Highly luminescent, biocompatible ytterbium(iii) complexes as near-infrared fluorophores for living cell imaging

Article abstract of DOI:10.1039/c8sc00259b

Herein, we report the design and synthesis of biocompatible Yb³⁺ complexes for near-infrared (NIR) living cell imaging. Upon excitation at either the visible (Soret band) or red region (Q band), these β-fluorinated Yb³⁺ complexes display high NIR luminescence (quantum yields up to 23% and 13% in dimethyl sulfoxide and water, respectively) and have higher stabilities and prolonged decay lifetimes (up to 249 μs) compared to the β-non-fluorinated counterparts. This renders the β-fluorinated Yb³⁺ complexes as a new class of biological optical probes in both steady-state imaging and time-resolved fluorescence lifetime imaging (FLIM). NIR confocal fluorescence images showed strong and specific intracellular Yb³⁺ luminescence signals when the biocompatible Yb³⁺ complexes were uptaken into the living cells. Importantly, FLIM measurements showed an intracellular lifetime distribution between 100 and 200 μs, allowing an effective discrimination from cell autofluorescence, and afforded high signal-to-noise ratios as firstly demonstrated in the NIR region. These results demonstrated the prospects of NIR lanthanide complexes as biological probes for NIR steady-state fluorescence and time-resolved fluorescence lifetime imaging.

Full text of DOI:10.1039/c8sc00259b

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Highly Luminescent, Biocompatible Ytterbium(III) Complexes as
Near-Infrared Fluorophores for Living Cell Imaging
Yingying Ning,a Juan Tang,a Yi-Wei Liu,a Jing Jing*,b Yuansheng Sun,c and Jun-Long Zhang*,a
Received 00th January 20xx,
Accepted 00th January 20xx
Herein, we report the design and synthesis of biocompatible Yb3+ complexes for near-infrared (NIR) living cell imaging.
DOI: 10.1039/x0xx00000x
Upon excitation at either the visible (Soret band) or red region (Q band), these
NIR luminescence (quantum yields up to 23% and 13% in dimethyl sulfoxide and water, respectively) and possess higher
stabilities and elongated decay lifetimes (up to 249 μs) compared to the -non-fluorinated counterparts. This renders the
-fluorinated Yb3+ complexes as a new class of biological optical probes in both steady-state imaging and time-resolved
β
-fluorinated Yb3+ complexes displayed high
www.rsc.org/
β
β
fluorescence lifetime imaging (FLIM). NIR confocal fluorescence images showed the strong and specific intracellular Yb3+
luminescent signals when the biocompatible Yb3+ complexes were uptaken into the living cells. Importantly, FLIM
measurements showed an intracellular lifetime distribution between 100-200 μs, allowing an effective discrimination from
cell autofluorescence, and afforded high signal-to-noise ratio as firstly demonstrated in NIR region. These results
demonstrated the perspective of NIR lanthanide complexes as biological probes for the NIR steady-state fluorescence
imaging and time-resolved fluorescence lifetime imaging.
green (Cy),15 benzo-2,1,3-thiadiazole based molecules (FEB,16
IR-FGP,17 CH105518) and flavylium polymethine fluorophore
(Flav)19 series, but can often be accompanied by low quantum
Introduction
Near-infrared (700-1700 nm) optical imaging has received
increasing attention due to its ability to provide deeper tissue
penetration, better signal-to-noise ratio and higher imaging
resolution, which facilitates real-time visualization in clinical
diagnoses and surgical operation.1-5 In this field, the critical
chemical challenge is to develop NIR absorbent and emitting
probes with good biocompatibility, high brightness and
photostability. To meet this challenge, nanomaterials including
yields. Alternatively, encouraged by the success of lanthanide
(Ln) coordination chemistry in visible optical imaging (Eu3+,
Tb3+),20-24 inorganic chemists have envisaged to expand the
optical window to the NIR region by using NIR emissive Lns
such as Nd3+, Sm3+ and Yb3+. Toward this goal, Maury and co-
workers reported triazac-yclononane Sm3+ and Yb3+ complexes
for two-photon NIR-to-NIR imaging.25-27 Eliseeva and
coworkers developed
a
polynuclear Sm3+ dendrimer for
conjugated polymers,6 surface-modified carbon nanotubes,7
quantum dots,8,
9
combined visible and NIR cell imaging.28 Pecoraro, Petoud and
coworkers demonstrated Ln3+ (Ln = Nd, Yb) encapsulated
sandwich metallacrowns for combined cell fixation and
counter staining29 and necrotic cell imaging.30 Wong and co-
metal-organic frameworks10 and
upconversion nanoparticles,11 have been employed for in vivo
applications and demonstrated the advantages of NIR imaging
over traditional optical imaging in visible region. As
nanomaterials may suffer from uncertain cytotoxicity and non-
defined metabolism,12 the development of non-toxic and
highly luminescent molecules with NIR optical imaging
capabilities is of great importance. For small organic
fluorophores, the design of “donor-acceptor-donor” type
workers also prepared
a
series of NIR emissive Yb3+
complexes31-33 and Yb3+ porphyrinate with conjugated
rhodamine B for two-photon living cell imaging but in the
visible region.34 The features of NIR Ln complexes including
large stokes shifts, elemental characteristic emissions35-38 and
long decay lifetimes39-41 render them complementary to
organic fluorophores, but low quantum yield (<2.5%)25-34
arising from the forbidden f-f transitions and quench effect of
high vibration X-H bond (X = C, N and O) is still a general issue
remained to be addressed.
chromophores or
shift the absorption and emission to NIR region,13,
π-conjugation extensions can effectively red-
14
as
exemplified by organic molecules such as the indocyanine
To enhance the NIR emission of Yb3+, pioneer works have
a.Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare
Earth Materials Chemistry and Applications, College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, P. R. China..
