Supporting Information

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

Supporting Information

for

Amphiphilic Bisporphyrin and its YbIII Complex: Improved Cellular Uptake

and Development of a Bifunctional Photodynamic Therapeutic and

Near-Infrared Tumor Imaging Agent

Feng-Lei Jiang, Chun-Ting Poon, Wai-Kwok Wong,* Ho-Kee Koon, Nai-Ki Mak,*

Chun Yu Choi, Daniel W. J. Kwong,* and Yi Liu*

General: All chemicals used were obtained from Aldrich Chemical Company. Elec-

tronic absorption spectra in the UV/Vis region were recorded on a Varian Cary 100

UV-visible spectrophotometer. Steady-state visible photoluminescence (PL) and exci

-tation spectra were recorded on a Photon Technology International (PTI) Alphascan

spectrofluorimeter. Near-infrared (NIR) emission was detected using a liquid nitrogen-

cooled InSb IR detector (EG & G) with a preamplifier and recorded by a lock-in ampli-

fier system as the third harmonics. NMR spectra were recorded on a Varian INOVA

400 MHz spectrometer. 1H NMR chemical shifts were referenced to deuterated chloro-

form (internal standard) and then recalculated to TMS (d 0.00). 31P{1H} NMR spectra

were referenced to external 85% H3PO4. High-resolution mass spectra, reported as

m/z, were obtained on a Autoflex Bruker MALDI-TOF system.

Experimental details

Cells. The Sarcoma 180 cell line was obtained from Prof. K.N. Leung of the Bio-

chemistry Department, Chinese University of Hong Kong and maintained in Roswell

Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% fetal calf

serum (Gibco, USA) and antibiotics PSN (50 U/mL penicillin G, 50 µg/mL streptomycin

and 100 µg/mL neomycin). Cells were incubated at 37 °C in a humidified 5% CO2

incubator.

Singlet oxygen measurement. Four sample solutions of diphenylisobenzofuran

(DPBF) in methanol (5×10-5 M) containing, respectively, no sensitizer (control sample),

H2TMPyP (2 x 10-6 M), 1 (10-6 M) and Yb.1 (10-6 M) were prepared in dark. Each sam-

ple container was covered with aluminum foil with a yellow filter (with cutoff wave-

length <500 nm) on one side. The samples were then exposed to light (50 W) through

the filter. After irradiation, visible spectra of the sample solutions were measured spec-

trophotometrically. The normalized absorbances of DPBF at 415 nm in these samples

were reported as a function of the photo-irradiation time.

Near-infrared (NIR) luminescence lifetime measurement. The NIR luminescence

lifetime of Yb.1 in water was measured using the following setup. The excitation

source was a 425 nm pulse laser from optical parametric oscillator (OPO, from Magic-

Prism-Opoteck) pumped by the third harmonic of a Quantel Brilliant B Nd-YAG laser,

with the luminescence signal being collected by a lens, using appropriate optical filters,

and then dispersed through a 0.5 m focal length monochromator. The NIR decay sig-

nal, detected by an Oriel 77343 photomultiplier tube and monitored by a Agilent infini-

um 54830B 600 MHz oscilloscope, was fitted to a monoexponential function to obtain

the reported first-order lifetime.

DNA photocleavage assay. DNA photocleavage activities of the bisporphyrins, 1

and Yb.1 , were measured using the plasmid DNA relaxation assay. The plasmid DNA

(pBluescript), enriched with the supercoiled conformer (i.e., Form I), and the one-

phor-all plus buffer (10 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium

acetate, pH 7.5) was vortexed. Aliquots of the DNA were pipetted into different Eppen-

dorf tubes. Various amounts of autoclaved water (control sample) or test samples

were added into the Eppendorf tubes to give a final volume of 20 µL in each sample

tube. The sample mixtures were then photo -irradiated at 400-450 nm for 45 min using

