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Supplementary Information
Photostable fluorescent organic dots with aggregation-induced
emission (AIE dots) for noninvasive long-term cell tracing
Kai Li1,2,*, Wei Qin3,*, Dan Ding2, Nikodem Tomczak1, Junlong Geng2, Rongrong Liu1,
Jianzhao Liu3, Xinhai Zhang1, Hongwei Liu1, Bin Liu1,2 & Ben Zhong Tang1,3,4
1
Institute of Materials Research and Engineering, 3, Research Link, Singapore 117602.
2
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4
Engineering Drive 4, Singapore 117576. 3 Department of Chemistry, Division of Biomedical
Engineering, Institute for Advanced Study, State Key Laboratory of Molecular Neuroscience,
and Institute of Molecular Functional Materials, The Hong Kong University of Science &
Technology, Clear Water Bay, Kowloon, Hong Kong, China.
4
Guangdong Innovative
Research Team, State Key Laboratory of Luminescent Materials and Devices, South China
University of Technology, Guangzhou, China, 510640.
* Both authors contributed equally to this work. Correspondence and requests for materials
should be addressed to B.L. ([email protected]) or B.Z.T. ([email protected]).
Supplementary Information contains:
Supplementary Figures S1-S13
Supplementary Tables S1-S3
1
Supplementary Methods
Supplementary References
Supplementary Figure S1. Synthetic routes to TPAFN and TPETPAFN.
2
12H
8H
*
4H
7.8
4H
7.6
7.4
7.2
7.0
6.8
Chemical shift (nm)
Supplementary Figure S2. 1H NMR spectrum of TPAFN in CDCl3.
36H
*
8H
4H
4H
4H
7.8
7.6
7.4
7.2
7.0
6.8
Chemical shift (ppm)
Supplementary Figure S3. 1H NMR spectrum of TPETPAFN in CDCl3.
3
qw-2CN-TPA, MW=564; t-bpmp, LP200;
tan110614_5 24 (0.798) Cn (Cen,2, 90.00, Ht); Sb (99,40.00 ); Sm (SG, 2x3.00); Cm (13:29)
TOF LD+
2.99e3
564.2508
%
100
565.2565
566.2662
250.0938
523.3075
273.1214
319.7897
337.1792
409.1683
433.2142
0
200
225
250
275
563.2476
300
325
350
375
400
425
450
524.3126
511.2240
475
500
525
550
587.2493 605.8540 638.5781
575
600
625
650
700.8080 730.3456 761.9014
675
700
725
750
775
813.3851 827.2590
800
825
m/z
850
Supplementary Figure S4. High resolution mass spectrum (MALDI-TOF) of TPAFN.
qw-2CN-2ph-TPE, MW=1073; DHB, LP200;
tan110330_6 8 (0.264) Cn (Cen,2, 90.00, Ht); Sb (99,40.00 ); Sm (SG, 2x3.00); Cm (1:20)
TOF LD+
1.82e3
1072.4502
100
%
1073.4526
1074.4594
1075.4829
1075.8931
420.3396 494.7103 537.2281
0
300
400
500
586.0114
600
722.4651
700
995.4218 1071.4375
845.4288
800
900
1000
1077.9133
1100
1225.8075 1298.6310
1200
1300
1421.7800 1505.0836
1400
1500
1635.3408
1600
1697.0752
m/z
1700
Supplementary Figure S5. High resolution mass spectrum (MALDI-TOF) of
TPETPAFN.
4
TPETPAFN
TPAFN
LUMO
LUMO
HOMO
HOMO
LUMO
-2.42 eV
-2.42 eV
Eg = 2.48 eV
Eg = 2.61 eV
HOMO
LUMO
-4.90 eV
-5.03 eV
HOMO
Supplementary Figure S6. HOMO and LUMO energy levels of TPAFN and
TPETPAFN. Molecular orbital amplitude plots of HOMO and LUMO energy levels of
TPAFN and TPETPAFN calculated using B3LYP/6-31G(d) basis set in Gaussian 03 program.
Eg (energy gap) = LUMO -HOMO.
60 μm
Supplementary Figure S7. Cell autofluorescence. Fluorescence/transmission overlay
image of the MCF-7 cancer cells without incubation with Tat-AIE dots.
5
a
b
20 μm
20 μm
Supplementary Figure S8. Distribution of fluorescent dots in cell cytoplasm.
Fluorescence/transmission overlay images of MCF-7 cancer cells labeled by (a) Tat-AIE dots
and (b) Qtracker® 655.