43
been done by Seitz,42,
Charbonniere44 and others,45-47
including replacing the high vibration energy X-H bond (X = C,
O, N) with X-D/F bond, shortening the radiative lifetime of
lanthanide and tuning the ligand to lanthanide sensitization
process. Recently, we used perfluorinated porphyrins as
b.School of Chemistry, Beijing Institute of Technology, Beijing 100081, P. R. China.
c. ISS Inc, Champaign, IL 61822, USA.
Electronic Supplementary Information (ESI) available: [detailed photophysical and
cell experiment data; 1H and 19F NMR spectra, UV-visible absorption spectra; IR
spectra and MS spectra]. See DOI: 10.1039/x0xx00000x
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antenna ligand, and deuterated Kläui ligand as ancillary ligand under air-saturated conditions. They also possess much better
to construct the sandwiched Yb3+ complexes by removing the photostability, as compared to their
-hydrogenated
β
C-H bond close to the Yb3+ center.48 These Yb3+ complexes counterparts. As the excitation wavelength of the Yb3+
presented high NIR luminescence (900-1150 nm) with the complexes is below 700 nm, which might be disadvantage for
unprecedented quantum yields up to ca. 25% in CH2Cl2 (65% in thick tissue bioimaging, we herein demonstrated the first
CD2Cl2), long decay lifetimes of ca. 200 ꢀs in CH2Cl2 (700 ꢀs in biological application in NIR living cell imaging. NIR confocal
CD2Cl2) and large extinction coefficients in both visible and red imaging microscopy showed the strong and specific
2
regions (105-106 M-1cm-1 for Soret band and 104-105 M-1cm-1 luminescence assigned to the F5/2→2F7/2 transition of Yb3+ in
for Q band). These features render these Yb3+ complexes living cells upon excitation at ca. 408 and 600 nm, with
prospective NIR probes for both steady-state imaging and preferential localization in the lysosome. Intracellular FLIM
time-resolved fluorescence lifetime imaging (FLIM). However, measurements on the microsecond scale demonstrated the
endowing these Yb3+ complexes with biocompatibility is still use of Yb-1–5 as NIR FLIM probes with high signal-to-noise
challenging due to their low solubility and cellular uptake after ratio. Therefore, this work opens access to the design of
fluorination. Moreover, avoiding new X-H (X = C, N and O) biological NIR probes with capability of both steady-state
bonds close to Yb3+ to dramatically quench NIR luminescence is fluorescence and time-resolved fluorescence lifetime imaging.
also difficult in subsequent modifications. To address these
issues, we herein report the synthesis of biocompatible Yb3+
Results and discussion
Synthesis and characterization of Yb3+ complexes
We designed biocompatible Yb3+ complexes through three
synthetic strategies (Scheme 1). First, complexes Yb-1–3 were
obtained through modification of the meso-phenyl with water
complexes of
β
-fluorinated porphyrinates (Yb-1–5) through
-peripheral positions.
modification of the meso-phenyl and
β
Photophysical studies revealed that these Yb3+ complexes are
the brightest NIR emissive biological fluorophores up to date,
with quantum yields of 9-23% in DMSO and 5-13% in H2O and
decay lifetimes of 84-249 μs in DMSO and 56-173 μs in H2O
Scheme 1. Synthetic routes for biocompatible
2 | J. Name., 2012, 00, 1-3
β
-fluorinated Yb3+ complexes Yb-1–5 and
β
-hydrogenated analogues Yb-2c–5c studied in this work.
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soluble groups such as glycosyl, carboxyl and respectively. Upon excitation at the Soret or Q band, Yb-1–5
triphenylphosphonium, respectively. As previously reported, exhibited the strong and characteristic Yb3+ emission at ca.
2
the substitution of meso-phenyl groups has slight effects on 900−1200 nm, assigned to the F5/2→2F7/2 transition. The two
the Yb3+ NIR emission.48 As shown in scheme 1a, these peaks centered at ca. 918 and 957 nm are assigned as hot
complexes were synthesized from the condensation of water- bands as they dramatically decreased at low temperature (Fig.
soluble benzylaldehyde (or pentafluorobenzylaldehyde) and S72), while the other four are the f-f transition peaks. The
3,4-difluoropyrrole to porphyrin free bases. Consequent quantum yields (
reaction of ytterbium acetylacetonate hydrate Yb(acac)3 with in H2O under air-saturated condition, with long lifetimes (τobss)
-fluorinated porphyrin free bases afforded the desired Yb3+ of ca. 84-249 μs in DMSO and 56-173 μs in H2O, much higher
complexes Yb-1–3, after hydrolysis or ionization (see the than their -hydrogenated counterparts (ΦYb and τobs typically
below 2.5% and 38 μs in H2O, respectively). Taking Yb-4 as an
example (Fig. 1b), the Yb in H2O reaches 10%, with a τobs up to
ΦYb = 2.3%,
35 μs in H2O). With an increasing molecular
hydrophilicity (Table S1), a decreasing quantum yield and
C-F bonds, which is important to maintain the high NIR lifetime were observed due to solvation quenching. Among Yb-
luminescence. Further methylation of the amine to 1–5 Yb-2 showed the highest ΦYb (13%) and longest τobs (173
quaternary ammonium afforded Yb-4 49 19F-NMR spectrum μs) in H2O. In degassed H2O, Yb-1-4 and Yb-2c-4c exhibited the
and high-resolution electrospray ionization mass spectrometry decay lifetimes of 26-175 μs and quantum yields of 1.4-13%
demonstrated -C-F bond intact after 1,3-dipolar cycloaddition. (Table 1), similar to those in the air-saturated solutions. In
Third, as our continued interest in porpholactone,50 we degassed DMSO, the decay lifetimes and quantum yields are
reduced -lactone moiety to porpholactol, which is available slightly higher, within the average difference of 10%. However,
to attach a trimethyl-N-ethylammonium moiety, and afforded for Yb-5 and Yb-5c, the excited state lifetimes and NIR
Yb-5 (scheme 1c). As controls, we also synthesized -non- luminescence quantum yields increased dramatically in
fluorinated Yb3+ complexes
Yb-2c–5c based on degassed solution. For example, the decay lifetimes of Yb-5
tetraphenylporphyrin analogues following the similar were 84(4) μs in DMSO and 64(2) μs in water and increased to
procedures. To better compare -fluorination effect, all 130(6) and 78(3) μs, respectively, in the absence of oxygen.