a tranilluminator (Vilber Lourmat) equipped with 4 x 15 W light tubes (Aqua Lux) with

maximum emission at 455 nm. After photoirradiation, 2 µL of the 6x sample dye solu-

tion (which contained 20% glycerol, 0.25% bromophenol blue and 0.25% xylene cyan-

ol FF) was added to each Eppendorf tube and mixed well by centrifugation. The sam-

ple mixtures were loaded onto a 0.8% (v/v) agarose gel (13 cm x 10 cm), with 1x TBE

buffer (89 mM Tris-borate, 1 mM EDTA, pH 8) as supporting electrolyte, and electro-

phorezed at 1.3 Vcm-1 for 3 h using a mini gel set (CBS Scientific Co., Model MGU-

502T). After electrophoresis, the gel was stained with 0.5 µg/mL ethidium bromide

solution for 30 min and then destained with deionized water for 10 min. The resulting

gel image was viewed under 365 nm and captured digitally using a gel documentation

system (BioRad). The DNA cleavage activity was calculated as follows: % Cleavage

Activity = %Form II(sample) − %Form II(control). In the singlet oxygen and hydroxyl

radical quencher experiments, DNA photocleavage assays were carried out for 1 and

Yb.1 (both at 20 µM concentration) in the presence of 50- to 5000-fold molar excess of

quencher. L-histidine and mannitol were used as singlet oxygen and hydroxyl radical

quenchers, respectively.

Flow cytometric analysis of cellular uptake of porphyrins. Sarcoma 180 cells

were incubated separately with 4 µM of H2TMPyP, 1 and Yb.1 for different durations,

namely, 3 h, 6 h or 22 h, in dark. The cells were then trypsinized, washed with PBS

and fixed with paraformaldehyde (3% in PBS). The fluorescence profiles of these cells

were analyzed using FACSCalibur (Becton Dickinson). The 488 nm laser line was

used for excitation and the fluorescence signal was detected using the FL-3 channel

(long pass filter). At least 10 000 events were counted.

Spectrophotometric analysis of cellular uptake of porphyrins. Sarcoma 180 cells

were incubated separately with 4 µM of H2TMPyP, 1 and Yb.1 for 22 h in dark. The

treated cells were collected and washed thoroughly with PBS. The number of cells in

each treated cell suspension was counted under microscope and then diluted to a cell

density of 3.3 × 105 cells/mL in PBS. The absorption spectra (from 350-500 nm) of

these porphyrin-treated cell suspensions containing identical cell number were mea-

sured spectrophotometrically. The amounts of porphyrins taken up by these cells were

estimated semi-quantitatively based on single -point calibration using the absorbance

of a 5 µM standard solution prepared by dissolving H2TMPyP, 1 and Yb.1 in PBS,

respectively. The concentrations of different porphyrins taken up per 106 cells were

calculated.

Confocal microscopic analysis of the porphyrin-treated cells. Subcellular locali-

zations of 1 and Yb.1 were studied using an Olympus FV1000 confocal microscope.

A 488 nm argon-ion laser line was used for excitation of the organelle probes (Mito-

Tracker Green FM dye M7514 and LysoTracker Green DND-26 L7526, Molecular

Probes) and a diode laser line at 405 nm was used for excitation of 1 and Yb.1. Emis-

sions of the organelle probes and the porphyrins were detected using variable band

pass filter sets at 500-600 nm and 630-730 nm, respectively. Except otherwise stated,

the pinhole size of 80-100 µm was selected to exclude fluorescence light emitted from

out-of-focus planes above and below the focusing plane. A 40x objective was used for

image capturing. Images were processed and analyzed using the FV10-ASW

software (Olympus).