400
R1
Blank
1st
5th
7th
Counts
300
200
100
0
0
10
10
1
10
2
10
3
10
4
FL1 Log
Supplementary Figure S9. Tracing of living cells using Tat-AIE dots with low
concentration. Flow cytometry histograms of the MCF-7 cells labeled with 0.2 nM Tat-AIE
dots after subculturing for designated passages. The untreated MCF-7 cells (blank) were used
as a control.
6
Cell viability (%)
100
80
60
40
20
0
1
2
8
Tat-AIE dot Concentration (nM)
Supplementary Figure S10. Cytotoxicity of Tat-AIE dots. Metabolic viability of MCF-7
breast cancer cells (red) and C6 glioma cells (blue) after incubation with the Tat-AIE dots at
different concentrations for 72 h.
80 μm
Supplementary Figure S11. Localization of Tat-AIE dots in tumor tissue. The
fluorescence image of sectioned tumor tissue collected from the mouse at 9 days post
injection of Tat-AIE dot-labeled C6 glioma cells.
7
6
5
TPA cross section (10 GM)
7
5
4
3
2
1
0
790
820
850
880
910
940
970
Wavelength (nm)
Supplementary Figure S12. Two-photon property. Two-photon absorption spectrum of
Tat-AIE dots in water.
60 μm
Supplementary Figure S13. Two-photon image. Two-photon excited fluorescence image
of the tumor tissue at 150 μm depth. λex = 800 nm.
8
Supplementary Table S1. Optical properties of TPETPAFN and TPAFN.
TPAFN
TPETPAFN
λab (nm)a
 (M-1 cm-1)b
Eg (eV)c
484
497
2.39×104
3.68×104
2.25(2.61)
2.16(2.48)
λem (nm)d
soln (ΦF,s)e
aggr
film (ΦF,f)f
αAIEg
652(2.32)
660(0.59)
18.3
89.0
655
671
649(42.5)
663(52.5)
Absorption maximum (λab) in THF. bMolar absorptivity () in THF. cHOMO-LUMO band
gap (Eg) calculated from the onset of the absorption spectrum, HOMO = highest occupied
molecular orbital, LUMO = lowest unoccupied molecular orbital. The values in the
parentheses are derived from theoretical DFT calculations. dEmission maximum (λem) in THF
solutions (soln, 1 μM), THF/water mixtures (aggr; 1:9 v/v; 1 μM), and solid thin films. eΦF,s
is fluorescence quantum yield in THF solution, which is determined by using
4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (ΦF,s = 43% in
methanol) as standard. fΦF,f is the fluorescence quantum efficiency in thin film state measured
by a calibrated integrating sphere. gAIE factor αAIE = ΦF,f/ΦF,s.
a
P2
P1
P3
Supplementary Table S2. Summary of dihedral angles (o) for TPAFN.
Dihedral angles (o)
-34.7
C3-C4-C7-C10
Angles between planes
≈66
≈66
≈76
P1-P2
P1-P3
P2-P3
9
P2
P1
P3
Supplementary Table S3. Summary of dihedral angles (o) for TPETPAFN.
Dihedral angles (o)
-35.2
-50.6
-49.1
-48.5
-50.1
C3-C4-C7-C10
C45-C46-C48-C59
C45-C46-C49-C65
C43-C42-C45-C46
C46-C45-C47-C64
Angles between planes
≈64
≈66
≈74
P1-P2
P1-P3
P2-P3
Supplementary Methods
Materials.
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (DSPE-PEG2000) was a gift from Lipoid GmbH (Ludwigshafen, Germany).
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)-2000]
(DSPE-PEG2000-NH2) was a commercial product of Avanti Polar Lipids, Inc. Qtracker® 655
cell labeling kit was purchased from Life Technologies, Invitrogen, Singapore. Fluoromount®
aqueous
mounting
medium,
Dulbecco's
10
Modified
Eagle
Medium
(DMEM),
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), tetrahydrofuran
(THF), diphenylamine,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide
(MTT), penicillin-streptomycin solution, fetal bovine serum (FBS) and trypsin-EDTA
solution were purchased from Sigma-Aldrich. THF was distilled from sodium benzophenone
ketyl under dry nitrogen immediately prior to use. All reactions and manipulations were
carried out under nitrogen gas with the use of standard inert atmosphere. Cell penetrating
peptide derived from transactivator of transcription proteins, HIV-1 Tat (49-57) with
C-terminus modified with cysteine (RKKRRQRRRC), was a commercial product customized
by GenicBio, China. Milli-Q water was supplied by Milli-Q Plus System (Millipore
Corporation, Breford, USA). MCF-7 breast cancer cells and rat C6 glioma cells were
provided by American Type Culture Collection.
