the liver and bladder already 30 min. post injection. This

suggests that the interaction between the micelles and the 99mTc tricarbonyl moiety is strong and

that it does not seem to be significantly metabolised by the liver (as significant change is

observed in the late SPECT images).


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In vivo SPECT/CT images of mice injected with

CONTROL: In vivo SPECT/CT images of mice injected with

different times post-injection.

30 min

30 min

SPECT/CT images of mice injected with 99mTc-TC-MQD at different times

SPECT/CT images of mice injected with [99m

Tc(OH2)3(CO)3]+

.

150 min

1h 24 h

at different times.

at

24 h


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Labelling of micelles with fac-[Re(OH2)3(CO)3]+. Aqueous solutions of fac-[Re(OH2)3(CO)3]

+

(1-2.5 mM) were added to solutions of MQD in water (3-750 nM). A control solution of MQD

without fac-[Re(OH2)3(CO)3]+ was also prepared in the same way. The mixture was vortexed for

30 s and then heated at 90°C for 20 min. The mixture was allowed to cool down to room

temperature and then the solution was transferred to a Nanosep 100k centrifugal device and

centrifuged at 4000 rpm for 6 min. The retentate was washed with water (300 µL) five times

and after the final centrifugation the MQD micelles were removed from the membrane surface

by the addition of water (300 µL). The retentate was liophilised and analyzed by FT-IR. The

amount of Zn, Cd and Re was analyzed by ICP-AES.

Synthesis of c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] (1). The Pt(IV) complex was prepared

according to a previous literature procedure [3]. Briefly, cis-[Pt(NH3)2Cl2] is oxidized by

hydrogen peroxide to generate c,c,t-[Pt(NH3)2Cl2(OH)2] and then is reacted with succinic

anhydride (1:4 stequiometry) to yield the desired compound.

Irradiations. Mixtures of MQDs (100 nM) and c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] (50,

100, 200, 300, 500 µM and 1mM) in D2O were freshly prepared in vials and irradiated with 480

nm and 630 nm light for 1 h. In each case a control solution of c,c,t-

[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] without QD was also prepared and irradiated in the same

way. These studies were conducted with a LED light source (Prizmatix, MWLLS-11, Fibre

coupled 11 LED Multi-Wavelength LED light source) equipped with a Polymer Optical Fibre

(POF, ∅ = 1500 µm, fiber length ∼ 1 m) to enable delivery of light to the sample in the vial.

The fibre optic was placed ~ 20 mm above the solution in the vial and the power (ca.15 mW cm-

2 for 480 nm; 30 mW cm-2 for 630 nm) was measured with a Fieldmate power meter (Coherent,

OP2-VIS head). All sample preparations and irradiations were carried out in darkness.

NMR studies. 1H NMR spectra were acquired for c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] and

mixtures of c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] and MQD in D2O, before and after

irradiation with LED light. Measurements were carried out at 298 K with a Bruker 500 MHz

(1H) spectrometer. Processing was carried out using Mnova software.

XPS studies. XPS experiments were performed in a SPECS Sage HR 100 spectrometer with a

non-monochromatic X-ray source (Magnesium Kα line of 1253.6 eV energy and 250 W), placed

perpendicular to the analyzer axis and calibrated using the 3d5/2 line of Ag with a full width at

half maximum (FWHM) of 1.1 eV. The selected resolution for the spectra was 15 eV of Pass

Energy and 0.15 eV/step for the detailed spectra of the Pt 4f peaks. The deconvolution of the Pt


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4f peaks was carried out at 36 s of X-ray exposure. The deconvolution allowed to estimate the

ratio of the Pt(II) and Pt(IV) states. All measurements were made in an ultra high vacuum

(UHV) chamber at a pressure below 5 × 10-8 mbar. In the fittings Gaussian-Lorentzian functions

were used where the FWHM of all the peaks were constrained while the peak positions and

areas were set free.

Transmision electron microscopy (TEM). TEM studies were conducted on a JEOL JEM-2011

electron microscope operating at 200kV. The sample was prepared by depositing a drop of a

solution of nanocrystal onto a copper specimen grid coated with a holey ultrathin carbon film

and allowing it to dry.

Dynamic light scattering measurements. Particle size analysis was measured with a

NanoSizer (Malvern Nano-Zs, UK).

UV-vis and fluorescence studies. Measurements were made with a Varian Cary 5000 UV-vis

Spectrophotometer and a HORIBA Jobin-Yvon fluorimeter (F1-1065) equipped with a Xe 450

W arc lamp. Excitation was at 480 nm with bandwidths of 5 nm for excitation and emission.

Temperature was maintained at 25°C.

FT-IR studies. Spectra in KBr were acquired in a Thermo Nicolet FT-IR spectrometer.

ICP-AES analysis. The samples were analysed for Cd and Zn using an optical emission

spectrophotometer with inductively coupled plasma (Horiba Jobin Yvon, Activa). A quartz

Meinhard concentric nebulizer was used with a Scott-type spray chamber and a standard quartz

sheath connection between the spray chamber and the torch. Instrumental parameters were the

following: RF power (1200 W), sample flow rate (1.0 mL min-1) and plasma gas flow and

nebulizer gas flow of 12 and 0.95 L min-1, respectively. For analytical detection, the

wavelengths used for Cd and Zn were (214.438, 226.502, 228.802) and (202.551, 206.200,

213.856), respectively.