complexes used deuterated Kläui’s ligand as ancillary ligand.51,
ΦYb) of Yb-1-5 are 9-23% in DMSO and 5-13%
β
β
details in the experimental section).
The second approach is 1,3-dipolar cycloaddition of
β
-
Φ
peripheral of Yb3+ perfluorinated porphyrin with azomethine 140 μs, which is 4-fold higher than that of Yb-4c
(
ylides (scheme 1b). The advantage of this approach is to τobs
directly functionalize -periphery without the elimination of
=
β
β-
a
,
.
β
β
β
(
)
β
52
1
Their structures were characterized by H, 19F-NMR, UV–vis
and IR spectroscopes and mass spectrometry. Detailed
synthetic procedures and characterizations are presented in
the experimental section and electronic supplementary
information (Figs. S1-60).
Photophysical properties of Yb3+ complexes in the NIR region
The absorption spectra of Yb-1–3 present similar profiles in
H2O with 0.1% DMSO (Fig. 1a) and display intense Soret bands
centered at ca. 410 nm and Q bands between 500-620 nm,
indicating the subtle effect of substitution of meso-phenyl
group on the electronic structures. Through
β-modification,
the Q(0,0) bands of Yb-4 and Yb-5 extend to 603 and 620 nm,
respectively, accompanied with increasing extinction
coefficients. This closely resembles the absorption spectra of
chlorin-type porphyrinoids with one saturated pyrrole ring.
Compared to Yb-1–5, β-hydrogenated counterparts display
similar absorption with slightly bathochromic shifts (Fig. S61).
NIR luminescence of Yb-1–5 and Yb-2c–5c were measured in
air-saturated and degassed dimethyl sulfoxide (DMSO) and
H2O, and the data are summarized in Table 1 and Figs. S62-71.
The quantum yields were determined using a comparative
method
with
the
reference
of
5,10,15,20-
tetraphenylporphyrin-Yb(III)-
Fig.
1 a) Normalized absorption spectra of Yb-1–5 in water with 0.1% DMSO; b)
[(cyclopentadienyl)tris(di(ethyl)phosphito)cobaltate]
(YbTPP(LOEt)), ΦΔ = 0.024 in CH2Cl2).53 Experimental relative
errors of the lifetimes and quantum yields are ±5% and ±10%
emission spectra of Yb-4 and Yb-4c in water (0.1% DMSO, λex = 410 nm, A410nm = 0.1).
Inset: decay curves of Yb-4 and Yb-4c in water monitored at 980 nm.
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Table 1. Decay lifetime (τobs) monitored at 980 nm and quantum yields (ΦYb) of Yb-1-5 and Yb-2c-5c in air-saturated and degassed DMSO and H2Oa,b
Comp.
DMSO
H2Oc
Air-equil.
Degassed
τobs (μs)
Air-equil.
Degassed
τobs(μs)
161(5)
249(10)
205(2)
173(4)
84(4)
Φ
Yb(%)
Φ
16(2)
23(3)
Yb (%)
τobs(μs)
95(2)
173(5)
56(2)
140(5)
64(2)
38(1)
25(1)
35(1)
32(2)
Φ
Yb (%)
τobs(μs)
96(3)
175(6)
58(3)
142(5)
78(3)
41(2)
26(2)
38(2)
42(3)
ΦYb (%)
7.7(0.5)
Yb-1
Yb-2
16(2)
23(2)
20(1)
17(2)
9.0(1)
165(4)
251(8)
213(5)
187(8)
130(6)
52(2)
7.6(0.4)
13(1)
13(1)
Yb-3
21(2)
5.1(0.2)
10(1)
5.2(0.3)
10(1)
Yb-4
18(2)
Yb-5
13(1)
6.0(0.3)
2.5(0.2)
1.4(0.1)
2.3(0.2)
1.6(0.2)
7.1(0.4)
2.6(0.2)
1.4(0.1)
2.4(0.1)
2.5(0.2)
Yb-2c
Yb-3c
Yb-4c
Yb-5c
52(2)
6.2(0.2)
5.6(0.2)
5.4(0.2)
4.0(0.3)
6.0(0.2)
5.6(0.2)
5.5(0.2)
5.6(0.2)
49(1)
51(2)
49(2)
50(2)
41(2)
52(2)
a
Standard error values are given in parentheses; they refer to the reproducibility of the measurements. The estimated uncertainties in the quantum yield are 15%.
b
Experimental relative errors: τobs, ±5%; ΦYb ±10%. Quantum yields were determined using a comparative method and referenced to Yb(TPP)(LOEt) (ΦΔ = 2.4% in
CH2Cl2). c Measured with 0.1% DMSO.