Characterization.
1
H and
13
C NMR spectra were measured on a Bruker AV 300
spectrometer in CDCl3 using tetramethylsilane (TMS; δ = 0) as the internal reference. High
resolution mass spectra (HRMS) were recorded on a GCT premier CAB048 mass
spectrometer operating in MALDI-TOF mode. Elemental analysis was performed on a
ThermoFinnigan Flash EA1112. Fluorescence quantum efficiencies of the organic
compounds in films were measured using a calibrated integrating sphere. The UV-vis spectra
were recorded on a Milton Roy 5 Spectronic 3000 Array spectrophotometer. The
fluorescence spectra were measured using a fluorometer (LS-55, Perkin Elmer, USA). The
average particle size and size distribution were determined by laser light scattering with a
particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at
11
24°C. The morphology of AIE dots was studied by high-resolution transmission electron
microscope (HR-TEM, JEM-2010F, JEOL, Japan).
Preparation of bis(4-bromophenyl)fumaronitrile(2)s1. A cooled (-78 °C) solution of
sodium methoxide (25% solution in methanol) (43 mmol, 9.83 mL) in methanol (15 mL) was
added dropwise over a period of 30 min to a cooled (-78 °C) solution of iodine (5.08 g, 20
mmol), 4-bromophenylacetonitrile (1) (3.92 g, 20 mmol) and ethyl ether (70 mL). After
stirring for 3 h at -78 °C, the mixture was allowed to warm to 0 °C and stirred for another 4 h
at 0 °C. The mixture was quenched upon addition of HCl(aq) (3%, v/v, 60 mL) and the
resulted precipitates were filtered, rinsed with water (3 × 100 mL), Na2S2O5(aq) (5%, v/v, 3 ×
50 mL) and water (3 × 100 mL). The product was obtained as pale yellow solids in 86% yield
(3.34 g) after recrystallization from ethanol. 1H NMR (300 MHz, CDCl3), δ (TMS, ppm):
7.73–7.66 (m, 8H); 13C NMR (75 MHz, CDCl3), δ (TMS, ppm): 133.4, 131.2, 130.8, 127.5,
125.3, 116.8; HRMS (MALDI-TOF, m/z): [M]+ calcd. for C16H879Br2N2, 385.9054; found,
385.9055; calcd. for C16H879Br81BrN2, 387.9034; found, 387.9034; calcd. for C16H881Br2N2,
389.9013; found, 389.9035.
Preparation of 1-(4-bromophenyl)-1,2,2-triphenylethylene (6)s2. A 2.5 M solution of
n-butyllithium in hexane (9.75 mmol, 3.9 mL) was added to a solution of diphenylmethane
(1.85 g, 11 mmol) in anhydrous tetrahydrofuran (60 mL) at 0 °C. After stirring for 0.5 h at
0 °C, 4-bromobenzophenone (4) (2.35 g, 9 mmol) was added at -78 °C, and the resulting
mixture was stirred for 5 h at -78 °C, allowing the temperature to gradually rise to 24°C. The
reaction was then quenched with an aqueous solution of ammonium chloride and the mixture
was extracted with chloroform (250 mL). The organic layer was evaporated after drying with
12
anhydrous MgSO4. The resulting crude alcohol (5) was dissolved in toluene (100 mL) in a
250 mL Schlenk flask fitted with a Dean-Stark trap. The p-toluenesulfonic acid (50 mg, 0.29
mmol) was added, and the mixture was refluxed for 2.5 h before cooling to 24°C. The solvent
was evaporated and the crude product was purified by silica gel column chromatography
using hexane/chloroform (v/v = 30/1) as eluent to yield 6 as white powders in 86% yield
(3.18 g). 1H NMR (300 MHz, CDCl3), δ (TMS, ppm): 7.21 (d, 2H, J = 8.4 Hz), 7.13–7.08 (m,
9H), 7.03–6.98 (m, 6H), 6.89 (d, 2H, J = 8.4 Hz); HRMS (MALDI-TOF, m/z): [M]+ calcd.
for C26H1979Br, 410.0670; found, 410.0669; calcd. for C26H1981Br, 412.0650; found,
412.0656.
Preparation
of
N-(4-(1,2,2-triphenylvinyl)phenyl)benzenamine
(7)
.