Photodynamic treatment (PDT) assay. Sarcoma 180 cells (10 000 cells/well)

were incubated with various concentrations of H2TMPyP, 1 and Yb.1 (0-8 µM) for 22 h

in 96-well flat bottom tissue culture plates. Then the treated cells were centrifuge

washed with fresh medium and exposed to yellow light (0-16 J/cm2) from a 400 W

tungsten lamp fitted with heat-isolation filter and 500 nm long-pass filter at an intensity

of 5.6 mW/cm2, as measured with a power meter (OPHIR). The cells were incubated

for another 22 h. Viability was then assessed with the 3-(4, 5-dimethylthiazol-2-yl)-2,

5-diphenyl-tetrazolium bromide (MTT) reduction assay. The optical density (OD) of

dissolved formazan crystal was measured using the iEMS Analyzer (Lab-system,

Type 1401) at 570 and 690 nm. The percentage of cytotoxicity was calculated by

using the following equation:

cytotoxicity (%) = (ODcontrol group – ODtreatment group)/ODcontrol group × 100,

where OD = OD570-690 nm.

Flow cytometric analysis of apoptotic cells. Sarcoma 180 cells were incubated

with 1 and Yb.1 (4 µM) for 22 h. The cells were then washed 3 times with fresh medi-

um and irradiated with yellow light (12 J/cm2, > 500 nm). After 12 to 24 h of incubation,

the cells were washed with PBS and fixed in 70% ethanol for at least 1 h at 4°C. The

fixed cells were then stained with propidium iodide (40 µg/mL) containing buffer (1

mg/mL RNase A and 0.1% Triton X-100 in PBS) for 30 min. Fluorescence intensities

of the stained cells were analyzed using the FACSCalibur Flow Cytometer (Becton

Dickinson). The 488 nm laser line was used for excitation, and the fluorescence signal

was collected using the FL-2 channel. At least 10 000 events were counted. DNA con-

tent was analyzed using Cell Quest Software and ModfitLT Version 3.0 Software.

Nucleus staining. Sarcoma 180 cells were treated with 1 (4 µM) for 22 h, washed

with fresh medium and irradiated with light (> 500 nm, 12 J/cm2) and incubated for 3 to

22 h. Cells from each sample of the control and the PDT-treated groups were collect-

ed, washed with PBS three times, fixed with paraformaldehyde (2% in PBS, 20 min)

and methanol (-20 °C, 20 min). The cells were washed with PBS again and stained

with fluorescence dye Hoechst 33258 (20 µg/mL in PBS, 15 min). The fluorescence

image of the cells was observed and photographed using a Zeiss Axioplan fluores-

cence microscope with 330-380 nm excitation from an attached 50 W mercury lamp

combined with a 400 nm dichroic mirror and the 420 nm long-pass filter. Apoptotic

cells with the characteristic pattern of chromatin condensation and fluorescence were

counted.

Preparation of 1: The synthetic route is presented below.

N

NH N

HNN

N

N

OH

N

NH N

HNO

Br

NN

NN

O

Yb

Co

OOO

(MeO)2P P(OMe)2(MeO)2P

O

N

NH N

HN

N

N

N

NHN

HNN

O

O

N

NH N

HN

N

N

N

NHN

HNN

O

O

N

NH N

HN

N

N

N

Cl

K2CO3Dry DMF

60 oC

NN

NN

O

Yb

Co

OOO

(MeO)2P P(OMe)2(MeO)2P

Br

NN

NN

O

Yb

Co

OOO

(MeO)2P P(OMe)2(MeO)2P

Cl

Cl

O

N

NH N

HN

N

N

N

Cl

Cl

Cl

1 CH3I , DMF2 Ion exchange column

1 CH3I , DMF2 Ion exchange column

N

NH N

HNN

N

N

OHK2CO3Dry DMF60 oC

p-BrC3O-TPPH2 [Yb(p-BrC3O-TPP)(LOMe)]

p-OH-TPy P

H2TPPC3TPyPYbTPPC3TMPyP

1Yb.1

p-BrC3O-TPPH2[a], [Yb(p-BrC3O-TPP)(LOMe)][a] and p-OH-TPyP[b] were synthe-

sized according to literature procedures [(a) F.-L. Jiang, W.-K. Wong, X.-J. Zhu, G. -J.