Stability in aqueous media
In DMSO (Fig. S73) and H2O (Fig. S74), the absorption of Yb3+
complexes showed negligible change after 48 h, indicating
they are stable and no decomposition was observed under
such conditions. We also measured the 2D NMR of Yb-4 in
CH3OH as an example (Fig. S75) and only one single peak was
observed. This demonstrated that there is no H/D exchange of
the deuterated ligand in protonated solvents. To further
demonstrate the potential ability of Yb3+ porphyrinates as
imaging agents, we investigated the stability of these Yb3+
complexes by monitoring the luminescence in the
presence/absence of light in water, phosphate buffer (PBS),
and fetal bovine serum (FBS). Interestingly, we found that β-
fluorinated compounds are more stable than their -non-
β
fluorinated analogues. Yb-4 showed high dark stability over 48
h (Fig. 2), and the NIR luminescence did not significantly
change over a long period of time under continuous irradiation
(405 nm laser, 0.2 Wcm-2). At pH= 5 (lysosome pH), Yb-4 was
photostable over
4 h, while Yb-4c showed obvious
photobleaching (55%). These results suggest that
fluorination enhances the stability of Yb3+ porphyrinates in
aqueous media.54
β-
Lipophilicity
Fig. 2 a) Spectroscopic evaluation of Yb-4 photostability in water, PBS and FBS with 0.1%
DMSO under irradiation of a 405 nm laser (0.2 Wcm-2
(inset: dark stability); b)
spectroscopic evaluation of Yb-4 and Yb-4c stabilities in pH = 5 PBS with 0.1% DMSO
under irradiation of
405 nm laser (0.2 Wcm-2). Emission intensity values were
)
To investigate the
logarithm of n-octanol/water partition coefficient (log P) for
Yb-1–5 and Yb-2c–5c were measured. As shown in Table S1,
fluorinated complexes exhibits the higher log P values than the
β-fluorination effect on the lipophilicity, the
a
referenced to the emission intensity of Yb3+ (980 nm) at 0 h.
β
-
corresponding
indicated that
β
-hydrogenated complexes. The results
β
-fluorination increases the lipophilicity of the
Cytotoxicity
Yb3+ complexes. Additionally, the log P values of Yb-3 and Yb-
3c are significantly lower than other single-charged complexes.
This provides useful information to understand the cellular
uptake and subcellular localization in the context.
We measured their cytotoxicity in HeLa cells. The cell viability
was over 80% in the dark even at a concentration of 20 μM
(Fig. S76). Upon light irradiation for 30 min (400–700 nm, 6.5
mW/cm2), Yb-1–4 showed negligible photocytotoxicity toward
HeLa cells at a concentration of 10 μM (cell viability > 80%),
4 | J. Name., 2012, 00, 1-3
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while Yb-5 showed an IC50 value (the dye concentration NIR steady-state fluorescence imaging
required to kill 50% of the cells) as low as 1.0 μM (Fig. S77).
Cell imaging experiments (Fig. 3) were performed at a 10 μM
concentration of Yb-1–4 using an Alba5 FLIM/FFS confocal
microscopy imaging system (ISS Inc., Champaign, Illinois, USA)
(Fig. S79). A stock solution of Yb3+ complex in chromatographic
grade, anhydrous DMSO and the concentration is 2 mM. The
solution was diluted to a final concentration of 10 μM by the
complete growth medium. The Yb3+ NIR signal was collected in
Singlet oxygen detection experiment revealed that
thephotocytotoxiciy of Yb-5 may be ascribed to the singlet
oxygen generation upon light irradiation (Fig. S78). This is
resulted from the inefficient energy transfer from the triplet
state of perfluorinated porpholactol (ca. 11494 cm-1)55 to the
excited state of Yb3+ (ca. 10250 cm-1).56-58 The singlet oxygen
quantum yield of Yb-5 was further estimated to be 0.36,
channel
1 using a single photon avalanche photodiode
referenced to the tetraphenylporphyrin (TPP,
ΦΔ=0.55 in
detector (SPAD) manufactured by Excelitas (model SPCM-
AQRH-15, quantum efficiency > 25% at 950 nm), while that
from LysoTracker® Green DND-26 was collected in channel 2
using a GaAsP photomultiplier tube detector manufactured by
Hamamatsu (Model H7422p-40). The detection wavelength of
SPAD is up to 1050 nm, with typically low dark noise (50 cps).
CHCl3),59 by the emission centered at approximately 1270 nm
derived from the 1Δg → 3Σg transition of 1O2 (Fig. S78). The
virtues of Yb-5 including strong absorption in the NIR region
and high singlet oxygen efficiency indicated its potential
application as
a dual-modal imaging and photodynamic
therapy probe in the future study.
Fig. 3 NIR confocal images performed on HeLa cells incubated with 10 μM of the corresponding Yb3+ complexes for 12 h followed by 30 min incubation with 75 nM LysoTracker
Green. a) Bright field; b) NIR signal arising from Yb3+ in channel 1 (λex, 408 nm; λem, 935/170 nm bandpass); c) visible signal arising from LysoTracker Green in channel 2 (λex, 470 nm;
λem, 530/43 nm bandpass); d) merged b and c showing colozalisation (P = 0.75 for Yb-2, 0.78 for Yb-3, 0.67 for Yb-4, 0.29 for Yb-4c ). Scale bar: 10 μm.