1-(4-Bromophenyl)-1,2,2-triphenylethylene (6) (2.05 g, 5 mmol) and aniline (0.6 g, 6.5 mmol,
0.6 mL), tri-tert-butylphosphine (16.2 mg, 0.08 mmol), Pd2(dba)3 (64 mg, 0.07 mmol) and
sodium tert-butoxide (625 mg, 6.5 mmol) were mixed with dry toluene (30 mL) in a
two-necked round bottom flask containing a stir bar. The mixture was stirred at 110 °C for 24
h. After solvent removal, water (30 mL) and chloroform (200 mL) were added. The organic
layer was separated and washed with brine, dried over anhydrous MgSO4 and evaporated to
dryness under reduced pressure. The crude product was purified by column chromatography
on silica gel using hexane/chloroform (v/v = 5/1) as eluent to afford 7 as pale yellow solids in
78.3% yield (1.65 g). 1H NMR (300 MHz, CDCl3), δ (TMS, ppm): 7.48–7.44 (d, 2H),
7.33–7.28 (t, 4H). 7.14–7.01 (m, 21H), 6.96–6.92 (t, 1H), 5.75 (s, 1H);
13
C NMR (75 MHz,
CDCl3), δ (TMS, ppm): 144.12, 144.01, 143.95, 142.68, 141.37, 140.65, 139.85, 136.14,
132.40, 131.43, 131.36, 131.34, 129.24, 127.68, 127.57, 127.55, 126.32, 126.20, 126.15,
13
120.96, 117.89, 116.36; HRMS (MALDI-TOF, m/z): [M]+ calcd. for C32H25N, 423.1987;
found, 423.1738.
Preparation
of
TPAFN
(10)s3,s4.
The
compound
was
prepared
from
bis(4-bromophenyl)fumaronitrile (2) (194 mg, 0.5 mmol), diphenylamine (8) (338 mg, 2
mmol), Cs2CO3 (1.14 g, 3.5 mmol), Pd(OAc)2 (11.2 mg, 0.05 mmol), and
tri-tert-butylphosphine (30.3 mg, 0.15 mmol), following the same procedure described for the
synthesis of TPETPAFN to afford 10 as a red solid in 50% yield (141 mg). 1H NMR (300
MHz, CDCl3), δ (TMS, ppm): 7.68 (d, J = 8.9 Hz, 4H), 7.33 (t, J = 7.8 Hz 8H), 7.18–7.12 (m,
12H), 7.05 (d, J = 8.9 Hz, 4H); 13C NMR (75 MHz, CDCl3), δ (TMS, ppm): 150.28, 146.31,
129.81, 129.64, 125.91, 124.70, 124.48, 120.31, 117.72; HRMS (MALDI-TOF, m/z): [M]+
calcd. for C40H28N4, 564.2314; found, 564.2508.
Preparation of TPAFN and TPETPAFN nanoaggregates. Stock solutions of the
compounds in THF with a concentration of 10-5 M were prepared. Aliquots of the stock
solution were transferred to 10 mL volumetric flasks. After appropriate amounts of THF were
added, water was added dropwise under vigorous stirring to furnish 10-6 M solutions with
different water contents (0–90 vol%). The PL measurements of the resultant solutions were
then performed immediately.
Two-photon absorption measurements. The two-photon absorption spectrum was
measured using two-photon induced fluorescence (TPIF) spectroscopys5. The Rhodamine 6G
solution and Tat-AIE dot suspension were degassed before measurements. The samples were
excited with laser pulses of 100 fs produced by the mode-locked Ti:Sapphire laser
(Spectraphysics Tsunami) with a repetition rate of 82 MHz, and a femtosecond optical
14
parametric amplifier (OPA) was used within the spectral range of 800-960 nm. The emission
spectra for the Tat-AIE dot aqueous suspensions were collected at a 90o angle by a high
numerical aperture lens and directed to a spectrometer’s entrance slit. Rhodamine 6G in
methanol was used as the reference. Two-photon absorption cross sections were further
calculated from the following equations6:
2
1

F21c1n1
F1 2 c 2 n2
Where δ1 and δ2 are the TPA cross sections, F1 and F2 are the TPIF intensities, η1 and η2 are
the fluorescence quantum yields, c1 and c2 are the concentrations, n1 and n2 are the refractive
indexes of solvents (1 corresponds to Rhodamine 6G, 2 is Tat-AIE dot).
Cell culture. MCF-7 breast cancer cells and C6 glioma cells were cultured in DMEM
containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C in a humidified
environment containing 5% CO2. Before experiment, the cells were pre-cultured until
confluence was reached.