Zhou, W.-Y. Wong, P.-L. Wu, H.-L. Tam, K.-W. Cheah, C. Ye, Y. Liu, Eur. J. Inorg.

Chem. 2007, 21, 3365-3374; (b) C. Casas, B. Saint-Jalmes, C. Loup, C. J. Lacey, B.

Munier, J. Org. Chem. 1993, 58, 2913-2917].

p-OH-TPyP (30 mg, 0.047 mmol) was dissolved in freshly distilled DMF (10 mL)

and stirred with anhydrous K2CO3 (300 mg, excess). Then p-BrC3O-TPPH2 (56 mg,

0.075 mmol) was added. The resulting mixture was stirred at 60 oC at N2 atmosphere

for overnight. The reaction progress was monitored by TLC. The mixture was cooled

to room temperature. After 20 mL of chloroform was added, it was washed with deion-

ized water to remove all the DMF, concentrated and purified by column chromatogra-

phy on silica gel using chloroform/methanol (v/v, 50:1) as eluent. The second band

gave the precursor H2TPPC3TPyP. Yield: 50 mg, 81%. HRMS: 1305.5168 (M+1). 1H

NMR (CDCl3): d -2.86 (s, 2H), -2.76 (s, 2H), 2.67 (m, 2H), 4.64 (m, 4H), 7.41 (m, 4H),

7.67-7.81 (m, 9H), 8.11 (m, 4H), 8.15-8.24 (m, 12H), 8.78-8.86 (m, 12 H), 8.91 (m, 2H),

8.98-9.04 (m, 8H). UV-Vis: (in toluene) ?max/nm [log(e/dm3mol-1cm-1)] 421 (5.41), 516

(4.08), 550 (3.71), 591 (3.53), 647 (3.31).

H2TPPC3TPyP was reacted with CH3I in DMF for 24 h, and then passed through

an ion exchange column so as to replace the iodide counter ion with chloride. The

solvent was removed in vacuo and the resulting solid gave 1 in quantitative yield.

HRMS: 1349.5631 (M-3I). 1H NMR (CDCl3): d -3.00 (s, 2H), -2.92 (s, 2H), 2.60 (m,

2H), 4.64 (m, 4H), 4.70 (m, 9H), 7.55 (m, 4H), 7.78-7.83 (m, 9H), 8.18 (m, 12H),

8.82-9.146 (m, 22 H), 9.44 (m, 4H). UV-Vis: (in H2O) ?max/nm [log(e/dm3mol-1cm-1)]

422 (5.43), 517 (4.28), 554 (4.04), 589 (3.95), 650 (3.30).

Preparation of Yb.1

The precursor YbTPPC3TPyP was prepared according to the method for

H2TPPC3TPyP with [Yb(p-BrC3O-TPP)(LOMe)] and p-OH-TPyP as starting materials.

Yield: 35%. HRMS: 1926.4215 (M+). 1H NMR (CDCl3): d -4.61 (s, 5H), -2.72 (s, 2H),

3.42 (m, 2H), 5.30 (t, J = 5.6 Hz, 2H), 5.72 (t, J = 5.6 Hz, 2H), 6.44 (s, 18H), 7.90 (d, J

= 8.4 Hz, 2H), 8.17 (d, J = 4.4 Hz, 4H), 8.24 (d, J = 4.4 Hz, 2H), 8.50 (d, J = 8.4 Hz,

2H), 8.70-8.78 (m, 8H), 8.93-9.11 (m, 12H), 9.30 (d, J = 4.4 Hz, 2H), 9.41-9.46 (m, 3H),

10.22 (s, 1H), 10.60 (s, 3H), 15.38 (m, 8H), 16.71-16.96 (m, 4H). 31P{1H} NMR

(CDCl3): d 69.0 ppm. UV-Vis: (in toluene) ?max/nm [log(e/dm3mol-1cm-1)] 429 (5.87),

515 (4.38), 558 (4.48), 597 (4.11), 647 (3.48).