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As shown in Fig. 3, upon excitation at Soret band (408 nm laser, Further modifications are required to achieve different
µWcm-2), we observed intense intracellular NIR intracellular localization profile.60-63 As controls, incubation
4
luminescence in channel 1 using 935/170 nm bandpass for Yb- HeLa cells with Yb-2c–4c showed much less intense NIR
2
2–4, assigned to F5/2→2F7/2 transition from Yb3+. However, we luminescence than Yb-2–4, as shown in Figs. 3 and S81, even
could not observe such luminescence for Yb-1 (Fig. S80). The ICP showed much higher cellular uptake of Yb-2c–4c than Yb-
results of inductively coupled plasma spectrometry (ICP) 2–4 (Table S2). These results suggested that
showed that 0.1-0.2 µg/mL Yb3+ can be detected for Yb-2–4
improves NIR luminescence in living cells, consistent to the in
while <0.02 µg/mL for Yb-1 (Table S2), suggesting that Yb-1 vitro photophysical properties.
could not be cellular uptaken under this condition. The exact
The excitation wavelength for the Yb3+ complexes could be
β-fluorination
,
mechanism why Yb-1 could not enter living cells is not clear, further extended to the red region upon excitation at Q band
but the results indicated the importance of choice of (Fig. S82). Taking Yb-4 as an example (Fig. 4), we also observed
biocompatible conjugates on the design of Yb3+ NIR probes.
The images of Yb-2–4 show high signal-to-noise ratios, as localization with LysoTracker® Green under 620 nm excitation
high NIR intracellular luminescent signals with good co-
the background fluorescence is dramatically reduced in the (~ 4 µWcm-2). This reveals that the excitation wavelength can
NIR region. These complexes preferentially colocalized in the be extended from visible to far red region by biomimetic
lysosome, as revealed by good colocalization of NIR modification.
β-
fluorescence with the fluorescence of lysosome tracker.
Fig. 4 NIR confocal images performed on living HeLa cells incubated with 10 μM Yb-4 for 12 h followed by 30 min incubation with 75 nM LysoTracker Green. a) Bright field; b) NIR
signal arising from Yb3+ in channel 1 (λex, 620 nm; λem, 935/170 nm bandpass); (c) visible signal arising from LysoTracker Green in channel 2 (λex, 470 nm; λem, 530/43 nm bandpass);
d) merged b and c showing colocalization (P = 0.52). Scale bar: 10 μm.
with different viscosity and polarity under air-saturated and
degassed condition as an example.69
NIR time-resolved fluorescence lifetime imaging
Time-resolved FLIM has been widely used in biomedical
sciences fields, as it is insensitive to the concentration of
fluorophore, excitation intensity, and photobleaching. This
allows visualizing cellular events that are difficult to observe by
steady-state intensity imaging.64, 65 Although the application of
luminescent lanthanides such as Eu3+ and Tb3+ as visible
emitter in FLIM have been extensively demonstrated,23, 24, 66-68
FLIM in NIR region still remained unexplored so far. Given the
long lifetimes and high luminescence of
β
-fluorinated Yb3+
complexes, they are potential probe candidates for NIR FLIM
on the μs scale, which can facilitate discrimination from cell
autofluorescence (ns) and enhance signal-to-noise ratios.
In this work, confocal FLIM experiments were performed
using 408 nm pulsed laser excitation (Figs. S83-84) and the
details were described in experimental section. As shown in Fig.
5, Yb-4 shows a lifetime distribution between 100-200 μs in
cells, much longer than that of Yb-4c (20-40 μs). More
importantly, the lifetime of Yb-4 varies in different subcellular
locations, probably due to the different intracellular
microenvironments such as the polarity, viscosity and
lipophilicity.
Fig. 5 NIR time-resolved images of living HeLa cells incubated with 10 μM Yb-4 and Yb-
4c (λex, 408 nm; λem, 935/170 nm bandpass; dwell time, 4 ms). Scale bar: 10 μm.
Table 2. Decay lifetime (τobs) of Yb-4 monitored at 980 nm in different solvents (λex
= 410 nm).a
τobs (μs)
Nb
ET
Solvent
Viscosity (cP)
Air-equil.
Degassed
Methanol
Ethanol
0.54
1.02
4.31
2.04
934
0.765
0.655
0.602
0.552
—
68(2)
86(4)
74(3)
99(2)
t-butanol
i-Propanol
Glycerol
117(4)
110(5)
192(4)
127(3)
120(3)
195(5)
To verify the hypothesis that NIR decay lifetime of Yb-4 is
sensitive to intracellular microenvironments, we investigated
the decay lifetimes of Yb-4 (Table 2) in a variety of solvents
a
Standard error values are given in parentheses; they refer to the
reproducibility of the measurements. Experimental relative errors: τobs, ±5%. b
Refers to Reichardt’s solvent polarity parameter.70
6 | J. Name., 2012, 00, 1-3
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As shown in Table 2, experimental results showed that the polyformaldehyde, were purchased from J&K Scientific and
degassed solvents slightly elongated the decay lifetime of Yb3+ used as received. Anhydrous CH2Cl2 was distilled from calcium
(ca. 10%). The excited state lifetimes in such solvents also hydride and H2O were obtained from Milli-Q integral.
exhibited close correlation with the changes of solvent
viscosity (Fig. S85) and polarity (Fig. S86), even in degassed Benzaldehyde, Kläui’s ligand and porphyrinates
solvents. Additionally, we further measured the Yb3+ lifetimes
4-(2,3,4,6-Tetraacetyl-glucopyranobseynlz)aldehyde75 and p-
chloromethylbenzaldehyde76 was synthesized according to
in water with the addition of bovine albumin (BSA). In the
presence of BSA (Table S3 and Fig. S87), the Yb3+ luminescence
literature. Other modified-benzaldehyde were all purchased
intensity and decay lifetime both enhanced. This may be due
from J&K Scientific and used as received.