Cell culture and cytotoxicity studies. PC3 cells were obtained from the American Type Tissue

Collection (LGC Promochem) and cultured in Ham's F-12K (Kaighn's) medium (Gibco®)

supplemented with 10% fetal bovine serum (Invitrogen) at 37ºC and 5% CO2. Cells were

passaged at ~70 % confluence and a low passage number was maintained using cryopreserved

stocks stored in FBS supplemented with 10 % DMSO (Sigma Aldrich).

The effect of drug formulations on cell viability was measured using the Sulforhodamine B

assay [4].


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Cells were plated at 3000 cells/well in 96-well plates and allowed to adhere for 24 h. The

formulations (different mixtures of c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] and MQDs and the

corresponding controls before and after irradiation for 1h with the LED (480nm) were diluted

1/10 in media and incubated with cells at 37ºC. After 72h, cells were fixed by addition of ice

cold 25% trichloroacetic acid (TCA) solution prior to staining with Sulforhodamine B (SRB)

dye solution. Plates were washed with 1% glacial acetic acid, air-dried and resuspended in 10

mM Tris buffer, pH 10.5 before reading absorbance at 550 nm. Curve fitting and generation of

IC50 values was carried out using GraphPad Prism 4 software from six replicates.

References

[1] T. Jin, F. Fujii, H. Sakata, M. Tamura, and M. Kinjo, Chem. Commun., 2005, 4300.

[2] W.W.Yu, L. Qu, W.Guo, and X.Peng, Chem. Mater., 2003, 15, 2854.

[3] K.R. Barnes, A. Kutikov, and S.J. Lippard, Chem. Biol., 2004, 11, 557.

[4] V. Vichai, K. Kirtikara, Nat. Protoc., 2006, 1, 1112.


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Hydrodynamic Diameter (nm)

Vo

lum

e (%

)

1 10 100 10000

5

10

15

20

25

Fig S1. Transmission electron micrograph (TEM) (top) and hydrodynamic size

distributions (bottom) measured by DLS imaging of the MQDs.


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Fig S2. UV-vis (red) and photoluminescence (red) spectrum of the MQDs in water

(top). Second derivative of the absorption spectra showing the position of the first

exciton peak (bottom).

(red) and photoluminescence (red) spectrum of the MQDs in water

Second derivative of the absorption spectra showing the position of the first

(red) and photoluminescence (red) spectrum of the MQDs in water

Second derivative of the absorption spectra showing the position of the first


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a) b)

c) d)

Fig. S3. X-ray photoelectron spectra of the water-soluble MQDs showing the: a) Cd 3d, b) Se

2d, c) Zn 2p and d) S 2p peaks.


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Fig. S4. FT-IR spectrum of MQDs

[Re(OH2)3(CO)3]+.

MQDs before (black) and after (blue) reaction with

before (black) and after (blue) reaction with fac-


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Fig S5. Photoluminescence spectra of

Photoluminescence spectra of MQDs before and after addition of 1.


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(A)

(B)

Fi g S6. 1H NMR spectra (500 MHz) of :

different time intervals in PBS (pH 7.4, 37

H NMR spectra (500 MHz) of : (A) 1 + ascorbic acid (10 mM) and (B) 1 + glutathione at

different time intervals in PBS (pH 7.4, 37 °C).

+ glutathione at


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(A)

(B)

Fig S7. 1H NMR spectra (500 MHz) of

480 nm (15 mW/cm2) and (B) 630 nm (20 mW/cm

H NMR spectra (500 MHz) of 1 and 1 + MQDs before and after 1 h irradiation with

630 nm (20 mW/cm2).

+ MQDs before and after 1 h irradiation with (A)


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% Survival

0 20 40 60 80 100 120

Irradiated mixture (MQD + 1)

non irradiated mixture (MQD + 1)

irradiated 1

non irradiated 1

irradiated MQD

non irradiated MQD

----

----

----

----

----

----

----

----

-

[1] = 50 µM

% Survival

0 20 40 60 80 100 120



irradiated 1

non irradiated 1

irradiated MQD

non irradiated MQD

----

----

----

----

----

----

----

----

-

[1] = 30 µM

% Survival

0 20 40 60 80 100 120



irradiated 1

non irradiated 1

irradiated MQD

non irradiated MQD

----

----

----

----

----

----

----

----

-

[1] = 20 µM

Fig. S8. Cytotoxicity for the MQDs, 1 and 1 + MQDs in the dark and following 1 h

irradiation with a low power (14 mW) LED light (480 nm). [MQD] = 4 nM.


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Time (h)1 3 5

--------------------------------------------------------------------------------------------------

[MQD] (nM)

% S

urv

ival

1 2 4 60

20

40

60

80

100

120

--------------------------------------------------------------------------------------------------

[1] (µµµµM)

% S

urv

ival

10 20 30 500

20

40

60

80

100

120

2

MQD-N

H

MQD-COOH 3

MQD-OCH

Fig. S9. Cytotoxicity for the 1 + MQDs mixtures under different conditions: [MQD] =

1-4 nM, [1] = 10 µM, irradiation time = 1 h (top left), [MQD] = 4 nM, [1] = 10 µM,

irradiation time = 1-5 h (top right), [MQD] = 4 nM, [1] = 10-50 µM, irradiation time =

1 h (bottom left), [MQD] = 4 nM prepared with PEGylated phospholipids with -NH2, -

COOH and -OCH3 groups, [1] = 10 µM, irradiation time = 1 h (bottom right).


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