Yb.1 was prepared with the method as that of 1. HRMS: 1971.4975 (M-3I). 1H

NMR (DMSO): d -4.94 (s, 5H), -3.08 (s, 2H), 3.30 (m, 2H), 3.92 (s, 6H), 4.68 (s, 3H),

5.22 (t, J = 5.6 Hz, 2H), 5.63 (t, J = 5.6 Hz, 2H), 6.37 (s, 18H), 7.51 (m, 6H), 7.96-8.28

(m, 10H), 8.43-9.45 (m, 14H), 10.34 (m, 1H), 10.70 (m, 3H), 15.50 (m, 8H), 16.82-

17.19 (m, 4H). 31P{1H} NMR (DMSO): d 67.9 ppm. UV/Vis: (in H2O) ?max/nm

[log(e/dm3mol-1cm-1)] 427 (5.33), 523 (4.00), 561 (4.18), 598 (3.84), 649 (3.00).

300 350 400 450 500 550 600 650 700 750

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Abs

Wavelength (nm)

Yb.1 1

Figure S1. Absorption spectra of 1 and Yb.1 in water.

300 400 500 600 700 800

0

200000

400000

600000

800000

1000000

1200000

1400000

Pho

tolu

min

esce

nce

Inte

nsity

(a. u

.)

Wavelength (nm)

Em: 1 Ex: 1 Ex: Yb.1 Em: Yb.1

431 nm

431 nm

661 nm

661 nm

Figure S2. Steady-state excitation (Ex) and fluorescence spectra (Em) of 1 µM aqueous

solutions of 1 and Yb.1.

-50 0 50 100 150 200

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

Inte

nsity

(a. u

.)

Time (microsecond)

NIR luminescence decay curve Fitted NIR luminescence decay curve

Figure S3. Near-infrared (NIR) luminescence decay curve of Yb.1 monitored at 1000 nm. The

data was fitted with a monoexponential function, y(t) = A1exp(−t/τ) + yo, to obtain a fitted first-

order lifetime τ of 10.13 ± 0.05 µs (R2 = 0.96).

a) b)

c) d)

Figure S4. Gel images of DNA photocleavage assay of 1 and Yb.1 at different concentrations

and in the presence of L-histidine, a singlet oxygen quencher. Photo-irradiation conditions:

?irrad = 455 nm; duration, 45 min. a) DNA photocleavage activity of 1 at different concentra-

tions. Lane 1: supercoiled DNA (Form I) alone; lane 2: 1 µM; lane 3: 5 µM; lane 4: 10 µM; lane

5: 15 µM; lane 6: 20 µM; lane 7: 25 µM. b) DNA photocleavage activity of Yb.1 at different

concentrations. Lane 1: DNA control; lane 2: 1 µM; lane 3: 5 µM; lane 4: 10 µM; lane 5: 15 µM;

lane 6: 20 µM; lane 7: 25 µM. c) DNA photocleavage activity of 1 (20 µM) in the presence of

increasing concentration of L-histidine. Lane 1: DNA control; lane 2: 1 alone; lane 3: 1 + 1 mM

L-histidine; lane 4: 1 + 2 mM L-histidine; lane 5: 1 + 10 mM L-histidine; lane 6: 1 + 20 mM

L-histidine; lane 7: 1 + 100 mM L-histidine. d) DNA photocleavage activity of Yb.1 (20 µM) in

the presence of increasing concentration of L-histidine. Lane 1: DNA control; lane 2: Yb.1;

lane 3: Yb.1 + 1 mM L-histidine; lane 4: Yb.1 + 2 mM L-histidine; lane 5: Yb.1 + 10 mM

L-histidine; lane 6: Yb.1 + 20 mM L-histidine; lane 7: Yb.1 + 100 mM L-histidine.