β-octafluorinated
ytterbium porphyrin (YbF28TPP),48 β-hexafluorinated ytterbium
to the increased hydrophobic inclusion and decreased solvent
quenching effect after protein association,71 while quenching
of the excited state of chromophore through charge transfer
process have not been observed.62, 63, 72 These results revealed
that the solvation effect is complicated herein and it seems
that not just only one factor influences the Yb3+ luminescence
lifetime. The viscosity, polarity and oxygen concentration may
all influence the ground and excited states of the porphyrin
and Yb3+, and thus varies the Yb3+ luminescence lifetime.69, 72-74
porpholactone
[(cyclopentadienyl)tris(di(methyl-d3)phosphito)
(YbF26TPPL),50
sodium
cobaltate]
(NaLOCD3) (D atom > 99%)52 were synthesized according to
literature methods.
Synthesis of Yb-1
Yb-1 was synthesized from 2,3,7,8,12,13,17,18-octafluoro-
5,10,15,20-tetrakis[4-(2,3,4,6-tetraacetyl-glucosyl)-
phenyl]porphyrin (L1) (Scheme S1). The synthesis of L1 was
75
carried out according to similar procedures in literature.
Conclusions
2,6,-Difluoropyrrole (103 mg, 1.0 mmol) and 4-(2,3,4,6-
tetraacetyl-glucopyranobseynlz)aldehyde (453 mg, 1.0 mmol)
were dissolved in 250 mL methylene chloride. The mixture was
stirred for 10 min after which a few drops of BF3`Et2O was
added. The mixture was then stirred overnight at room
temperature. 2,3-Dicyano-5,6-dichlorobenzoquinone (DDQ,
0.23 g, 1.0 mmol) was added. After reaction for 1 h, silica gel
(2g) was added to the dark solution and all solvent was
evaporated. The absorbed products were placed on the top of
a silica gel column. The product was obtained by using ethyl
acetate/petroleum ether (v/v = 2:1) as eluent. Then L1 (200
mg, 0.2 mmol), Yb(acac)3•3H2O (500 mg, 1 mmol) and 1,2,4-
trichlorobenzene (TCB) were added to a Schlenk tube and
refluxed overnight. After cooling to room temperature, the
reaction mixtures were transferred to a silica column, TCB was
first eluted by petroleum ether, and then the unreacted free
base ligand was eluted by CH2Cl2, corresponding Yb3+ complex
was obtained by using CH2Cl2/MeOH (v/v = 5:1) as eluent and
used directly to the next step. The obtained Ln complexes
(acac as ancillary ligand) and 3 equiv. NaLOCD3 were dissolved in
mixing solvent of CHCl3/CH3OH (v/v = 1:1, 5 mL). The mixture
was refluxed for 8 h. After cooling to room temperature, the
reaction mixtures were transferred to a silica column, L1-
Taken together, we reported three synthetic approaches to
biocompatible Yb3+ complexes, starting from the extremely
hydrophobic Yb3+ perfluorinated porphyrinates, for NIR living
cell imaging. These
β
-fluorinated Yb3+ probes possess the
following features: 1) high luminescence (5-13% quantum
yields in H2O), 2) enhanced stabilities, and 3) long decay
lifetimes (>100 us), which are advantageous for FLIM on the
microsecond scale. Combined with the additional advantages
such as tunable excitation wavelength (visible to far red region)
and large extinction coefficients of porphyrinoids ligands, they
are perspective candidates for NIR molecular probes for real-
time visualization in clinical diagnoses and surgical operations,
which is currently under investigation in our laboratory. This
work paves the way for molecular probes for NIR imaging in
the range of 900-1100 nm biological window by constructing
NIR Ln molecular complexes.
Experimental Section
General information
Unless otherwise stated, all reactions were performed under
an inert atmosphere of nitrogen. UV-vis spectra were recorded
on an Agilent 8453 UV-vis spectrometer equipped with an
Yb(III)-LOCD
(
Yb-L1
)
was obtained by using ethyl
3
acetate/petroleum ether (v/v = 4:1) as eluent. Then Yb-L1 and
o
o
Agilent 89090A thermostat (±0.1 C) at 25 C. IR spectra were
recorded on a Spectrum Spotlight 200 FT-IR microscopy. Mass
spectra were recorded on Bruker APEX IV FT-ICR mass
spectrometer (ESI) or AB Sciex MALDI-TOF mass spectrometer.
Simulated mass spectra were obtained at the website
http://www.chemcalc.org. NMR spectra were recorded on
Bruker ARX400 400MHz or AVANCE III 500MHz NMR
spectrophotometer. For the optical measurements in liquid
solution, spectroscopic-grade dimethyl sulfoxide was used as
purchased from Alfa-Aesar. Anhydrous 1,2,4-trichlorobenzene
(TCB), diisobutyl aluminium hydride (DIBAL-H), sarcosine,
CH3ONa (10 mg) was dissolved in mixing solvent of
CH2Cl2/CH3OH (v/v
= 1:1, 10 mL) and stirred at room
temperature for 3 h. Then the reaction mixtures were
transferred to a silica column, the product Yb-1 was obtained
by using CH3OH as eluent.