Cytotoxicity of Tat-AIE dots. The metabolic activities of MCF-7 breast cancer cells and C6
glioma cells were evaluated using methylthiazolyldiphenyltetrazolium bromide (MTT) assays.
MCF-7 breast cancer cells and C6 glioma cells were seeded in 96-well plates (Costar, IL,
USA) at an intensity of 6 × 104 cells/mL, respectively. After 24 h incubation, the old medium
was replaced by Tat-AIE dot suspension at concentrations of 1, 2, and 8 nM, and the cells
were then incubated for 72 h. To eliminate the UV-vis absorption interference of the Tat-AIE
dots at 570 nm, the cells incubated with the Tat-AIE dots without post-treatment by MTT
were used as the control. After 72 h, the wells were washed twice with 1× PBS buffer and
100 µL of freshly prepared MTT (0.5 mg/mL) solution in culture medium was added into
15
each well. The MTT medium solution was carefully removed after 3 h incubation in the
incubator. DMSO (100 µL) was then added into each well and the plate was gently shaken
for 10 min at 24°C to dissolve all the precipitates formed. The absorbance of MTT at 570 nm
was monitored by the microplate reader (Genios Tecan). Cell viability was expressed by the
ratio of the absorbance of the cells incubated with Tat-AIE dot suspension to that of the cells
incubated with culture medium only.
Ex vivo one-photon and two-photon excited fluorescence imaging. In the ex vivo tumor
imaging experiment, the mice were euthanized with CO2 and the tumors were collected at 9 d
post injection and fixed in 4% paraformaldehyde for two-photon excited 3D fluorescence
imaging. To prepare sectioned tumor tissue, the tumor was resected and fixed in 4%
paraformaldehyde for 2 h. The tumor was then incubated in 20% sucrose/PBS overnight,
embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek) and cut into thin
sections (6 µm) with a microtome at -24 oC (Leica CM 1900 Rapid Sectioning Cryostat).
The whole tumor and sectioned tumor tissue were imaged using Leica TCS SP 5X and
multiphoton microscope equipped with two-photon Chameleon Ultra II (excitation at 800 nm
at ~39 mW with a 550-780 nm bandpass filter). Two-photon excited fluorescence images of
consecutive layers with approximately 3 μm interval were recorded to generate 3D colored
projection to demonstrate the penetration depth of Tat-AIE dots in the tumor. The one-photon
excited fluorescence images were taken upon excitation at 560 nm with a 600-780 nm
bandpass filter.
Calculation of Tat-AIE dot concentration
16
Freeze-drying of the Tat-AIE dot stock solution (2 mL) yielded 0.3 mg of powders. As the
Tat-AIE dots are stable in water, the density of the dot suspension could be estimated as ~1
g/cm3. As the average size of Tat-AIE dots determined from HR-TEM is ~29 nm, the
concentration of the Tat-AIE dots in stock can be calculated from the following equation:
Total number of Tat - AIE dots in 2 mL of suspension

0.3  10 -3 g
1g / mL
Total Volume of Tat - AIE dots

 2.3  1013
4
Average Volume of Each dot
  (14.5  10 -7 ) 3 mL
3
Finally, the concentration of Tat-AIE dots in stock solution was calculated as following:
2.3  1013
23
-1
[Tat - AIE dots]  6.02  10 -3mol  19 nM
2  10 L
Supplementary References
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S2. Banerjee, M., Emond, S. J., Lindeman, S. V. & Rathore, R. Practical synthesis of
unsymmetrical
tetraarylethylenes
and
their
application
for
the
preparation
of
[triphenylethylene-spacer-triphenylethylene] triads. J. Org. Chem. 72, 8054-8061 (2007).
S3. Yeh, H. C., Yeh, S. J. & Chen, C. T. Readily synthesised arylamino fumaronitrile for
non-doped red organic light-emitting diodes. Chem. Commun. 2632-2633 (2003).
S4. Palayangoda, S. S., Cai, X., Adhikari, R. M. & Neckers, D. C. Carbazole-based
donor-acceptor compounds: Highly fluorescent organic nanoparticles. Org. Lett. 10, 281-284
(2008).
S5. Xu, C. & Webb, W. W. Measurement of two-photon excitation cross sections of molecular
fluorophores with data from 690 to 1050 nm. J. Opt. Soc. Am. B 13, 481-491 (1996).
17
S6. Oulianov, D. A., Tomov, I. V., Dvornikov, A. S. & Rentzepis, P. M. Observations on the
measurement of two-photon absorption cross-section. Opt. Commun. 191, 235-243 (2001).
18