Figure S5. Flow cytometric analysis of cellular uptake of H2TMPyP, 1 and Yb.1. Sarcoma 180

cells were incubated with H2TMPyP, 1 and Yb.1 (4 µM) for 0 h (control, black line), 3 h (blue

line), 6 h (green line) or 22 h (red line) in dark. The cells were then washed with PBS. A mini-

mum of 10,000 cells per sample was analyzed using the excitation and emission wavelengths

of 488 nm and 650 nm, respectively. The X-axis represents the red-fluorescence intensity of

the treated cells.

340 360 380 400 420 440 460 480 500 520

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Abs

Wavelength (nm)

5 µM of H2TMPyP in water

H2TMPyP-loaded

cell suspension

a

340 360 380 400 420 440 460 480 500 520

0.00

0.01

0.02

0.03

0.04

0.05

Abs

Wavelength (nm)

5 µM of 1 in water 1-loaded

cell suspension

b

340 360 380 400 420 440 460 480 500 520

0.00

0.01

0.02

0.03

0.04

0.05

c

Abs

Wavelength (nm)

5 µM of Yb.1 in water Yb.1-loaded

cell suspension

Figure S6. Absorption spectra of the porphyrin-loaded cell suspensions: (a) H2TMPyP, (b) 1,

and (c) Yb.1. Sarcoma 180 cells were incubated with H2TMPyP, 1 and Yb.1 in the dark for 22

h, centrifuge washed, resuspended in PBS and then diluted to a cell density of 3.33×105 cells

per mL. The absorption spectra of these porphyrin-treated cell suspensions were measured.

The absorption spectra of their authentic compounds dissolved in water (5 µM) are also given

for comparison.

Figure S7. Confocal images of Sarcoma 180 cells treated with H2TMPyP and two organelle-

specific probes, (a) LysoTracker and (b) MitoTracker. Sarcoma 180 cells were incubated with

4 µM of H2TMPyP for 22 h. The cells were then stained with the mitochondria or lysosome

probes as described in Figure 5. Upper panel: confocal (left) and bright field (right) images.

Red dots represent the fluorescence from H2TMPyP and the green dots represent the fluo-

rescence from the organelle probes. Yellow dots represent the overlapping fluorescent signals

from H2TMPyP and the organelle probes. Scale bar: 20 µm. Note: Since the amount of

H2TMPyP absorbed by the cells (and hence the fluorescence intensity emitted by H2TMPyP)

was lower than the 1 and Yb.1, a pinhole size of 500 µm was used to increase the amount of

fluorescence light entering the detector of the confocal microscope. Hence, the yellow dots

(as indicated by an arrow) appear in (a) may be the result of overlapping of both focused and

light emitted from out-of-focus planes above and below the focusing plane.

Figure S8. Detection of apoptotic cells after PDT. Drug control: cells treated with test com-

pound but no light; Light control: cells treated with light but no test compound. Sarcoma 180

cells were treated with 1 (4 µM) for 22 h, washed with fresh medium and irradiated with light

(>500 nm, 12 J/cm2). After light irradiation, the cells were incubated for 12 to 24 h and then

stained with propidium iodide. A minimum of 10,000 cells per sample was analyzed by flow

cytometry.

Figure S9. Fluorescence microscopic images of Sarcoma 180 cells stained with Hoechst

33258. The Sarcoma 180 cells were treated with 1 (4 µM) for 22 h, washed with fresh medium

and then photo-irradiated (>500 nm, 12 J/cm2). After photo-irradiation, the cells were incu-

bated for 3 to 22 h. The cells were then stained with Hoechst 33258 for 15 min, washed with

PBS for at least 3 times and immediately examined under a fluorescence microscope.