Synthesis of Yb-2
Yb-2 was synthesized from 2,3,7,8,12,13,17,18-octafluoro-5-
pentafluorophenyl-10,15,20-tri[(methyl-p-formyl)-phenyl]
porphyrin (L2) (Scheme S2). The synthesis procedure of L2 and
L2-Yb(III)-LOCD
(
Yb-L2) are similar to that described for L1 and
3
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Yb-L1 but the starting materials are 2,6,-difluoropyrrole (103 The synthetic procedures for Yb-2c were similar to that for Yb-
mg, 1.0 mmol), pentafluorobenzaldehyde (147 mg, 0.75 mmol) , starting from 5-pentafluorophenyl-10,15,20-tri[(methyl-p-
and methyl p-formylbenzoate (41 mg, 0.25 mmol). Then Yb-L2 formyl)-phenyl] porphyrin (L2c). The synthesis procedure of
was dissolved in mixing solvent of tetrahydrofuran/CH3OH (v/v L2c and L2c-Yb(III)-LOCD Yb-L2c) are similar to that described
2
(
3
= 1:1, 10 mL), with the addition of 1 mL 10% KOH solution. The for L2 and Yb-L2 but the starting materials are pyrrole (67 mg,
reaction mixtures were stirred at 50 oC overnight and the 1.0 mmol), pentafluorobenzaldehyde (147 mg, 0.75 mmol) and
solution was neutralized with 0.2 M HCl. After evaporating of methyl p-formylbenzoate (41 mg, 0.25 mmol) (Scheme S6).
the organic solvent, the product Yb-2 was obtained by
centrifugalization and further recrystallization from CH3OH.
Synthesis of Yb-3c
The synthetic procedures for Yb-3c were similar to that for Yb-
Synthesis of Yb-3
3
.
The starting 5,10,15,20-tetrakis(p-chloromethylpheny1)
porphyrin L3c was synthesized according to literature
methods76 and the intermediate product L3c-Yb(III)-NaLOCD3
(
)
Yb-3 was synthesized from 2,3,7,8,12,13,17,18-octafluoro-
5,10,15,20-tetrakis(p-chloromethylphenyl) porphyrin
(
L3
)
was synthesized similarly to Yb-L3 (Scheme S7).
(
(
Scheme S3). The synthesis procedure of L3 and L3-Yb(III)-LOCD
3
Yb-L3) are similar to that described for L1 and Yb-L1 but the
Synthesis of Yb-4c
starting materials are 2,6,-difluoropyrrole (103 mg, 1.0 mmol)
and p-chloromethylbenzaldehyde (154 mg, 1.0 mmol). Then The synthetic procedures for Yb-4c were similar to that for Yb-
Yb-L3 and excess of triphenylphosphine (50 mg) was dissolved The starting porphyrin was synthesized according to
in N,N-dimethylformamide and refluxed for 24 h. Then the literature methods31, 41 and the intermediate product Yb-L4c
4.
solvent was removed and the product Yb-3 was obtained by was synthesized similarly to Yb-L4 (Scheme S8).
recrystallization from ice methanol.
Synthesis of Yb-5c
Synthesis of Yb-4
The synthetic procedures for Yb-5c were similar to that for Yb-
. The starting ytterbium porpholactone was synthesized
according to literature methods50 and the intermediate
5
A toluene solution of the YbF28TPP (50 mg, 0.025 mmol),
sarcosine (43 mg, 0.5 mmol), and polyoxymethylene (40 mg)
was heated at reflux for 24 h.49, 77 After being cooled to room
temperature, all solvent was evaporated. The reaction
products were applied on the top of a silica gel column, and
product Yb-L5c was obtained similarly to Yb-L5 (Scheme S9).
Photphysical properties measurements
Yb-L4 was obtained by using ethyl acetate/petroleum ether Emission, excitation spectra and lifetime were measured on an
(v/v = 1:5) as eluent. Then Yb-L4 was dissolved in 10 mL Edinburgh Analytical Instruments FLS980 lifetime and steady
toluene, with the addition of 2 mL CH3I. The reaction mixtures state spectrometer equipped with a 450 W Xe lamp, a 60 W
were stirred at 50 oC for 48 h, then the product (Yb-4) was microsecond flash lamp, PMT R928 for visible emission
obtained by filtration and washing with toluene and hexane spectrum, HAMAMATSU R5509-73 PMT with C9940-02 cooler
(
Scheme S4).
for NIR emission spectrum and luminescence lifetime.
Excitation and emission spectra were corrected for
instrumental functions (including the correction for detector,
gratings etc). All luminescence decays were exponentially tail-
fitted by monoexponential functions without deconvolution
for the negligible instrumental reference function in the NIR
region.
Synthesis of Yb-5
The YbF26TPPL (50 mg, 0.025 mmol) was dissolved in dry CH2Cl2
(10 mL) and the solution is cooled to 78 oC. DIBAL-H (20% in
hexane, 0.2 mL, 7.0 equiv) was added by syringe and the
reaction mixture was warmed to room temperature and
allowed to stir for an additional hour.78 The reaction was then
quenched by addition of a few drops of H2O and evaporated to
Quantum yields determination
dryness. The Yb3+ porpholactol product Yb-L5 were obtained Quantum yields in solution were determined using
by using CH2Cl2/petroleum ether (v/v = 2:1) as eluent. Then Yb- comparative method and the equation:
L5 and excessive 2-bromoethanol were dissolved in dry CH2Cl2 Φs
r=(Gs/Gr)*(ηs2/ηr2), where the subscripts r and s denote
(10 mL) and few drops of BF3·Et2O was added. The reaction reference and sample respectively, is the quantum yield, G
/
Φ
Φ
was allowed to stir for 24 h at room temperature. Then the is the slope from the plot of integrated emission intensity vs
reaction solution was evaporated and the obtained solid was absorbance, and η is the refractive index of the solvent.79 The
dissolved in CH3CN, with the addition of 1 mL trimethylamine. reference was YbTPP(LOEt) in CH2Cl2 (ΦΔ = 0.024).53 Generally,
o
The reaction was refluxed at 90 C overnight. After cooling to YbTPP(LOEt) and solution of Yb3+ complex with 4 different
room temperature, the reaction solution was evaporated and concentration was firstly prepared in corresponding solvents.
the product (Yb-5) was obtained by using CH2Cl2/CH3OH (v/v = The absorbance of all the solutions at 410 nm were recorded
5:1) as eluent (Scheme S5).
on an Agilent 8453 UV-vis spectrometer equipped with an
o
Agilent 89090A thermostat (± 0.1 C) and NIR emissions were
Synthesis of Yb-2c
recorded on an Edinburgh Analytical Instruments FLS980
8 | J. Name., 2012, 00, 1-3
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lifetime and steady state spectrometer with the excitation CV stands for the viability of cells, As, Ac and Ab stand for the
wavelength at 410 nm under an identical condition. According absorbance of cells containing the studied complexes, cell
to the ratio of the slope GYb-x/Gr (x = 1-5, 2c-5c), the relative control (no treated cells) and blank control (wells containing
quantum yield of Yb3+ complexes could be obtained according neither cells nor the studied complexes).
to the equation described above. The absorbance of all the
samples and references are below 0.1 and absorbance values
Light-induced cytotoxicity assay
undergo background correction by subtracting average over
HeLa cells were seeded in flat-bottomed 96-well plates, 104
range from 800-820 nm. The integrated emission intensity
which integrate from 880 nm to 1150 nm have been
subtracting the blank (integrated emission intensity with the
only pure solvent under an identical condition). The estimated
error for the quantum yield measurements is 15%. For the
quantum yield measured in degassed solution, solutions of
Yb3+ complexes with 4 different concentration were degassed
via 5 freeze-pump-thaw cycles. Then the measurement and
calculation were the same as above.
cells per well, with 200 μL complete culture media in the dark
for 24 h. Cells were incubated with varying concentrations
from 0 to 16 μΜ complexes for another 24 h in the dark while
wells containing no cells are set as the controls. After washed
three times with PBS, the cells were irradiated for 30 min in
100 μL PBS under the light irradiation (400–700 nm) with the
same dose of light (6.5 mW/cm2) for 30 min. Then PBS was
replaced by 200 μL fresh culture media. After cultured for 24 h,
the cells were washed three times with PBS. Then 10 μL Cell
Counting Kit-8 (CCK-8) solution and 90 μL PBS were added per
well. After 2 hours, the absorbance at 450 nm was read by 96-
well plate reader. The viability of HeLa cells was calculated by
using Equation (2) (see above).
Singlet oxygen quantum yield measurement
Measurements were taken at 410 nm excitation in air-
saturated solutions at room temperature with TPP (ΦΔ = 0.55)
in CHCl3 as reference.59 The maxima absorption of the
sensitizer at corresponding wavelength was generally kept
below 0.2.
Inductively coupled plasma (ICP) spectrometry experiments for
cellular uptake quantification
To quantify the concentration of in cells, 1 × 106 cells were
seeded in a six-well microplate. After 36 h of attachment, the
cells were incubated with 10 μM of corresponding Yb3+
complexes for 12 h at 37 °C. Cells were trypsinized and
resuspended in nitric acid overnight. Then the solution was
heated at 100°C for 4 h before adding water to achieve a final
volume of 10 mL. The measurements were taken on an
inductively coupled plasma-atomic emission spectrometer
(Prodigy 7, Leeman).
Dermination of the octanol-water partition coefficients (log P)
Equal volume (2000 mL) of n-octanol and water were
thoroughly mixed by an oscillator and separated after 24 h.
Yb3+ complex (0.1 mg each) was then dissolved in 40 mL of the
separated n-octanol and the solution and 40 mL of water
(previously separated from the mixture) was added. The new
water-octanol system was allowed to equilibrate for additional
24h. After separating, both fractions were analyzed by UV-vis
spectra. The log P values were calculated by the following
equation80
Colocalization assay
log P = log (Coctanol/Cwater
)
(1)
where Coctanol and Cwater refer to the concentration of Yb3+
HeLa cells were placed onto 0.1 mM poly-D-lysine coated
glasses in complete media and the cells were incubated for 24
h. A stock solution of Yb3+ complex in chromatographic grade,
anhydrous DMSO was prepared as 2 mM. The solution was
diluted to a final concentration of 10 μM by complete growth
medium. Stock solutions of Lyso Tracker Green DND-26 were
prepared as 1 mM, and the stock solution was diluted to the
working concentrations in complete medium (Lyso Tracker: 75
nM). After incubation of 10 μM Yb3+ complex for 12 h followed
by 30 min incubation with 75 nM solution of lysotracker green,
cells were washed with PBS buffer twice before confocal
experiments.
complexes in the n-octanol and water, respectively.
Cell culture
All HeLa cells were incubated in complete medium (Dulbecco’s
modified Eagle’s Medium, supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin-streptomycin) at 37 °C in
atmosphere containing 5% CO2.
Dark cytotoxicity assay
HeLa cells were seeded in flat-bottomed 96-well plates, 104
cells per well, with 200 μL complete culture media in the dark
for 24 h. Cells were incubated with 20 μΜ complexes for
another 24 h in the dark while wells containing no cells are set
as the controls. After washed three times with PBS, 10 μL Cell
Counting Kit-8 (CCK-8) solution and 90 μL PBS were added per
well. After 2 hours, the absorbance at 450 nm was read by 96-
well plate reader. The viability of HeLa cells was calculated by
the following equation:
NIR confocal images
The ISS Alba5 FLIM/FFS confocal system (ISS Inc.) was used to
acquire the confocal images. The system was attached the
Nikon TE2000 inverted microscope, equipped with the Nikon
60X / 1.2 NA water immersion objective lens. Both steady-
state and time-resolved (lifetime) confocal images were
acquired by the ISS VistaVision software and the ISS FastFLIM
CV = (As – Ab)/(Ac – Ab)×100%
(2)
data acquisition unit.
A
408-nm diode laser or YSL
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